Methods for cancer detection and monitoring by means of personalized detection of circulating tumor DNA

Abstract

The invention provides methods for detecting single nucleotide variants in breast cancer, bladder cancer, or colorectal cancer. Additional methods and compositions, such as reaction mixtures and solid supports comprising clonal populations of nucleic acids, are provided. For example, provided here is a method for monitoring and detection of early relapse or metastasis of breast cancer, bladder cancer, or colorectal cancer, comprising generating a set of amplicons by performing a multiplex amplification reaction on nucleic acids isolated from a sample of blood or urine or a fraction thereof from a patient who has been treated for a breast cancer, bladder cancer, or colorectal cancer, wherein each amplicon of the set of amplicons spans at least one single nucleotide variant locus of a set of patient-specific single nucleotide variant loci associated with the breast cancer, bladder cancer, or colorectal cancer; and determining the sequence of at least a segment of each amplicon of the set of amplicons that comprises a patient-specific single nucleotide variant locus, wherein detection of one or more patient-specific single nucleotide variants is indicative of early relapse or metastasis of breast cancer, bladder cancer, or colorectal cancer.

Description

(19) *EP004737593A2* (11) EP 4 737 593 A2 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: 06.05.2026 Bulletin 2026 / 19 (21) Application number: 25225504.7 (22) Date of filing: 12.04.2019 (51) International Patent Classification (IPC): C12Q 1 / 6886 (2018.01) (52) Cooperative Patent Classification (CPC): C12Q 1 / 6886; C12Q 2600 / 106; C12Q 2600 / 118; C12Q 2600 / 156 (84) Designated Contracting States: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR (30) Priority: 14.04.2018 US 201862657727 P 09.05.2018 US 201862669330 P 03.07.2018 US 201862693843 P 06.08.2018 US 201862715143 P 16.10.2018 US 201862746210 P 11.12.2018 US 201862777973 P 12.02.2019 US 201962804566 P (62) Document number(s) of the earlier application(s) in accordance with Art. 76 EPC: 19721181.6 / 3 781 714 (71) Applicant: Natera, Inc. San Carlos, CA 94070 (US) (72) Inventors: • ZIMMERMANN, Bernhard San Carlos, California 94070 (US) • SALARI, Raheleh San Carlos, California 94070 (US) • SWENERTON, Ryan San Carlos, California 94070 (US) • WU, Hsin-Ta San Carlos, California 94070 (US) • SETHI, Himanshu San Carlos, California 94070 (US) (74) Representative: Marks & Clerk LLP 15 Fetter Lane London EC4A 1BW (GB) Remarks: •This application was filed on 19‑12‑2025 as a divisional application to the application mentioned under INID code 62. •Claims filed after the date of filing of the application / after the date of receipt of the divisional application (Rule 68(4) EPC). (54) METHODS FOR CANCER DETECTION AND MONITORING BY MEANS OF PERSONALIZED DETECTION OF CIRCULATING TUMOR DNA (57) The invention provides methods for detecting single nucleotide variants in breast cancer, bladder can- cer, or colorectal cancer. Additional methods and com- positions, such as reaction mixtures and solid supports comprising clonal populations of nucleic acids, are pro- vided. For example, provided here is a method for mon- itoring and detection of early relapse or metastasis of breast cancer, bladder cancer, or colorectal cancer, com- prising generating a set of amplicons by performing a multiplex amplification reaction on nucleic acids isolated fromasampleof bloodor urineor a fraction thereof froma patient who has been treated for a breast cancer, bladder cancer, or colorectal cancer, wherein each amplicon of the set of amplicons spans at least one single nucleotide variant locus of a set of patient-specific single nucleotide variant loci associated with the breast cancer, bladder cancer, or colorectal cancer; and determining the se- quence of at least a segment of each amplicon of the set of amplicons that comprises a patient-specific single nucleotide variant locus, wherein detection of one or more patient-specific single nucleotide variants is indi- cative of early relapse or metastasis of breast cancer, bladder cancer, or colorectal cancer. EP 4 73 7 59 3 A 2 Processed by Luminess, 75001 PARIS (FR) (Cont. next page) 2 EP 4 737 593 A2 Description CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 62 / 657,727 filed April 14, 2018; U.S. Provisional Application No. 62 / 669,330 filed May 9, 2018; U.S. Provisional Application No. 62 / 693,843 filed July 3, 2018; U.S. Provisional Application No. 62 / 715,143 filed August 6, 2018; U.S. Provisional Application No. 62 / 746,210 filed October 16, 2018;U.S. Provisional ApplicationNo. 62 / 777,973 filedDecember 11, 2018; andU.S. Provisional Application No. 62 / 804,566 filed February 12, 2019. Each of these applications cited above is hereby incorporated by reference in its entirety. BACKGROUND OF THE INVENTION

[0002] Detection of early relapse or metastasis of cancers has traditionally relied on imaging and tissue biopsy. The biopsy of tumor tissue is invasive and carries risk of potentially contributing to metastasis or surgical complications, while imaging-based detection is not sufficiently sensitive to detect relapse or metastasis in an early stage. Better and less invasive methods are needed for detecting relapse or metastasis of cancers. SUMMARY OF THE INVENTION

[0003] One aspect of the invention described herein relates to amethod for monitoring and detection of early relapse or metastasis of cancer (e.g., breast cancer, bladder cancer, or colorectal cancer), comprising generating a set of amplicons by performing a multiplex amplification reaction on nucleic acids isolated from a sample of blood or urine or a fraction thereof fromapatientwhohasbeen treated foracancer (e.g., breast cancer, bladder cancer, or colorectal cancer),wherein each amplicon of the set of amplicons spans at least one single nucleotide variant locus of a set of patient-specific single nucleotide variant loci associated with the cancer (e.g., breast cancer, bladder cancer, or colorectal cancer); and determining the sequence of at least a segment of each amplicon of the set of amplicons that comprises a patient- specific single nucleotide variant locus,wherein detection of one ormore (or two ormore, or three ormore, or four ormore, or five or more, or six or more, or seven or more, or eight or more, or nine or more, or ten or more) patient-specific single nucleotide variants is indicative of early relapse or metastasis of cancer (e.g., breast cancer, bladder cancer, or colorectal cancer).

[0004] In addition to breast cancer, bladder cancer, and colorectal cancer, the methods described herein can also be used for monitoring and detection of early relapse or metastasis of other types of cancer, such as: acute lymphoblastic leukemia; acute myeloid leukemia; adrenocortical carcinoma; AIDS-related cancers; AIDS-related lymphoma; anal cancer; appendix cancer; astrocytomas; atypical teratoid / rhabdoid tumor; basal cell carcinoma; brain stem glioma; brain tumor (including brain stem glioma, central nervous system atypical teratoid / rhabdoid tumor, central nervous system embryonal tumors, astrocytomas, craniopharyngioma, ependymoblastoma, ependymoma, medulloblastoma, medulloe- pithelioma, pineal parenchymal tumors of intermediate differentiation, supratentorial primitive neuroectodermal tumors and pineoblastoma); bronchial tumors; Burkitt lymphoma; cancer of unknown primary site; carcinoid tumor; carcinoma of unknown primary site; central nervous system atypical teratoid / rhabdoid tumor; central nervous system embryonal tumors; cervical cancer; childhood cancers; chordoma; chronic lymphocytic leukemia; chronic myelogenous leukemia; chronicmyeloproliferative disorders; colon cancer; craniopharyngioma; cutaneousT-cell lymphoma; endocrine pancreas islet cell tumors; endometrial cancer; ependymoblastoma; ependymoma; esophageal cancer; esthesioneuroblastoma; Ewing sarcoma; extracranial germ cell tumor; extragonadal germ cell tumor; extrahepatic bile duct cancer; gallbladder cancer; gastric (stomach) cancer; gastrointestinal carcinoid tumor; gastrointestinal stromal cell tumor; gastrointestinal stromal tumor (GIST); gestational trophoblastic tumor; glioma; hairy cell leukemia; head and neck cancer; heart cancer; Hodgkin lymphoma; hypopharyngeal cancer; intraocular melanoma; islet cell tumors; Kaposi sarcoma; kidney cancer; Langerhans cell histiocytosis; laryngeal cancer; lip cancer; liver cancer; malignant fibrous histiocytoma bone cancer; medulloblastoma; medulloepithelioma; melanoma; Merkel cell carcinoma; Merkel cell skin carcinoma; mesothelioma; metastatic squamous neck cancer with occult primary; mouth cancer; multiple endocrine neoplasia syndromes; multiple myeloma; multiple myeloma / plasma cell neoplasm; mycosis fungoides; myelodysplastic syndromes; myeloproliferative neoplasms; nasal cavity cancer; nasopharyngeal cancer; neuroblastoma; Non-Hodgkin lymphoma; nonmelanoma skin cancer; non-small cell lung cancer; oral cancer; oral cavity cancer; oropharyngeal cancer; osteosarcoma; other brain and spinal cord tumors; ovarian cancer; ovarian epithelial cancer; ovarian germ cell tumor; ovarian low malignant potential tumor; pancreatic cancer; papillomatosis; paranasal sinus cancer; parathyroid cancer; pelvic cancer; penile cancer; pharyngeal cancer; pineal parenchymal tumorsof intermediate differentiation; pineoblastoma; pituitary tumor; plasmacell neoplasm / multiple myeloma; pleuropulmonary blastoma; primary central nervous system (CNS) lymphoma; primary hepatocellular liver cancer; prostate cancer; rectal cancer; renal cancer; renal cell (kidney) cancer; renal cell cancer; 3 EP 4 737 593 A2 5 10 15 20 25 30 35 40 45 50 55 respiratory tract cancer; retinoblastoma; rhabdomyosarcoma; salivary gland cancer; Sezary syndrome; small cell lung cancer; small intestine cancer; soft tissue sarcoma; squamous cell carcinoma; squamous neck cancer; stomach (gastric) cancer; supratentorial primitive neuroectodermal tumors; T-cell lymphoma; testicular cancer; throat cancer; thymic carcinoma; thymoma; thyroid cancer; transitional cell cancer; transitional cell cancer of the renal pelvis and ureter; trophoblastic tumor; ureter cancer; urethral cancer; uterine cancer; uterine sarcoma; vaginal cancer; vulvar cancer; Waldenstrom macroglobulinemia; or Wilm’s tumor.

[0005] In someembodiments, nucleic acids are isolated froma tumor of the patient and somaticmutations are identified in the tumor for the set of patient-specific single nucleotide variant loci before determining the sequence of at least a segment of eachampliconof the set of amplicons for the sampleof bloodor urineor fraction thereof, andwherein thesingle nucleotide variants .

[0006] In someembodiments, themethodcomprisingcollectingandsequencingbloodor urinesamples from thepatient longitudinally.

[0007] In some embodiments, at least 2 or at least 5 SNVs are detected and the presence of the at least 2 or at least 5 SNVs is indicative of early relapse or metastasis of breast cancer, bladder cancer, or colorectal cancer.

[0008] In some embodiments, the breast cancer, bladder cancer, or colorectal cancer is a stage 1 or stage 2 breast cancer, bladder cancer, or colorectal cancer. In some embodiments, the breast cancer, bladder cancer, or colorectal cancer is a stage 3 or stage 4 breast cancer, bladder cancer, or colorectal cancer.

[0009] In some embodiments, the individual has been treatedwith surgery before isolation of the blood or urine sample.

[0010] In some embodiments, the individual has been treated with chemotherapy before isolation of the blood or urine sample.

[0011] In some embodiments, the individual has been treated with an adjuvant or neoadjuvant before isolation of the blood or urine sample.

[0012] In some embodiments, the individual has been treated with radiotherapy before isolation of the blood or urine sample.

[0013] In some embodiments, the method further comprises comprising administering a compound to the individual, where the compound is known to be specifically effective in treating breast cancer, bladder cancer, or colorectal cancer having one or more of the determined single nucleotide variants.

[0014] In some embodiments, the method further comprises determining the variant allele frequency for each of the single nucleotide variants from the sequence determination.

[0015] In someembodiments, a breast cancer, bladder cancer, or colorectal cancer treatment plan is identifiedbasedon the variant allele frequency determinations.

[0016] In some embodiments, the method further comprises administering a compound to the individual, where the compound is known to be specifically effective in treating breast cancer, bladder cancer, or colorectal cancer havingoneof the single nucleotide variants with a variable allele frequency greater than at least one half of the other single nucleotide variants that were determined.

[0017] In some embodiments, the sequence is determined by high throughput DNA sequencing of the plurality of single nucleotide variance loci.

[0018] In some embodiments, the method further comprises detecting a clonal single nucleotide variant in the breast cancer, bladder cancer, or colorectal cancer by determining the variant allele frequency for each of the SNV loci based on thesequenceof theplurality of copiesof theseriesofamplicons,whereinahigher relativeallele frequencycompared to the other single nucleotide variants of the plurality of single nucleotide variant loci is indicative of a clonal single nucleotide variant in the breast cancer, bladder cancer, or colorectal cancer.

[0019] In some embodiments, themethod further comprises administering a compound to the individual that targets the one or more clonal single nucleotide variants, but not the other single nucleotide variants.

[0020] In some embodiments, a variant allele frequency of greater than 1.0% is indicative a clonal single nucleotide variant.

[0021] In some embodiments, the method further comprises forming an amplification reaction mixture by combining a polymerase, nucleotide triphosphates, nucleic acid fragments fromanucleic acid library generated from thesample, anda set of primers that eachbindswithin 150basepairs of a single nucleotide variant loci, or a set of primer pairs that each span a region of 160 base pairs or less comprising a single nucleotide variant loci, and subjecting the amplification reaction mixture to amplification conditions to generate the set of amplicons.

[0022] In some embodiments, determining whether a single nucleotide variant is present in the sample, comprises identifying a confidence value for each allele determination at each of the set of single nucleotide variance loci based at least in part on a depth of read for the loci.

[0023] In some embodiments, a single nucleotide variant call is made if the confidence value for the presence a single nucleotide variant is greater than 90%.

[0024] In some embodiments, a single nucleotide variant call is made if the confidence value for the presence a single nucleotide variant is greater than 95%. 4 EP 4 737 593 A2 5 10 15 20 25 30 35 40 45 50 55

[0025] In some embodiments, the set of single nucleotide variance loci comprises all of the single nucleotide variance loci identified in the TCGA and COSMIC data sets for breast cancer, bladder cancer, or colorectal cancer.

[0026] In some embodiments, the set of single nucleotide variance sites comprises all of the single nucleotide variance sites identified in the TCGA and COSMIC data sets for breast cancer, bladder cancer, or colorectal cancer.

[0027] In some embodiments, themethod is performedwith a depth of read for the set of single nucleotide variance loci of at least 1,000.

[0028] In some embodiments, the set of single nucleotide variant loci comprises 25 to 1000 single nucleotide variance loci known to be associated with breast cancer, bladder cancer, or colorectal cancer.

[0029] In some embodiments, an efficiency and an error rate per cycle are determined for each amplification reaction of the multiplex amplification reaction of the single nucleotide variance loci, and the efficiency and the error rate are used to determine whether a single nucleotide variant at the set of single variant loci is present in the sample.

[0030] In some embodiments, the amplification reaction is a PCR reaction and the annealing temperature is between 1 and 15 °C greater than the melting temperature of at least 50% of the primers of the set of primers.

[0031] In some embodiments, the amplification reaction is a PCR reaction and the length of the annealing step in the PCR reaction is between 15 and 120 minutes.

[0032] In some embodiments, the amplification reaction is a PCR reaction and the length of the annealing step in the PCR reaction is between 15 and 120 minutes.

[0033] In some embodiments, the primer concentration in the amplification reaction is between 1 and 10 nM.

[0034] In some embodiments, the primers in the set of primers, are designed to minimize primer dimer formation.

[0035] In some embodiments, the amplification reaction is a PCR reaction, the annealing temperature is between 1 and 15 °C greater than themelting temperature of at least 50% of the primers of the set of primers, the length of the annealing step in thePCRreaction isbetween15and120minutes, theprimer concentration in theamplification reaction isbetween1 and 10 nM, and the primers in the set of primers, are designed to minimize primer dimer formation.

[0036] In some embodiments, the multiplex amplification reaction is performed under limiting primer conditions.

[0037] Another aspect of the inventiondescribedherein relates to composition comprising circulating tumornucleic acid fragments comprising a universal adapter, wherein the circulating tumor nucleic acids originated from a breast cancer, bladder cancer, or colorectal cancer.

[0038] In some embodiments, the circulating tumor nucleic acids originated froma sample of blood or urine or a fraction thereof, of an individual with breast cancer, bladder cancer, or colorectal cancer.

[0039] Another aspect of the invention described herein relates to composition comprising a solid support comprising a plurality of clonal populations of nucleic acids, wherein the clonal populations comprise amplicons generated from a sample of circulating free nucleic acids, wherein the circulating tumor nucleic acids originated from a breast cancer, bladder cancer, or colorectal cancer.

[0040] In some embodiments, the circulating free nucleic acids originated from a sample of blood or urine or a fraction thereof, of an individual with breast cancer, bladder cancer, or colorectal cancer.

[0041] In some embodiments, the nucleic acid fragments in different clonal populations comprise the same universal adapter.

[0042] In some embodiments, the clonal populations of nucleic acids are derived fromnucleic acid fragments froma set of samples from two or more individuals.

[0043] In someembodiments, thenucleicacid fragments compriseoneof a seriesofmolecular barcodescorresponding to a sample in the set of samples.

[0044] Afurtheraspectof the inventiondescribedherein relates toamethod formonitoringanddetectionofearly relapse or metastasis of breast cancer, bladder cancer, or colorectal cancer, comprising selecting a set of at least 8 or 16 patient- specific single nucleotide variant loci based on somatic mutations identified in a tumor sample of a patient who has been diagnosed with a breast cancer, bladder cancer, or colorectal cancer; longitudinally collecting one or more blood or urine samples from the patient after the patient has been treatedwith surgery, first-line chemotherapy, and / or adjuvant therapy; generating a set of amplicons by performing amultiplex amplification reaction on nucleic acids isolated fromeach blood or urine sample or a fraction thereof, wherein each amplicon of the set of amplicons spans at least one single nucleotide variant locus of the set of patient-specific single nucleotide variant loci associated with the breast cancer, bladder cancer, or colorectal cancer; and determining the sequence of at least a segment of each amplicon of the set of amplicons that comprises a patient-specific single nucleotide variant locus, wherein detection of one or more (or two or more, or three or more, or four or more, or five or more, or six or more, or seven or more, or eight or more, or nine or more, or ten or more) patient-specific single nucleotide variants from the blood or urine sample is indicative of early relapse or metastasis of breast cancer, bladder cancer, or colorectal cancer.

[0045] A further aspect of the invention described herein relates to amethod for treating breast cancer, bladder cancer, or colorectal cancer, comprising treating a patient who has been diagnosed with a breast cancer, bladder cancer, or colorectal cancer with surgery, first-line chemotherapy, and / or adjuvant therapy; longitudinally collecting one or more blood or urine samples from the patient; generating a set of amplicons by performing amultiplex amplification reaction on 5 EP 4 737 593 A2 5 10 15 20 25 30 35 40 45 50 55 nucleic acids isolated fromeach blood or urine sample or a fraction thereof, wherein each amplicon of the set of amplicons spans at least one single nucleotide variant locus of a set of at least 8 or 16 patient-specific single nucleotide variant loci associated with the breast cancer, bladder cancer, or colorectal cancer, which have been selected based on somatic mutations identified in a tumor sample of the patient; determining the sequence of at least a segment of each amplicon of the set of amplicons that comprises a patient-specific single nucleotide variant locus, wherein detection of one ormore (or two or more, or three or more, or four or more, or five or more, or six or more, or seven or more, or eight or more, or nine or more, or tenormore) patient-specific single nucleotide variants from thebloodor urine sample is indicative of early relapse or metastasis of breast cancer, bladder cancer, or colorectal cancer; and administering a compound to the individual, where the compound is known to be effective in treating breast cancer, bladder cancer, or colorectal cancer having one or more of the single nucleotide variants detected from the blood or urine sample.

[0046] A further aspect of the invention described herein relates to a method for monitoring or predicting response to treatment of breast cancer, bladder cancer, or colorectal cancer, comprising longitudinally collecting one ormore blood or urine samples from a patient that is undergoing treatment of a breast cancer, bladder cancer, or colorectal cancer; generating a set of amplicons by performing amultiplex amplification reaction on nucleic acids isolated fromeach blood or urine sample or a fraction thereof, wherein each amplicon of the set of amplicons spans at least one single nucleotide variant locus of a set of at least 8 or 16 patient-specific single nucleotide variant loci associated with the breast cancer, bladder cancer, or colorectal cancer,whichhavebeenselectedbasedonsomaticmutations identified in a tumor sampleof the patient; and determining the sequence of at least a segment of each amplicon of the set of amplicons that comprises a patient-specificsinglenucleotidevariant locus,whereindetectionofoneormore (or twoormore,or threeormore,or fouror more, or five or more, or six or more, or seven or more, or eight or more, or nine or more, or ten or more) patient-specific single nucleotide variants from the blood or urine sample is indicative of poor response to the treatment of breast cancer, bladder cancer, or colorectal cancer.

[0047] In some embodiments, the methods described herein comprise detecting ctDNA in plasma of breast cancer patients before therapy, and / or during neoadjuvant therapy (e.g., after cycle 1, cycle 2, cycle 3, cycle 4, etc.). In some embodiments, a treatment plan is definedbased on ctDNAconcentration determination (e.g. presence / absence) and rate of decline during neo-adjuvant therapy.

[0048] In some embodiments, themethods described herein comprise assessing ctDNA presence and levels for every cancer patient (i.e. targeting mutations that are actually present in the tumor). In some embodiment, the methods described herein comprise detecting 2 or more, 4 or more, 10 or more, 16 or more, 32 or more, 50 or more, 64 or more, or 100 or more of mutations that are actually present in a patients tumor(s).

[0049] According to some embodiments of the present invention, at least 50%, or least 60%, or at least 70%, or at least 80%, or at least 90%, or about 100% of patients that will have metastatic recurrence (e.g. after neo-adjuvant therapy and surgery) have ctDNA detectable at baseline.

[0050] According to some embodiments of the present invention, at least 50%, or least 60%, or at least 70%, or at least 80%, or at least 90%, or about 100% of patients that will have metastatic recurrence (e.g. after neo-adjuvant therapy and surgery) have ctDNA detectable after cycle 1 of neoadjuvant therapy.

[0051] According to some embodiments of the present invention, at least 50%, or least 60%, or at least 70%, or at least 80%, or at least 90%, or about 100% of patients that will have metastatic recurrence (e.g. after neo-adjuvant therapy and surgery) have ctDNA detectable after cycle 2 of neoadjuvant therapy.

[0052] According to some embodiments of the present invention, at least 50%, or least 60%, or at least 70%, or at least 80%, or at least 90%, or about 100% of patients that will have metastatic recurrence (e.g. after neo-adjuvant therapy and surgery) have ctDNA detectable after neoadjuvant therapy and before surgery.

[0053] According to some embodiments of the present invention, at least 50%, or least 60%, or at least 70%, or at least 80%, or at least 90%, or about 100% of patients that will have metastatic recurrence (e.g. after neo-adjuvant therapy and surgery) have ctDNA detectable after surgery.

[0054] According to some embodiments of the present invention, at least 50%, or least 60%, or at least 70%, or at least 80%,or at least 90%,or about 100%ofpatients that havectDNAdetectable (e.g. after surgery)will havemetastaticwithout further treatment recurrence (e.g. after neo-adjuvant therapy and surgery).

[0055] According to some embodiments of the present invention, at least 50%, or least 60%, or at least 70%, or at least 80%,orat least 90%,orabout100%ofpatients that have increasingctDNA levelsbetweenbaselineandcycle1, or cycle2, etc., will have metastatic relapse after surgery if no additional treatment is administered.

[0056] In someembodiments, themethods described herein comprise detecting occurrence, recurrence, ormetastasis of certain subtypes of cancers, including certain subtypes of breast cancer. In someembodiments, themethods described herein comprise detecting occurrence, recurrence, or metastasis of HR+ / HER2‑ tumor including HR+ / HER2‑ breast cancer (e.g., hormone receptor positive- ERα+ and / or PR+). HR+ tumors typically are less aggressive and have a favorable prognosis with a 5-year survival rate of over 90%.

[0057] In someembodiments, themethods described herein comprise detecting occurrence, recurrence, ormetastasis of HER2+ tumor including HER2+ breast cancer (human epidermal growth factor receptor 2 positive). HER2+ tumors are 6 EP 4 737 593 A2 5 10 15 20 25 30 35 40 45 50 55 generally more invasive, have a worse prognosis, and are more likely to recur and metastasize than HR+ / HER2‑ breast cancers.

[0058] In someembodiments, themethods described herein comprise detecting occurrence, recurrence, ormetastasis of HR‑ / HER2‑ tumor including HR‑ / HER2‑ breast cancer (TNBC or triple negative BC). Triple-negative breast cancers (TNBCs) donot expressERα, PR, orHER2. These tumors tend to be themost aggressive andhave theworst prognosis of all the breast cancer subtypes.

[0059] In some embodiments, the method described herein is capable of detecting patient-specific single nucleotide variants in at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of patients having early relapse or metastasis of cancer.

[0060] In some embodiments, the method described herein is capable of detecting patient-specific single nucleotide variantsat least 80%,at least 85%,at least 90%,at least 95%,orat least 98%ofpatientshavingearly relapseormetastasis of HER2+ breast cancer.

[0061] In some embodiments, the method described herein is capable of detecting patient-specific single nucleotide variants least 80%,at least 85%,at least 90%,at least 95%,or at least 98%ofpatientshavingearly relapseormetastasisof triple negative breast cancer.

[0062] In some embodiments, the method described herein is capable of detecting patient-specific single nucleotide variants in at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of patients having early relapse or metastasis of HR+ / HER2‑ breast cancer.

[0063] In some embodiments, the method described herein is capable of detecting patient-specific single nucleotide variants in patients having early relapse or metastasis of cancer at least 100 days, at least 150 days, at least 200 days, at least 250 days, or at least 300 days prior to clinical relapse or metastasis of cancer detectable by imaging, and / or at least 100 days, at least 150 days, at least 200 days, at least 250 days, or at least 300 days prior to elevation of CA15‑3 level.

[0064] In some embodiments, the method described herein is capable of detecting patient-specific single nucleotide variants in patients having early relapse ormetastasis ofHER2+breast cancer at least 100 days, at least 150 days, at least 200 days, at least 250 days, or at least 300 days prior to clinical relapse ormetastasis of HER2+ breast cancer detectable by imaging, and / or at least 100 days, at least 150 days, at least 200 days, at least 250 days, or at least 300 days prior to elevation of CA15‑3 level.

[0065] In some embodiments, the method described herein is capable of detecting patient-specific single nucleotide variants in patients having early relapse ormetastasis of triple negative breast cancer at least 100 days, at least 150 days, at least 200 days, at least 250 days, or at least 300 days prior to clinical relapse or metastasis of triple negative breast cancer detectable by imaging, and / or at least 100 days, at least 150 days, at least 200 days, at least 250 days, or at least 300 days prior to elevation of CA15‑3 level.

[0066] In some embodiments, the method described herein is capable of detecting patient-specific single nucleotide variants in patients having early relapse ormetastasis of HR+ / HER2-breast cancer at least 100 days, at least 150 days, at least 200 days, at least 250 days, or at least 300 days prior to clinical relapse or metastasis of HR+ / HER2‑ breast cancer detectable by imaging, and / or at least 100 days, at least 150 days, at least 200 days, at least 250 days, or at least 300 days prior to elevation of CA15‑3 level.

[0067] In someembodiments, themethoddescribedherein doesnot detect patient-specific single nucleotide variants in at least 95%,at least 98%, at least 99%, at least 99.5%, at least 99.8%,or at least 99.9%of patients lackingearly relapseor metastasis of cancer.

[0068] In someembodiments, themethoddescribedherein doesnot detect patient-specific single nucleotide variants in at least 95%,at least 98%, at least 99%, at least 99.5%, at least 99.8%,or at least 99.9%of patients lackingearly relapseor metastasis of HER2+ breast cancer.

[0069] In someembodiments, themethoddescribedherein doesnot detect patient-specific single nucleotide variants in at least 95%,at least 98%, at least 99%, at least 99.5%, at least 99.8%,or at least 99.9%of patients lackingearly relapseor metastasis of triple negative breast cancer.

[0070] In someembodiments, themethoddescribedherein doesnot detect patient-specific single nucleotide variants in at least 95%,at least 98%, at least 99%, at least 99.5%, at least 99.8%,or at least 99.9%of patients lackingearly relapseor metastasis of HR+ / HER2‑ breast cancer.

[0071] In some embodiments, themethod described herein has a specificity of at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or at least 99.9% in detecting early relapse or metastasis of cancer when two or more patient-specific single nucleotide variants are detected above a predetermined confidence threshold (e.g., 0.95, 0.96, 0.97, 0.98, or 0.99).

[0072] In some embodiments, themethod described herein has a specificity of at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or at least 99.9% in detecting early relapse ormetastasis ofHER2+breast cancerwhen two or more patient-specific single nucleotide variants are detected above a predetermined confidence threshold (e.g., 0.95, 0.96, 0.97, 0.98, or 0.99).

[0073] In some embodiments, themethod described herein has a specificity of at least 95%, at least 98%, at least 99%, 7 EP 4 737 593 A2 5 10 15 20 25 30 35 40 45 50 55 at least 99.5%, at least 99.8%, or at least 99.9% in detecting early relapse or metastasis of triple negative breast cancer when two or more patient-specific single nucleotide variants are detected above a predetermined confidence threshold (e.g., 0.95, 0.96, 0.97, 0.98, or 0.99).

[0074] In some embodiments, themethod described herein has a specificity of at least 95%, at least 98%, at least 99%, at least 99.5%,at least 99.8%,orat least 99.9% indetectingearly relapseormetastasisofHR+ / HER2‑breast cancerwhen two or more patient-specific single nucleotide variants are detected above a predetermined confidence threshold (e.g., 0.95, 0.96, 0.97, 0.98, or 0.99).

[0075] In someembodiments, themethoddescribed herein detects patient-specific single nucleotide variants in at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of patients having early relapse or metastasis of muscle invasive bladder cancer (MIBC).

[0076] In some embodiments, the method described herein detects patient-specific single nucleotide variants in patients having early relapse or metastasis of cancer at least 100 days, at least 150 days, at least 200 days, or at least 250 days prior to clinical relapse or metastasis of MIBC detectable by imaging.

[0077] In someembodiments, themethoddescribedherein doesnot detect patient-specific single nucleotide variants in at least 95%,at least 98%, at least 99%, at least 99.5%, at least 99.8%,or at least 99.9%of patients lackingearly relapseor metastasis of MIBC.

[0078] In some embodiments, themethod described herein has a specificity of at least 95%, at least 98%, at least 99%, at least 99%, at least 99.5%, at least 99.8%, or at least 99.9% in detecting early relapse ormetastasis ofMIBCwhen two or more patient-specific single nucleotide variants are detected above a predetermined confidence threshold (e.g., 0.95, 0.96, 0.97, 0.98, or 0.99).

[0079] In addition or alternative to single-nucleotide variants, the methods described herein can also be based on the detection of other genomic variants, such as indels, multiple nucleotide variants, and / or gene fusions.

[0080] Accordingly, an additional aspect of the invention described herein relates to a method for monitoring and detection of early relapse or metastasis of breast cancer, bladder cancer, or colorectal cancer, comprising selecting a plurality of genomic variant loci (e.g., SNV, indel,multiple nucleotide variant, andgene fusion) basedon somaticmutations identified ina tumor sampleofapatientwhohasbeendiagnosedwithabreast cancer, bladder cancer, or colorectal cancer; longitudinally collectingoneormorebloodor urine samples from thepatient after thepatient hasbeen treatedwith surgery, first-line chemotherapy, and / or adjuvant therapy; generating a set of amplicons by performing a multiplex amplification reaction onnucleic acids isolated fromeachbloodor urine sample or a fraction thereof,wherein eachampliconof the set of amplicons spans at least one genomic variant locus of the set of patient-specific genomic variant loci associated with the breast cancer, bladder cancer, or colorectal cancer; and determining the sequence of at least a segment of each amplicon of the set of amplicons that comprises a patient-specific genomic variant locus,wherein detection of one ormore (or two or more, or three ormore, or four ormore, or five ormore, or six ormore, or seven ormore, or eight ormore, or nine ormore, or ten ormore) patient-specific genomic variants from the blood or urine sample is indicative of early relapse ormetastasis of breast cancer, bladder cancer, or colorectal cancer.

[0081] An additional aspect of the invention described herein relates to a method for treating breast cancer, bladder cancer, or colorectal cancer, comprising treatingapatientwhohasbeendiagnosedwithabreast cancer, bladder cancer, or colorectal cancer with surgery, first-line chemotherapy, and / or adjuvant therapy; longitudinally collecting one or more blood or urine samples from the patient; generating a set of amplicons by performing amultiplex amplification reaction on nucleic acids isolated fromeach blood or urine sample or a fraction thereof, wherein each amplicon of the set of amplicons spansat least onegenomic variant locus (e.g., SNV, indel,multiple nucleotide variant, andgene fusion) of a set of at least 8 or 16 patient-specific genomic variant loci associated with the breast cancer, bladder cancer, or colorectal cancer, which havebeenselected basedon somaticmutations identified in a tumor sample of the patient; determining the sequenceof at least a segment of each amplicon of the set of amplicons that comprises a patient-specific genomic variant locus, wherein detection of one ormore (or two ormore, or three ormore, or four ormore, or five ormore, or six ormore, or seven ormore, or eight or more, or nine or more, or ten or more) patient-specific genomic variants from the blood or urine sample is indicative of early relapse or metastasis of breast cancer, bladder cancer, or colorectal cancer; and administering a compound to the individual, where the compound is known to be effective in treating breast cancer, bladder cancer, or colorectal cancer having one or more of the genomic variants detected from the blood or urine sample.

[0082] Anadditional aspect of the inventiondescribedherein relates toamethod formonitoringorpredicting response to treatment of breast cancer, bladder cancer, or colorectal cancer, comprising longitudinally collecting one ormore blood or urine samples from a patient that is undergoing treatment of a breast cancer, bladder cancer, or colorectal cancer; generating a set of amplicons by performing amultiplex amplification reaction on nucleic acids isolated fromeach blood or urine sample or a fraction thereof, wherein each amplicon of the set of amplicons spans at least one genomic variant locus (e.g., SNV, indel, multiple nucleotide variant, and gene fusion) of a set of at least 8 or 16 patient-specific genomic variant loci associated with the breast cancer, bladder cancer, or colorectal cancer, which have been selected based on somatic mutations identified in a tumor sample of the patient; anddetermining the sequenceof at least a segment of eachamplicon of the set of amplicons that comprises a patient-specific genomic variant locus,wherein detection of one ormore (or two or 8 EP 4 737 593 A2 5 10 15 20 25 30 35 40 45 50 55 more, or three ormore, or four ormore, or five ormore, or six ormore, or seven ormore, or eight ormore, or nine ormore, or tenormore)patient-specificgenomicvariants from thebloodorurinesample is indicativeofpoor response to the treatment of breast cancer, bladder cancer, or colorectal cancer.

[0083] Inadditionoralternative topatient-specificgenomicvariants, themethodsdescribedherein canalsobebasedon the detection of recurring cancer-associated mutations (e.g., hotspot cancer mutations, drug resistant markers, cancer panel mutations) that are recurring in many cancer patients.

[0084] Accordingly, an additional aspect of the invention described herein relates to a method for monitoring and detection of early relapse or metastasis of breast cancer, bladder cancer, or colorectal cancer, comprising selecting a plurality of recurring cancer-associated mutations; longitudinally collecting one or more blood or urine samples from the patient after thepatient hasbeen treatedwith surgery, first-line chemotherapy, and / or adjuvant therapy; generatinga set of amplicons by performing amultiplex amplification reaction on nucleic acids isolated from each blood or urine sample or a fraction thereof, wherein each amplicon of the set of amplicons spans at least one of the set of recurring mutations associated with the breast cancer, bladder cancer, or colorectal cancer; and determining the sequence of at least a segment of each amplicon of the set of amplicons that comprises a recurring cancer-associated mutation, wherein detection of one ormore (or two ormore, or three ormore, or four ormore, or five ormore, or six ormore, or seven ormore, or eight or more, or nine or more, or ten or more) recurring cancer-associatedmutations from the blood or urine sample is indicative of early relapse or metastasis of breast cancer, bladder cancer, or colorectal cancer.

[0085] An additional aspect of the invention described herein relates to a method for treating breast cancer, bladder cancer, or colorectal cancer, comprising treatingapatientwhohasbeendiagnosedwithabreast cancer, bladder cancer, or colorectal cancer with surgery, first-line chemotherapy, and / or adjuvant therapy; longitudinally collecting one or more blood or urine samples from the patient; generating a set of amplicons by performing amultiplex amplification reaction on nucleic acids isolated fromeach blood or urine sample or a fraction thereof, wherein each amplicon of the set of amplicons spans at least one recurring cancer-associated mutation (e.g., hotspot cancer mutation, drug resistant marker, cancer panel mutation) of a set of at least 8 or 16 recurring mutations associated with the breast cancer, bladder cancer, or colorectal cancer; determining the sequence of at least a segment of each amplicon of the set of amplicons that comprises a recurring cancer-associatedmutation,wherein detectionof oneormore (or twoormore, or threeormore, or four ormore, or five or more, or six or more, or seven or more, or eight or more, or nine or more, or ten or more) recurring cancer- associated mutations from the blood or urine sample is indicative of early relapse or metastasis of breast cancer, bladder cancer, or colorectal cancer; andadministeringa compound to the individual,where the compound is known tobeeffective in treating breast cancer, bladder cancer, or colorectal cancer having one or more of the recurring cancer-associated mutations detected from the blood or urine sample.

[0086] Anadditional aspect of the inventiondescribedherein relates toamethod formonitoringorpredicting response to treatment of breast cancer, bladder cancer, or colorectal cancer, comprising longitudinally collecting one ormore blood or urine samples from a patient that is undergoing treatment of a breast cancer, bladder cancer, or colorectal cancer; generating a set of amplicons by performing amultiplex amplification reaction on nucleic acids isolated fromeach blood or urine sample or a fraction thereof, wherein each amplicon of the set of amplicons spans at least one recurring cancer- associatedmutation (e.g., hotspot cancermutation, drug resistantmarker, cancer panelmutation)of aset of at least 8or16 recurring mutations associated with the breast cancer, bladder cancer, or colorectal cancer; and determining the sequence of at least a segment of each amplicon of the set of amplicons that comprises a recurring cancer-associated mutation,wherein detection of one ormore (or twoormore, or three ormore, or four ormore, or five ormore, or six ormore, or sevenormore, or eight ormore, or nineormore, or tenormore) recurring cancer-associatedmutations from thebloodor urine sample is indicative of poor response to the treatment of breast cancer, bladder cancer, or colorectal cancer.

[0087] In addition or alternative to initially identifying somatic mutations from a tumor sample of a patient who has been diagnosedwith a breast cancer, bladder cancer, or colorectal cancer, themethods described herein can also be based on the identifying somatic mutations from other biological samples of the patients, such as blood, serum, plasma, urine, hair, tears, saliva, skin, fingernails, feces, bile, lymph, cervical mucus, or semen.

[0088] Accordingly, an additional aspect of the invention described herein relates to a method for monitoring and detection of early relapse or metastasis of breast cancer, bladder cancer, or colorectal cancer, comprising selecting a plurality of genomic variant loci (e.g., SNV, indel,multiple nucleotide variant, andgene fusion) basedon somaticmutations identified in a biological sample comprising cancer-associated mutations (e.g., blood, serum, plasma, urine, hair, tears, saliva, skin, fingernails, feces, bile, lymph, cervical mucus, or semen) of a patient who has been diagnosed with a breast cancer, bladder cancer, or colorectal cancer; longitudinally collecting one ormore blood or urine samples from the patient after the patient has been treated with surgery, first-line chemotherapy, and / or adjuvant therapy; generating a set of amplicons by performing amultiplex amplification reaction on nucleic acids isolated from each blood or urine sample or a fraction thereof, wherein each amplicon of the set of amplicons spans at least one genomic variant locus of the set of patient-specific genomic variant loci associated with the breast cancer, bladder cancer, or colorectal cancer; and determining the sequence of at least a segment of each amplicon of the set of amplicons that comprises a patient- specific genomic variant locus,wherein detectionof oneormore (or twoormore, or threeormore, or four ormore, or fiveor 9 EP 4 737 593 A2 5 10 15 20 25 30 35 40 45 50 55 more, or six or more, or seven ormore, or eight or more, or nine or more, or ten or more) patient-specific genomic variants from the blood or urine sample is indicative of early relapse or metastasis of breast cancer, bladder cancer, or colorectal cancer.

[0089] An additional aspect of the invention described herein relates to a method for treating breast cancer, bladder cancer, or colorectal cancer, comprising treatingapatientwhohasbeendiagnosedwithabreast cancer, bladder cancer, or colorectal cancer with surgery, first-line chemotherapy, and / or adjuvant therapy; longitudinally collecting one or more blood or urine samples from the patient; generating a set of amplicons by performing amultiplex amplification reaction on nucleic acids isolated fromeach blood or urine sample or a fraction thereof, wherein each amplicon of the set of amplicons spansat least onegenomic variant locus (e.g., SNV, indel,multiple nucleotide variant, andgene fusion) of a set of at least 8 or 16 patient-specific genomic variant loci associated with the breast cancer, bladder cancer, or colorectal cancer, which have been selected based on somatic mutations identified in a biological sample of the patient comprising cancer- associated mutations (e.g., blood, serum, plasma, urine, hair, tears, saliva, skin, fingernails, feces, bile, lymph, cervical mucus, or semen); determining thesequenceof at least a segment of eachampliconof the set of amplicons that comprises apatient-specificgenomicvariant locus,whereindetectionof oneormore (or twoormore, or threeormore, or four ormore, or five ormore, or six ormore, or seven ormore, or eight ormore, or nine ormore, or ten ormore) patient-specific genomic variants from the blood or urine sample is indicative of early relapse or metastasis of breast cancer, bladder cancer, or colorectal cancer; andadministering acompound to the individual,where thecompound is known tobeeffective in treating breast cancer, bladder cancer, or colorectal cancer having one ormore of the genomic variants detected from the blood or urine sample.

[0090] Anadditional aspect of the inventiondescribedherein relates toamethod formonitoringorpredicting response to treatment of breast cancer, bladder cancer, or colorectal cancer, comprising longitudinally collecting one ormore blood or urine samples from a patient that is undergoing treatment of a breast cancer, bladder cancer, or colorectal cancer; generating a set of amplicons by performing amultiplex amplification reaction on nucleic acids isolated fromeach blood or urine sample or a fraction thereof, wherein each amplicon of the set of amplicons spans at least one genomic variant locus (e.g., SNV, indel, multiple nucleotide variant, and gene fusion) of a set of at least 8 or 16 patient-specific genomic variant loci associated with the breast cancer, bladder cancer, or colorectal cancer, which have been selected based on somatic mutations identified in a biological sample of the patient comprising cancer-associated mutations (e.g., blood, serum, plasma, urine, hair, tears, saliva, skin, fingernails, feces, bile, lymph, cervical mucus, or semen); and determining the sequence of at least a segment of each amplicon of the set of amplicons that comprises a patient-specific genomic variant locus, wherein detection of one ormore (or two ormore, or three ormore, or four ormore, or five ormore, or six ormore, or seven or more, or eight or more, or nine or more, or ten or more) patient-specific genomic variants from the blood or urine sample is indicative of poor response to the treatment of breast cancer, bladder cancer, or colorectal cancer.

[0091] Other embodiments and features and advantages of the disclosed inventionswill be apparent from the following detailed description and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0092] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publicationwith color drawing(s)will be provided by theOfficeupon request andpayment of the necessary fee.

[0093] The presently disclosed embodimentswill be further explainedwith reference to the attached drawings, wherein like structures are referred to by like numerals throughout the several views. The drawings shown are not necessarily to scale, with emphasis instead generally being placed upon illustrating the principles of the presently disclosed embodi- ments. FIG. 1 is a workflow Diagram. FIG. 2 Top panel: the number of SNVs per sample; bottom panel: the working assays, sorted by driver category. FIG. 3. Measured cfDNA concentration. Each data point refers to a plasma sample. FIG. 4. Samples showing good correlation between tissue VAF measurements determined previously (x axis) and here using mPCR-NGS (y axis). Each sample is shown in a separate box, and the VAF data points are colored by tissue subsection. FIG. 5. Samples showing poor correlation between tissue VAF measurements determined previously (x axis) and here using mPCR-NGS (y axis). Each sample is shown in a separate box, and the VAF data points are colored by tissue subsection. FIG. 6A-B. Depth of read histogram as a function of the resulting call. Top: the assay did not detect the expected plasma SNV. Bottom: the assay detected the expected plasma SNV. FIG. 7. Number of SNVs detected in plasma by histological type. FIG. 8. SNV detection (left) and sample detection (right) in plasma by tumor stage. FIG. 9. Plasma VAF as a function of tumor stage and SNV clonality. 10 EP 4 737 593 A2 5 10 15 20 25 30 35 40 45 50 55 FIG. 10. Number of SNVs detected in plasma from each sample as a function of the cfDNA input amount. FIG. 11.PlasmaVAFasa function of average tumorVAF.Average tumorVAFwas calculatedacross all the tumor sub- sections analyzed from each tumor. FIG. 12 shows the clonal ratios (red to blue) andmutant variant allele frequency (MutVAF) of each detected SNV. The total SNVs detected fromeach sample are placed in a single column and the samples are categorized by tumor stage (pTNMstage). Samples with no detected SNVs are included. The clonal ratio is defined as the ratio between the number of tumor subsections in which SNV was observed and the total number of subsections analyzed from that tumor. FIG. 13 shows the clonal status (blue for clonal and red for subclonal) andmutant variant allele frequency (MutVAF) of each detected SNV. The total SNVs detected from each sample are placed in a single column and the samples are categorized by tumor stage (pTNMstage). Samples with no detected SNVs are included. The clonal status was determined by PyCloneCluster using whole exome equencing data from the tumor tissue. FIG. 14 shows the clonal status (blue for clonal and red for subclonal) andmutant variant allele frequency (MutVAF) of each detected SNV where the top panel shows only the clonal SNVs and the bottom panel shows only the subclonal SNVs. The total SNVs detected from each sample are placed in a single column and the samples are categorized by tumor stage (pTNMstage). Samples with no detected SNVs are included. The clonal status was determined by PyCloneCluster using whole exome equencing data from the tumor tissue. FIG. 15 shows the number of SNVs detected in plasma as a function of histological type and tumor size. The histological type and tumor stage were determined by the pathology report. Each data point is colored by size, where red denotes the largest tumor size and blue denotes the smallest tumor size. FIG. 16 is a table of cfDNA analysis showing DNA concentration, genome copy equivalents into library prep, plasma hemolysis grade, and cDNA profile in all samples. FIG. 17 is a table of SNVs detected in the plasma for each sample. FIG. 18 is a table of additional SNVs detected in plasma. FIG. 19 is an example of detected assays and their background allele fractions for a plasma sample at relapse time (LTX103). FIG. 20A-B: Schematic of Clinical and Molecular Protocols. FIG. 21: Study Overview. FIG. 22: Patient Summaries for 36 Months of Surveillance and Plasma Collection. FIG. 23A-B: Relapse risk stratified by post-operative ctDNA status. FIG. 24A-B: Post-therapy Relapse Risk Stratified by Postoperative ctDNA Status. FIG. 25: Effectiveness of adjuvant therapy in preventing relapse. FIG. 26A-B: Time to Release Based on Radiology and ctDNA. FIG. 27A-D: Early Detection of Relapse and Prediction of Treatment Response. FIG. 28: Schematic of Clinical Sample Collection. FIG. 29: Plasma Sequencing QC. FIG. 30A-F: Early Relapse Detection. FIG. 31: Relapse-Free Survival and ctDNA Status at Diagnosis and Post Cystectomy. FIG. 32A-B: Neoadjuvant Treatment Response. FIG. 33: Signatera (RUO) Process. FIG. 34: Plasma Sequencing QC. FIG. 35: Sensitivity of Single SNV Detection. FIG. 36: Expected Input vs Observed VAF with Signatera (RUO). FIG. 37: Patient Summary for the breast cancer study in Example 6. FIG.38A-H: is a tableof information regarding thesamplesanalyzed in thestudyofExample6.FIG.38A ispart 1of the table. FIG. 38B is a continuation of the table. FIG. 38C is a continuation of the table. FIG. 38D is a continuation of the table. FIG. 38E is a continuation of the table. FIG. 38F is a continuation of the table. FIG. 38G is a continuation of the table. FIG. 38H is a continuation of the table. FIG. 39: Demographics of the patients in the breast cancer study of Example 6. 50 patientsWES raw data (with driver variants for 35 patients) was received. 218 plasma samples at variable number of time points (between 1 and 8) were received. 108 extra extracted DNA samples were received. Relapse status was also collected. Blood samples were collected post adjuvant therapy with 6-month time intervals. FIG. 40: Summary of WES analysis and pool design for breast cancer study in Example 6. Pool A is based on the Signateramethod.Pool Bcontains 25patients and is indicatedwith anasterisk in theboxandwhisker plot. 19patients in Pool B had low tumor purity. 6 patients had extra early stage HER2‑ tumors. Pool B contains driver variants. FIG. 41: Plasma samples for the breast cancer study in Example 6. The median plasma volume was 4mL. Median DNA input was 26 ng. Median DNA input is lower than CRC and MIBC samples (45 ng and 66ng respectively). FIG. 42: Sequencing quality control depicting median process error rate per type andmedian assay depth of read for 11 EP 4 737 593 A2 5 10 15 20 25 30 35 40 45 50 55 the breast cancer study in Example 6. In total, 326 plasma sequencing samples were processed. Mutation calling FP rate is estimated to be 0.28%. FIG. 43: Plasma sample reruns for the breast cancer study in Example 6. 319 sequenced samples with 214 unique plasma samples are represented for 49 patients. FIG. 44: Results fromPool A for the breast cancer study in Example 6. Out of 49 patients, 11were baseline positive. 3 haveonly one timepoint. The remaining8patients staypositiveall the time.PoolBanddriver producedsimilar results. Driver information: 16 relapse samples have drivermutations. 11 relapse samples have at least one assaywith driver. FIG. 45: Summary of 16 Patients with ctDNA detected. FIG. 46: Graphical depiction of data corresponding to patient CD047 (TNBC) in FIG. 38. FIG. 47: Graphical depiction of data corresponding to patient CD033 (TNBC) in FIG. 38. FIG. 48: Graphical depiction of data corresponding to patient CD037 (HER2+) in FIG. 38. FIG. 49: Graphical depiction of data corresponding to patient CD040 (HER2+)in FIG. 38. FIG. 50: Graphical depiction of data corresponding to patient CD048 (HER2‑) in FIG. 38. FIG. 51: Graphical depiction of data corresponding to patient CD005 (HER2‑) in FIG. 38. FIG. 52: Graphical depiction of data corresponding to patient CD036 (HER2‑) in FIG. 38. FIG. 53: Graphical depiction of data corresponding to patient CD044 (HER2‑) in FIG. 38. FIG. 54: Graphical depiction of data corresponding to patient CD049 in FIG. 38. FIG. 55: Graphical depiction of data corresponding to patient CD029 in FIG. 38. FIG. 56: Graphical depiction of data corresponding to patient CD026 in FIG. 38. FIG. 57: Graphical depiction of data corresponding to patient CD017 in FIG. 38. FIG. 58: Graphical depiction of data corresponding to patient CD031 in FIG. 38. HW: SHC2, PKD1, COLEC12. FIG.59:Graphical depictionofdatacorresponding topatientCD025 inFIG.38. In thispatient, ctDNAwasobserved for a mutation in FGF9 for 2 consecutive time points. This patient may experience a relapse in the near future. FIG. 60: Patient recruitment and Collection of Clinical Samples. For the 49 BC women monitored in this study, collected tumor tissue and serial plasma samples were analyzed using Signatera™ RUO workflow in a blinded manner. Exomic alterations were determined through paired-end sequencing of FFPE tumor-tissue specimens and matched normal DNA. Patient specific panels including 16 somatic mutations identified from WES were designed. Plasma samples were processed using their corresponding custom panels. 208 samples were analyzed for ctDNA detection. FIG. 61A-C: Summary overview and results of ctDNA analysis. (A) Summary of each patient’s (n=49) treatment regimen along with results of serial plasma samples (n=208) analyzed. (B) Summary table showing total patients in eachbreast cancer subtype, number relapsed, percent detectedbyctDNAanalysis, andmedian lead time indays. (C) Comparison of molecular and clinical relapse colored by breast cancer subtype HR+, HER2+, TNBC using paired Wilcoxon signed rank test (p-value <0.001). FIG. 62A-B: CtDNAdetection in serial plasma samples predicts recurrence free survival (A) Recurrence-free survival according to the detection of ctDNA in any follow-up plasma samples post-surgery [HR:35.84 (7.9626 - 161.32] p- value <0.001. (B) Recurrence-free survival according to the detection of ctDNA in the first post-surgical plasma sample [HR: 11.784 (4.2784 - 32.457]. Data are from n = 49 patients with p-value < 0.001. FIG. 63: (A-E) Plasma levels of ctDNA across multiple plasma time points for five breast cancer patients (one per panel). Primary tumor and matched normal whole-exome sequencing identified patient-specific somatic mutations. Using the analytically-validated Signatera™ RUO workflow, each patient specific assay was designed to target 16 somatic SNVand INDEL variants using massively parallel sequencing (median depth >100,000X per target). Mean VAFsaredenotedbydarkbluecircleandsolid line representaverageVAFprofileover time.The lead time iscalculated by difference in clinical relapse andmolecular relapse. CA15‑3 levels is graphed over time and the baseline levels are marked in light blue shade. (F) Summary of VAFs and number of targets detected atmolecular and clinical relapse for all ctDNA positive samples, excluding patients with only one time point. FIG. 64A-C:Signatera variant selection strategy for the 49patient specific panels. (Top) Tumor tissueVAFdistribution in patients’ custom panel. Different colors represents different subtypes: HER2‑ (dark blue), triple negative (orange), and HER2+ (green). (Middle) The number of inferred clonal and subclonal variants in patients’ custom panel. The median number of clonal variants in 49 custom panel is 13 out of 16. (Bottom) The number of inferred clonal and subclonal variants in patients’ WES data. FIG. 65: (A-L) Plasma levels of ctDNA across multiple plasma time points for 12 (11 relapsed and 1 non-relapsed) breast cancer patients. Primary tumor and matched normal whole-exome sequencing identified patient-specific somaticmutations.Using theanalytically-validatedSignatera™workflow,eachpatient specificassaywasdesigned to target 16somaticSNVand INDELvariantsusingmassivelyparallel sequencing (mediandepth>100,000Xper target). Mean VAFs are denoted by dark blue circle and solid line represent average VAF profile over time. The lead time is calculatedbydifference in clinical relapseandmolecular relapse.CA15‑3 levels is graphedover timeand thebaseline levels are marked in light blue shade. 12 EP 4 737 593 A2 5 10 15 20 25 30 35 40 45 50 55 FIG. 66: Distribution of VAFs and Mutant Counts. In total, 251 targets were detected in ctDNA positive plasma samples. The VAF of detected targets ranged from 0.01% to 64%, with a median of 0.82%. We used the observed mutant VAFand total number ofDNAmolecules in each sample to calculate thenumber of tumormolecules present in the patient’s plasma sample. Number of detectedmutant molecules in the 251 positive targets ranged from 1 to 6500 mutant molecules, with a median of 39 molecules. FIG. 67A-D: Signatera quality control process:Quality control was performedat every step of theworkflow. In total out of 215 plasma samples 208 passed our sample QC process, and out of 784 unique assays designed there were 767 which passed our assay QC (corresponding to total of 3237 assays passing out of 3328 across all samples). A) Extracted cfDNA per mL. cfDNA extracted from each plasma sample was quantified by Quant-iT High Sensitivity dsDNA Assay Kit. Samples with quantified cfDNA amount <5ng were flagged WARNING. Extracted cfDNA per mL ranged between 1 to 21.4 ng with the median of 4.7 ng. B) Library prep DNA input amount. Up to 66ng of cfDNA from eachplasmasamplewasusedas input into libraryprepprotocol. LibraryDNA inputamount rangedbetween1 to66ng with the median of 25.02. The purified libraries were QC’ed before proceeding to the next step. C) Sequencing coverage.Assayswith coverage less than5000xwereexcluded fromanalyses.Subsequently, sampleswith less than 8passing assays failed sequencing coverageQC.Themedian depth of read for the assays passed coverageQCwas 110,000x.D)Sampleconcordance. Inorder to tracksample integrity,SNP tracerswereused tomeasureconcordance between patient’s samples. For each plasma samples, a genotyping concordance score was calculated in compar- ison to its corresponding matched normal genotyping data. Samples are considered to be from the same patients whenat least 85%of their SNPshad identical genotypes.Sixplasmasamples identified to beswappedwereexcluded from ctDNA analyses. FIG. 68A-B: Analytical Validation Results. (A) Single target detection sensitivity. An analytical sensitivity of ~60% for mutation detection at ~0.03% spiked-in tumor DNA was achieved with Signatera. (B) Estimated sample-level sensitivities for Signatera when at least two mutations are detected from a set of 16 target variants. FIG. 69: After screening and recruitment patients were followed up with 6 monthly blood samples. HER2 status was determined by immunohistochemical and fluorescence in situ hybridization assays. A patient was considered to have HER2-positive cancer if either assaywas positive. NACT: neoadjuvant chemotherapy; ACT: adjuvant chemotherapy. FIG. 70: Workflow diagram for the muscle invasive bladder cancer study in Example 9. FIG. 71A-G: Patient summary for themuscle invasive bladder cancer study in Example 9. FIG. 71A shows the rate of synonymous and non-synonymous mutations called from WES. One patient’s tumor was hypermutated with a mutational burden of 126mutations / Mb and displayed a POLD1mutation which previously has been associated with hyper-mutators (Campbell, B. B. et al. Comprehensive Analysis of Hypermutation in Human Cancer. Cell 171, 1042‑1056.e10 (2017). FIG. 71B shows the relative contribution of bladder cancer associatedmutational signatures. FIG. 71C shows mutations in frequently mutated genes in bladder cancer (TCGA) (Robertson, A. G. et al. Comprehensive Molecular Characterization of Muscle-Invasive Bladder Cancer. Cell 171, 540‑556.e25 (2017)). FIG.71Dshowsdeleteriousmutations inDNAdamage response (DDR)associatedgenesmutated inmore than5%of the 68 samples. FIG. 71E shows total number of deleterious DDR mutations. FIG. 71F shows clinical and histopathological characteristics. FIG. 71G shows summarized ctDNA status. FIG. 72:Diagramoutlining the clinical protocol and sampling schedule for themuscle invasive bladder cancer study in Example 9. FIG. 73: Diagram outlining the Signatera™ workflow. FIG. 74: Longitudinal representation of ctDNA results for all analyzed samples corresponding to the muscle invasive bladder cancer study in Example 9. Patients are separated into three groups based on ctDNA status: the upper panel shows patients ctDNA positive before and after cystectomy (CX); the middle panel shows patient ctDNA positive before CX only; the bottom panel shows ctDNA negative patient Horizontal lines represent each patients disease course and circles represent ctDNAstatus, red circles indicate sampleswith at least 2 positive assays. Treatment and imaging information is indicated for each patient. FIG. 75A-E: Graphical depiction of prognostic value of ctDNA detection for themuscle invasive bladder cancer study in Example 9. Kaplan-Meier survival analysis showing probability of recurrence-free survival (RFS) and overall survival (OS) stratified by ctDNA status before chemotherapy (FIG. 75A), before cystectomy (CX) (FIG. 75B), and after cystectomy (CX) (FIG. 75C). FIG. 75D shows association between disease recurrence and ctDNA status before chemotherapy, before cystectomy and after cystectomy as well as disease recurrence and lymph node status before cystectomy. FIG. 75E shows association between ctDNA status before cystectomy (CX) and pathology status at cystectomy (CX). Assessment of statistical significance was performed usingWilcoxon rank-sum test for continuous variables and Fisher’s Exact test for categorical variables. FIG.76:GraphsshowingctDNAchanges in individual diseasecourses for themuscle invasivebladder cancer study in Example9.FIG.76shows representationofdetaileddiseasecourses, applied treatmentsandassociated longitudinal ctDNAanalyses fromselectedpatients. ctDNAstatus, applied treatment and imaging results arepresentedaccording to the legend. Positive lead times for ctDNA based recurrence detection are indicated. 13 EP 4 737 593 A2 5 10 15 20 25 30 35 40 45 50 55 FIG. 77: Graphs showing time differences between molecular recurrence (ctDNA positivity) and clinical recurrence (radiographic imaging positive) for the muscle invasive bladder cancer study in Example 9. P-value was calculated using a paired Wilcoxon rank-sum test. FIG. 78A-H: Graphs showing predictive markers of chemotherapy response for the muscle invasive bladder cancer study in Example 9. FIG. 78A shows association between disease recurrence and response to chemotherapy. FIG. 78B shows relative signature 5 contribution for all patients stratified by response to chemotherapy and ERCC2 mutationstatus, respectively.FIG.78Cshows fractionof patients responding to therapy in relation toERCC2mutation status. FIG. 78D RNA subtype figures_NEW figure. FIG. 78E shows association between ctDNA and response to chemotherapy for patients being ctDNA negative throughout the whole disease course, patients where ctDNA level drops down to zero and patients where ctDNA level remains to be positive. FIG. 78F shows the level of ctDNA for all patients with detectable ctDNA before, during and after chemotherapy. Patients are grouped by response to chemotherapy and recurrence status is indicated. FIG. 79A-D:Graphs showing the total number of identifiedmutations per patient in relation to theERCC2 status or the number of damaging DNA damage response (DDR) mutations for the muscle invasive bladder cancer study in Example 9. FIG. 80: Graphs depicting genomic heterogeneity between primary tumor and metastatic relapse for the muscle invasive bladder cancer study in Example 9. Whole exome sequencing (WES) data of the primary tumors was compared to ctDNA. WES data from plasma samples with high ctDNA variant allele frequency (VAF) detected at metastatic relapse. Genomic positions with mutations identified in either plasma or tumor exome data were investigated for base counts. Resulting allele frequencies identified in plasma and tumor exome data are shown. Individual mutations are color coded according to statistical probability (strength) of themutation call. Venn diagrams represent the number of mutations identified exclusively in the tumor, plasma or in both. FIG. 81: Graphs depicting variance allele frequency (VAF%) at different days relative to cystectomy (CX) from 8 patients from the muscle invasive bladder cancer study in Example 9. FIG. 82: Graphs showing ctDNA level in plasma from 10 patients previously analyzed by ddPCR compared to ultra- deep sequencing for the muscle invasive bladder cancer study in Example 9. FIG.83A-E:Graphsshowingclinical, histopathological andmolecular parameters for all 125patients.FIG.83Ashows the relativecontributionof thefivemostprevalent colorectal cancerassociatedmutational signatures.FIG.83Bshows the rate of synonymous and non-synonymous mutations called from WES. FIG. 83C depicts a graph showing mutations in frequently mutated genes in colorectal cancer (TCGA) {Cancer Genome Atlas, 2012 #52}. FIG. 83D shows clinical and histopathological characteristics. FIG. 83E shows a graph summarizing pre‑ and post-OP ctDNA status. FIG. 84: Diagram showing patient enrollment, sample collection, and definitions of the patient subgroups used to address the defined clinical questions. Abbreviations: ctDNA, circulating tumor DNA; CT-scan, computed tomogra- phy scan; post-op, postoperative; TTR, time to recurrence. FIG. 85A-C: Graphs showing quality control (QC) tests of workflow for whole exome sequencing of the patient samples. 793 (99%) out of 795 plasma samples passed the sample QC process. 194 samples (from 70 patients) run with SNP tracer to check the concordance between the plasma sample and its corresponding tissue biopsy. All 194 plasma samples passed the concordance QC. FIG. 85A shows library preparation DNA input amount. Up to 66ng of cell-freeDNA (cfDNA) fromeachplasmasamplewasusedas input into librarypreparationprotocol. LibraryDNA input amount ranged between 1 to 66 ng with the median of 45.66. The purified libraries were quality controlled before proceeding to thenext step.Onesample failed library preparationQC.FIG. 85Bshowssequencing coverage.Assays with coverage less than 5000xwere excluded fromanalyses. Subsequently, sampleswith less than 8 passing assays failed sequencing coverageQC.One sample failed the sequencing coverage requirement. Themedian depth of read for the assays passed coverage QC was 105,000x. FIG. 85C shows sequencing error rate measured in all plasma samples. Average transition error rate is 5e‑5 and average transversion error rate is 8e‑6. FIG. 86 shows circulating tumor DNA (ctDNA) results and dynamics for each individual patient. FIG. 87A-F shows ctDNA status pre-operative (pre-op), at day 30 post-operative and during adjuvant chemotherapy (ACT). FIG. 87A shows pre-op detection of ctDNA. FIG. 87B shows recurrence rate. FIG. 87C shows Kaplan-Meier estimatesofTTR for94stage I-III patients, stratifiedbypostoperativeday30ctDNAstatus.FIG.87DshowsACTeffect on ctDNA-positive patients, assessed by recurrence rate and longitudinal ctDNA status. FIG. 87E shows recurrence rate stratified by ctDNA status at first visit post-ACT. FIG. 87F shows Kaplan-Meier estimates of TTR for 58 ACT treated patients, stratified by ctDNA status at first post-ACT visit. FIG. 88 shows pre-operative detection of carcinoembryonic antigen (CEA) in 125 stage i-III CRC patients. FIG. 89 shows a schematic overview of ctDNA profiling results of plasma samples included in the day 30 ctDNA analysis ordered by recurrence status and disease stage. Patients marked by a (s) have synchronous CRC. Plasma marked with ** are positive in the second pool only (n=1) FIG. 90A-B shows a schematic overview of ctDNA profiling results of a subset of plasma samples included in the day 14 EP 4 737 593 A2 5 10 15 20 25 30 35 40 45 50 55 30 ctDNAanalysis and receiving ACTordered by recurrence status and disease stage. Patientsmarked by a (s) have synchronous CRC. FIG. 91 shows a schematic overview of ctDNA profiling results of plasma samples included in the longitudinal post- ACT ctDNA analysis ordered by recurrence status, post-operative ctDNA status, and length of follow-up. Patients marked by a (s) have synchronous CRC (n=2). Plasma samples marked with ** are positive in the second pool only (n=1). FIG. 92 shows a schematic overviewof CEAprofiling results of plasma samples included in the longitudinal post-ACT ctDNA analysis ordered by recurrence status, post-operative ctDNA status, and length of follow-up. Patients marked by a (s) have synchronous CRC (n=2). Plasma marked with ** are positive in the second pool only (n=1). FIG. 93A-D: Graphs showing the association between ctDNA status and recurrence subsequent to definitive treatment. FIG. 93A shows the recurrence rate stratified by longitudinal ctDNA status. FIG. 93B shows Kaplan- Meier estimates of TTR for 75 patients with longitudinal samples, stratified by longitudinal ctDNA status. FIG. 93C shows a graph comparing time to radiological and ctDNA recurrence. FIG. 93D shows that ctDNA variance allele frequency (VAF) in plasma increased towards the radiologic relapse. Early timepoints before andduringACTomitted. FIG. 94: Schematic overview of ctDNA profiling results of longitudinal plasma samples from relapsing vs. non- relapsing patients. Patients with just one positive plasma sample during surveillance is considered positive. FIG. 95: Schematic overview of CEA profiling results of longitudinal serum samples from relapsing vs. non-relapsing patients. Patients with just one positive plasma sample during surveillance is considered positive. FIG. 96: Graph comparing time to radiological and CEA recurrence. FIG. 97A-C: Detection of actionable mutation in recurrence patients. FIG. 97A shows percentage of ctDNA+ recurrence patients with actionable mutations detected during surveillance. First ctDNA+ sample (Left column) and all ctDNA+ plasma samples (Right column). FIG. 97B shows actionable variants called in blood. Correlation between mean blood VAFs calculated using Signatera ctDNA+ assays and variance allele frequencies (VAFs) of actionable mutations, plotted with logarithmic scales on both the horizontal and vertical axes. FIG. 97C shows serial ctDNA profiling of two representative recurrence patients with actionable mutations. FIG. 98: Schematic comparison of current standard of care and potential ctDNA guided post-operative patient management. FIG. 99: Graph showing ctDNA decreased by adjuvant chemotherapy (ACT). FIG. 100A-B: Graphs showing the association between ctDNA status and recurrence subsequent to definitive treatment.FIG.100Ashows the recurrence ratestratifiedby longitudinal ctDNAstatus,andKaplan-Meier estimatesof TTR for 58 patients with longitudinal samples, stratified by longitudinal ctDNA analysis. FIG. 100B shows the recurrence rate stratified by CEA analysis, and Kaplan-Meier estimates of TTR for 58 patients with longitudinal samples, stratified by CEA analysis.

[0094] The above-identified figures are provided by way of representation and not limitation. DETAILED DESCRIPTION OF THE INVENTION

[0095] Methods and compositions provided herein improve thedetection, diagnosis, staging, screening, treatment, and management of cancer (e.g., breast cancer, bladder cancer, or colorectal cancer). Methods provided herein, in illustrative embodiments analyze single nucleotide variant mutations (SNVs) in circulating fluids, especially circulating tumor DNA. The methods provide the advantage of identifying more of the mutations that are found in a tumor and clonal as well as subclonal mutations, in a single test, rather than multiple tests that would be required, if effective at all, that utilize tumor samples. The methods and compositions can be helpful on their own, or they can be helpful when used along with other methods for detection, diagnosis, staging, screening, treatment, andmanagement of cancer (e.g., breast cancer, bladder cancer, or colorectal cancer), for example to help support the results of these other methods to provide more confidence and / or a definitive result.

[0096] Accordingly, provided herein in one embodiment, is a method for determining the single nucleotide variants present in a cancer (e.g., breast cancer, bladder cancer, or colorectal cancer) bydetermining the single nucleotide variants present in a ctDNA sample from an individual, such as an individual having or suspected of having cancer (e.g., breast cancer, bladder cancer, or colorectal cancer) using a ctDNA SNV amplification / sequencing workflow provided herein.

[0097] The terms "cancer" and "cancerous" refer to or describe the physiological condition in animals that is typically characterized by unregulated cell growth. A "tumor" comprises oneormore cancerous cells. There are severalmain types of cancer. Carcinoma is a cancer that begins in the skin or in tissues that line or cover internal organs. Sarcoma is a cancer that begins in bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissue. Leukemia is a cancer that starts in blood-forming tissue, such as the bone marrow, and causes large numbers of abnormal blood cells to be produced andenter the blood. Lymphomaandmultiplemyelomaare cancers that begin in the cells of the immune system. Central nervous system cancers are cancers that begin in the tissues of the brain and spinal cord. 15 EP 4 737 593 A2 5 10 15 20 25 30 35 40 45 50 55

[0098] In some embodiments, the cancer comprises an acute lymphoblastic leukemia; acute myeloid leukemia; adrenocortical carcinoma; AIDS-related cancers; AIDS-related lymphoma; anal cancer; appendix cancer; astrocytomas; atypical teratoid / rhabdoid tumor; basal cell carcinoma; bladder cancer; brain stem glioma; brain tumor (including brain stem glioma, central nervous system atypical teratoid / rhabdoid tumor, central nervous system embryonal tumors, astrocytomas, craniopharyngioma, ependymoblastoma, ependymoma, medulloblastoma, medulloepithelioma, pineal parenchymal tumorsof intermediatedifferentiation, supratentorial primitiveneuroectodermal tumorsandpineoblastoma); breast cancer; bronchial tumors; Burkitt lymphoma; cancer of unknown primary site; carcinoid tumor; carcinoma of unknown primary site; central nervous system atypical teratoid / rhabdoid tumor; central nervous system embryonal tumors; cervical cancer; childhood cancers; chordoma; chronic lymphocytic leukemia; chronic myelogenous leukemia; chronic myeloproliferative disorders; colon cancer; colorectal cancer; craniopharyngioma; cutaneous T-cell lymphoma; endocrine pancreas islet cell tumors; endometrial cancer; ependymoblastoma; ependymoma; esophageal cancer; esthesioneuroblastoma; Ewing sarcoma; extracranial germ cell tumor; extragonadal germ cell tumor; extrahepatic bile duct cancer; gallbladder cancer; gastric (stomach) cancer; gastrointestinal carcinoid tumor; gastrointestinal stromal cell tumor; gastrointestinal stromal tumor (GIST); gestational trophoblastic tumor; glioma; hairy cell leukemia; head and neck cancer; heart cancer; Hodgkin lymphoma; hypopharyngeal cancer; intraocular melanoma; islet cell tumors; Kaposi sarcoma; kidney cancer; Langerhans cell histiocytosis; laryngeal cancer; lip cancer; liver cancer; malignant fibrous histiocytoma bone cancer; medulloblastoma; medulloepithelioma; melanoma; Merkel cell carcinoma; Merkel cell skin carcinoma; mesothelioma; metastatic squamous neck cancer with occult primary; mouth cancer; multiple endocrine neoplasia syndromes; multiple myeloma; multiple myeloma / plasma cell neoplasm; mycosis fungoides; myelodysplastic syndromes; myeloproliferative neoplasms; nasal cavity cancer; nasopharyngeal cancer; neuroblastoma; Non-Hodgkin lymphoma; nonmelanoma skin cancer; non-small cell lung cancer; oral cancer; oral cavity cancer; oropharyngeal cancer; osteosarcoma; other brain and spinal cord tumors; ovarian cancer; ovarian epithelial cancer; ovarian germ cell tumor; ovarian low malignant potential tumor; pancreatic cancer; papillomatosis; paranasal sinus cancer; parathyroid cancer; pelvic cancer; penile cancer; pharyngeal cancer; pineal parenchymal tumors of intermediate differentiation; pineoblas- toma; pituitary tumor; plasma cell neoplasm / multiple myeloma; pleuropulmonary blastoma; primary central nervous system (CNS) lymphoma; primary hepatocellular liver cancer; prostate cancer; rectal cancer; renal cancer; renal cell (kidney) cancer; renal cell cancer; respiratory tract cancer; retinoblastoma; rhabdomyosarcoma; salivary gland cancer; Sezary syndrome; small cell lung cancer; small intestine cancer; soft tissue sarcoma; squamous cell carcinoma; squamous neck cancer; stomach (gastric) cancer; supratentorial primitive neuroectodermal tumors; T-cell lymphoma; testicular cancer; throat cancer; thymic carcinoma; thymoma; thyroid cancer; transitional cell cancer; transitional cell cancer of the renal pelvis and ureter; trophoblastic tumor; ureter cancer; urethral cancer; uterine cancer; uterine sarcoma; vaginal cancer; vulvar cancer; Waldenstrom macroglobulinemia; or Wilm’s tumor.

[0099] In another embodiment, provided herein is amethod for detecting cancer (e.g., breast cancer, bladder cancer, or colorectal cancer) in a sample of blood or a fraction thereof from an individual, such as an individual suspected of having a cancer, that includes determining the single nucleotide variants present in a sample by determining the single nucleotide variants present in a ctDNAsampleusinga ctDNASNVamplification / sequencingworkflowprovidedherein. Thepresence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15SNVson the lowendof the range, and 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, or 50 SNVs on the high end of the range, in the sample at the plurality of single nucleotide loci is indicative of the presence of cancer (e.g., breast cancer, bladder cancer, or colorectal cancer).

[0100] In another embodiment, provided herein is amethod for detecting a clonal single nucleotide variant in a tumor of an individual (e.g., breast cancer, bladder cancer, or colorectal cancer). The method includes performing a ctDNA SNV amplification / sequencing workflow as provided herein, and determining the variant allele frequency for each of the SNV loci basedon the sequenceof the plurality of copies of the series of amplicons. Ahigher relative allele frequency compared to the other single nucleotide variants of the plurality of single nucleotide variant loci is indicative of a clonal single nucleotide variant in the tumor. Variant allele frequencies are well known in the sequencing art. Support for this embodiment, is provided, for example in FIGs. 12‑14.

[0101] In certain embodiments, themethod further includes determining a treatment plan, therapy and / or administering a compound to the individual that targets the one ormore clonal single nucleotide variants. In certain examples, subclonal and / or other clonal SNVs are not targeted by therapy. Specific therapies and associated mutations are provided in other sections of this specification and are known in the art. Accordingly, in certain examples, the method further includes administering a compound to the individual, where the compound is known to be specifically effective in treating cancer (e.g., breast cancer, bladder cancer, or colorectal cancer) havingoneormoreof the determined single nucleotide variants.

[0102] In certainaspectsof thisembodiment, a variant allele frequencyof greater than0.25%,0.5%,0.75%,1.0%,5%or 10% is indicative a clonal single nucleotide variant. These cutoffs are supported by the data in tabular form FIG. 20A-B.

[0103] In certain examples of this embodiment, the cancer is a stage 1a, 1b, or 2a breast cancer, bladder cancer, or colorectal cancer. In certain examples of this embodiment, the cancer is a stage 1a or 1b breast cancer, bladder cancer, or colorectal cancer. In certain examples of the embodiment, the individual is not subjected to surgery. In certain examples of the embodiment, the individual is not subjected to a biopsy. 16 EP 4 737 593 A2 5 10 15 20 25 30 35 40 45 50 55

[0104] In someexamples of this embodiment, a clonal SNV is identified or further identified if other testing such as direct tumor testing suggest an on-test SNV is a clonal SNV, for any SNVon test that has a variable allele frequency greater than at least one quarter, one third, one half, or three quarters of the other single nucleotide variants that were determined.

[0105] In some embodiments, methods herein for detecting SNVs in ctDNA can be used instead of direct analysis of DNA from a tumor. Results provided herein demonstrate that SNVs that are much more likely to be clonal SNVs have higher VAFs (See e.g. FIGs. 12‑14).

[0106] In certain examples of any of themethod embodiments provided herein herein, before a targeted amplification is performedonctDNA froman individual, data is providedonSNVs that are found ina tumor from the individual. Accordingly, in these embodiments, a SNV amplification / sequencing reaction is performed on one or more tumor samples from the individual. In this methods, the ctDNA SNV amplification / sequencing reaction provided herein is still advantageous because it provides a liquid biopsy of clonal and subclonal mutations. Furthermore, as provided herein, clonal mutations can be more unambiguously identified in an individual that has cancer (e.g., breast cancer, bladder cancer, or colorectal cancer), if a high VAF percentage, for example, more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10% VAF in a ctDNA sample from the individual is determined for an SNV.

[0107] In certain embodiment, method provided herein can be used to determinewhether to isolate and analyze ctDNA fromcirculating freenucleic acids froman individualwith cancer (e.g., breast cancer, bladder cancer, or colorectal cancer). First, it is determined whether the cancer is breast cancer, bladder cancer, or colorectal cancer. If the cancer is a breast cancer, bladder cancer, or colorectal cancer, circulating freenucleic acidsare isolated from individual. Themethod in some examples, further includes determining the stage of the cancer.

[0108] In some methods, provided herein are inventive compositions and / or solid supports. A composition comprising circulating tumor nucleic acid fragments comprising a universal adapter, wherein the circulating tumor nucleic acids originated from breast cancer, bladder cancer, or colorectal cancer.

[0109] In some embodiments, provided herein is an inventive composition that includes circulating tumor nucleic acid fragments comprising auniversal adapter, wherein the circulating tumor nucleic acids originated froma sample of bloodor a fraction thereof, of an individual with cancer (e.g., breast cancer, bladder cancer, or colorectal cancer). These methods typically include formation of ctDNA fragment that include a universal adapter. Furthermore, such methods typically include the formationofasolid support especially asolid support for high throughput sequencing, that includesaplurality of clonal populations of nucleic acids, wherein the clonal populations comprise amplicons generated from a sample of circulating free nucleic acids, wherein the ctDNA. In illustrative embodiments based on the surprising results provided herein, the ctDNA originated from cancer (e.g., breast cancer, bladder cancer, or colorectal cancer).

[0110] Similarly, provided herein as an embodiment of the invention is a solid support comprising a plurality of clonal populations of nucleic acids, wherein the clonal populations comprise nucleic acid fragments generated from a sample of circulating free nucleic acids from a sample of blood or a fraction thereof, from an individual with cancer (e.g., breast cancer, bladder cancer, or colorectal cancer).

[0111] In certain embodiments, the nucleic acid fragments in different clonal populations comprise the same universal adapter. Such a composition is typically formed during a high throughput sequencing reaction in methods of the present invention.

[0112] Theclonal populations of nucleic acids canbederived fromnucleic acid fragments fromaset of samples from two or more individuals. In these embodiments, the nucleic acid fragments comprise one of a series of molecular barcodes corresponding to a sample in the set of samples.

[0113] Detailed analytical methods are provided herein as SNVMethods 1 and SNVMethod 2 in the analytical section herein. Any of the methods provided herein can further include analytical steps provided herein. Accordingly, in certain examples, themethods for determiningwhether a single nucleotide variant is present in the sample, includes identifying a confidence value for each allele determination at each of the set of single nucleotide variance loci, which can be based at least in part on a depth of read for the loci. The confidence limit can be set at least 75%, 80%, 85%, 90%, 95%, 96%, 96%, 98%, or 99%. The confidence limit can be set at different levels for different types of mutations.

[0114] Themethod can performed with a depth of read for the set of single nucleotide variance loci of at least 5, 10, 15, 20, 25, 50, 100, 150, 200, 250, 500, 1,000, 10,000, 25,000, 50,000, 100,000, 250,000, 500,000, or 1 million.

[0115] In certainembodiments, amethodof anyof theembodiments herein includesdetermininganefficiencyand / or an error rate per cycle are determined for each amplification reaction of the multiplex amplification reaction of the single nucleotidevariance loci.Theefficiencyand theerror ratecan thenbeused todeterminewhetherasinglenucleotidevariant at theset of single variant loci is present in thesample.Moredetailedanalytical stepsprovided inSNVMethod2provided in the analytical method can be included as well, in certain embodiments.

[0116] In illustrative embodiments, of any of the methods herein the set of single nucleotide variance loci includes all of the single nucleotide variance loci identified in the TCGA and COSMIC data sets for cancer (e.g., breast cancer, bladder cancer, or colorectal cancer).

[0117] In certain embodiments of anyof themethodsherein the set of single nucleotide variant loci include2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 250, 500, 1000, 2500, 5000, or 10,000 single nucleotide variance loci known to be 17 EP 4 737 593 A2 5 10 15 20 25 30 35 40 45 50 55 associatedwith cancer (e.g., breast cancer, bladder cancer, or colorectal cancer) on the low end of the range, and , 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 250, 500, 1000, 2500, 5000, 10,000, 20,000 and 25,000 on the high end of the range.

[0118] In any of the methods for detecting SNVs herein that include a ctDNA SNVamplification / sequencing workflow, improvedamplification parameters formultiplexPCRcanbeemployed. For example,wherein theamplification reaction is a PCR reaction and the annealing temperature is between 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10°C greater than the melting temperature on the low end of the range, and 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15° on the high end the range for at least 10, 20, 25, 30, 40, 50, 06, 70, 75, 80, 90, 95 or 100% the primers of the set of primers.

[0119] In certain embodiments, wherein the amplification reaction is a PCR reaction the length of the annealing step in thePCRreaction isbetween10, 15, 20,30, 45, and60minuteson the lowendof the range,and15, 20, 30,45, 60, 120, 180, or 240minutes on the high endof the range. In certain embodiments, the primer concentration in the amplification, suchas the PCR reaction is between 1 and 10 nM. Furthermore, in exemplary embodiments, the primers in the set of primers, are designed to minimize primer dimer formation.

[0120] Accordingly, in an example of any of the methods herein that include an amplification step, the amplification reaction is a PCR reaction, the annealing temperature is between 1 and 10 °C greater than the melting temperature of at least 90% of the primers of the set of primers, the length of the annealing step in the PCR reaction is between 15 and 60 minutes, the primer concentration in the amplification reaction is between 1 and 10 nM, and the primers in the set of primers, are designed to minimize primer dimer formation. In a further aspect of this example, the multiplex amplification reaction is performed under limiting primer conditions.

[0121] In another embodiment, providedherein is amethod for supportinga cancer (e.g., breast cancer, bladder cancer, or colorectal cancer) diagnosis for an individual, such as an individual suspected of having cancer (e.g., breast cancer, bladder cancer, or colorectal cancer), from a sample of blood or a fraction thereof from the individual, that includes performing a ctDNASNVamplification / sequencingworkflow as provided herein, to determinewhether one ormore single nucleotide variants are present in the plurality of single nucleotide variant loci. In this embodiment, the following elements, statements, guidelines or rules apply: theabsenceof a single nucleotide variant supports adiagnosis of stage1a, 1b, or 2a adenocarcinoma, the presence of a single nucleotide variant supports a diagnosis of squamous cell carcinoma or a stage 2bor 3aadenocarcinoma, and / or thepresenceof tenormoresingle nucleotide variants supportsadiagnosisof squamous cell carcinoma or a stage 2b or 3 adenocarcinoma.

[0122] These results identify analysis using a ctDNA SNV amplification / sequencing workflow of lung ADC and SCC samples from an individual as a valuable method for identifying SNVs found in an ADC tumor, especially for stage 2b and 3a ADC tumors, and especially an SCC tumor at any stage (See e.g. FIG. 15 and FIG. 20A-B).

[0123] In certain embodiments, methods herein for detecting SNVs can be used to direct a therapeutic regimen. Therapies are available and under development that target specific mutations associated with ADC and SCC (Nature Review Cancer. 14:535‑551 (2014). For example, detection of an EGFRmutation at L858R or T790M can be informative for selecting a therapy. Erlotinib, gefitinib, afatinib, AZK9291, CO‑1686, and HM61713 are current therapies approved in the U.S. or in clinical trials, that target specific EGFRmutations. In another example, a G12D, G12C, or G12Vmutation in KRAScanbeused to direct an individual to a therapy of a combination of Selumetinib plus docetaxel. As another example, a mutation of V600E in BRAF can be used to direct a subject to a treatment of Vemurafenib, dabrafenib, and trametinib.

[0124] A sample analyzed inmethods of the present invention, in certain illustrative embodiments, is a blood sample, or a fraction thereof.Methods provided herein, in certain embodiments, are specially adapted for amplifyingDNA fragments, especially tumor DNA fragments that are found in circulating tumor DNA (ctDNA). Such fragments are typically about 160 nucleotides in length.

[0125] It is known in the art that cell-free nucleic acid (cfNA), e.g cfDNA, can be released into the circulation via various forms of cell death such as apoptosis, necrosis, autophagy and necroptosis. The cfDNA, is fragmented and the size distribution of the fragments varies from 150‑350 bp to > 10000 bp. (see Kalnina et al. World JGastroenterol. 2015Nov 7; 21(41): 11636‑11653). For example the size distributions of plasma DNA fragments in hepatocellular carcinoma (HCC) patients spanneda rangeof 100‑220bp in lengthwithapeak in count frequencyat about166bpand thehighest tumorDNA concentration in fragments of 150‑180 bp in length (see: Jiang et al. Proc Natl Acad Sci USA 112:E1317-E1325).

[0126] Inan illustrativeembodiment thecirculating tumorDNA(ctDNA) is isolated frombloodusingEDTA‑2Na tubeafter removal of cellular debris and platelets by centrifugation. The plasma samples can be stored at ‑80oC until the DNA is extractedusing, for example,QIAampDNAMini Kit (Qiagen,Hilden,Germany), (e.g.Hamakawaet al., Br JCancer. 2015; 112:352‑356). Hamakava et al. reported median concentration of extracted cell free DNA of all samples 43.1 ng per ml plasma (range 9.5‑1338 ng ml / ) and a mutant fraction range of 0.001‑77.8%, with a median of 0.90%.

[0127] In certain illustrative embodiments the sample is a tumor. Methods are known in the art for isolating nucleic acid from a tumor and for creating a nucleic acid library from such aDNA sample given the teachings here. Furthermore, given the teachings herein, a skilled artisanwill recognize how to create a nucleic acid library appropriate for themethods herein from other samples such as other liquid samples where the DNA is free floating in addition to ctDNA samples.

[0128] Methods of the present invention in certain embodiments, typically include a step of generating and amplifying a 18 EP 4 737 593 A2 5 10 15 20 25 30 35 40 45 50 55 nucleic acid library from the sample (i.e. library preparation). The nucleic acids from the sample during the library preparation step can have ligation adapters, often referred to as library tags or ligation adaptor tags (LTs), appended, where the ligationadapterscontainauniversal primingsequence, followedbyauniversal amplification. Inanembodiment, thismaybedoneusingastandardprotocol designed tocreatesequencing librariesafter fragmentation. Inanembodiment, theDNAsamplecanbeblunt ended, and thenanAcanbeaddedat the3’ end.AY-adaptorwithaT-overhangcanbeadded and ligated. In some embodiments, other sticky ends can be used other than an A or Toverhang. In some embodiments, other adaptors can be added, for example looped ligation adaptors. In some embodiments, the adaptors may have tag designed for PCR amplification.

[0129] Anumber of the embodiments provided herein, include detecting the SNVs in a ctDNA sample. Suchmethods in illustrative embodiments, include an amplification step and a sequencing step (Sometimes referred to herein as a "ctDNA SNV amplification / sequencing workflow). In an illustrative example, a ctDNA amplification / sequencing workflow can include generating a set of amplicons by performing a multiplex amplification reaction on nucleic acids isolated from a sample of blood or a fraction thereof from an individual, such as an individual suspected of having cancer, for example breast cancer, bladder cancer, or colorectal cancer, wherein each amplicon of the set of amplicons spans at least one single nucleotide variant loci of a set of single nucleotide variant loci, such as an SNV loci known to be associated with cancer (e.g., breast cancer, bladder cancer, or colorectal cancer); anddetermining thesequenceof at least a segment of at each amplicon of the set of amplicons, wherein the segment comprises a single nucleotide variant loci. In this way, this exemplary method determines the single nucleotide variants present in the sample.

[0130] Exemplary ctDNA SNVamplification / sequencing workflows in more detail can include forming an amplification reaction mixture by combining a polymerase, nucleotide triphosphates, nucleic acid fragments from a nucleic acid library generated from the sample, and a set of primers that each binds an effective distance froma single nucleotide variant loci, or aset of primerpairs that eachspananeffective region that includesasinglenucleotidevariant loci. Thesinglenucleotide variant loci, in exemplary embodiments, is one known to be associated with cancer, for example breast cancer, bladder cancer, or colorectal cancer. Then, subjecting the amplification reaction mixture to amplification conditions to generate a set of amplicons comprising at least one single nucleotide variant loci of a set of single nucleotide variant loci, preferably known to be associated with cancer (e.g., breast cancer, bladder cancer, or colorectal cancer); and determining the sequence of at least a segment of each amplicon of the set of amplicons, wherein the segment comprises a single nucleotide variant loci.

[0131] Theeffectivedistanceofbindingof theprimerscanbewithin1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11,12,13,14, 15,20,25,30, 35, 40, 45, 50, 75, 100, 125, or 150 base pairs of a SNV loci. The effective range that a pair of primers spans typically includes anSNVand is typically 160 base pairs or less, and can be 150, 140, 130, 125, 100, 75, 50 or 25 base pairs or less. In other embodiments, the effective range that a pair of primers spans is 20, 25, 30, 40, 50, 60, 70, 75, 100, 110, 120, 125, 130, 140, or 150nucleotides fromanSNV loci on the lowendof the range, and25, 30, 40,50, 60, 70, 75, 100, 110, 120, 125, 130, 140, or 150, 160, 170, 175, or 200 on the high end of the range.

[0132] Further details regarding methods of amplification that can be used in a ctDNA SNV amplification / sequencing workflow to detect SNVs for use in methods of the invention are provided in other sections of this specification. SNV Calling Analytics

[0133] During performance of the methods provided herein, nucleic acid sequencing data is generated for amplicons created by the tiled multiplex PCR. Algorithm design tools are available that can be used and / or adapted to analyze this data to determine within certain confidence limits, whether a mutation, such as a SNV is present in a target gene.

[0134] Sequencing Reads can be demultiplexed using an in-house tool and mapped using the Burrows-Wheeler alignment software, Bwamem function (BWA, Burrows-Wheeler Alignment Software (see Li H. andDurbinR. (2010) Fast and accurate long-read alignment with Burrows-Wheeler Transform. Bioinformatics, Epub. [PMID: 20080505]) on single end mode using pear merged reads to the hg19 genome. Amplification statistics QC can be performed by analyzing total reads, number of mapped reads, number of mapped reads on target, and number of reads counted.

[0135] In certain embodiments, anyanalyticalmethod for detectinganSNV fromnucleic acid sequencingdatadetection can be usedwithmethods of the inventionmethods of the invention that include a step of detecting anSNVor determining whether an SNV is present. In certain illustrative embodiments, methods of the invention that utilize SNV METHOD 1 below are used. In other, evenmore illustrative embodiments, methods of the invention that include a step of detecting an SNV or determining whether an SNV is present at an SNV loci, utilize SNV METHOD 2 below.

[0136] SNV METHOD 1:For this embodiment, a background errormodel is constructed using normal plasma samples, whichwere sequencedon the samesequencing run toaccount for run-specific artifacts. In certainembodiments, 5, 10, 15, 20, 25, 30, 40,50, 100, 150, 200, 250, ormore than250normal plasmasamplesareanalyzedon thesamesequencing run. In certain illustrative embodiments, 20, 25, 40, or 50 normal plasma samples are analyzed on the same sequencing run. Noisy positions with normal median variant allele frequency greater than a cutoff are removed. For example this cutoff in certain embodiments is > 0.1%, 0.2%, 0.25%, 0.5%, 1%, 2%, 5%, or 10%. In certain illustrative embodiments noisy 19 EP 4 737 593 A2 5 10 15 20 25 30 35 40 45 50 55 positions with normal medial variant allele frequency greater than 0.5% are removed. Outlier samples were iteratively removed from themodel toaccount for noiseandcontamination. In certainembodiments, sampleswithaZscoreofgreater than 5, 6, 7, 8, 9, or 10 are removed from the data analysis. For each base substitution of every genomic loci, the depth of readweightedmean and standard deviation of the error are calculated. Tumor or cell-free plasma samples’ positions with at least 5 variant reads and a Z-score of 10 against the background error model for example, can be called as a candidate mutation.

[0137] SNV METHOD 2: For this embodiment Single Nucleotide Variants (SNVs) are determined using plasma ctDNA data. ThePCRprocess ismodeled as a stochastic process, estimating the parameters using a training set andmaking the final SNV calls for a separate testing set. The propagation of the error across multiple PCR cycles is determined, and the mean and the variance of the background error are calculated, and in illustrative embodiments, background error is differentiated from real mutations.

[0138] The following parameters are estimated for each base: p = efficiency (probability that each read is replicated in each cycle) pe = error rate per cycle for mutation type e (probability that an error of type e occurs) X0 = initial number of molecules

[0139] Asa read is replicatedover thecourseofPCRprocess, themoreerrorsoccur.Hence, theerror profileof the reads is determinedby thedegreesof separation from theoriginal read.We refer to a readas kth generation if it has gone through k replications until it has been generated.

[0140] Let us define the following variables for each base: Xij = number of generation i reads generated in the PCR cycle j Yij = total number of generation i reads at the end of cycle j Xije = number of generation i reads with mutation e generated in the PCR cycle j

[0141] Moreover, in addition to normal molecules X0, if there are additional feX0 molecules with the mutation e at the beginning of the PCR process (hence fe / (1+fe) will be the fraction of mutated molecules in the initial mixture).

[0142] Given the total number of generation i‑1 reads at cycle j‑1, the number of generation i reads generated at cycle j has a binomial distribution with a sample size of Yi‑1,j‑1 and probability parameter of p.Hence, E(Xij, |Yi‑1,j‑1, p) = p Yi‑1,j‑1 and Var(Xij, |Yi‑1,j‑1, p)= p(1-p) Yi‑1,j‑1.

[0143] Wealso have .Hence, by recursion, simulation or similar methods, we can determineE(Xij,). Similarly, we can determine Var(Xij) = E(Var(Xij, | p)) + Var(E(Xij, | p)) using the distribution ofp.

[0144] finally,E(Xije |Yi-1,j‑1,pe)=peYi‑1,j‑1andVar(Xije |Yi‑1,j‑1, p)=pe (1-pe)Yi‑1,j‑1,andwecanuse these to computeE (Xije) and Var(Xije).

[0145] In certain embodiments, SNV Method 2 is performed as follows: a) Estimate a PCR efficiency and a per cycle error rate using a training data set; b) Estimate a number of startingmolecules for the testing data set at each base using the distribution of the efficiency estimated in step (a); c) If needed, update the estimate of the efficiency for the testing data set using the starting number of molecules estimated in step (b); d) Estimate the mean and variance for the total number of molecules, background error molecules and real mutation molecules (for a searchspaceconsistingof an initial percentageof realmutationmolecules) using testingset dataand parameters estimated in steps (a), (b) and (c); e) Fit a distribution to the number of total error molecules (background error and real mutation) in the total molecules, and calculate the likelihood for each real mutation percentage in the search space; and f) Determine the most likely real mutation percentage and calculate the confidence using the data from in step (e).

[0146] A confidence cutoff can be used to identify an SNVat an SNV loci. For example, a 90%, 95%, 96%, 97%, 98%, or 99% confidence cutoff can be used to call an SNV. Exemplary SNV METHOD 2 Algorithm

[0147] The algorithm starts by estimating the efficiency and error rate per cycle using the training set. Let n denote the total number of PCR cycles.

[0148] The number of readsRb at each base b can be approximated by (1+pb)n X0,where pb is the efficiency at base b. 20 EP 4 737 593 A2 5 10 15 20 25 30 35 40 45 50 55 Then (Rb / X0)1 / n can be used to approximate 1+pb. Then, we can determine the mean and the standard variation of pb across all training samples, to estimate the parameters of the probability distribution (such as normal, beta, or similar distributions) for each base.

[0149] Similarly the number of error e readsRbe at each base b can be used to estimate pe. After determining themean and the standard deviation of the error rate across all training samples, we approximate its probability distribution (such as normal, beta, or similar distributions) whose parameters are estimated using this mean and standard deviation values.

[0150] Next, for the testing data, we estimate the initial starting copy at each base as where f(.) is an estimated distribution from the training set. where f(.) is an estimated distribution from the training set.

[0151] Hence, we have estimated the parameters that will be used in the stochastic process. Then, by using these estimates, we can estimate the mean and the variance of the molecules created at each cycle (note that we do this separately for normal molecules, error molecules, and mutation molecules).

[0152] Finally, by using a probabilistic method (such as maximum likelihood or similar methods), we can determine the best fe value that fits the distribution of the error, mutation, and normal molecules the best. More specifically, we estimate the expected ratio of the error molecules to total molecules for various fe values in the final reads, and determine the likelihood of our data for each of these values, and then select the value with the highest likelihood.

[0153] Primer tails can improve the detection of fragmented DNA from universally tagged libraries. If the library tag and theprimer-tails contain ahomologoussequence, hybridization canbe improved (for example,melting temperature (Tm) is lowered) and primers can be extended if only a portion of the primer target sequence is in the sample DNA fragment. In some embodiments, 13 or more target specific base pairs may be used. In some embodiments, 10 to 12 target specific basepairsmaybeused. In someembodiments, 8 to 9 target specific basepairsmaybeused. In someembodiments, 6 to 7 target specific base pairs may be used.

[0154] In one embodiment, Libraries are generated from the samples above by ligating adaptors to the ends of DNA fragments in the samples, or to the ends ofDNA fragments generated fromDNA isolated from the samples. The fragments can then be amplified using PCR, for example, according to the following exemplary protocol:

[0155] 95°C, 2 min; 15 x [95°C, 20 sec, 55°C, 20 sec, 68°C, 20 sec], 68°C 2 min, 4°C hold.

[0156] Manykitsandmethodsareknown in theart forgenerationof librariesofnucleicacids that includeuniversalprimer binding sites for subsequent amplification, for example clonal amplification, and for subsequence sequencing. To help facilitate ligation of adapters library preparation and amplification can include end repair and adenylation (i.e. A-tailing). Kits especially adapted for preparing libraries from small nucleic acid fragments, especially circulating free DNA, can be useful for practicingmethods provided herein. For example, theNEXTflexCell Free kits available fromBiooScientific () or theNatera Library PrepKit (available fromNatera, Inc. SanCarlos, CA) . However, such kitswould typically bemodified to include adaptors that are customized for the amplification and sequencing steps of themethods provided herein. Adaptor ligation can be performed using commercially available kits such as the ligation kit found in the AGILENT SURESELECT kit (Agilent, CA).

[0157] Target regions of the nucleic acid library generated from DNA isolated from the sample, especially a circulating free DNA sample for themethods of the present invention, are then amplified. For this amplification, a series of primers or primer pairs, which can include between 5, 10, 15, 20, 25, 50, 100, 125, 150, 250, 500, 1000, 2500, 5000, 10,000, 20,000, 25,000, or 50,000 on the low end of the range and 15, 20, 25, 50, 100, 125, 150, 250, 500, 1000, 2500, 5000, 10,000, 20,000, 25,000, 50,000, 60,000, 75,000, or 100,000primerson theupper endof the range, that eachbind to oneof a series of primer binding sites.

[0158] Primer designs can be generated with Primer3 (Untergrasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M, Rozen SG (2012) "Primer3 - new capabilities and interfaces." Nucleic Acids Research 40(15):e115 and Koressaar T, Remm M (2007) "Enhancements and modifications of primer design program Primer3." Bioinformatics 23(10):1289‑91) source code available at primer3.sourceforge.net). Primer specificity can be evaluated by BLASTand added to existing primer design pipeline criteria: Primer specificities can be determined using the BLASTn program from the ncbi-blast‑2.2.29+ package. The task option "blastn-short" can be used to map the primers against hg19 human genome. Primer designs can be determined as "specific" if the primer has less than 100 hits to the genome and the top hit is the target complementary primer binding region of the genome and is at least two scores higher than other hits (score is defined by BLASTn program). This can be done in order to have a unique hit to the genome and to not have many other hits throughout the genome.

[0159] The final selected primers can be visualized in IGV (JamesT.Robinson, Helga Thorvaldsdottir,WendyWinckler, Mitchell Guttman, Eric S. Lander, GadGetz, Jill P.Mesirov. IntegrativeGenomicsViewer. Nature Biotechnology 29, 24‑26 (2011)) and UCSC browser (KentWJ, Sugnet CW, Furey TS, Roskin KM, Pringle TH, Zahler AM, Haussler D. The human genome browser at UCSC. Genome Res. 2002 Jun;12(6):996‑1006 ) using bed files and coverage maps for validation. 21 EP 4 737 593 A2 5 10 15 20 25 30 35 40 45 50 55

[0160] Methodsof the present invention, in certain embodiments, include forminganamplification reactionmixture. The reaction mixture typically is formed by combining a polymerase, nucleotide triphosphates, nucleic acid fragments from a nucleic acid library generated from the sample, a set of forward and reverse primers specific for target regions that contain SNVs. The reaction mixtures provided herein, themselves forming in illustrative embodiments, a separate aspect of the invention.

[0161] An amplification reaction mixture useful for the present invention includes components known in the art for nucleic acidamplification, especially forPCRamplification.For example, the reactionmixture typically includesnucleotide triphosphates, a polymerase, and magnesium. Polymerases that are useful for the present invention can include any polymerase that can be used in an amplification reaction especially those that are useful in PCR reactions. In certain embodiments, hot start Taq polymerases are especially useful. Amplification reaction mixtures useful for practicing the methods provided herein, such as AmpliTaq Gold master mix (Life Technologies, Carlsbad, CA), are available commer- cially.

[0162] Amplification (e.g. temperature cycling) conditions for PCR are well known in the art. The methods provided herein can includeanyPCRcyclingconditions that result inamplificationof targetnucleicacidssuchas targetnucleicacids from a library. Non-limiting exemplary cycling conditions are provided in the Examples section herein.

[0163] There are many workflows that are possible when conducting PCR; some workflows typical to the methods disclosed herein are provided herein. The steps outlined herein are not meant to exclude other possible steps nor does it imply that any of the steps described herein are required for the method to work properly. A large number of parameter variations or other modifications are known in the literature, and may be made without affecting the essence of the invention.

[0164] In certain embodiments of the method provided herein, at least a portion and in illustrative examples the entire sequence of an amplicon, such as an outer primer target amplicon, is determined. Methods for determining the sequence of anamplicon are known in theart. Anyof the sequencingmethods known in theart, e.g. Sanger sequencing, canbeused for such sequence determination. In illustrative embodiments high throughput next-generation sequencing techniques (also referred to herein as massively parallel sequencing techniques) such as, but not limited to, those employed in MYSEQ (ILLUMINA), HISEQ (ILLUMINA), ION TORRENT (LIFE TECHNOLOGIES), GENOME ANALYZER ILX (ILLU- MINA), GS FLEX+ (ROCHE 454), can be used for sequencing the amplicons produced by the methods provided herein.

[0165] High throughput genetic sequencers are amenable to the use of barcoding (i.e., sample tagging with distinctive nucleic acid sequences) soas to identify specific samples from individuals thereby permitting the simultaneous analysis of multiple samples in a single run of the DNA sequencer. The number of times a given region of the genome in a library preparation (or other nucleic preparation of interest) is sequenced (number of reads) will be proportional to the number of copiesof that sequence in thegenomeof interest (or expression level in thecaseof cDNAcontainingpreparations).Biases in amplification efficiency can be taken into account in such quantitative determination. Target Genes

[0166] Target genes of the present invention in exemplary embodiments, are cancer-related genes, and in many illustrative embodiments, cancer-related genes. A cancer-related gene (for example, a cancer-related gene or a bladder cancer-related gene or a colorectal cancer-related gene) refers to a gene associatedwith an altered risk for a cancer (e.g. breast cancer, bladder cancer, or colorectal cancer) or an altered prognosis for a cancer. Exemplary cancer-related genes that promote cancer include oncogenes; genes that enhance cell proliferation, invasion, or metastasis; genes that inhibit apoptosis; and pro-angiogenesis genes. Cancer-related genes that inhibit cancer include, but are not limited to, tumor suppressor genes; genes that inhibit cell proliferation, invasion, or metastasis; genes that promote apoptosis; and anti- angiogenesis genes.

[0167] Anembodimentof themutationdetectionmethodbeginswith theselectionof the regionof thegene thatbecomes the target. The regionwith knownmutations is used to develop primers formPCR-NGS to amplify and detect themutation.

[0168] Methods provided herein can be used to detect virtually any type of mutation, especially mutations known to be associated with cancer and most particularly the methods provided herein are directed to mutations, especially SNVs, associatedwith cancer, specifically breast cancer, bladder cancer, or colorectal cancer. Exemplary SNVs can be in one or more of the following genes: EGFR, FGFR1, FGFR2, ALK, MET, ROS1, NTRK1, RET, HER2, DDR2, PDGFRA, KRAS, NF1, BRAF, PIK3CA, MEK1, NOTCH1, MLL2, EZH2, TET2, DNMT3A, SOX2, MYC, KEAP1, CDKN2A, NRG1, TP53, LKB1, and PTEN, which have been identified in various lung cancer samples as being mutated, having increased copy numbers, or being fused to other genes and combinations thereof (Non-small-cell lung cancers: a heterogeneous set of diseases. Chen et al. Nat. Rev. Cancer. 2014 Aug 14(8):535‑551). In another example, the list of genes are those listed above, where SNVs have been reported, such as in the cited Chen et al. reference. 22 EP 4 737 593 A2 5 10 15 20 25 30 35 40 45 50 55 Amplification (e.g. PCR) Reaction Mixtures:

[0169] Methodsof the present invention, in certain embodiments, include forminganamplification reactionmixture. The reaction mixture typically is formed by combining a polymerase, nucleotide triphosphates, nucleic acid fragments from a nucleic acid library generated from the sample, a series of forward target-specific outer primers and a first strand reverse outer universal primer. Another illustrative embodiment is a reaction mixture that includes forward target-specific inner primers instead of the forward target-specific outer primers and amplicons from a first PCR reaction using the outer primers, instead of nucleic acid fragments from the nucleic acid library. The reactionmixtures provided herein, themselves forming in illustrative embodiments, a separate aspect of the invention. In illustrative embodiments, the reactionmixtures are PCR reaction mixtures. PCR reaction mixtures typically include magnesium.

[0170] In some embodiments, the reaction mixture includes ethylenediaminetetraacetic acid (EDTA), magnesium, tetramethyl ammoniumchloride (TMAC), or anycombination thereof. In someembodiments, theconcentrationofTMAC is between 20 and 70 mM, inclusive. While not meant to be bound to any particular theory, it is believed that TMAC binds to DNA, stabilizes duplexes, increases primer specificity, and / or equalizes the melting temperatures of different primers. In some embodiments, TMAC increases the uniformity in the amount of amplified products for the different targets. In some embodiments, the concentration of magnesium (such as magnesium frommagnesium chloride) is between 1 and 8mM.

[0171] The large number of primers used for multiplex PCR of a large number of targets may chelate a lot of the magnesium (2 phosphates in the primers chelate 1 magnesium). For example, if enough primers are used such that the concentration of phosphate from the primers is ~9 mM, then the primers may reduce the effective magnesium concentration by ~4.5 mM. In some embodiments, EDTA is used to decrease the amount of magnesium available as a cofactor for the polymerase since high concentrations of magnesium can result in PCR errors, such as amplification of non-target loci. In someembodiments, theconcentrationofEDTA reduces theamount of availablemagnesium tobetween 1 and 5 mM (such as between 3 and 5 mM).

[0172] In some embodiments, the pH is between 7.5 and 8.5, such as between 7.5 and 8, 8 and 8.3, or 8.3 and 8.5, inclusive. In someembodiments,Tris is usedat, for example, a concentrationof between10and100mM,suchasbetween 10 and 25 mM, 25 and 50 mM, 50 and 75 mM, or 25 and 75 mM, inclusive. In some embodiments, any of these concentrationsofTris areusedatapHbetween7.5and8.5. In someembodiments, acombinationofKCland (NH4)2SO4 is used, such as between 50 and 150mMKCl and between 10 and 90mM (NH4)2SO4, inclusive. In some embodiments, the concentration of KCl is between 0 and 30 mM, between 50 and 100 mM, or between 100 and 150mM, inclusive. In some embodiments, the concentration of (NH4)2SO4 is between10and50mM,50and90mM,10and20mM,20and40mM,40 and 60 mM, or 60 and 80 mM (NH4)2SO4, inclusive. In some embodiments, the ammonium [NH4+] concentration is between0and160mM, suchasbetween0 to 50, 50 to 100, or 100 to160mM, inclusive. In someembodiments, the sumof the potassium and ammonium concentration ([K+] + [NH4+]) is between 0 and 160mM, such as between 0 to 25, 25 to 50, 50 to150, 50 to75, 75 to100, 100 to125, or125 to160mM, inclusive.Anexemplarybufferwith [K+]+ [NH4+]=120mM is20 mMKCland50mM(NH4)2SO4. In someembodiments, thebuffer includes25 to75mMTris, pH7.2 to8,0 to50mMKCl, 10 to 80mMammoniumsulfate, and 3 to 6mMmagnesium, inclusive. In someembodiments, the buffer includes 25 to 75mM Tris pH 7 to 8.5, 3 to 6mMMgCl2, 10 to 50mMKCl, and 20 to 80mM (NH4)2SO4, inclusive. In some embodiments, 100 to 200 Units / mL of polymerase are used. In some embodiments, 100 mM KCl, 50 mM (NH4)2SO4, 3 mM MgCl2, 7.5 nM of each primer in the library, 50 mM TMAC, and 7 ul DNA template in a 20 ul final volume at pH 8.1 is used.

[0173] In some embodiments, a crowding agent is used, such as polyethylene glycol (PEG, such as PEG 8,000) or glycerol. In some embodiments, the amount of PEG (such as PEG 8,000) is between 0.1 to 20%, such as between 0.5 to 15%, 1 to 10%, 2 to 8%, or 4 to 8%, inclusive. In someembodiments, the amount of glycerol is between0.1 to 20%, suchas between 0.5 to 15%, 1 to 10%, 2 to 8%, or 4 to 8%, inclusive. In some embodiments, a crowding agent allows either a low polymerase concentration and / or a shorter annealing time to be used. In some embodiments, a crowding agent improves the uniformity of the DOR and / or reduces dropouts (undetected alleles). Polymerases In some embodiments, a polymerase with proof-reading activity, a polymerase without (or with negligible) proof-reading activity, or a mixture of a polymerase with proof-reading activity and a polymerase without (or with negligible) proof-reading activity is used. In some embodiments, a hot start polymerase, a non-hot start polymerase, or amixture of a hot start polymerase and a non- hot start polymerase is used. In some embodiments, a HotStarTaq DNA polymerase is used (see, for example, QIAGEN catalog No. 203203). In some embodiments, AmpliTaq Gold® DNA Polymerase is used. In some embodiments a PrimeSTAR GXL DNA polymerase, a high fidelity polymerase that provides efficient PCR amplification when there is excess template in the reaction mixture, and when amplifying long products, is used (Takara Clontech, Mountain View, CA). In some embodiments, KAPATaq DNAPolymerase or KAPATaq HotStart DNA Polymerase is used; they are based on the single-subunit, wild-type Taq DNA polymerase of the thermophilic bacterium Thermus aquaticus. KAPATaq and KAPATaq HotStart DNA Polymerase have 5’‑3’ polymerase and 5’‑3’ exonuclease activities, but no 3’ to 5’ exonuclease (proofreading) activity (see, for example, KAPA BIOSYSTEMS catalog No. BK1000). In some embodiments, Pfu DNA polymerase is used; it is a highly thermostable DNA polymerase from the hyperthermophilic archaeum Pyrococcus furiosus. The enzyme catalyzes the template-dependent polymerization of nucleotides into duplex DNA in the 5’→3’ 23 EP 4 737 593 A2 5 10 15 20 25 30 35 40 45 50 55 direction. Pfu DNA Polymerase also exhibits 3’→5’ exonuclease (proofreading) activity that enables the polymerase to correct nucleotide incorporation errors. It has no 5’→3’ exonuclease activity (see, for example, Thermo Scientific catalog No. EP0501). In some embodiments Klentaq1 is used; it is a Klenow-fragment analog of Taq DNA polymerase, it has no exonuclease or endonuclease activity (see, for example, DNA POLYMERASE TECHNOLOGY, Inc, St. Louis, Missouri, catalogNo.100). In someembodiments, thepolymerase isaPHUSIONDNApolymerase, suchasPHUSIONHighFidelity DNA polymerase (M0530S, New England BioLabs, Inc.) or PHUSION Hot Start Flex DNA polymerase (M0535S, New EnglandBioLabs, Inc.). In someembodiments, thepolymerase isaQ5®DNAPolymerase, suchasQ5®High-FidelityDNA Polymerase (M0491S, New England BioLabs, Inc.) or Q5® Hot Start High-Fidelity DNA Polymerase (M0493S, New EnglandBioLabs, Inc.). In someembodiments, thepolymerase is aT4DNApolymerase (M0203S,NewEnglandBioLabs, Inc.).

[0174] In some embodiment, between 5 and 600 Units / mL (Units per 1 mL of reaction volume) of polymerase is used, such as between 5 to 100, 100 to 200, 200 to 300, 300 to 400, 400 to 500, or 500 to 600 Units / mL, inclusive. PCR Methods

[0175] In some embodiments, hot-start PCR is used to reduce or prevent polymerization prior to PCR thermocycling. Exemplary hot-start PCR methods include initial inhibition of the DNA polymerase, or physical separation of reaction components reaction until the reaction mixture reaches the higher temperatures. In some embodiments, slow release of magnesium is used. DNA polymerase requires magnesium ions for activity, so the magnesium is chemically separated from the reaction by binding to a chemical compound, and is released into the solution only at high temperature. In some embodiments, non-covalent binding of an inhibitor is used. In this method a peptide, antibody, or aptamer are non- covalently bound to the enzyme at low temperature and inhibit its activity. After incubation at elevated temperature, the inhibitor is released and the reaction starts. In some embodiments, a cold-sensitive Taq polymerase is used, such as a modified DNA polymerase with almost no activity at low temperature. In some embodiments, chemical modification is used. In this method, a molecule is covalently bound to the side chain of an amino acid in the active site of the DNA polymerase. The molecule is released from the enzyme by incubation of the reaction mixture at elevated temperature. Once the molecule is released, the enzyme is activated.

[0176] In someembodiments, the amount to template nucleic acids (suchasanRNAorDNAsample) is between20and 5,000 ng, such as between 20 to 200, 200 to 400, 400 to 600, 600 to 1,000; 1,000 to 1,500; or 2,000 to 3,000 ng, inclusive.

[0177] In some embodiments a QIAGEN Multiplex PCR Kit is used (QIAGEN catalog No. 206143). For 100 x 50 µl multiplex PCR reactions, the kit includes 2x QIAGENMultiplex PCRMaster Mix (providing a final concentration of 3 mM MgCl2, 3 x0.85ml), 5xQ-Solution (1 x2.0ml), andRNase-FreeWater (2 x1.7ml). TheQIAGENMultiplexPCRMasterMix (MM) contains a combination of KCl and (NH4)2SO4 as well as the PCR additive, Factor MP, which increases the local concentration of primers at the template. Factor MP stabilizes specifically bound primers, allowing efficient primer extension by HotStarTaq DNA Polymerase. HotStarTaq DNA Polymerase is a modified form of Taq DNA polymerase andhasnopolymeraseactivityat ambient temperatures. In someembodiments,HotStarTaqDNAPolymerase isactivated by a 15-minute incubation at 95°C which can be incorporated into any existing thermal-cycler program.

[0178] In some embodiments, 1x QIAGEN MM final concentration (the recommended concentration), 7.5 nM of each primer in the library, 50mMTMAC, and 7 ul DNA template in a 20 ul final volume is used. In some embodiments, the PCR thermocycling conditions include 95°C for 10 minutes (hot start); 20 cycles of 96°C for 30 seconds; 65°C for 15 minutes; and 72°C for 30 seconds; followed by 72°C for 2 minutes (final extension); and then a 4°C hold.

[0179] In some embodiments, 2x QIAGEN MM final concentration (twice the recommended concentration), 2 nM of eachprimer in the library, 70mMTMAC, and7ulDNA template in a20ul total volume is used. In someembodiments, up to 4 mM EDTA is also included. In some embodiments, the PCR thermocycling conditions include 95°C for 10 minutes (hot start); 25 cycles of 96°C for 30 seconds; 65°C for 20, 25, 30, 45, 60, 120, or 180 minutes; and optionally 72°C for 30 seconds); followed by 72°C for 2 minutes (final extension); and then a 4°C hold.

[0180] Another exemplary set of conditions includes a semi-nested PCR approach. The first PCR reaction uses 20 ul a reaction volumewith 2xQIAGENMMfinal concentration, 1.875nMof eachprimer in the library (outer forward and reverse primers), and DNA template. Thermocycling parameters include 95°C for 10 minutes; 25 cycles of 96°C for 30 seconds, 65°C for 1minute, 58°C for 6minutes, 60°C for 8minutes, 65°C for 4minutes, and72°C for 30seconds; and then72°C for 2 minutes, and then a 4°C hold. Next, 2 ul of the resulting product, diluted 1:200, is used as input in a second PCR reaction. This reaction uses a 10 ul reaction volume with 1x QIAGEN MM final concentration, 20 nM of each inner forward primer, and 1 uMof reverse primer tag. Thermocycling parameters include 95°C for 10minutes; 15 cycles of 95°C for 30 seconds, 65°C for1minute, 60°C for5minutes,65°C for5minutes, and72°C for30seconds;and then72°C for2minutes, and thena 4°C hold. The annealing temperature can optionally be higher than themelting temperatures of some or all of the primers, as discussed herein (see U.S. Patent Application No. 14 / 918,544, filed Oct. 20, 2015, which is herein incorporated by reference in its entirety).

[0181] Themelting temperature (Tm) is the temperature at which one-half (50%) of a DNA duplex of an oligonucleotide 24 EP 4 737 593 A2 5 10 15 20 25 30 35 40 45 50 55 (such as a primer) and its perfect complement dissociates and becomes single strand DNA. The annealing temperature (TA) is the temperature one runs thePCRprotocol at. For priormethods, it is usually 5°Cbelow the lowestTmof the primers used, thus close to all possible duplexes are formed (such that essentially all the primer molecules bind the template nucleic acid). While this is highly efficient, at lower temperatures there aremore unspecific reactions bound to occur. One consequence of having too low a TA is that primersmay anneal to sequences other than the true target, as internal single- base mismatches or partial annealing may be tolerated. In some embodiments of the present inventions, the TA is higher thanTm,whereat agivenmoment only a small fractionof the targetshaveaprimer annealed (suchasonly~1‑5%). If these get extended, they are removed from the equilibrium of annealing and dissociating primers and target (as extension increases Tm quickly to above 70°C), and a new ~1‑5% of targets has primers. Thus, by giving the reaction a long time for annealing, one can get ~100% of the targets copied per cycle.

[0182] In various embodiments, the annealing temperature is between1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 °Cand2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 15 °C on the high end of the range, greater than the melting temperature (such as the empirically measured or calculated Tm) of at least 25, 50, 60, 70, 75, 80, 90, 95, or 100% of the non-identical primers. In various embodiments, the annealing temperature is between1and15 °C (suchasbetween1 to10, 1 to 5, 1 to 3, 3 to 5, 5 to 10,5 to8,8 to10,10 to12, or12 to15 °C, inclusive)greater than themelting temperature (suchas theempiricallymeasured or calculated Tm) of at least 25; 50; 75; 100; 300; 500; 750; 1,000; 2,000; 5,000; 7,500; 10,000; 15,000; 19,000; 20,000; 25,000; 27,000; 28,000; 30,000; 40,000; 50,000; 75,000; 100,000; or all of the non-identical primers. In various embodiments, the annealing temperature is between 1 and 15 °C (such as between 1 to 10, 1 to 5, 1 to 3, 3 to 5, 3 to 8, 5 to 10, 5 to 8, 8 to 10, 10 to 12, or 12 to 15 °C, inclusive) greater than the melting temperature (such as the empirically measured or calculatedTm) of at least 25%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, or all of the non-identical primers, and the length of the annealing step (per PCR cycle) is between 5 and 180 minutes, such as 15 and 120 minutes, 15 and 60 minutes, 15 and 45 minutes, or 20 and 60 minutes, inclusive. Exemplary Multiplex PCR Methods

[0183] In various embodiments, long annealing times (as discussed herein and exemplified in Example 10) and / or low primer concentrationsareused. In fact, in certainembodiments, limiting primer concentrationsand / or conditionsareused. In variousembodiments, the lengthof theannealing step is between15, 20, 25, 30, 35, 40, 45, or 60minuteson the lowend of the range and 20, 25, 30, 35, 40, 45, 60, 120, or 180minutes on the high end of the range. In various embodiments, the length of the annealing step (per PCR cycle) is between 30 and 180 minutes. For example, the annealing step can be between 30 and 60 minutes and the concentration of each primer can be less than 20, 15, 10, or 5 nM. In other embodiments the primer concentration is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 nMon the lowend of the range, and 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, and 50 on the high end of the range.

[0184] Athigh level ofmultiplexing, thesolutionmaybecomeviscousdue to the largeamount of primers in solution. If the solution is tooviscous,onecan reduce theprimer concentration toanamount that is still sufficient for theprimers tobind the template DNA. In various embodiments, between 1,000 and 100,000 different primers are used and the concentration of each primer is less than 20 nM, such as less than 10 nM or between 1 and 10 nM, inclusive. Detection of Copy number Variation (CNV)

[0185] In addition to SNVs and indels, methods for monitoring and detection of early relapse andmetastasis described herein can also benefit from detection of CNVs.

[0186] In one aspect, the present invention generally relates, at least in part, to improved methods of determining the presence or absence of copy number variations, such as deletions or duplications of chromosome segments or entire chromosomes. The methods are particularly useful for detecting small deletions or duplications, which can be difficult to detect with high specificity and sensitivity using prior methods due to the small amount of data available from the relevant chromosome segment. The methods include improved analytical methods, improved bioassay methods, and combina- tions of improved analytical and bioassay methods. Methods of the invention can also be used to detect deletions or duplications that are only present in a small percentage of the cells or nucleic acid molecules that are tested. This allows deletions or duplications to be detected prior to the occurrence of disease (such as at a precancerous stage) or in the early stages of disease, such as before a large number of diseased cells (such as cancer cells) with the deletion or duplication accumulate. The more accurate detection of deletions or duplications associated with a disease or disorder enable improved methods for diagnosing, prognosticating, preventing, delaying, stabilizing, or treating the disease or disorder. Several deletions or duplications are known to be associated with cancer or with severe mental or physical handicaps.

[0187] Inanotheraspect, thepresent inventiongenerally relates, at least in part, to improvedmethodsofdetectingsingle nucleotide variations (SNVs). These improved methods include improved analytical methods, improved bioassay methods, and improved methods that use a combination of improved analytical and bioassay methods. The methods in certain illustrative embodiments are used to detect, diagnose, monitor, or stage cancer, for example in samples where 25 EP 4 737 593 A2 5 10 15 20 25 30 35 40 45 50 55 the SNV is present at very low concentrations, for example less than 10%, 5%, 4%, 3%, 2.5%, 2%, 1%, 0.5%, 0.25%, or 0.1% relative to the total number of normal copies of the SNV locus, such as circulating free DNA samples. That is, these methods in certain illustrative embodiments are particularly well suited for samples where there is a relatively low percentage of amutation or variant relative to the normal polymorphic alleles present for that genetic loci. Finally, provided herein aremethods that combine the improvedmethods for detecting copy number variations with the improvedmethods for detecting single nucleotide variations.

[0188] Successful treatment of a disease such as cancer often relies on early diagnosis, correct staging of the disease, selection of an effective therapeutic regimen, and close monitoring to prevent or detect relapse. For cancer diagnosis, histological evaluation of tumor material obtained from tissue biopsy is often considered the most reliable method. However, the invasive nature of biopsy-based sampling has rendered it impractical for mass screening and regular follow up. Therefore, the present methods have the advantage of being able to be performed non-invasively if desired for relatively low cost with fast turnaround time. The targeted sequencing that may be used by the methods of the invention requires less reads than shotgun sequencing, such as a fewmillion reads instead of 40million reads, thereby decreasing cost. The multiplex PCR and next generation sequencing that may be used increase throughput and reduces costs.

[0189] In someexemplaryembodiments, analysis ofAAI patterns in ctDNAprovidemoredetailed insights into theclonal architecture of tumors to help predict their therapeutic responses and optimize treatment strategies. Therefore, in certain embodiments,mmPCR-NGSpanels are selected that target clinically actionable CNVs andSNVs. Such panels in certain illustrativeembodiments, areparticularlyuseful for patientswith cancerswhereCNVs representasubstantial proportionof the mutation load, as is common in breast, ovarian, and lung cancer.

[0190] In some embodiments, the methods are used to detect a deletion, duplication, or single nucleotide variant in an individual. A sample from the individual that contains cells or nucleic acids suspected of having a deletion, duplication, or singlenucleotidevariantmaybeanalyzed. Insomeembodiments, thesample is froma tissueororgansuspectedofhaving adeletion, duplication, or single nucleotide variant, suchas cells or amass suspectedof being cancerous. Themethods of the invention can be used to detect deletion, duplication, or single nucleotide variant that are only present in one cell or a small number of cells in a mixture containing cells with the deletion, duplication, or single nucleotide variant and cells without the deletion, duplication, or single nucleotide variant. In someembodiments, cfDNAor cfRNA fromablood sample from the individual is analyzed. In some embodiments, cfDNAor cfRNA is secreted by cells, such as cancer cells. In some embodiments, cfDNA or cfRNA is released by cells undergoing necrosis or apoptosis, such as cancer cells. Themethods of the invention can be used to detect deletion, duplication, or single nucleotide variant that are only present in a small percentage of the cfDNA or cfRNA. In some embodiments, one or more cells from an embryo are tested.

[0191] In addition to determining the presence or absence of copy number variation, one or more other factors can be analyzed if desired. These factors can be used to increase the accuracy of the diagnosis (such as determining the presence or absence of cancer or an increased risk for cancer, classifying the cancer, or staging the cancer) or prognosis. These factors canalsobeused to select a particular therapyor treatment regimen that is likely to beeffective in the subject. Exemplary factors include thepresenceorabsenceofpolymorphismsormutation; altered (increasedordecreased) levels of total or particular cfDNA, cfRNA, microRNA (miRNA); altered (increased or decreased) tumor fraction; altered (increased or decreased) methylation levels, altered (increased or decreased) DNA integrity, altered (increased or decreased) or alternative mRNA splicing.

[0192] The following sections describe methods for detecting deletions or duplications using phased data (such as inferred or measured phased data) or unphased data; samples that can be tested; methods for sample preparation, amplification, and quantification; methods for phasing genetic data; polymorphisms, mutations, nucleic acid alterations, mRNA splicing alterations, and changes in nucleic acid levels that can be detected; databases with results from the methods, other risk factors and screeningmethods; cancers that can be diagnosed or treated; cancer treatments; cancer models for testing treatments; and methods for formulating and administering treatments. Exemplary Methods for Determining Ploidy Using Phased Data

[0193] Some of the methods of the invention are based in part on the discovery that using phased data for detecting CNVs decreases the false negative and false positive rates compared to using unphased data. This improvement is greatest for samples with CNVs present in low levels. Thus, phase data increases the accuracy of CNV detection compared tousingunphaseddata (suchasmethods that calculateallele ratiosatoneormore loci or aggregateallele ratios to giveanaggregated value (suchasanaverage value) over a chromosomeor chromosomesegmentwithout considering whether the allele ratios at different loci indicate that the same or different haplotypes appear to be present in an abnormal amount). Using phased data allows amore accurate determination to bemade of whether differences betweenmeasured and expected allele ratios are due to noise or due to the presence of a CNV. For example, if the differences between measured and expected allele ratios at most or all of the loci in a region indicate that the same haplotype is over- represented, then aCNV ismore likely to bepresent. Using linkagebetweenalleles in a haplotypeallowsone to determine whether the measured genetic data is consistent with the same haplotype being overrepresented (rather than random 26 EP 4 737 593 A2 5 10 15 20 25 30 35 40 45 50 55 noise). In contrast, if the differences between measured and expected allele ratios are only due to noise (such as experimental error), then in someembodiments, about half the time the first haplotype appears to be overrepresented and about the other half of the time, the second haplotype appears to be overrepresented.

[0194] In some embodiments, phased genetic data is used to determine if there is an overrepresentation of the number of copies of a first homologous chromosomesegment as compared to a second homologous chromosomesegment in the genome of an individual (such as in the genome of one or more cells or in cfDNA or cfRNA). Exemplary overrepresenta- tions include the duplication of the first homologous chromosome segment or the deletion of the second homologous chromosome segment. In some embodiments, there is not an overrepresentation since the first and homologous chromosome segments are present in equal proportions (such as one copy of each segment in a diploid sample). In someembodiments, calculated allele ratios in a nucleic acid sample are compared to expected allele ratios to determine if there isanoverrepresentationasdescribed further below. In this specification thephrase "afirst homologouschromosome segment as compared to a secondhomologous chromosomesegment"meansa first homolog of a chromosomesegment and a second homolog of the chromosome segment.

[0195] In someembodiments, themethod includesobtainingphasedgenetic data for the first homologouschromosome segment comprising the identity of the allele present at that locus on the first homologous chromosome segment for each locus in a set of polymorphic loci on the first homologous chromosome segment, obtaining phased genetic data for the second homologous chromosome segment comprising the identity of the allele present at that locus on the second homologous chromosome segment for each locus in the set of polymorphic loci on the second homologous chromosome segment, and obtaining measured genetic allelic data comprising, for each of the alleles at each of the loci in the set of polymorphic loci, the amount of each allele present in a sample of DNA or RNA from one or more target cells and one or more non-target cells from the individual. In some embodiments, the method includes enumerating a set of one or more hypotheses specifying the degree of overrepresentation of the first homologous chromosome segment; calculating, for each of the hypotheses, expected genetic data for the plurality of loci in the sample from the obtained phased genetic data for one or more possible ratios of DNA or RNA from the one or more target cells to the total DNA or RNA in the sample; calculating (such as calculating on a computer) for each possible ratio of DNA or RNAand for each hypothesis, the data fit between the obtained genetic data of the sample and the expected genetic data for the sample for that possible ratio of DNA or RNA and for that hypothesis; ranking one or more of the hypotheses according to the data fit; and selecting the hypothesis that is ranked the highest, thereby determining the degree of overrepresentation of the number of copies of the first homologous chromosome segment in the genome of one or more cells from the individual.

[0196] In some embodiments, the method involves obtaining phased genetic data using any of the methods described herein or any known method. In some embodiments, the method involves simultaneously or sequentially in any order (i) obtaining phased genetic data for the first homologous chromosome segment comprising the identity of the allele present at that locus on the first homologous chromosome segment for each locus in a set of polymorphic loci on the first homologous chromosomesegment, (ii) obtaining phased genetic data for the secondhomologous chromosomesegment comprising the identity of the allele present at that locus on the second homologous chromosome segment for each locus in thesetof polymorphic loci on thesecondhomologouschromosomesegment, and (iii) obtainingmeasuredgenetic allelic data comprising the amount of each allele at each of the loci in the set of polymorphic loci in a sample of DNA from one or more cells from the individual.

[0197] In some embodiments, themethod involves calculating allele ratios for one ormore loci in the set of polymorphic loci that are heterozygous in at least one cell from which the sample was derived. In some embodiments, the calculated allele ratio for a particular locus is the measured quantity of one of the alleles divided by the total measured quantity of all the alleles for the locus. In some embodiments, the calculated allele ratio for a particular locus is themeasured quantity of one of the alleles (such as the allele on the first homologous chromosome segment) divided by the measured quantity of one or more other alleles (such as the allele on the second homologous chromosome segment) for the locus. The calculated allele ratiosmaybe calculated using any of themethods described herein or any standardmethod (such as any mathematical transformation of the calculated allele ratios described herein).

[0198] In someembodiments, themethod involves determining if there is anoverrepresentation of the number of copies of thefirst homologouschromosomesegmentbycomparingoneormorecalculatedallele ratios for a locus toanallele ratio that is expected for that locus if the first and second homologous chromosome segments are present in equal proportions. In some embodiments, the expected allele ratio assumes the possible alleles for a locus have an equal likelihood of being present. In some embodiments in which the calculated allele ratio for a particular locus is the measured quantity of one of the alleles divided by the total measured quantity of all the alleles for the locus, the corresponding expected allele ratio is 0.5 for a biallelic locus, or 1 / 3 for a triallelic locus. In someembodiments, the expectedallele ratio is the same for all the loci, such as 0.5 for all loci. In some embodiments, the expected allele ratio assumes that the possible alleles for a locus can have a different likelihood of being present, such as the likelihood based on the frequency of each of the alleles in a particular population that the subject belongs in, such as a population based on the ancestry of the subject. Such allele frequencies are publicly available (see, e.g., HapMap Project; Perlegen Human Haplotype Project; web at ncbi.nlm.nih.gov / projects / SNP / ; Sherry ST,WardMH, KholodovM, et al. dbSNP: the NCBI database of genetic variation. 27 EP 4 737 593 A2 5 10 15 20 25 30 35 40 45 50 55 Nucleic Acids Res. 2001 Jan 1;29(1):308‑11, which are each incorporated by reference in its entirety). In some embodiments, the expected allele ratio is the allele ratio that is expected for the particular individual being tested for a particular hypothesis specifying the degree of overrepresentation of the first homologous chromosome segment. For example, theexpectedallele ratio for aparticular individualmaybedeterminedbasedonphasedorunphasedgenetic data from the individual (such as from a sample from the individual that is unlikely to have a deletion or duplication such as a noncancerous sample) or data from one or more relatives from the individual.

[0199] In some embodiments, a calculated allele ratio is indicative of an overrepresentation of the number of copies of the first homologous chromosome segment if either (i) the allele ratio for themeasured quantity of the allele present at that locus on the first homologous chromosome divided by the total measured quantity of all the alleles for the locus is greater than the expected allele ratio for that locus, or (ii) the allele ratio for themeasured quantity of the allele present at that locus on the second homologous chromosome divided by the total measured quantity of all the alleles for the locus is less than the expected allele ratio for that locus. In some embodiments, a calculated allele ratio is only considered indicative of overrepresentation if it is significantly greater or lower than the expected ratio for that locus. In some embodiments, a calculated allele ratio is indicative of no overrepresentation of the number of copies of the first homologous chromosome segment if either (i) the allele ratio for the measured quantity of the allele present at that locus on the first homologous chromosomedividedby the totalmeasuredquantity of all thealleles for the locus is less thanor equal to theexpectedallele ratio for that locus, or (ii) the allele ratio for the measured quantity of the allele present at that locus on the second homologous chromosome divided by the total measured quantity of all the alleles for the locus is greater than or equal to the expected allele ratio for that locus. In some embodiments, calculated ratios equal to the corresponding expected ratio are ignored (since they are indicative of no overrepresentation).

[0200] In various embodiments, one or more of the followingmethods is used to compare one ormore of the calculated allele ratios to the corresponding expected allele ratio(s). In some embodiments, one determines whether the calculated allele ratio is aboveor below theexpectedallele ratio for aparticular locus irrespectiveof themagnitudeof thedifference. In some embodiments, one determines themagnitude of the difference between the calculated allele ratio and the expected allele ratio for aparticular locus irrespectiveofwhether thecalculatedallele ratio is aboveorbelow theexpectedallele ratio. In some embodiments, one determines whether the calculated allele ratio is above or below the expected allele ratio and the magnitude of the difference for a particular locus. In some embodiments, one determines whether the average or weighted average value of the calculated allele ratios is above or below the average or weighted average value of the expected allele ratios irrespective of the magnitude of the difference. In some embodiments, one determines the magnitude of the difference between the average or weighted average value of the calculated allele ratios and the averageorweightedaveragevalueof theexpectedallele ratios irrespectiveofwhether theaverageorweightedaverageof the calculated allele ratio is above or below the average or weighted average value of the expected allele ratio. In some embodiments, one determines whether the average or weighted average value of the calculated allele ratios is above or below the average or weighted average value of the expected allele ratios and the magnitude of the difference. In some embodiments, one determines an average or weighted average value of the magnitude of the difference between the calculated allele ratios and the expected allele ratios.

[0201] In someembodiments, themagnitudeof thedifferencebetween thecalculatedallele ratioand theexpectedallele ratio for one or more loci is used to determine whether the overrepresentation of the number of copies of the first homologous chromosome segment is due to a duplication of the first homologous chromosome segment or a deletion of the second homologous chromosome segment in the genome of one or more of the cells.

[0202] In some embodiments, an overrepresentation of the number of copies of the first homologous chromosome segment is determined to be present if one or more of following conditions is met. In some embodiments, the number of calculated allele ratios that are indicative of an overrepresentation of the number of copies of the first homologous chromosome segment is above a threshold value. In some embodiments, the number of calculated allele ratios that are indicative of no overrepresentation of the number of copies of the first homologous chromosome segment is below a threshold value. In some embodiments, the magnitude of the difference between the calculated allele ratios that are indicative of an overrepresentation of the number of copies of the first homologous chromosome segment and the corresponding expected allele ratios is above a threshold value. In some embodiments, for all calculated allele ratios that are indicative of overrepresentation, the sum of the magnitude of the difference between a calculated allele ratio and the corresponding expected allele ratio is above a threshold value. In some embodiments, the magnitude of the difference between the calculated allele ratios that are indicative of no overrepresentation of the number of copies of the first homologous chromosome segment and the corresponding expected allele ratios is below a threshold value. In some embodiments, the average or weighted average value of the calculated allele ratios for themeasured quantity of the allele present on thefirst homologouschromosomedividedby the totalmeasuredquantity of all thealleles for the locus isgreater than the average or weighted average value of the expected allele ratios by at least a threshold value. In some embodiments, the average or weighted average value of the calculated allele ratios for the measured quantity of the allele present on the secondhomologous chromosomedividedby the totalmeasuredquantity of all thealleles for the locus is less than the average or weighted average value of the expected allele ratios by at least a threshold value. In some 28 EP 4 737 593 A2 5 10 15 20 25 30 35 40 45 50 55 embodiments, the data fit between the calculated allele ratios and allele ratios that are predicted for an overrepresentation of the number of copies of the first homologous chromosomesegment is belowa threshold value (indicative of a gooddata fit). In some embodiments, the data fit between the calculated allele ratios and allele ratios that are predicted for no overrepresentation of the number of copies of the first homologous chromosome segment is above a threshold value (indicative of a poor data fit).

[0203] In some embodiments, an overrepresentation of the number of copies of the first homologous chromosome segment is determined to be absent if one or more of following conditions is met. In some embodiments, the number of calculated allele ratios that are indicative of an overrepresentation of the number of copies of the first homologous chromosome segment is below a threshold value. In some embodiments, the number of calculated allele ratios that are indicative of no overrepresentation of the number of copies of the first homologous chromosome segment is above a threshold value. In some embodiments, the magnitude of the difference between the calculated allele ratios that are indicative of an overrepresentation of the number of copies of the first homologous chromosome segment and the corresponding expected allele ratios is below a threshold value. In some embodiments, the magnitude of the difference between the calculated allele ratios that are indicative of no overrepresentation of the number of copies of the first homologous chromosome segment and the corresponding expected allele ratios is above a threshold value. In some embodiments, the average or weighted average value of the calculated allele ratios for themeasured quantity of the allele presenton thefirst homologouschromosomedividedby the totalmeasuredquantity of all thealleles for the locusminus the average or weighted average value of the expected allele ratios is less than a threshold value. In some embodiments, the average or weighted average value of the expected allele ratios minus the average or weighted average value of the calculated allele ratios for themeasured quantity of the allele present on the second homologous chromosome divided by the total measured quantity of all the alleles for the locus is less than a threshold value. In some embodiments, the data fit between the calculatedallele ratios andallele ratios that are predicted for an overrepresentation of the number of copies of the first homologous chromosome segment is above a threshold value. In some embodiments, the data fit between the calculated allele ratios and allele ratios that are predicted for no overrepresentation of the number of copies of the first homologous chromosome segment is below a threshold value. In some embodiments, the threshold is determined from empirical testing of samples known to have a CNV of interest and / or samples known to lack the CNV.

[0204] In some embodiments, determining if there is an overrepresentation of the number of copies of the first homologous chromosome segment includes enumerating a set of one or more hypotheses specifying the degree of overrepresentation of the first homologous chromosome segment. On exemplary hypothesis is the absence of an overrepresentation since the first and homologous chromosome segments are present in equal proportions (such as one copy of each segment in a diploid sample). Other exemplary hypotheses include the first homologous chromosome segment being duplicated one or more times (such as 1, 2, 3, 4, 5, or more extra copies of the first homologous chromosome compared to the number of copies of the second homologous chromosome segment). Another exemplary hypothesis includes the deletion of the second homologous chromosome segment. Yet another exemplary hypothesis is the deletion of both the first and the second homologous chromosome segments. In some embodiments, predicted allele ratios for the loci that are heterozygous in at least one cell are estimated for each hypothesis given the degree of overrepresentation specified by that hypothesis. In some embodiments, the likelihood that the hypothesis is correct is calculated by comparing the calculated allele ratios to the predicted allele ratios, and the hypothesis with the greatest likelihood is selected.

[0205] In some embodiments, an expected distribution of a test statistic is calculated using the predicted allele ratios for each hypothesis. In some embodiments, the likelihood that the hypothesis is correct is calculated by comparing a test statistic that is calculated using the calculated allele ratios to the expected distribution of the test statistic that is calculated using the predicted allele ratios, and the hypothesis with the greatest likelihood is selected.

[0206] In some embodiments, predicted allele ratios for the loci that are heterozygous in at least one cell are estimated given the phased genetic data for the first homologous chromosome segment, the phased genetic data for the second homologous chromosome segment, and the degree of overrepresentation specified by that hypothesis. In some embodiments, the likelihood that the hypothesis is correct is calculated by comparing the calculated allele ratios to the predicted allele ratios; and the hypothesis with the greatest likelihood is selected. Use of Mixed Samples

[0207] It will be understood that for many embodiments, the sample is a mixed sample with DNA or RNA from one or more target cells and one or more non-target cells. In some embodiments, the target cells are cells that have a CNV, such as a deletion or duplication of interest, and the non-target cells are cells that do not have the copy number variation of interest (such as a mixture of cells with the deletion or duplication of interest and cells without any of the deletions or duplicationsbeing tested). In someembodiments, the target cells are cells that areassociatedwithadiseaseor disorder or an increased risk for disease or disorder (such as cancer cells), and the non-target cells are cells that are not associated withadiseaseordisorderoran increased risk for diseaseordisorder (suchasnoncancerouscells). In someembodiments, 29 EP 4 737 593 A2 5 10 15 20 25 30 35 40 45 50 55 the target cells all have the same CNV. In some embodiments, two or more target cells have different CNVs. In some embodiments, one or more of the target cells has a CNV, polymorphism, or mutation associated with the disease or disorder or an increased risk for disease or disorder that is not found it at least one other target cell. In some such embodiments, the fraction of the cells that are associated with the disease or disorder or an increased risk for disease or disorder out of the total cells from a sample is assumed to be greater than or equal to the fraction of the most frequent of these CNVs, polymorphisms, or mutations in the sample. For example if 6% of the cells have a K-rasmutation, and 8% of the cells have a BRAF mutation, at least 8% of the cells are assumed to be cancerous.

[0208] In someembodiments, the ratio ofDNA (orRNA) from theoneormore target cells to the total DNA (orRNA) in the sample is calculated. In someembodiments, a set of one ormore hypotheses specifying the degree of overrepresentation of the first homologous chromosome segment are enumerated. In some embodiments, predicted allele ratios for the loci that are heterozygous in at least one cell are estimated given the calculated ratio of DNA or RNA and the degree of overrepresentation specified by that hypothesis are estimated for each hypothesis. In some embodiments, the likelihood that the hypothesis is correct is calculated by comparing the calculated allele ratios to the predicted allele ratios, and the hypothesis with the greatest likelihood is selected.

[0209] In some embodiments, an expected distribution of a test statistic calculated using the predicted allele ratios and the calculated ratio of DNA or RNA is estimated for each hypothesis. In some embodiments, the likelihood that the hypothesis is correct is determined by comparing a test statistic calculated using the calculated allele ratios and the calculated ratio of DNA or RNA to the expected distribution of the test statistic calculated using the predicted allele ratios and the calculated ratio of DNA or RNA, and the hypothesis with the greatest likelihood is selected.

[0210] In some embodiments, themethod includes enumerating a set of one ormore hypotheses specifying the degree of overrepresentation of the first homologous chromosome segment. In some embodiments, the method includes estimating, for each hypothesis, either (i) predicted allele ratios for the loci that are heterozygous in at least one cell given thedegreeof overrepresentationspecifiedby that hypothesisor (ii) for oneormorepossible ratiosofDNAorRNA,an expecteddistributionofa test statistic calculatedusing thepredictedallele ratiosand thepossible ratioofDNAorRNA from the one or more target cells to the total DNA or RNA in the sample. In some embodiments, a data fit is calculated by comparing either (i) the calculated allele ratios to the predicted allele ratios, or (ii) a test statistic calculated using the calculatedallele ratiosand thepossible ratioofDNAorRNA to theexpecteddistributionof the test statistic calculatedusing the predicted allele ratios and the possible ratio of DNAorRNA. In someembodiments, one ormore of the hypotheses are ranked according to the data fit, and the hypothesis that is ranked the highest is selected. In some embodiments, a technique or algorithm, such as a search algorithm, is used for one or more of the following steps: calculating the data fit, ranking thehypotheses, or selecting thehypothesis that is ranked thehighest. In someembodiments, the data fit is a fit to a beta-binomial distribution or a fit to a binomial distribution. In some embodiments, the technique or algorithm is selected from the group consisting of maximum likelihood estimation, maximum a-posteriori estimation, Bayesian estimation, dynamic estimation (such as dynamic Bayesian estimation), and expectation-maximization estimation. In some embodi- ments, the method includes applying the technique or algorithm to the obtained genetic data and the expected genetic data.

[0211] In some embodiments, the method includes creating a partition of possible ratios that range from a lower limit to an upper limit for the ratio of DNA or RNA from the one or more target cells to the total DNA or RNA in the sample. In some embodiments, a set of one or more hypotheses specifying the degree of overrepresentation of the first homologous chromosome segment are enumerated. In some embodiments, the method includes estimating, for each of the possible ratios of DNA or RNA in the partition and for each hypothesis, either (i) predicted allele ratios for the loci that are heterozygous in at least one cell given the possible ratio of DNAorRNAand the degree of overrepresentation specified by that hypothesis or (ii) an expected distribution of a test statistic calculated using the predicted allele ratios and the possible ratio ofDNAorRNA. In someembodiments, themethod includes calculating, for eachof thepossible ratiosofDNAorRNA in the partition and for each hypothesis, the likelihood that the hypothesis is correct by comparing either (i) the calculated allele ratios to the predicted allele ratios, or (ii) a test statistic calculated using the calculated allele ratios and the possible ratio of DNA or RNA to the expected distribution of the test statistic calculated using the predicted allele ratios and the possible ratio of DNA or RNA. In some embodiments, the combined probability for each hypothesis is determined by combining the probabilities of that hypothesis for each of the possible ratios in the partition; and the hypothesis with the greatest combined probability is selected. In some embodiments, the combined probability for each hypothesis is determining by weighting the probability of a hypothesis for a particular possible ratio based on the likelihood that the possible ratio is the correct ratio.

[0212] In some embodiments, a technique selected from the group consisting of maximum likelihood estimation, maximum a-posteriori estimation, Bayesian estimation, dynamic estimation (such as dynamic Bayesian estimation), and expectation-maximization estimation is used to estimate the ratio of DNA or RNA from the one or more target cells to the totalDNAorRNA in thesample. In someembodiments, the ratioofDNAorRNA from theoneormore target cells to the total DNAorRNA in thesample isassumed tobe thesame for twoormore (or all) of theCNVsof interest. In someembodiments, the ratioofDNAorRNA from theoneormore target cells to the totalDNAorRNA in thesample is calculated foreachCNVof 30 EP 4 737 593 A2 5 10 15 20 25 30 35 40 45 50 55 interest. Exemplary Methods for Using Imperfectly Phased Data

[0213] It will be understood that for many embodiments, imperfectly phased data is used. For example, it may not be known with 100% certainty which allele is present for one or more of the loci on the first and / or second homologous chromosome segment. In some embodiments, the priors for possible haplotypes of the individual (such as haplotypes based on population based haplotype frequencies) are used in calculating the probability of each hypothesis. In some embodiments, the priors for possible haplotypes are adjusted by either using anothermethod to phase the genetic data or by using phased data from other subjects (such as prior subjects) to refine population data used for informatics based phasing of the individual.

[0214] In some embodiments, the phased genetic data comprises probabilistic data for two or more possible sets of phased genetic data, wherein each possible set of phased data comprises a possible identity of the allele present at each locus in the set of polymorphic loci on the first homologous chromosome segment and a possible identity of the allele present at each locus in the set of polymorphic loci on the second homologous chromosome segment. In some embodiments, the probability for at least one of the hypotheses is determined for each of the possible sets of phased genetic data. In some embodiments, the combined probability for the hypothesis is determined by combining the probabilities of the hypothesis for each of the possible sets of phased genetic data; and the hypothesis with the greatest combined probability is selected.

[0215] Any of the methods disclosed herein or any known method may be used to generate imperfectly phased data (such as using population based haplotype frequencies to infer the most likely phase) for use in the claimed methods. In someembodiments, phaseddata isobtainedbyprobabilistically combininghaplotypesof smaller segments.Forexample, possible haplotypes can be determined based on possible combinations of one haplotype from a first region with another haplotype from another region from the same chromosome. The probability that particular haplotypes from different regions are part of the same, larger haplotype block on the same chromosome can be determined using, e.g., population based haplotype frequencies and / or known recombination rates between the different regions.

[0216] In some embodiments, a single hypothesis rejection test is used for the null hypothesis of disomy. In some embodiments, the probability of the disomy hypothesis is calculated, and the hypothesis of disomy is rejected if the probability is belowagiven threshold value (suchas less than1 in1,000). If thenull hypothesis is rejected, this couldbedue to errors in the imperfectly phased data or due to the presence of a CNV. In some embodiments, more accurate phased data isobtained (suchasphaseddata fromanyof themolecular phasingmethodsdisclosedherein toobtainactual phased data rather than bioinformatics-based inferred phased data). In some embodiments, the probability of the disomy hypothesis is recalculated using the more accurate phased data to determine if the disomy hypothesis should still be rejected. Rejection of this hypothesis indicates that a duplication or deletion of the chromosome segment is present. If desired, the false positive rate can be altered by adjusting the threshold value. Further Exemplary Embodiments for Determining Ploidy Using Phased Data

[0217] In illustrative embodiments, provided herein is a method for determining ploidy of a chromosomal segment in a sampleof an individual. Themethod includes the following steps: receivingallele frequencydata comprising theamount of eachallele present in the sample at each loci in a set of polymorphic loci on the chromosomal segment; generating phased allelic information for the set of polymorphic loci by estimating the phase of the allele frequency data; generating individual probabilities of allele frequencies for the polymorphic loci for different ploidy states using the allele frequency data; generating joint probabilities for the set of polymorphic loci using the individual probabilities and the phased allelic information; and selecting, based on the joint probabilities, a best fit model indicative of chromosomal ploidy, thereby determining ploidy of the chromosomal segment.

[0218] As disclosed herein, the allele frequency data (also referred to herein as measured genetic allelic data) can be generated by methods known in the art. For example, the data can be generated using qPCR or microarrays. In one illustrative embodiment, the data is generated using nucleic acid sequence data, especially high throughput nucleic acid sequence data.

[0219] In certain illustrative examples, the allele frequency data is corrected for errors before it is used to generate individual probabilities. In specific illustrative embodiments, the errors that are corrected include allele amplification efficiency bias. In other embodiments, the errors that are corrected include ambient contamination and genotype contamination. In some embodiments, errors that are corrected include allele amplification bias, sequencing errors, ambient contamination and genotype contamination.

[0220] In certain embodiments, the individual probabilities are generated using a set of models of both different ploidy states and allelic imbalance fractions for the set of polymorphic loci. In these embodiments, and other embodiments, the joint probabilities are generated by considering the linkage between polymorphic loci on the chromosome segment. 31 EP 4 737 593 A2 5 10 15 20 25 30 35 40 45 50 55

[0221] Accordingly, in one illustrative embodiment that combines some of these embodiments, provided herein is a method for detecting chromosomal ploidy in a sample of an individual, that includes the following steps: receiving nucleic acid sequence data for alleles at a set of polymorphic loci on a chromosome segment in the individual; detecting allele frequencies at the set of loci using the nucleic acid sequence data; correcting for allele amplification efficiency bias in the detected allele frequencies to generate corrected allele frequencies for the set of polymorphic loci; generating phased allelic information for the set of polymorphic loci by estimating the phase of the nucleic acid sequence data; generating individual probabilities of allele frequencies for the polymorphic loci for different ploidy states by comparing the corrected allele frequencies to a set of models of different ploidy states and allelic imbalance fractions of the set of polymorphic loci; generating joint probabilities for theset of polymorphic loci bycombining the individual probabilitiesconsidering the linkage between polymorphic loci on the chromosome segment; and selecting, based on the joint probabilities, the best fit model indicative of chromosomal aneuploidy.

[0222] As disclosed herein, the individual probabilities can be generated using a set of models or hypothesis of both different ploidy states and average allelic imbalance fractions for the set of polymorphic loci. For example, in a particularly illustrative example, individual probabilities are generated bymodeling ploidy states of a first homolog of the chromosome segment andasecondhomologof the chromosomesegment. Theploidy states that aremodeled include the following: (1) all cells have no deletion or amplification of the first homolog or the second homolog of the chromosome segment; (2) at least some cells have a deletion of the first homolog or an amplification of the second homolog of the chromosome segment; and (3) at least some cells have a deletion of the second homolog or an amplification of the first homolog of the chromosome segment.

[0223] It will be understood that the above models can also be referred to as hypothesis that are used to constrain a model. Therefore, demonstrated above are 3 hypothesis that can be used.

[0224] Theaverageallelic imbalance fractionsmodeledcan includeany rangeof averageallelic imbalance that includes the actual average allelic imbalance of the chromosomal segment. For example, in certain illustrative embodiments, the range of average allelic imbalance that is modeled can be between 0, 0.1, 0.2, 0.25, 0.3, 0.4, 0.5, 0.6, 0.75, 1, 2, 2.5, 3, 4, and 5% on the low end, and 1, 2, 2.5, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70 80 90, 95, and 99% on the high end. The intervals for themodelingwith the range canbe any interval depending on the computing power used and the time allowed for the analysis. For example, 0.01, 0.05, 0.02, or 0.1 intervals can be modeled.

[0225] In certain illustrativeembodiments, thesamplehasanaverageallelic imbalance for thechromosomal segmentof between 0.4% and 5%. In certain embodiments, the average allelic imbalance is low. In these embodiments, average allelic imbalance is typically less than 10%. In certain illustrative embodiments, the allelic imbalance is between 0.25, 0.3, 0.4, 0.5, 0.6, 0.75, 1, 2, 2.5, 3, 4, and 5% on the low end, and 1, 2, 2.5, 3, 4, and 5% on the high end. In other exemplary embodiments, the average allelic imbalance is between 0.4, 0.45, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0% on the low end and 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 3.0, 4.0, or 5.0%on thehighend.For example, theaverageallelic imbalanceof the sample in an illustrative example is between 0.45 and 2.5%. In another example, the average allelic imbalance is detected with a sensitivity of 0.45, 0.5, 0.6, 0.8, 0.8, 0.9, or 1.0%. That is, the testmethod is capable of detecting chromosomal aneuploidy down to anAAI of 0.45, 0.5, 0.6, 0.8, 0.8, 0.9, or 1.0%. In An exemplary samplewith lowallelic imbalance inmethods of the present invention include plasma samples from individuals with cancer having circulating tumor DNA or plasma samples from pregnant females having circulating fetal DNA.

[0226] It will be understood that for SNVs, the proportion of abnormal DNA is typically measured using mutant allele frequency (number of mutant alleles at a locus / total number of alleles at that locus). Since the difference between the amounts of two homologs in tumours is analogous, wemeasure the proportion of abnormal DNA for aCNVby the average allelic imbalance (AAI), defined as |(H1 - H2)| / (H1 + H2), where Hi is the average number of copies of homolog i in the sample and Hi / (H1 + H2) is the fractional abundance, or homolog ratio, of homolog i. The maximum homolog ratio is the homolog ratio of the more abundant homolog.

[0227] Assay drop-out rate is the percentage of SNPs with no reads, estimated using all SNPs. Single allele drop-out (ADO) rate is the percentage of SNPs with only one allele present, estimated using only heterozygous SNPs. Genotype confidencecanbedeterminedbyfittingabinomial distribution to thenumberof readsateachSNP thatwereB-allele reads, and using the ploidy status of the focal region of the SNP to estimate the probability of each genotype.

[0228] For tumor tissue samples, chromosomal aneuploidy (exemplified in this paragraph by CNVs) can be delineated by transitions between allele frequency distributions. In plasma samples of cancer patients, individuals suspected of having cancer, individuals who previously were diagnosed with cancer, or as a cancer screen for at-risk individuals or the general population, CNVs can be identified by amaximum likelihood algorithm that searches for plasmaCNVs in regions known to exhibit aneuploidy in cancer, and / or where the tumor sample from the same individual also has CNVs. In illustrative embodiments, the algorithm uses haplotype phase information of the individual whose sample is being analyzed for the presence of circulating tumor DNA to fit measured and corrected test sample allele counts to expected allele counts, for example using a joint distribution mode. Such haplotype phase information can be deduced from any sample froman individual that includesmostly, or at least 60, 70, 80, 90, 95, 96, 97, 98, 99%orall normal cell DNA, suchas, but not limited to, a buffy coat sample, a saliva sample, or a skin sample, fromparental genotypic information, or by denovo 32 EP 4 737 593 A2 5 10 15 20 25 30 35 40 45 50 55 haplotype phasing, which could be achieved by a variety of methods (See e.g., Snyder, M., et al., Haplotype-resolved genomesequencing: experimentalmethodsandapplications.NatRevGenet 16, 344‑358 (2015)), suchashaplotypingby dilution (Kaper, F., et al., Whole-genome haplotyping by dilution, amplification, and sequencing. Proc Natl Acad Sci U SA 110, 5552‑5557 (2013)) or long-read sequencing (Kuleshov, V. et al. Whole-genome haplotyping using long reads and statistical methods. Nat Biotech 32, 261‑266 (2014)). This algorithm can model expected allelic frequencies across all allelic imbalance ratios at 0.025% intervals for three sets of hypotheses: (1) all cells are normal (no allelic imbalance), (2) some / all cells have a homolog 1 deletion or homolog 2 amplification, or (3) some / all cells have a homolog 2 deletion or homolog 1 amplification. The likelihood of each hypothesis can be determined at each SNP using a Bayesian classifier based on a beta binomial model of expected and observed allele frequencies at all heterozygous SNPs, and then the joint likelihood across multiple SNPs can be calculated, in certain illustrative embodiments taking linkage of the SNP loci into consideration, asexemplifiedherein. In fact, in illustrative embodiments normal cell haplotypephase information obtained as disclosed above, is used by the algorithm to fit the measured and typically corrected test sample allele counts to expected allele counts using a joint distribution model The maximum likelihood hypothesis can then be selected.

[0229] Consider a chromosomal region with an average of N copies in the tumor, and let c denote the fraction of DNA in plasma derived from the mixture of normal and tumour cells in a disomic region. AAI is calculated as:

[0230] In certain illustrative examples, the allele frequency data is corrected for errors before it is used to generate individual probabilities. Different types of error and / or bias correction are disclosed herein. In specific illustrative embodiments, the errors that are corrected are allele amplification efficiency bias. In other embodiments, the errors that are corrected include sequencing errors, ambient contamination and genotype contamination. In some embodiments, errors that are corrected include allele amplification bias, sequencing errors, ambient contamination and genotype contamination.

[0231] It will be understood that allele amplification efficiency bias can be determined for an allele as part of an experiment or laboratory determination that includes an on test sample, or it can be determined at a different time using a set of samples that include the allele whose efficiency is being calculated. Ambient contamination and genotype contamination are typically determined on the same run as the on-test sample analysis.

[0232] In certain embodiments, ambient contamination and genotype contamination are determined for homozygous alleles in the sample. It will be understood that for any given sample from an individual some loci in the sample, will be heterozygous and others will be homozygous, even if a locus is selected for analysis because it has a relatively high heterozygosity in thepopulation. It is advantageous insomeembodiments, todetermineploidyofachromosomal segment using heterozygous loci for an individual, whereas ambient and genotype contamination can be calculated using homozygous loci.

[0233] In certain illustrative examples, the selecting is performed by analyzing amagnitude of a difference between the phased allelic information and estimated allelic frequencies generated for the models.

[0234] In illustrative examples, the individual probabilities of allele frequencies are generated based on a beta binomial model of expected and observed allele frequencies at the set of polymorphic loci. In illustrative examples, the individual probabilities are generated using a Bayesian classifier.

[0235] In certain illustrative embodiments, the nucleic acid sequence data is generated by performing high throughput DNA sequencing of a plurality of copies of a series of amplicons generated using a multiplex amplification reaction, wherein each amplicon of the series of amplicons spans at least one polymorphic loci of the set of polymorphic loci and wherein each of the polymeric loci of the set is amplified. In certain embodiments, the multiplex amplification reaction is performed under limiting primer conditions for at least ½ of the reactions. In some embodiments, limiting primer concentrations are used in 1 / 10, 1 / 5, ¼, 1 / 3, ½, or all of the reactions of the multiplex reaction. Provided herein are factors to consider to achieve limiting primer conditions in an amplification reaction such as PCR.

[0236] In certain embodiments, methods provided herein detect ploidy for multiple chromosomal segments across multiple chromosomes. Accordingly, the chromosomal ploidy in these embodiments is determined for a set of chromo- some segments in the sample. For these embodiments, highermultiplex amplification reactions are needed. Accordingly, for these embodiments themultiplex amplification reaction can include, for example, between 2,500 and 50,000multiplex reactions. In certain embodiments, the following rangesofmultiplex reactionsare performed: between100, 200, 250, 500, 1000, 2500, 5000, 10,000, 20,000, 25000, 50000 on the low end of the range and between 200, 250, 500, 1000, 2500, 5000, 10,000, 20,000, 25000, 50000, and 100,000 on the high end of the range.

[0237] In illustrativeembodiments, thesetof polymorphic loci isaset of loci thatare known toexhibit highheterozygosity. However, it is expected that for any given individual, some of those loci will be homozygous. In certain illustrative 33 EP 4 737 593 A2 5 10 15 20 25 30 35 40 45 50 55 embodiments,methods of the invention utilize nucleic acid sequence information for both homozygous and heterozygous loci for an individual. The homozygous loci of an individual are used, for example, for error correction, whereas heterozygous loci are used for the determination of allelic imbalance of the sample. In certain embodiments, at least 10% of the polymorphic loci are heterozygous loci for the individual.

[0238] As disclosed herein, preference is given for analyzing target SNP loci that are known to be heterozygous in the population. Accordingly, in certain embodiments, polymorphic loci are chosen wherein at least 10, 20, 25, 50, 75, 80, 90, 95, 99, or 100% of the polymorphic loci are known to be heterozygous in the population.

[0239] As disclosed herein, in certain embodiments the sample is a plasma sample from a pregnant female.

[0240] In some examples, the method further comprises performing the method on a control sample with a known average allelic imbalance ratio. The control can have an average allelic imbalance ratio for a particular allelic state indicative of aneuploidy of the chromosomesegment, of between 0.4 and10% tomimic an average allelic imbalance of an allele in a sample that is present in low concentrations, such aswould be expected for a circulating freeDNA froma tumor.

[0241] In some embodiments, PlasmArt controls, as disclosed herein, are used as the controls. Accordingly, in certain aspects the is a sample generated by a method comprising fragmenting a nucleic acid sample known to exhibit a chromosomal aneuploidy into fragments that mimic the size of fragments of DNA circulating in plasma of the individual. In certain aspects a control is used that has no aneuploidy for the chromosome segment.

[0242] In illustrative embodiments, data from one or more controls can be analyzed in the method along with a test sample. The controls for example, can include a different sample from the individual that is not suspected of containing Chromosomal aneuploidy, or a sample that is suspected of containing CNVor a chromosomal aneuploidy. For example, where a test sample is a plasma sample suspected of containing circulating free tumor DNA, the method can be also be performed for a control sample from a tumor from the subject along with the plasma sample. As disclosed herein, the control sample can be prepared by fragmenting a DNA sample known to exhibit a chromosomal aneuploidy. Such fragmenting can result in aDNAsample thatmimics theDNAcomposition of an apoptotic cell, especiallywhen the sample is from an individual afflicted with cancer. Data from the control sample will increase the confidence of the detection of Chromosomal aneuploidy.

[0243] In certain embodiments of the methods of determining ploidy, the sample is a plasma sample from an individual suspected of having cancer. In these embodiments, the method further comprises determining based on the selecting whether copy number variation is present in cells of a tumor of the individual. For these embodiments, the sample canbe a plasma sample from an individual. For these embodiments, the method can further include determining, based on the selecting, whether cancer is present in the individual.

[0244] These embodiments for determining ploidy of a chromosomal segment, can further include detecting a single nucleotide variant at a single nucleotide variance location in a set of single nucleotide variance locations, wherein detecting either a chromosomal aneuploidy or the single nucleotide variant or both, indicates the presence of circulating tumor nucleic acids in the sample.

[0245] Theseembodiments can further include receivinghaplotype informationof thechromosomesegment for a tumor of the individual and using the haplotype information to generate the set of models of different ploidy states and allelic imbalance fractions of the set of polymorphic loci.

[0246] As disclosed herein, certain embodiments of the methods of determining ploidy can further include removing outliers from the initial or corrected allele frequency data before comparing the initial or the corrected allele frequencies to the set of models. For example, in certain embodiments, loci allele frequencies that are at least 2 or 3 standard deviations above or below themean value for other loci on the chromosome segment, are removed from the data before being used for the modeling.

[0247] Asmentioned herein, it will be understood that for many of the embodiments provided herein, including those for determining ploidy of a chromosomal segment, imperfectly or perfectly phased data is preferably used. It will also be understood, that provided herein are a number of features that provide improvements over prior methods for detecting ploidy, and that many different combinations of these features could be used.

[0248] In certain embodiments provided herein are computer systems and computer readable media to perform any methods of the present invention. These include systems and computer readable media for performing methods of determining ploidy. Accordingly, and as non-limiting examples of system embodiments, to demonstrate that any of the methods provided herein can be performed using a systemand a computer readablemediumusing the disclosure herein, in another aspect, provided herein is a system for detecting chromosomal ploidy in a sample of an individual, the system comprising: an input processor configured to receiveallelic frequencydata comprising theamount of eachallele present in the sample at each loci in a set of polymorphic loci on the chromosomal segment; a modeler configured to: generate phased allelic information for the set of polymorphic loci by estimating thephase of the allele frequency data; andgenerate individual probabilities of allele frequencies for the polymorphic loci for different ploidy states using the allele frequency data; and generate joint probabilities for the set of polymorphic loci using the individual probabilities and the phased allelic information; and a hypothesis manager configured to select, based on the joint probabilities, a best fit model indicative of chromosomal ploidy, thereby determining ploidy of the chromosomal segment. 34 EP 4 737 593 A2 5 10 15 20 25 30 35 40 45 50 55

[0249] In certain embodiments of this systemembodiment, the allele frequency data is data generated by a nucleic acid sequencingsystem. In certainembodiments, thesystem further comprisesanerror correctionunit configured tocorrect for errors in the allele frequency data, wherein the corrected allele frequency data is used by the modeler for to generate individual probabilities. In certain embodiments the error correction unit corrects for allele amplification efficiency bias. In certain embodiments, the modeler generates the individual probabilities using a set of models of both different ploidy states and allelic imbalance fractions for the set of polymorphic loci. The modeler, in certain exemplary embodiments generates the joint probabilities by considering the linkage between polymorphic loci on the chromosome segment.

[0250] In one illustrative embodiment, provided herein is a system for detecting chromosomal ploidy in a sample of an individual, that includes the following: an input processor configured to receive nucleic acid sequence data for alleles at a set of polymorphic loci on a chromosome segment in the individual and detect allele frequencies at the set of loci using the nucleic acid sequence data; an error correction unit configured to correct for errors in the detected allele frequencies and generate corrected allele frequencies for the set of polymorphic loci; a modeler configured to: generate phased allelic information for the set of polymorphic loci by estimating the phase of the nucleic acid sequence data; generate individual probabilities of allele frequencies for the polymorphic loci for different ploidy states by comparing the phased allelic information to a set of models of different ploidy states and allelic imbalance fractions of the set of polymorphic loci; and generate joint probabilities for the set of polymorphic loci by combining the individual probabilities considering the relative distance between polymorphic loci on the chromosome segment; and a hypothesis manager configured to select, based on the joint probabilities, a best fit model indicative of chromosomal aneuploidy.

[0251] In certain exemplary system embodiments provided herein the set of polymorphic loci comprises between 1000 and 50,000 polymorphic loci. In certain exemplary system embodiments provided herein the set of polymorphic loci comprises 100 known heterozygosity hot spot loci. In certain exemplary system embodiments provided herein the set of polymorphic loci comprise 100 loci that are at or within 0.5kb of a recombination hot spot.

[0252] In certain exemplary systemembodiments provided herein thebest fitmodel analyzes the following ploidy states of a first homolog of the chromosome segment and a second homolog of the chromosome segment: (1) all cells have no deletion or amplification of the first homologor the secondhomologof the chromosomesegment; (2) someor all cells have a deletion of the first homolog or an amplification of the second homolog of the chromosome segment; and (3) some or all cells have a deletion of the second homolog or an amplification of the first homolog of the chromosome segment.

[0253] In certain exemplary system embodiments provided herein the errors that are corrected comprise allelic amplification efficiency bias, contamination, and / or sequencing errors. In certain exemplary system embodiments provided herein the contamination comprises ambient contamination and genotype contamination. In certain exemplary system embodiments provided herein the ambient contamination and genotype contamination is determined for homo- zygous alleles.

[0254] In certain exemplary system embodiments provided herein the hypothesis manager is configured to analyze a magnitude of a difference between the phased allelic information and estimated allelic frequencies generated for the models. In certainexemplary systemembodimentsprovidedherein themodeler generates individual probabilities of allele frequencies based on a beta binomial model of expected and observed allele frequencies at the set of polymorphic loci. In certain exemplary system embodiments provided herein themodeler generates individual probabilities using a Bayesian classifier.

[0255] In certain exemplary system embodiments provided herein the nucleic acid sequence data is generated by performing high throughput DNA sequencing of a plurality of copies of a series of amplicons generated using a multiplex amplification reaction, wherein each amplicon of the series of amplicons spans at least one polymorphic loci of the set of polymorphic loci and wherein each of the polymeric loci of the set is amplified. In certain exemplary system embodiments provided herein, wherein themultiplex amplification reaction is performed under limiting primer conditions for at least½ of the reactions. In certain exemplary system embodiments provided herein, wherein the sample has an average allelic imbalance of between 0.4% and 5%.

[0256] In certain exemplary system embodiments provided herein, the sample is a plasma sample from an individual suspected of having cancer, and the hypothesis manager is further configured to determine, based on the best fit model, whether copy number variation is present in cells of a tumor of the individual.

[0257] In certainexemplary systemembodimentsprovidedherein the sample is aplasmasample froman individual and the hypothesis manager is further configured to determine, based on the best fit model, that cancer is present in the individual. In theseembodiments, thehypothesismanager canbe further configured todetect asinglenucleotidevariant at a single nucleotide variance location in a set of single nucleotide variance locations, wherein detecting either a chromosomal aneuploidy or the single nucleotide variant or both, indicates the presence of circulating tumor nucleic acids in the sample.

[0258] In certain exemplary systemembodiments provided herein, the input processor is further configured to receiving haplotype information of the chromosome segment for a tumor of the individual, and the modeler is configured to use the haplotype information to generate the set of models of different ploidy states and allelic imbalance fractions of the set of polymorphic loci. 35 EP 4 737 593 A2 5 10 15 20 25 30 35 40 45 50 55

[0259] In certain exemplary system embodiments provided herein, the modeler generates the models over allelic imbalance fractions ranging from 0% to 25%.

[0260] It will be understood that any of themethods provided herein can be executed by computer readable code that is stored on noontransitory computer readablemedium. Accordingly, provided herein in one embodiment, is a nontransitory computer readablemedium for detecting chromosomal ploidy in a sample of an individual, comprising computer readable code that, when executed by a processing device, causes the processing device to: receive allele frequency data comprising the amount of each allele present in the sample at each loci in a set of polymorphic loci on the chromosomal segment; generate phasedallelic information for the set of polymorphic loci by estimating thephaseof the allele frequency data; generate individual probabilities of allele frequencies for the polymorphic loci for different ploidy states using the allele frequency data; generate joint probabilities for the set of polymorphic loci using the individual probabilities and the phased allelic information; and select, based on the joint probabilities, a best fit model indicative of chromosomal ploidy, thereby determining ploidy of the chromosomal segment.

[0261] In certain computer readable medium embodiments, the allele frequency data is generated from nucleic acid sequence data. certain computer readable medium embodiments further comprise correcting for errors in the allele frequency data and using the corrected allele frequency data for the generating individual probabilities step. In certain computer readable medium embodiments the errors that are corrected are allele amplification efficiency bias. In certain computer readablemediumembodiments the individual probabilities are generated using a set ofmodels of both different ploidy states and allelic imbalance fractions for the set of polymorphic loci. In certain computer readable medium embodiments the joint probabilities are generated by considering the linkage between polymorphic loci on the chromo- some segment.

[0262] In one particular embodiment, provided herein is a nontransitory computer readable medium for detecting chromosomal ploidy inasampleofan individual, comprisingcomputer readable code that,whenexecutedbyaprocessing device, causes the processing device to: receive nucleic acid sequence data for alleles at a set of polymorphic loci on a chromosome segment in the individual; detect allele frequencies at the set of loci using the nucleic acid sequence data; correcting for allele amplification efficiency bias in the detected allele frequencies to generate corrected allele frequencies for the set of polymorphic loci; generate phasedallelic information for the set of polymorphic loci by estimating the phaseof the nucleic acid sequence data; generate individual probabilities of allele frequencies for the polymorphic loci for different ploidystatesbycomparing thecorrectedallele frequencies toasetofmodelsof different ploidystatesandallelic imbalance fractionsof theset of polymorphic loci; generate joint probabilities for thesetof polymorphic loci bycombining the individual probabilities considering the linkagebetweenpolymorphic loci on thechromosomesegment; andselect, basedon the joint probabilities, the best fit model indicative of chromosomal aneuploidy.

[0263] In certain illustrative computer readable medium embodiments, the selecting is performed by analyzing a magnitude of a difference between the phased allelic information and estimated allelic frequencies generated for the models.

[0264] In certain illustrative computer readable medium embodiments the individual probabilities of allele frequencies are generated based on a beta binomial model of expected and observed allele frequencies at the set of polymorphic loci.

[0265] It will be understood that any of the method embodiments provided herein can be performed by executing code stored on nontransitory computer readable medium. Exemplary Embodiments for Detecting Cancer

[0266] In certain aspects, the present invention provides a method for detecting cancer. The sample, it will be understood can be a tumor sample or a liquid sample, such as plasma, from an individual suspected of having cancer. Themethodsareespecially effectiveat detectinggeneticmutations suchassingle nucleotidealterations suchasSNVs, or copynumberalterations, suchasCNVs in sampleswith low levels of thesegenetic alterationsasa fractionof the totalDNA in a sample. Thus the sensitivity for detecting DNA or RNA from a cancer in samples is exceptional. The methods can combine any or all of the improvements provided herein for detectingCNVandSNV to achieve this exceptional sensitivity.

[0267] Accordingly, in certain embodiments provided herein, is a method for determining whether circulating tumor nucleic acids are present in a sample in an individual, and a nontransitory computer readable medium comprising computer readable code that, when executed by a processing device, causes the processing device to carry out the method.Themethod includes the followingsteps:analyzing thesample todetermineaploidyataset of polymorphic loci on achromosomesegment in the individual; anddetermining the level of averageallelic imbalancepresentat thepolymorphic loci based on the ploidy determination, wherein an average allelic imbalance equal to or greater than 0.4%, 0.45%, 0.5%, 0.6%,0.7%,0.75%,0.8%,0.9%,or1%is indicativeof thepresenceof circulating tumornucleicacids, suchasctDNA, in the sample.

[0268] In certain illustrative examples, an average allelic imbalance greater than 0.4, 0.45, or 0.5% is indicative the presence of ctDNA. In certain embodiments the method for determining whether circulating tumor nucleic acids are present, further comprises detecting a single nucleotide variant at a single nucleotide variance site in a set of single 36 EP 4 737 593 A2 5 10 15 20 25 30 35 40 45 50 55 nucleotide variance locations, wherein detecting either an allelic imbalance equal to or greater than 0.5% or detecting the single nucleotide variant, or both, is indicative of the presence of circulating tumor nucleic acids in the sample. It will be understood that any of themethods provided for detecting chromosomal ploidy or CNV can be used to determine the level of allelic imbalance, typically expressed as average allelic imbalance. It will be understood that any of the methoods provided herein for detecting an SNV can be used to detect the single nucleotide for this aspect of the present invention.

[0269] In certain embodiments the method for determining whether circulating tumor nucleic acids are present, further comprises performing the method on a control sample with a known average allelic imbalance ratio. The control, for example, can be a sample from the tumor of the individual. In some embodiments, the control has an average allelic imbalance expected for the sample under analysis. For example, an AAI between 0.5% and 5% or an average allelic imbalance ratio of 0.5%.

[0270] In certain embodiments, the analyzing step in themethod for determiningwhether circulating tumor nucleic acids are present, includes analyzing a set of chromosome segments known to exhibit aneuploidy in cancer. In certain embodiments, the analyzing step in the method for determining whether circulating tumor nucleic acids are present, includes analyzing between 1,000 and 50,000 or between 100 and 1000, polymorphic loci for ploidy. In certain embodiments, the analyzing step in the method for determining whether circulating tumor nucleic acids are present, includes analyzing between 100 and 1000 single nucleotide variant sites. For example, in these embodiments the analyzing step can include performing amultiplex PCR to amplify amplicons across the 1000 to 50,000 polymeric loci and the 100 to 1000 single nucleotide variant sites. This multiplex reaction can be set up as a single reaction or as pools of different subset multiplex reactions. Themultiplex reactionmethods provided herein, such as themassivemultiplex PCR disclosed herein provide an exemplary process for carrying out the amplification reaction to help attain improved multiplexing and therefore, sensitivity levels.

[0271] In certain embodiments, the multiplex PCR reaction is carried out under limiting primer conditions for at least 10%, 20%, 25%, 50%, 75%, 90%, 95%, 98%, 99%, or 100% of the reactions. Improved conditions for performing the massive multiplex reaction provided herein can be used.

[0272] In certain aspects, the above method for determining whether circulating tumor nucleic acids are present in a sample in an individual, and all embodiments thereof, can be carried out with a system. The disclosure provides teachings regarding specific functional and structural features to carry out the methods. As a non-limiting example, the system includes the following:

[0273] An input processor configured to analyze data from the sample to determine a ploidy at a set of polymorphic loci on a chromosome segment in the individual; and An modeler configured to determine the level of allelic imbalance present at the polymorphic loci based on the ploidy determination, wherein an allelic imbalance equal to or greater than 0.5% is indicative of the presence of circulating. Exemplary Embodiments for Detecting Single Nucleotide Variants

[0274] In certain aspects, provided herein are methods for detecting single nucleotide variants in a sample. The improved methods provided herein can achieve limits of detection of 0.015, 0.017, 0.02, 0.05, 0.1, 0.2, 0.3, 0.4 or 0.5 percent SNV in a sample. All the embodiments for detecting SNVs can be carried out with a system. The disclosure provides teachings regarding specific functional and structural features to carry out the methods. Furthermore, provided hereinareembodiments comprisinganontransitory computer readablemediumcomprisingcomputer readable code that, when executed by a processing device, causes the processing device to carry out the methods for detectings SNVs provided herein.

[0275] Accordingly, provided herein in one embodiment, is amethod for determiningwhether a single nucleotide variant is present at a set of genomic positions in a sample from an individual, themethod comprising: for each genomic position, generating an estimate of efficiency and a per cycle error rate for an amplicon spanning that genomic position, using a trainingdataset; receivingobservednucleotide identity information foreachgenomicposition in thesample; determininga set of probabilities of single nucleotide variant percentage resulting from one or more real mutations at each genomic position, by comparing theobservednucleotide identity informationat eachgenomicposition toamodel of different variant percentages using the estimated amplification efficiency and the per cycle error rate for each genomic position independently; and determining the most-likely real variant percentage and confidence from the set of probabilities for each genomic position.

[0276] In illustrative embodiments of the method for determining whether a single nucleotide variant is present, the estimate of efficiency and the per cycle error rate is generated for a set of amplicons that span the genomic position. For example, 2, 3, 4, 5, 10, 15, 20, 25, 50, 100 or more amplicons can be included that span the genomic position.

[0277] In illustrative embodiments of the method for determining whether a single nucleotide variant is present, the observed nucleotide identity information comprises an observed number of total reads for each genomic position and an observed number of variant allele reads for each genomic position.

[0278] In illustrative embodiments of the method for determining whether a single nucleotide variant is present, the 37 EP 4 737 593 A2 5 10 15 20 25 30 35 40 45 50 55 sample is a plasma sample and the single nucleotide variant is present in circulating tumor DNA of the sample.

[0279] In another embodiment provided herein is a method for estimating the percent of single nucleotide variants that are present in a sample from an individual. The method includes the following steps: at a set of genomic positions, generating anestimate of efficiency andaper cycle error rate for oneormoreamplicon spanning thosegenomic positions, using a training data set; receiving observed nucleotide identity information for each genomic position in the sample; generating an estimated mean and variance for the total number of molecules, background error molecules and real mutationmolecules for a search space comprising an initial percentage of realmutationmolecules using the amplification efficiency and the per cycle error rate of the amplicons; and determining the percentage of single nucleotide variants present in the sample resulting from real mutations by determining amost-likely real single nucleotide variant percentage by fitting a distribution using the estimated means and variances to an observed nucleotide identity information in the sample.

[0280] In illustrative examples of thismethod for estimating the percent of single nucleotide variants that are present in a sample, the sample is a plasmasample and the single nucleotide variant is present in circulating tumorDNAof the sample.

[0281] The training data set for this embodiment of the invention typically includes samples from one or preferably a group of healthy individuals. In certain illustrative embodiments, the training data set is analyzed on the same day or even on the same runasoneormoreon-test samples. For example, samples fromagroupof 2, 3, 4, 5, 10, 15, 20, 25, 30, 36, 48, 96, 100, 192, 200, 250, 500, 1000 ormore healthy individuals can be used to generate the training data set.Where data is available for larger number of healthy individuals, e.g. 96 or more, confidence increases for amplification efficiency estimateseven if runsareperformed inadvanceof performing themethod for on-test samples. ThePCRerror rate canuse nucleic acid sequence information generated not only for the SNVbase location, but for the entire amplified region around the SNV, since the error rate is per amplicon. For example, using samples from 50 individuals and sequencing a 20 base pair amplicon around the SNV, error frequency data from 1000 base reads can be used to determine error frequency rate.

[0282] Typically the amplification efficiency is estimating by estimating amean and standard deviation for amplification efficiency for an amplified segment and then fitting that to a distribution model, such as a binomial distribution or a beta binomial distribution. Error rates are determined for a PCR reaction with a known number of cycles and then a per cycle error rate is estimated.

[0283] In certain illustrativeembodiments, estimating thestartingmoleculesof the test dataset further includesupdating the estimate of the efficiency for the testing data set using the starting number of molecules estimated in step (b) if the observed number of reads is significantly different than the estimated number of reads. Then the estimate can be updated for a new efficiency and / or starting molecules.

[0284] The search space used for estimating the total number of molecules, background error molecules and real mutationmolecules can includeasearchspace from0.1%,0.2%,0.25%,0.5%,1%,2.5%,5%,10%,15%,20%,or 25%on the lowendand1%,2%,2.5%,5%,10%,12.5%,15%,20%,25%,50%,75%,90%,or95%on thehighendcopiesofabase at anSNVposition being theSNVbase. Lower ranges, 0.1%, 0.2%, 0.25%, 0.5%, or 1%on the lowendand1%, 2%, 2.5%, 5%, 10%, 12.5%, or 15% on the high end can be used in illustrative examples for plasma samples where the method is detecting circulating tumor DNA. Higher ranges are used for tumor samples.

[0285] A distribution is fit to the number of total error molecules (background error and real mutation) in the total molecules to calculate the likelihood or probability for each possible real mutation in the search space. This distribution could be a binomial distribution or a beta binomial distribution.

[0286] The most likely real mutation is determined by determining the most likely real mutation percentage and calculating the confidence using the data from fitting the distribution. As an illustrative example and not intended to limit the clinical interpretation of the methods provided herein, if the mean mutation rate is high then the percent confidence needed tomakeapositive determination of anSNV is lower. For example, if themeanmutation rate for anSNV in a sample using themost likely hypothesis is 5%and the percent confidence is 99%, then a positive SNV call would bemade.On the other hand for this illustrative example, if themeanmutation rate for anSNV in a sample using themost likely hypothesis is 1% and the percent confidence is 50%, then in certain situations a positive SNV call would not be made. It will be understood that clinical interpretation of the data would be a function of sensitivity, specificity, prevalence rate, and alternative product availability.

[0287] In one illustrative embodiment, the sample is a circulatingDNAsample, such as a circulating tumorDNAsample.

[0288] In another embodiment, provided herein is amethod for detecting one ormore single nucleotide variants in a test sample from an individual. The method according to this embodiment, includes the following steps: determining a median variant allele frequency for a plurality of control samples from each of a plurality of normal individuals, for each single nucleotide variant position in a set of single nucleotide variance positions based on results generated in a sequencing run, to identify selected single nucleotide variant positions having variant median allele frequencies innormal samplesbelowa thresholdvalueand todeterminebackgrounderror foreachof thesinglenucleotide variant positions after removing outlier samples for each of the single nucleotide variant positions; determining an observeddepth of readweightedmeanandvariance for the selected single nucleotide variant positions for the test sample based on data generated in the sequencing run for the test sample; and identifying using a computer, one or more single 38 EP 4 737 593 A2 5 10 15 20 25 30 35 40 45 50 55 nucleotide variant positions with a statistically significant depth of readweightedmean compared to the background error for that position, thereby detecting the one or more single nucleotide variants.

[0289] In certainembodimentsof thismethod for detectingoneormoreSNVs thesample isaplasmasample, thecontrol samples are plasma samples, and the detected one or more single nucleotide variants detected is present in circulating tumor DNA of the sample. In certain embodiments of this method for detecting one or more SNVs the plurality of control samplescomprisesat least 25samples. In certain illustrativeembodiments, theplurality of control samples isat least 5, 10, 15, 20, 25, 50, 75, 100, 200, or 250 samples on the low end and 10, 15, 20, 25, 50, 75, 100, 200, 250, 500, and 1000 samples on the high end.

[0290] In certain embodiments of this method for detecting one or more SNVs, outliers are removed from the data generated in the high throughput sequencing run to calculate the observed depth of read weighted mean and observed variance are determined. In certain embodiments of thismethod for detecting oneormoreSNVs thedepth of read for each single nucleotide variant position for the test sample is at least 100 reads.

[0291] In certain embodiments of thismethod for detecting oneormoreSNVs the sequencing run comprises amultiplex amplification reaction performed under limited primer reaction conditions. Improved methods for performing multiplex amplification reactions provided herein, are used to perform these embodiments in illustrative examples.

[0292] Not to be limited by theory, methods of the present embodiment utilize a background error model using normal plasma samples, that are sequenced on the same sequencing run as an on-test sample, to account for run-specific artifacts. Noisy positions with normal median variant allele frequencies above a threshold, for example > 0.1%, 0.2%, 0.25%, 0.5% 0.75%, and 1.0%, are removed.

[0293] Outlier samples are iteratively removed from the model to account for noise and contamination. For each base substitution of every genomic loci, the depth of read weighted mean and standard deviation of the error are calculated. In certain illustrative embodiments, samples, such as tumor or cell-free plasma samples, with single nucleotide variant positionswithat least a thresholdnumberof reads, for example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20,25, 50,100, 250,500, or 1000 variant reads and a1 Z-score greater than 2.5, 5, 7.5 or 10 against the background error model in certain embodiments, are counted as a candidate mutation.

[0294] In certain embodiments, a depth of read of greater than 100, 250, 500, 1,000, 2000, 2500, 5000, 10,000, 20,000, 25,0000, 50,000, or 100,000 on the low end of the range and 2000, 2500, 5,000, 7,500, 10,000, 25,000, 50,000, 100,000, 250,000 or 500,000 reads on the high end, is attained in the sequencing run for each single nucleotide variant position in the set of single nucleotide variant positions. Typically, the sequencing run is a high throughput sequencing run. Themean or median values generated for the on-test samples, in illustrative embodiments are weighted by depth of reads. Therefore, the likelihood that a variant allele determination is real in a sample with 1 variant allele detected in 1000 reads is weighed higher than a sample with 1 variant allele detected in 10,000 reads. Since determinations of a variant allele (i.e. mutation) are not made with 100% confidence, the identified single nucleotide variant can be considered a candidate variant or a candidate mutations. Exemplary Test Statistic for Analysis of Phased Data

[0295] An exemplary test statistic is described below for analysis of phased data from a sample known or suspected of being amixed sample containingDNAor RNA that originated from two ormore cells that are not genetically identical. Let f denote the fraction ofDNAorRNAof interest, for example the fraction ofDNAorRNAwith aCNVof interest, or the fraction ofDNAorRNA fromcellsof interest, suchascancer cells. In someembodiments for cancer testing, fdenotes the fractionof DNAorRNA fromcancer cells inamixtureof cancer andnormal cells, or fdenotes the fractionof cancer cells in amixtureof cancer and normal cells. Note that this refers to the fraction of DNA from cells of interest assuming two copies of DNA are given by each cell of interest. This differs from the DNA fraction from cells of interest at a segment that is deleted or duplicated.

[0296] The possible allelic values of eachSNPare denotedAandB. AA, AB, BA, andBBare used to denote all possible ordered allele pairs. In someembodiments, SNPswith ordered alleles ABor BAare analyzed. LetNi denote the number of sequence reads of the ith SNP, and Ai and Bi denote the number of reads of the ith SNP that indicate allele A and B, respectively. It is assumed:

[0297] The allele ratio Ri is defined: 39 EP 4 737 593 A2 5 10 15 20 25 30 35 40 45 50 55

[0298] Let T denote the number of SNPs targeted.

[0299] Without loss of generality, some embodiments focus on a single chromosome segment. As a matter of further clarity, in this specification the phrase "a first homologous chromosome segment as compared to a second homologous chromosome segment" means a first homolog of a chromosome segment and a second homolog of the chromosome segment. In somesuchembodiments, all of the targetSNPsarecontained in thesegment chromosomeof interest. Inother embodiments, multiple chromosome segments are analyzed for possible copy number variations. MAP Estimation

[0300] This method leverages the knowledge of phasing via ordered alleles to detect the deletion or duplication of the target segment. For each SNP i, define

[0301] Then define

[0302] Thedistributionsof theXiandSunder variouscopynumberhypotheses (suchashypotheses for disomy,deletion of the first or second homolog, or duplication of the first or second homolog) are described below. Disomy Hypothesis

[0303] Under the hypothesis that the target segment is not deleted or duplicated, where

[0304] If we assume a constant depth of read N, this gives us a Binomial distribution S with parameters Deletion Hypotheses

[0305] Under the hypothesis that the first homolog is deleted (i.e., an AB SNP becomes B, and a BA SNP becomes A), 40 EP 4 737 593 A2 5 10 15 20 25 30 35 40 45 50 55 then Ri has a Binomial distribution with parameters and T for AB SNPs, and and T for BA SNPs. Therefore,

[0306] If we assume a constant depth of read N, this gives a Binomial distribution S with parameters

[0307] Under the hypothesis that the second homolog is deleted (i.e., an AB SNP becomes A, and a BA SNP becomes B), then Ri has a Binomial distribution with parameters and T for AB SNPs, and and T for BA SNPs. Therefore,

[0308] If we assume a constant depth of read N, this gives a Binomial distribution S with parameters Duplication Hypotheses

[0309] Under thehypothesis that thefirst homolog isduplicated (i.e.,anABSNPbecomesAAB,andaBASNPbecomes BBA), thenRi has a Binomial distribution with parameters and T for AB SNPs, and and T for BA SNPs. Therefore,

[0310] If we assume a constant depth of read N, this gives us a Binomial distribution S with parameters

[0311] Under the hypothesis that the second homolog is duplicated (i.e., an AB SNP becomes ABB, and a BA SNP becomesBAA), thenRihas aBinomial distributionwith parameters andT for ABSNPs, and andT for BA 41 EP 4 737 593 A2 5 10 15 20 25 30 35 40 45 50 55 SNPs. Therefore,

[0312] If we assume a constant depth of read N, this gives a Binomial distribution S with parameters Classification

[0313] As demonstrated in the sections above, Xi is a binary random variable with

[0314] This allows one to calculate the probability of the test statistic S under each hypothesis. The probability of each hypothesis given themeasureddata canbecalculated. In someembodiments, thehypothesiswith thegreatest probability is selected. If desired, the distribution onScanbe simplifiedbyeither approximating eachNiwith a constant depth of reach N or by truncating the depth of reads to a constant N. This simplification gives

[0315] Thevalue for f canbeestimatebyselecting themost likely valueof fgiven themeasureddata, suchas thevalueof f that generates the best data fit using an algorithm (e.g., a search algorithm) such as maximum likelihood estimation, maximum a-posteriori estimation, or Bayesian estimation. In some embodiments, multiple chromosome segments are analyzed and a value forf is estimated based on the data for each segment. If all the target cells have these duplications or deletions, the estimated values for f based on data for these different segments are similar. In some embodiments, f is experimentally measured such as by determining the fraction of DNA or RNA from cancer cells based on methylation differences (hypomethylation or hypermethylation) between cancer and non-cancerous DNA or RNA. 42 EP 4 737 593 A2 5 10 15 20 25 30 35 40 45 50 55 Single Hypothesis Rejection

[0316] The distribution of S for the disomy hypothesis does not depend on f. Thus, the probability of the measured data canbe calculated for the disomy hypothesiswithout calculating f. A single hypothesis rejection test can be used for the null hypothesis of disomy. In some embodiments, the probability of S under the disomy hypothesis is calculated, and the hypothesis of disomy is rejected if the probability is below a given threshold value (such as less than 1 in 1,000). This indicates that a duplication or deletion of the chromosome segment is present. If desired, the false positive rate can be altered by adjusting the threshold value. Exemplary Methods for Analysis of Phased Data

[0317] Exemplarymethodsaredescribed below for analysis of data fromasample knownor suspectedof beingamixed sample containing DNA or RNA that originated from two or more cells that are not genetically identical. In some embodiments, phased data is used. In some embodiments, the method involves determining, for each calculated allele ratio,whether the calculatedallele ratio is aboveor below theexpectedallele ratio and themagnitudeof the difference for a particular locus. In some embodiments, a likelihood distribution is determined for the allele ratio at a locus for a particular hypothesis and the closer the calculated allele ratio is to the center of the likelihood distribution, the more likely the hypothesis is correct. In someembodiments, themethod involvesdetermining the likelihood that ahypothesis is correct for each locus. In some embodiments, the method involves determining the likelihood that a hypothesis is correct for each locus, and combining the probabilities of that hypothesis for each locus, and the hypothesis with the greatest combined probability is selected. In some embodiments, the method involves determining the likelihood that a hypothesis is correct for each locus and for each possible ratio of DNA or RNA from the one or more target cells to the total DNA or RNA in the sample. In someembodiments, a combinedprobability for eachhypothesis is determinedbycombining theprobabilities of that hypothesis for each locus and each possible ratio, and the hypothesis with the greatest combined probability is selected.

[0318] In one embodiment, the following hypotheses are considered: H11 (all cells are normal), H10 (presence of cells with only homolog 1, hence homolog 2 deletion),H01 (presence of cells with only homolog 2, hence homolog 1 deletion), H21 (presence of cells with homolog 1 duplication), H12 (presence of cells with homolog 2 duplication). For a fraction f of target cells such as cancer cells or mosaic cells (or the fraction of DNA or RNA from the target cells), the expected allele ratio for heterozygous (AB or BA) SNPs can be found as follows: Bias, Contamination, and Sequencing Error Correction:

[0319] TheobservationDsat theSNPconsists of the number of originalmapped readswith each allele present, nA0and nB0. Then, we can find the corrected reads nA and nB using the expected bias in the amplification of A and B alleles.

[0320] Let ca to denote the ambient contamination (such as contamination fromDNA in the air or environment) and r(ca) todenote theallele ratio for theambient contaminant (which is taken tobe0.5 initially).Moreover,cgdenotes thegenotyped contamination rate (such as the contamination fromanother sample), and r(cg) is the allele ratio for the contaminant. Let se (A,B)and se(B,A)denote the sequencing errors for calling one allele a different allele (such as by erroneously detecting an A allele when a B allele is present).

[0321] Onecan find theobserved allele ratioq(r, ca, r(ca) , cg , r(cg), se(A,B), se(B,A) ) for a givenexpected allele ratio rby 43 EP 4 737 593 A2 5 10 15 20 25 30 35 40 45 50 55 correcting for ambient contamination, genotyped contamination, and sequencing error.

[0322] Since the contaminant genotypes are unknown, population frequencies can be used to find P(r(cg)). More specifically, let p be the population frequency for one of the alleles (which may be referred to as a reference allele). Then, wehaveP(r(cg) =0) = (1-p)2, P(r(cg) = 0) =2p(1-p), andP(r(cg)=0) =p2.The conditional expectation over r(cg) canbeused to determine the E[q(r, ca, r(ca) , cg , r(cg), se(A,B), se(B,A)) ] . Note that the ambient and genotyped contamination are determined using the homozygous SNPs, hence they are not affected by the absence or presence of deletions or duplications. Moreover, it is possible to measure the ambient and genotyped contamination using a reference chromo- some if desired. Likelihood at each SNP:

[0323] The equation below gives the probability of observing nA and nB given an allele ratio r:

[0324] Let Ds denote the data for SNP s. For each hypothesis h ∈ { H11, H01, H10, H21, H12 }, one can let r=r(AB,h) or r=r(BA,h) in the equation (1) and find the conditional expectation over r(cg) to determine the observed allele ratioE[q(r, ca, r(ca) , cg , r(cg)) ]. Then, letting r= E[q(r, ca, r(ca) , cg , r(cg), se(A,B), se(B,A) ) ] in equation (2) one can determine P(Ds|h,f). Search Algorithm:

[0325] In some embodiments, SNPs with allele ratios that seem to be outliers are ignored (such as by ignoring or eliminatingSNPswith allele ratios that are at least 2 or 3 standard deviations above or below themean value). Note that an advantage identified for this approach is that in the presence of higher mosaicism percentage, the variability in the allele ratios may be high, hence this ensures that SNPs will not be trimmed due to mosaicism.

[0326] Let F = {f1, ...., fn} denote the search space for the mosaicism percentage (such as the tumor fraction). One can determine P(Ds|h,f) at each SNP s and f ∈ F, and combine the likelihood over all SNPs.

[0327] The algorithm goes over each f for each hypothesis. Using a search method, one concludes that mosaicism exists if there is a rangeF* of fwhere the confidence of the deletion or duplication hypothesis is higher than the confidence of the no deletion and no duplication hypotheses. In some embodiments, themaximum likelihood estimate forP(Ds|h,f) in F* is determined. If desired, the conditional expectation over f∈F*may be determined. If desired, the confidence for each hypothesis can be determined.

[0328] In some embodiments, a beta binomial distribution is used instead of binomial distribution. In some embodi- ments, a reference chromosome or chromosome segment is used to determine the sample specific parameters of beta binomial. Theoretical Performance using Simulations:

[0329] If desired, one can evaluate the theoretical performance of the algorithm by randomly assigning number of reference reads to a SNP with given depth of read (DOR). For the normal case, use p= 0.5 for the binomial probability parameter, and for deletions or duplications, p is revised accordingly. Exemplary input parameters for each simulation are as follows: (1) number of SNPs S (2) constant DOR D per SNP, (3) p, and (4) number of experiments. First Simulation Experiment:

[0330] This experiment focused onS∈ {500, 1000}, D ∈ {500, 1000} and p ∈ {0%, 1%, 2%, 3%, 4%, 5%}.Weperformed 1,000 simulation experiments in each setting (hence 24,000 experiments with phase, and 24,000 without phase). We simulated the number of reads from a binomial distribution (if desired, other distributions can be used). The false positive rate (in the case of p=0%) and false negative rate (in the case of p>0%) were determined both with or without phase information. Note that phase information is very helpful, especially for S=1000, D = 1000.Although forS=500, D=500, the algorithm has the highest false positive rates with or without phase out of the conditions tested.

[0331] Phase information is particularly useful for lowmosaicismpercentages (≤3%).Without phase information, a high level of false negatives were observed for p = 1% because the confidence on deletion is determined by assigning equal 44 EP 4 737 593 A2 5 10 15 20 25 30 35 40 45 50 55 chance toH10andH01,andasmall deviation in favor of onehypothesis is not sufficient to compensate for the low likelihood from the other hypothesis. This applies to duplications as well. Note also that the algorithm seems to bemore sensitive to depth of read compared to number of SNPs. For the results with phase information, we assume that perfect phase information is available for a high number of consecutive heterozygous SNPs. If desired, haplotype information can be obtained by probabilistically combining haplotypes on smaller segments. Second Simulation Experiment:

[0332] This experiment focused on S ∈ {100, 200, 300, 400, 500}, D ∈ {1000, 2000, 3000, 4000, 5000} and p ∈ {0%, 1%,1.5%, 2%,2.5%, 3%} and10000 randomexperiments at each setting. The false positive rate (in the caseof p=0%) and falsenegative rate (in the caseofp>0%)weredeterminedbothwith orwithout phase information. The falsenegative rate is below 10% forD ≥ 3000 andN ≥200 using haplotype information, whereas the same performance is reached forD=5000 and N≥400. The difference between the false negative rate was particularly stark for small mosaicism percentages. For example, when p=1%, a less than 20% false negative rate is never reached without haplotype data, whereas it is close to 0% forN ≥ 300 andD ≥ 3000. For p=3%, a 0% false negative rate is observed with haplotype data, whileN ≥ 300 andD ≥ 3000 is needed to reach the same performance without haplotype data. Exemplary Methods for Detecting Deletions and Duplications Without Phased Data

[0333] In some embodiments, unphased genetic data is used to determine if there is an overrepresentation of the number of copies of a first homologous chromosome segment as compared to a second homologous chromosome segment in the genome of an individual (such as in the genome of one or more cells or in cfDNA or cfRNA). In some embodiments, phasedgeneticdata is usedbut thephasing is ignored. In someembodiments, thesampleofDNAorRNA is amixed sample of cfDNAor cfRNA from the individual that includes cfDNAor cfRNA from two ormore genetically different cells. In someembodiments, themethodutilizes themagnitudeof thedifferencebetween the calculatedallele ratio and the expected allele ratio for each of the loci.

[0334] In some embodiments, the method involves obtaining genetic data at a set of polymorphic loci on the chromo- some or chromosome segment in a sample of DNA or RNA from one or more cells from the individual by measuring the quantity of each allele at each locus. In someembodiments, allele ratios are calculated for the loci that are heterozygous in at least one cell fromwhich the samplewas derived. In someembodiments, the calculated allele ratio for a particular locus is themeasured quantity of one of the alleles divided by the total measured quantity of all the alleles for the locus. In some embodiments, the calculated allele ratio for a particular locus is the measured quantity of one of the alleles (such as the allele on the first homologous chromosome segment) divided by themeasured quantity of one ormore other alleles (such as the allele on the second homologous chromosome segment) for the locus. The calculated allele ratios and expected allele ratios may be calculated using any of the methods described herein or any standard method (such as any mathematical transformation of the calculated allele ratios or expected allele ratios described herein).

[0335] In some embodiments, a test statistic is calculated based on the magnitude of the difference between the calculated allele ratio and the expected allele ratio for each of the loci. In some embodiments, the test statistic Δ is calculated using the following formula wherein δi is themagnitude of the difference between the calculated allele ratio and the expected allele ratio for the ith loci; wherein µi is the mean value of δi; and wherein is the standard deviation of δi.

[0336] For example, we can define δi as follows when the expected allele ratio is 0.5:

[0337] Values forµi andσi can be computed using the fact thatRi is a Binomial random variable. In some embodiments, 45 EP 4 737 593 A2 5 10 15 20 25 30 35 40 45 50 55 the standard deviation is assumed to be the same for all the loci. In some embodiments, the average or weighted average valueof the standarddeviationor anestimateof thestandarddeviation isused for the valueof . In someembodiments, the test statistic isassumed tohaveanormaldistribution.Forexample, thecentral limit theorem implies that thedistribution of Δ converges to a standard normal as the number of loci (such as the number of SNPs T) grows large.

[0338] In some embodiments, a set of one or more hypotheses specifying the number of copies of the chromosome or chromosome segment in the genome of one or more of the cells are enumerated. In some embodiments, the hypothesis that is most likely based on the test statistic is selected, thereby determining the number of copies of the chromosome or chromosome segment in the genome of one or more of the cells. In some embodiments, a hypotheses is selected if the probability that the test statistic belongs to a distribution of the test statistic for that hypothesis is above an upper threshold; oneormoreof thehypotheses is rejected if theprobability that the test statistic belongs to thedistributionof the test statistic for that hypothesis is belowan lower threshold; or a hypothesis is neither selectednor rejected if the probability that the test statistic belongs to the distribution of the test statistic for that hypothesis is between the lower threshold and the upper threshold, or if the probability is not determined with sufficiently high confidence. In some embodiments, an upper and / or lower threshold is determined fromanempirical distribution, suchasadistribution from trainingdata (suchas sampleswith a known copy number, such as diploid samples or samples known to have a particular deletion or duplication). Such an empirical distribution can be used to select a threshold for a single hypothesis rejection test. Note that the test statistic Δ is independent of S and therefore both can be used independently, if desired. Exemplary Methods for Detecting Deletions and Duplications Using Allele Distributions or Patterns

[0339] This section includes methods for determining if there is an overrepresentation of the number of copies of a first homologouschromosomesegmentascompared toasecondhomologouschromosomesegment. In someembodiments, the method involves enumerating (i) a plurality of hypotheses specifying the number of copies of the chromosome or chromosome segment that are present in the genome of one or more cells (such as cancer cells) of the individual or (ii) a plurality of hypotheses specifying the degree of overrepresentation of the number of copies of a first homologous chromosomesegment as compared to a secondhomologous chromosomesegment in the genomeof oneormore cells of the individual. In some embodiments, the method involves obtaining genetic data from the individual at a plurality of polymorphic loci (such as SNP loci) on the chromosome or chromosome segment. In some embodiments, a probability distributionof theexpectedgenotypesof the individual for eachof thehypotheses is created. In someembodiments, adata fit between the obtained genetic data of the individual and the probability distribution of the expected genotypes of the individual is calculated. In some embodiments, one or more hypotheses are ranked according to the data fit, and the hypothesis that is ranked the highest is selected. In some embodiments, a technique or algorithm, such as a search algorithm, is used for one or more of the following steps: calculating the data fit, ranking the hypotheses, or selecting the hypothesis that is ranked the highest. In some embodiments, the data fit is a fit to a beta-binomial distribution or a fit to a binomial distribution. In someembodiments, the technique or algorithm is selected from the group consisting ofmaximum likelihood estimation, maximum a-posteriori estimation, Bayesian estimation, dynamic estimation (such as dynamic Bayesianestimation), andexpectation-maximizationestimation. In someembodiments, themethod includesapplying the technique or algorithm to the obtained genetic data and the expected genetic data.

[0340] In some embodiments, the method involves enumerating (i) a plurality of hypotheses specifying the number of copies of the chromosome or chromosome segment that are present in the genome of one or more cells (such as cancer cells) of the individual or (ii) a plurality of hypothesesspecifying thedegreeof overrepresentationof thenumberof copiesof a first homologous chromosome segment as compared to a second homologous chromosome segment in the genome of oneormore cells of the individual. In someembodiments, themethod involvesobtaininggenetic data from the individual at a plurality of polymorphic loci (suchasSNP loci) on the chromosomeor chromosomesegment. In someembodiments, the genetic data includes allele counts for the plurality of polymorphic loci. In some embodiments, a joint distributionmodel is created for the expected allele counts at the plurality of polymorphic loci on the chromosome or chromosome segment for each hypothesis. In some embodiments, a relative probability for one or more of the hypotheses is determined using the joint distribution model and the allele counts measured on the sample, and the hypothesis with the greatest probability is selected.

[0341] In some embodiments, the distribution or pattern of alleles (such as the pattern of calculated allele ratios) is used to determine the presence or absence of a CNV, such as a deletion or duplication. If desired the parental origin of the CNV can be determined based on this pattern. Exemplary Counting Methods / Quantitative Methods

[0342] In some embodiments, one or more counting methods (also referred to as quantitative methods) are used to detect one or more CNS, such as deletions or duplications of chromosome segments or entire chromosomes. In some 46 EP 4 737 593 A2 5 10 15 20 25 30 35 40 45 50 55 embodiments, one or more counting methods are used to determine whether the overrepresentation of the number of copies of the first homologous chromosomesegment is due to a duplication of the first homologous chromosomesegment or a deletion of the secondhomologous chromosomesegment. In someembodiments, one ormore countingmethods are used to determine the number of extra copies of a chromosome segment or chromosome that is duplicated (such as whether there are 1, 2, 3, 4, or more extra copies). In some embodiments, one or more counting methods are used to differentiate a sample has many duplications and a smaller tumor fraction from a sample with fewer duplications and a larger tumor fraction. For example, one or more counting methods may be used to differentiate a sample with four extra chromosome copies and a tumor fraction of 10% froma samplewith two extra chromosome copies and a tumor fraction of 20%. Exemplary methods are disclosed, e.g. U.S. Publication Nos. 2007 / 0184467; 2013 / 0172211; and 2012 / 0003637; U.S. Patent Nos. 8,467,976; 7,888,017; 8,008,018; 8,296,076; and 8,195,415; U.S. Serial No. 62 / 008,235, filed June 5, 2014, andU.S.SerialNo. 62 / 032,785, filedAugust 4, 2014,whichareeachhereby incorporatedby reference in its entirety.

[0343] In some embodiment, the counting method includes counting the number of DNA sequence-based reads that map to one or more given chromosomes or chromosome segments. Some such methods involve creation of a reference value (cut-off value) for the number of DNA sequence readsmapping to a specific chromosomeor chromosome segment, wherein a number of reads in excess of the value is indicative of a specific genetic abnormality.

[0344] In some embodiments, the totalmeasured quantity of all the alleles for one ormore loci (such as the total amount of a polymorphic or non-polymorphic locus) is compared to a reference amount. In some embodiments, the reference amount is (i) a threshold value or (ii) an expected amount for a particular copy number hypothesis. In some embodiments, the reference amount (for the absence of aCNV) is the totalmeasured quantity of all the alleles for one ormore loci for one or more chromosomes or chromosomes segments known or expected to not have a deletion or duplication. In some embodiments, the reference amount (for the presence of a CNV) is the total measured quantity of all the alleles for one or more loci for oneormorechromosomesor chromosomessegmentsknownorexpected tohaveadeletionorduplication. In some embodiments, the reference amount is the total measured quantity of all the alleles for one or more loci for one or more reference chromosomes or chromosome segments. In some embodiments, the reference amount is the mean or median of the values determined for two ormore different chromosomes, chromosome segments, or different samples. In some embodiments, random (e.g., massively parallel shotgun sequencing) or targeted sequencing is used to determine the amount of one or more polymorphic or non-polymorphic loci.

[0345] In some embodiments utilizing a reference amount, the method includes (a) measuring the amount of genetic material on a chromosome or chromosome segment of interest; (b) comparing the amount from step (a) to a reference amount; and (c) identifying the presence or absence of a deletion or duplication based on the comparison.

[0346] In some embodiments utilizing a reference chromosome or chromosome segment, the method includes sequencingDNAorRNA fromasample to obtain aplurality of sequence tagsaligning to target loci. In someembodiments, the sequence tags are of sufficient length to be assigned to a specific target locus (e.g., 15‑100 nucleotides in length); the target loci are from a plurality of different chromosomes or chromosome segments that include at least one first chromosome or chromosome segment suspected of having an abnormal distribution in the sample and at least one second chromosomeor chromosomesegment presumed to be normally distributed in the sample. In someembodiments, the plurality of sequence tags are assigned to their corresponding target loci. In some embodiments, the number of sequence tags aligning to the target loci of the first chromosome or chromosome segment and the number of sequence tags aligning to the target loci of the second chromosome or chromosome segment are determined. In some embodi- ments, thesenumbersare compared todetermine thepresenceor absenceof anabnormal distribution (suchasadeletion or duplication) of the first chromosome or chromosome segment.

[0347] In some embodiments, the value of f (such as tumor fraction) is used in the CNV determination, such as to compare the observed difference between the amount of two chromosomes or chromosome segments to the difference that would be expected for a particular type of CNV given the value of f (see, e.g., US Publication No 2012 / 0190020; US Publication No 2012 / 0190021; US Publication No 2012 / 0190557; US Publication No 2012 / 0191358, which are each hereby incorporated by reference in its entirety). For example, the difference in theamount of a chromosomesegment that is duplicated in a tumor compared toadisomic referencechromosomesegment increasesas the tumor fraction increases. In someembodiments, themethod includes comparing the relative frequency of a chromosome or chromosome segment of interest to a reference chromosomes or chromosome segment (such as a chromosome or chromosome segment expected or known to be disomic) to the value of f to determine the likelihood of the CNV. For example, the difference in amounts between the first chromosomes or chromosome segment to the reference chromosome or chromosome segment can be compared to what would be expected given the value of f for various possible CNVs (such as one or two extra copies of a chromosome segment of interest).

[0348] The following prophetic examples illustrate the use of a counting method / quantitative method to differentiate between a duplication of the first homologous chromosome segment and a deletion of the second homologous chromosome segment. If one considers the normal disomic genome of the host to be the baseline, then analysis of a mixture of normal and cancer cells yields the average difference between the baseline and the cancer DNA in themixture. For example, imagine a case where 10% of the DNA in the sample originated from cells with a deletion over a region of a 47 EP 4 737 593 A2 5 10 15 20 25 30 35 40 45 50 55 chromosome that is targetedby theassay. In someembodiments, aquantitativeapproachshows that thequantity of reads corresponding to that region is expected to be 95%ofwhat is expected for a normal sample. This is becauseoneof the two target chromosomal regions in each of the tumor cells with a deletion of the targeted region is missing, and thus the total amount of DNAmapping to that region is 90% (for the normal cells) plus½ x 10% (for the tumor cells) = 95%. Alternately in someembodiments, anallelic approachshows that the ratioof allelesat heterozygous loci averaged19:20.Now imaginea case where 10% of the DNA in the sample originated from cells with a five-fold focal amplification of a region of a chromosome that is targetedby theassay. In someembodiments, aquantitativeapproachshows that thequantity of reads corresponding to that region is expected to be 125% of what is expected for a normal sample. This is because one of the two target chromosomal regions in each of the tumor cells with a five-fold focal amplification is copied an extra five times over the targeted region, and thus the total amount ofDNAmapping to that region is 90% (for the normal cells) plus (2+5) x 10% / 2 (for the tumor cells) = 125%.Alternately in someembodiments, an allelic approach shows that the ratio of alleles at heterozygous loci averaged 25:20. Note that when using an allelic approach alone, a focal amplification of five-fold over a chromosomal region inasamplewith10%cfDNAmayappear thesameasadeletionover thesame region inasamplewith 40% cfDNA; in these two cases, the haplotype that is under-represented in the case of the deletion appears to be the haplotypewithout aCNV in the casewith the focal duplication, and the haplotypewithout aCNV in the case of the deletion appears to be the over-represented haplotype in the case with the focal duplication. Combining the likelihoods produced by this allelic approach with likelihoods produced by a quantitative approach differentiates between the two possibilities. Exemplary Counting Methods / Quantitative Methods Using Reference Samples

[0349] An exemplary quantitative method that uses one or more reference samples is described in U.S. Serial No. 62 / 008,235, filed June 5, 2014 and U.S. Serial No. 62 / 032,785, filed August 4, 2014, which is hereby incorporated by reference in its entirety. In someembodiments, one ormore reference samplesmost likely to not haveanyCNVsononeor more chromosomes or chromosomes of interest (e.g., a normal sample) are identified by selecting the samples with the highest fractionof tumorDNA, selecting the sampleswith the z-score closest to zero, selecting thesampleswhere thedata fits the hypothesis corresponding to noCNVswith the highest confidence or likelihood, selecting the samples known to be normal, selecting the samples from individuals with the lowest likelihood of having cancer (e.g., having a low age, being a malewhen screening for breast cancer, having no family history,etc.), selecting the sampleswith thehighest input amount of DNA, selecting the samples with the highest signal to noise ratio, selecting samples based on other criteria believed to be correlated to the likelihood of having cancer, or selecting samples using some combination of criteria. Once the reference set is chosen, one canmake the assumption that these cases are disomic, and then estimate the per-SNPbias, that is, the experiment-specific amplification and other processing bias for each locus. Then, one can use this experiment- specificbias estimate to correct thebias in themeasurementsof the chromosomeof interest, suchas chromosome21 loci, and for the other chromosome loci as appropriate, for the samples that are not part of the subsetwhere disomy is assumed for chromosome 21. Once the biases have been corrected for in these samples of unknown ploidy, the data for these samples can thenbeanalyzedasecond timeusing thesameoradifferentmethod todeterminewhether the individuals are afflictedwith trisomy21.Forexample, aquantitativemethodcanbeusedon the remainingsamplesofunknownploidy, and a z-score can be calculated using the corrected measured genetic data on chromosome 21. Alternately, as part of the preliminary estimate of the ploidy state of chromosome 21, a tumor fraction for samples from an individual suspected of having cancer can be calculated. The proportion of corrected reads that are expected in the case of a disomy (the disomy hypothesis), and the proportion of corrected reads that are expected in the case of a trisomy (the trisomy hypothesis) can be calculated for a case with that tumor fraction. Alternately, if the tumor fraction was not measured previously, a set of disomy and trisomy hypotheses can be generated for different tumor fractions. For each case, an expected distribution of the proportion of corrected reads can be calculated given expected statistical variation in the selection andmeasurement of the various DNA loci. The observed corrected proportion of reads can be compared to the distribution of the expected proportion of corrected reads, and a likelihood ratio can be calculated for the disomy and trisomy hypotheses, for each of the samples of unknown ploidy. The ploidy state associated with the hypothesis with the highest calculated likelihood can be selected as the correct ploidy state.

[0350] In someembodiments, a subset of the sampleswith a sufficiently low likelihoodof having cancermaybe selected to act as a control set of samples. The subset can be a fixed number, or it can be a variable number that is based on choosing only those samples that fall below a threshold. The quantitative data from the subset of samples may be combined, averaged, or combined using aweighted averagewhere theweighting is based on the likelihood of the sample being normal. The quantitative data may be used to determine the per-locus bias for the amplification the sequencing of samples in the instant batch of control samples. The per-locus biasmay also include data from other batches of samples. The per-locus biasmay indicate the relative over‑ or under-amplification that is observed for that locus compared to other loci, making the assumption that the subset of samples do not contain any CNVs, and that any observed over or under- amplification is due to amplification and / or sequencing or other bias. The per-locus bias may take into account the GC content of the amplicon. The loci may be grouped into groups of loci for the purpose of calculating a per-locus bias. Once 48 EP 4 737 593 A2 5 10 15 20 25 30 35 40 45 50 55 the per-locus bias has been calculated for each locus in the plurality of loci, the sequencing data for one or more of the samples that are not in the subset of the samples, and optionally one or more of the samples that are in the subset of samples, may be corrected by adjusting the quantitative measurements for each locus to remove the effect of the bias at that locus.Forexample, ifSNP1wasobserved, in thesubset of patients, tohaveadepthof read that is twiceasgreat as the average, theadjustmentmay involve replacing thenumberof readscorresponding fromSNP1withanumber that is half as great. If the locus inquestion isaSNP, theadjustmentmay involvecutting thenumberof readscorresponding toeachof the alleles at that locus in half. Once the sequencing data for each of the loci in one ormore samples has been adjusted, itmay be analyzed using a method for the purpose of detecting the presence of a CNV at one or more chromosomal regions.

[0351] In an example, sample A is a mixture of amplified DNA originating from amixture of normal and cancerous cells that is analyzed using a quantitative method. The following illustrates exemplary possible data. A region of the q arm on chromosome22 is found toonly have90%asmuchDNAmapping to that regionasexpected; a focal region corresponding to theHER2gene is found to have 150%asmuchDNAmapping to that region as expected; and the p-armof chromosome 5 is found to have 105% asmuch DNAmapping to it as expected. A clinicianmay infer that the sample has a deletion of a region on the q arm on chromosome 22, and a duplication of the HER2 gene. The clinician may infer that since the 22q deletions are common in breast cancer, and that since cellswith a deletion of the 22q region onboth chromosomesusually do not survive, that approximately 20% of the DNA in the sample came from cells with a 22q deletion on one of the two chromosomes. The clinician may also infer that if the DNA from the mixed sample that originated from tumor cells originated from a set of genetically tumor cells whose HER2 region and 22q regions were homogenous, then the cells contained a five-fold duplication of the HER2 region.

[0352] In an example, Sample A is also analyzed using an allelic method. The following illustrates exemplary possible data.The twohaplotypesonsame regionon theqarmonchromosome22arepresent ina ratioof 4:5; the twohaplotypes in a focal region corresponding to the HER2 gene are present in ratios of 1:2; and the two haplotypes in the p-arm of chromosome 5 are present in ratios of 20:21. All other assayed regions of the genome have no statistically significant excess of either haplotype. A clinicianmay infer that the sample contains DNA from a tumor with a CNV in the 22q region, theHER2region, and the5parm.Basedon theknowledge that 22qdeletionsarevery common inbreast cancer, and / or the quantitativeanalysis showinganunder-representationof theamount ofDNAmapping to the22q regionof thegenome, the clinicianmay infer the existence of a tumorwith a 22qdeletion. Based on the knowledge that HER2amplifications are very common in breast cancer, and / or the quantitative analysis showing anover-representation of the amount ofDNAmapping to the HER2 region of the genome, the clinician may infer the existence of a tumor with a HER2 amplification. Exemplary Reference Chromosomes or Chromosome Segments

[0353] In some embodiments, any of the methods described herein are also performed on one or more reference chromosomes or chromosomes segments and the results are compared to those for one or more chromosomes or chromosome segments of interest.

[0354] In some embodiments, the reference chromosome or chromosome segment is used as a control for what would be expected for the absence of a CNV. In some embodiments, the reference is the same chromosome or chromosome segment fromoneormoredifferent samplesknownorexpected tonot haveadeletionor duplication in that chromosomeor chromosomesegment. In someembodiments, the reference is adifferent chromosomeor chromosomesegment from the sample being tested that is expected to bedisomic. In someembodiments, the reference is adifferent segment fromoneof the chromosomes of interest in the same sample that is being tested. For example, the reference may be one or more segments outside of the region of a potential deletion or duplication. Having a reference on the same chromosome that is being tested avoids variability between different chromosomes, such as differences in metabolism, apoptosis, histones, inactivation, and / or amplification between chromosomes. Analyzing segments without a CNVon the same chromosome as the one being tested can also be used to determine differences inmetabolism, apoptosis, histones, inactivation, and / or amplification between homologs, allowing the level of variability between homologs in the absence of a CNV to be determined for comparison to the results from a potential CNV. In some embodiments, the magnitude of the difference between the calculated and expected allele ratios for a potential CNV is greater than the correspondingmagnitude for the reference, thereby confirming the presence of a CNV.

[0355] In some embodiments, the reference chromosome or chromosome segment is used as a control for what would be expected for the presence of a CNV, such as a particular deletion or duplication of interest. In some embodiments, the reference is the same chromosome or chromosome segment from one or more different samples known or expected to have a deletion or duplication in that chromosome or chromosome segment. In some embodiments, the reference is a different chromosome or chromosome segment from the sample being tested that is known or expected to have aCNV. In someembodiments, themagnitude of the difference between the calculated andexpected allele ratios for a potential CNV is similar to (such as not significantly different) than the corresponding magnitude for the reference for the CNV, thereby confirming the presence of a CNV. In some embodiments, the magnitude of the difference between the calculated and expected allele ratios for a potential CNV is less than (such as significantly less) than the correspondingmagnitude for the 49 EP 4 737 593 A2 5 10 15 20 25 30 35 40 45 50 55 reference for the CNV, thereby confirming the absence of a CNV. In some embodiments, one or more loci for which the genotype of a cancer cell (or DNA or RNA from a cancer cell such as cfDNA or cfRNA) differs from the genotype of a noncancerous cell (or DNA or RNA from a noncancerous cell such as cfDNA or cfRNA) is used to determine the tumor fraction. The tumor fraction can be used to determine whether the overrepresentation of the number of copies of the first homologous chromosome segment is due to a duplication of the first homologous chromosome segment or a deletion of the second homologous chromosome segment. The tumor fraction can also be used to determine the number of extra copies of a chromosome segment or chromosome that is duplicated (such as whether there are 1, 2, 3, 4, or more extra copies), such as to differentiate a sample with four extra chromosome copies and a tumor fraction of 10% from a sample with twoextra chromosomecopies anda tumor fraction of 20%.The tumor fraction canalso be used to determine howwell the observed data fits the expected data for possible CNVs. In some embodiments, the degree of overrepresentation of a CNV is used to select a particular therapy or therapeutic regimen for the individual. For example, some therapeutic agents are only effective for at least four, six, or more copies of a chromosome segment.

[0356] In someembodiments, the oneormore loci used to determine the tumor fraction are ona reference chromosome or chromosomes segment, such as a chromosome or chromosome segment known or expected to be disomic, a chromosomeor chromosomesegment that is rarely duplicatedor deleted in cancer cells in general or in aparticular typeof cancer that an individual is known to have or is at increased risk of having, or a chromosomeor chromosome segment that is unlikely to be aneuploid (such segment that is expected to lead to cell death if deleted or duplicated). In some embodiments, any of the methods of the invention are used to confirm that the reference chromosome or chromosome segment is disomic in both the cancer cells and noncancerous cells. In someembodiments, one ormore chromosomes or chromosomes segments for which the confidence for a disomy call is high are used.

[0357] Exemplary loci that can be used to determine the tumor fraction include polymorphisms or mutations (such as SNPs) in a cancer cell (or DNA or RNA such as cfDNA or cfRNA from a cancer cell) that aren’t present in a noncancerous cell (orDNAorRNA fromanoncancerous cell) in the individual. In someembodiments, the tumor fraction is determinedby identifying those polymorphic loci where a cancer cell (or DNA or RNA from a cancer cell) has an allele that is absent in noncancerouscells (orDNAorRNAfromanoncancerouscell) inasample (suchasaplasmasampleor tumorbiopsy) from an individual; andusing the amount of the allele unique to the cancer cell at one ormoreof the identified polymorphic loci to determine the tumor fraction in the sample. In some embodiments, a noncancerous cell is homozygous for a first allele at the polymorphic locus, and a cancer cell is (i) heterozygous for the first allele and a second allele or (ii) homozygous for a second allele at the polymorphic locus. In some embodiments, a noncancerous cell is heterozygous for a first allele and a second allele at the polymorphic locus, and a cancer cell is (i) has one or two copies of a third allele at the polymorphic locus. In someembodiments, the cancer cells are assumedor known to only have one copy of the allele that is not present in the noncancerous cells. For example, if the genotype of the noncancerous cells is AA and the cancer cells is AB and 5% of the signal at that locus in asample is from theBallele and95% is from theAallele, then the tumor fractionof the sample is 10%. In some embodiments, the cancer cells are assumed or known to have two copies of the allele that is not present in the noncancerous cells. For example, if the genotype of the noncancerous cells is AAand the cancer cells is BBand 5%of the signal at that locus in a sample is from theBallele and95% is from theAallele, the tumor fraction of the sample is 5%. In some embodiments, multiple loci for which the cancer cells have an allele not in the noncancerous cells are analyzed to determinewhich of the loci in the cancer cells are heterozygous andwhich are homozygous. For example for loci in which the noncancerous cells are AA, if the signal from the B allele is ~5% at some loci and ~10% at some loci, then the cancer cells are assumed to be heterozygous at loci with ~5%B allele, and homozygous at loci with ~10%B allele (indicating the tumor fraction is ~10%).

[0358] Exemplary loci that can be used to determine the tumor fraction include loci for which a cancer cell and noncancerous cell have one allele in common (such as loci in which the cancer cell is AB and the noncancerous cell is BB, or the cancer cell is BBand the noncancerous cell is AB). The amount of A signal, the amount of B signal, or the ratio of A to B signal in amixed sample (containing DNAor RNA froma cancer cell and a noncancerous cell) is compared to the corresponding value for (i) a sample containingDNAorRNA fromonly cancer cells or (ii) a sample containingDNAorRNA from only noncancerous cells. The difference in values is used to determine the tumor fraction of the mixed sample.

[0359] In some embodiments, loci that can be used to determine the tumor fraction are selected based on the genotype of (i) a sample containing DNA or RNA from only cancer cells, and / or (ii) a sample containing DNA or RNA from only noncancerous cells. In some embodiments, the loci are selected based on analysis of the mixed sample, such as loci for which the absolute or relative amounts of each allele differs from what would be expected if both the cancer and noncancerous cells have the samegenotypeat aparticular locus. For example, if the cancer andnoncancerous cells have the samegenotype, the loci would be expected to produce 0%Bsignal if all the cells areAA, 50%Bsignal if all the cells are AB, or 100% B signal if all the cells are BB. Other values for the B signal indicate that the genotype of the cancer and noncancerous cells are different at that locus and thus that locus can be used to determine the tumor fraction.

[0360] In some embodiments, the tumor fraction calculated based on the alleles at one or more loci is compared to the tumor fraction calculated using one or more of the counting methods disclosed herein. 50 EP 4 737 593 A2 5 10 15 20 25 30 35 40 45 50 55 Exemplary Methods for Detecting a Phenotype or Analyzing Multiple Mutations

[0361] In some embodiments, themethod includes analyzing a sample for a set of mutations associated with a disease or disorder (such as cancer) or an increased risk for a disease or disorder. There are strong correlations between events within classes (such as M or C cancer classes) which can be used to improve the signal to noise ratio of a method and classify tumors into distinct clinical subsets. For example, borderline results for a fewmutations (such as a few CNVs) on one or more chromosomes or chromosomes segments considered jointly may be a very strong signal. In some embodiments, determining the presence or absence of multiple polymorphisms or mutations of interest (such as 2, 3, 4, 5, 8, 10, 12, 15, ormore) increases the sensitivity and / or specificity of the determination of the presence or absence of a disease or disorder such as cancer, or an increased risk for with a disease or disorder such as cancer. In some embodiments, the correlation between events across multiple chromosomes is used to more powerfully look at a signal compared to looking at each of them individually. The design of the method itself can be optimized to best categorize tumors. This may be incredibly useful for early detection and screening--vis-a-vis recurrence where sensitivity to one particular mutation / CNV may be paramount. In some embodiments, the events are not always correlated but have a probability of being correlated. In some embodiments, amatrix estimation formulation with a noise covariancematrix that has off diagonal terms is used.

[0362] In some embodiments, the invention features amethod for detecting a phenotype (such as a cancer phenotype) in an individual, wherein the phenotype is defined by the presence of at least one of a set of mutations. In some embodiments, themethod includes obtaining DNA or RNAmeasurements for a sample of DNA or RNA from one or more cells from the individual, wherein one ormore of the cells is suspected of having the phenotype; and analyzing theDNA or RNA measurements to determine, for each of the mutations in the set of mutations, the likelihood that at least one of the cells has that mutation. In some embodiments, the method includes determining that the individual has the phenotype if either (i) for at least oneof themutations, the likelihood that at least oneof the cells contains thatmutations is greater thana threshold, or (ii) for at least one of the mutations, the likelihood that at least one of the cells has that mutations is less than the threshold, and for aplurality of themutations, the combined likelihood that at least oneof the cells hasat least oneof the mutations is greater than the threshold. In someembodiments, oneormorecells haveasubset or all of themutations in the set of mutations. In some embodiments, the subset of mutations is associated with cancer or an increased risk for cancer. In some embodiments, the set of mutations includes a subset or all of the mutations in the M class of cancer mutations (Ciriello, Nat Genet. 45(10):1127‑1133, 2013, doi: 10.1038 / ng.2762, which is hereby incorporated by reference in its entirety). In some embodiments, the set of mutations includes a subset or all of the mutations in the C class of cancer mutations (Ciriello, supra). In some embodiments, the sample includes cell-free DNA or RNA. In some embodiments, the DNA or RNA measurements include measurements (such as the quantity of each allele at each locus) at a set of polymorphic loci on one or more chromosomes or chromosome segments of interest. Exemplary Combinations of Methods

[0363] To increase the accuracy of the results, two ormoremethods (such as any of themethods of the invention or any known method) for detecting the presence or absence of a CNV are performed. In some embodiments, one or more methods for analyzing a factor (such as any of the method described herein or any known method) indicative of the presence or absence of a disease or disorder or an increased risk for a disease or disorder are performed.

[0364] In some embodiments, standard mathematical techniques are used to calculate the covariance and / or correla- tion between two or more methods. Standard mathematical techniques may also be used to determine the combined probability of a particular hypothesis based on two or more tests. Exemplary techniques include meta-analysis, Fisher’s combined probability test for independent tests, Brown’s method for combining dependent p-values with known covariance, and Kost’s method for combining dependent p-values with unknown covariance. In cases where the likelihoods are determined by a first method in a way that is orthogonal, or unrelated, to the way in which a likelihood is determined for a second method, combining the likelihoods is straightforward and can be done by multiplication and normalization, or by using a formula such as:

[0365] Rcomb is the combined likelihood, and R1 and R2 are the individual likelihoods. For example, if the likelihood of trisomy frommethod1 is 90%, and the likelihood of trisomy frommethod2 is 95%, then combining theoutputs from the two methods allows the clinician to conclude that the fetus is trisomicwith a likelihood of (0.90)(0.95) / [(0.90)(0.95) + (1 - 0.90) (1 - 0.95)] = 99.42%. In cases where the first and the second methods are not orthogonal, that is, where there is a correlation between the two methods, the likelihoods can still be combined.

[0366] Exemplarymethods of analyzingmultiple factors or variables are disclosed in U.S. Patent No. 8,024,128 issued 51 EP 4 737 593 A2 5 10 15 20 25 30 35 40 45 50 55 onSeptember 20, 2011; U.S. PublicationNo. 2007 / 0027636, filed July 31, 2006; andU.S. PublicationNo. 2007 / 0178501, filed December 6, 2006, which are each hereby incorporated by reference in its entirety).

[0367] In various embodiments, the combined probability of a particular hypothesis or diagnosis is greater than 80, 85, 90, 92, 94, 96, 98, 99, or 99.9%, or is greater than some other threshold value. Limit of Detection

[0368] As demonstrated by experiments provided in the Examples section, methods provided herein are capable of detecting an average allelic imbalance in a sample with a limit of detection or sensitivity of 0.45% AAI, which is the limit of detection for aneuploidy of an illustrative method of the present invention. Similarly, in certain embodiments, methods provided herein are capable of detecting an average allelic imbalance in a sample of 0.45, 0.5, 0.6, 0.8, 0.8, 0.9, or 1.0%. That is, the test method is capable of detecting chromosomal aneuploidy in a sample down to an AAI of 0.45, 0.5, 0.6, 0.8, 0.8, 0.9, or 1.0%. As demonstrated by experiments provided in the Examples section, methods provided herein are capable of detecting the presence of an SNV in a sample for at least some SNVs, with a limit of detection or sensitivity of 0.2%, which is the limit of detection for at least some SNVs in one illustrative embodiment. Similarly, in certain embodiments, the method is capable of detecting an SNV with a frequency or SNV AAI of 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, 0.8, 0.9, or 1.0%. That is, the testmethod is capable of detecting anSNV in a sample down to a limit of detection of 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, 0.8, 0.9, or 1.0% of the total allele counts at the chromosomal locus of the SNV.

[0369] In some embodiments, a limit of detection of a mutation (such as an SNVor CNV) of amethod of the invention is less than or equal to 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, or 0.005%. In some embodiments, a limit of detection of a mutation (such as an SNV or CNV) of a method of the invention is between 15 to 0.005%, such as between 10 to 0.005%, 10 to 0.01%, 10 to 0.1%, 5 to 0.005%, 5 to 0.01%, 5 to 0.1%, 1 to 0.005%, 1 to 0.01%, 1 to 0.1%, 0.5 to 0.005%, 0.5 to 0.01%, 0.5 to 0.1%, or 0.1 to 0.01, inclusive.

[0370] In some embodiments, a limit of detection is such that amutation (such as an SNVor CNV) that is present in less thanor equal to 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, or 0.005%of theDNAorRNAmoleculeswith that locus in a sample (suchas asampleof cfDNAor cfRNA) isdetected (or is capableof beingdetected). For example, themutation canbedetectedeven if less thanor equal to10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, or 0.005%of theDNAorRNAmolecules that have that locushave that mutation in the locus (instead of, for example, awild-type or non-mutated version of the locus or a differentmutation at that locus). In some embodiments, a limit of detection is such that a mutation (such as an SNVor CNV) that is present in less than or equal to 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, or 0.005% of the DNA or RNAmolecules in a sample (such as a sample of cfDNA or cfRNA) is detected (or is capable of being detected). In some embodiments in which the CNV is a deletion, the deletion can be detected even if it is only present in less than or equal to 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, or 0.005% of the DNA or RNA molecules that have a region of interest that may or may not contain the deletion in a sample. In some embodiments inwhich theCNVisadeletion, thedeletioncanbedetectedeven if it is onlypresent in less thanorequal to10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, or 0.005%of theDNAorRNAmolecules in a sample. In someembodiments inwhich theCNV is aduplication, theduplicationcanbedetectedeven if theextraduplicatedDNAorRNA that is present is less thanorequal to 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, or 0.005%of theDNAorRNAmolecules that havea regionof interest thatmayormaynot be duplicated in a sample in a sample. In some embodiments in which the CNV is a duplication, the duplication can be detected even if the extra duplicated DNA or RNA that is present is less than or equal to 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, or 0.005% of the DNA or RNA molecules in a sample. Exemplary Samples

[0371] In some embodiments of any of the aspects of the invention, the sample includes cellular and / or extracellular genetic material from cells suspected of having a deletion or duplication, such as cells suspected of being cancerous. In some embodiments, the sample comprises any tissue or bodily fluid suspected of containing cells, DNA, or RNA having a deletion or duplication, such as tumors or other samples that include cancer cells, DNA, or RNA. The genetic measure- ments used as part of thesemethods can bemade on any sample comprisingDNA or RNA, for example but not limited to, tissue, blood, serum, plasma, urine, hair, tears, saliva, skin, fingernails, feces, bile, lymph, cervical mucus, semen, tumor, or other cells or materials comprising nucleic acids. Samples may include any cell type or DNA or RNA from any cell type maybeused (suchascells fromanyorganor tissuesuspectedofbeingcancerous,orneurons). In someembodiments, the sample includesnuclear and / ormitochondrialDNA. In someembodiments, the sample is fromanyof the target individuals disclosed herein. In some embodiments, the target individual cancer patient.

[0372] Exemplary samples include those containing cfDNA or cfRNA. In some embodiments, cfDNA is available for analysis without requiring the step of lysing cells. Cell-free DNAmay be obtained froma variety of tissues, such as tissues that are in liquid form, e.g., blood, plasma, lymph, ascites fluid, or cerebral spinal fluid. In some cases, cfDNA is comprised of DNAderived from fetal cells. In some cases, the cfDNA is isolated fromplasma that has been isolated fromwhole blood that has been centrifuged to remove cellular material. The cfDNAmay be amixture of DNA derived from target cells (such 52 EP 4 737 593 A2 5 10 15 20 25 30 35 40 45 50 55 as cancer cells) and non-target cells (such as non-cancer cells).

[0373] In someembodiments, thesamplecontainsor is suspected tocontainamixtureofDNA(orRNA), suchasmixture ofDNA(orRNA)originating fromcancer cells andDNA(orRNA)originating fromnoncancerous (i.e. normal) cells. In some embodiments, at least 0.5, 1, 3, 5, 7, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 92, 94, 95, 96, 98, 99, or 100%of the cells in the sample are cancer cells. In some embodiments, at least 0.5, 1, 3, 5, 7, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 92, 94, 95, 96, 98, 99, or 100% of the DNA (such as cfDNA) or RNA (such as cfRNA) in the sample is from cancer cell(s). In various embodiments, the percent of cells in the sample that are cancerouscells is between0.5 to 99%, suchasbetween1 to95%, 5 to95%, 10 to90%, 5 to70%,10 to70%,20 to90%,or 20 to70%, inclusive. In someembodiments, the sample is enriched for cancer cellsor forDNAorRNA fromcancer cells. In someembodiments inwhich thesample isenriched for cancer cells, at least 0.5, 1, 2, 3, 4, 5, 6, 7, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 92, 94, 95, 96, 98, 99, or 100%of the cells in the enriched sample are cancer cells. In someembodiments in which the sample is enriched for DNAor RNA from cancer cells, at least 0.5, 1, 2, 3, 4, 5, 6, 7, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 92, 94, 95, 96, 98, 99, or 100%of theDNAorRNA in the enriched sample is from cancer cell(s). In some embodiments, cell sorting (such as Fluorescent Activated Cell Sorting (FACS)) is used to enrich for cancer cells (Barteneva et. al., Biochim Biophys Acta., 1836(1):105‑22, Aug 2013. doi: 10.1016 / j.bbcan.2013.02.004. Epub 2013 Feb 24, and Ibrahim et al., Adv Biochem Eng Biotechnol. 106:19‑39, 2007, which are each hereby incorporated by reference in its entirety).

[0374] In some embodiments, the sample is enriched for fetal cells. In some embodiments in which the sample is enriched for fetal cells, at least 0.5, 1, 2, 3, 4, 5, 6, 7% or more of the cells in the enriched sample are fetal cells. In some embodiments, the percent of cells in the sample that are fetal cells is between 0.5 to 100%, such as between 1 to 99%, 5 to 95%,10 to95%,10 to95%,20 to90%,or 30 to70%, inclusive. In someembodiments, the sample is enriched for fetalDNA. Insomeembodiments inwhich thesample isenriched for fetalDNA,at least 0.5, 1, 2, 3, 4, 5, 6, 7%ormoreof theDNA in the enriched sample is fetal DNA. In some embodiments, the percent of DNA in the sample that is fetal DNA is between 0.5 to 100%, such as between 1 to 99%, 5 to 95%, 10 to 95%, 10 to 95%, 20 to 90%, or 30 to 70%, inclusive.

[0375] In some embodiments, the sample includes a single cell or includes DNA and / or RNA from a single cell. In some embodiments, multiple individual cells (e.g., at least 5, 10, 20, 30, 40, or 50 cells from the same subject or from different subjects) are analyzed in parallel. In some embodiments, cells from multiple samples from the same individual are combined,which reduces theamount ofworkcompared toanalyzing thesamplesseparately.Combiningmultiple samples can also allow multiple tissues to be tested for cancer simultaneously (which can be used to provide or more thorough screening for cancer or to determine whether cancer may have metastasized to other tissues).

[0376] In someembodiments, the sample contains a single cell or a small number of cells, such as2, 3, 5, 6, 7, 8, 9, or 10 cells. In some embodiments, the sample has between 1 to 100, 100 to 500, or 500 to 1,000 cells, inclusive. In some embodiments, the sample contains 1 to 10 picograms, 10 to 100 picograms, 100 picograms to 1 nanogram, 1 to 10 nanograms, 10 to 100 nanograms, or 100 nanograms to 1 microgram of RNA and / or DNA, inclusive.

[0377] In someembodiments, the sample is embedded in parafilm. In someembodiments, the sample is preservedwith a preservative such as formaldehyde and optionally encased in paraffin, which may cause cross-linking of the DNA such that less of it is available for PCR. In some embodiments, the sample is a formaldehyde fixed-paraffin embedded (FFPE) sample. In some embodiments, the sample is a fresh sample (such as a sample obtained with 1 or 2 days of analysis). In some embodiments, the sample is frozen prior to analysis. In some embodiments, the sample is a historical sample.

[0378] These samples can be used in any of the methods of the invention. Exemplary Sample Preparation Methods

[0379] In some embodiments, the method includes isolating or purifying the DNA and / or RNA. There are a number of standardprocedures known in theart toaccomplish suchanend. In someembodiments, the samplemaybecentrifuged to separate various layers. In some embodiments, the DNA or RNAmay be isolated using filtration. In some embodiments, the preparation of the DNA or RNA may involve amplification, separation, purification by chromatography, liquid liquid separation, isolation, preferential enrichment, preferential amplification, targetedamplification, or anyof anumberof other techniques either known in the art or described herein. In some embodiments for the isolation of DNA, RNase is used to degrade RNA. In some embodiments for the isolation of RNA, DNase (such as DNase I from Invitrogen, Carlsbad, CA, USA) is used todegradeDNA. In someembodiments, anRNeasymini kit (Qiagen), is used to isolateRNAaccording to the manufacturer’s protocol. In some embodiments, small RNAmolecules are isolated using themirVana PARIS kit (Ambion, Austin, TX, USA) according to themanufacturer’s protocol (Gu et al., J. Neurochem. 122:641‑649, 2012, , which is hereby incorporated by reference in its entirety). The concentration and purity of RNA may optionally be determined using Nanovue (GE Healthcare, Piscataway, NJ, USA), and RNA integrity may optionally be measured by use of the 2100 Bioanalyzer (Agilent Technologies, SantaClara, CA,USA) (Guet al., J. Neurochem. 122:641‑649, 2012, , which is hereby incorporated by reference in its entirety). In some embodiments, TRIZOL or RNAlater (Ambion) is used to stabilize RNA during storage.

[0380] In someembodiments, universal tagged adaptors are added tomake a library. Prior to ligation, sampleDNAmay 53 EP 4 737 593 A2 5 10 15 20 25 30 35 40 45 50 55 be blunt ended, and then a single adenosine base is added to the 3-prime end. Prior to ligation the DNA may be cleaved usinga restrictionenzymeor someother cleavagemethod.During ligation the3-primeadenosineof the sample fragments and the complementary 3-prime tyrosine overhang of adaptor can enhance ligation efficiency. In some embodiments, adaptor ligation is performed using the ligation kit found in the AGILENT SURESELECT kit. In some embodiments, the library is amplified using universal primers. In an embodiment, the amplified library is fractionated by size separation or by using products such as AGENCOURTAMPURE beads or other similar methods. In some embodiments, PCR amplifica- tion is used to amplify target loci. In some embodiments, the amplified DNA is sequenced (such as sequencing using an ILLUMINA IIGAX or HiSeq sequencer). In some embodiments, the amplified DNA is sequenced from each end of the amplifiedDNA to reducesequencingerrors. If there isasequenceerror inaparticular basewhensequencing fromoneend of theamplifiedDNA, there is less likely tobeasequenceerror in thecomplementarybasewhensequencing from theother side of the amplified DNA (compared to sequencing multiple times from the same end of the amplified DNA).

[0381] In some embodiments, whole genome application (WGA) is used to amplify a nucleic acid sample. There are a number ofmethods available forWGA: ligation-mediatedPCR (LM-PCR), degenerate oligonucleotide primerPCR (DOP- PCR), and multiple displacement amplification (MDA). In LM-PCR, short DNA sequences called adapters are ligated to blunt endsofDNA.Theseadapters contain universal amplification sequences,whichareused toamplify theDNAbyPCR. In DOP-PCR, random primers that also contain universal amplification sequences are used in a first round of annealing and PCR. Then, a second round of PCR is used to amplify the sequences further with the universal primer sequences. MDAuses thephi‑29polymerase,which isahighly processiveandnon-specificenzyme that replicatesDNAandhasbeen used for single-cell analysis. In some embodiments, WGA is not performed.

[0382] In some embodiments, selective amplification or enrichment are used to amplify or enrich target loci. In some embodiments, the amplification and / or selective enrichment techniquemay involve PCR such as ligationmediated PCR, fragment capture by hybridization,Molecular Inversion Probes, or other circularizing probes. In some embodiments, real- time quantitative PCR (RT-qPCR), digital PCR, or emulsion PCR, single allele base extension reaction followed by mass spectrometry are used (Hung et al., J Clin Pathol 62:308‑313, 2009, which is hereby incorporated by reference in its entirety). In some embodiments, capture by hybridization with hybrid capture probes is used to preferentially enrich the DNA. In some embodiments, methods for amplification or selective enrichment may involve using probes where, upon correct hybridization to the target sequence, the 3-prime end or 5-prime end of a nucleotide probe is separated from the polymorphic site of a polymorphic allele by a small number of nucleotides. This separation reduces preferential amplification of one allele, termed allele bias. This is an improvement over methods that involve using probes where the3-primeendor5-primeendof a correctly hybridizedprobearedirectly adjacent toor verynear to thepolymorphic site of an allele. In an embodiment, probes in which the hybridizing region may or certainly contains a polymorphic site are excluded. Polymorphic sites at the site of hybridization can cause unequal hybridization or inhibit hybridization altogether in some alleles, resulting in preferential amplification of certain alleles. These embodiments are improvements over other methods that involve targeted amplification and / or selective enrichment in that they better preserve the original allele frequencies of the sample at each polymorphic locus, whether the sample is pure genomic sample froma single individual or mixture of individuals

[0383] In some embodiments, PCR (referred to asmini-PCR) is used to generate very short amplicons (USApplication No. 13 / 683,604, filed Nov. 21, 2012, U.S. Publication No. 2013 / 0123120, U.S. Application No. 13 / 300,235, filed Nov. 18, 2011, U.S. Publication No 2012 / 0270212, filed Nov. 18, 2011, and U.S. Serial No. 61 / 994,791, filed May 16, 2014, which are each hereby incorporated by reference in its entirety). cfDNA (such as necroptically‑ or apoptotically-released cancer cfDNA) is highly fragmented. For fetal cfDNA, the fragment sizes are distributed in approximately aGaussian fashionwith ameanof 160bp, a standarddeviationof 15bp, aminimumsizeof about 100bp, andamaximumsizeof about 220bp.The polymorphic site of one particular target locus may occupy any position from the start to the end among the various fragmentsoriginating from that locus.BecausecfDNA fragmentsareshort, the likelihoodof bothprimer sitesbeingpresent the likelihood of a fragment of length L comprising both the forward and reverse primers sites is the ratio of the length of the amplicon to the length of the fragment. Under ideal conditions, assays in which the amplicon is 45, 50, 55, 60, 65, or 70 bp will successfully amplify from72%, 69%, 66%, 63%, 59%, or 56%, respectively, of available template fragmentmolecules. In certain embodiments that relate most preferably to cfDNA from samples of individuals suspected of having cancer, the cfDNA is amplified using primers that yield a maximum amplicon length of 85, 80, 75 or 70 bp, and in certain preferred embodiments 75 bp, and that have a melting temperature between 50 and 65°C, and in certain preferred embodiments, between 54‑60.5°C. The amplicon length is the distance between the 5-prime ends of the forward and reverse priming sites. Amplicon length that is shorter than typically used by those known in the art may result in more efficient measurements of the desired polymorphic loci by only requiring short sequence reads. In an embodiment, a substantial fractionof theampliconsare less than100bp, less than90bp, less than80bp, less than70bp, less than65bp, less than60 bp, less than 55 bp, less than 50 bp, or less than 45 bp.

[0384] In some embodiments, amplification is performed using direct multiplexed PCR, sequential PCR, nested PCR, doubly nested PCR, one-and-a-half sided nested PCR, fully nested PCR, one sided fully nested PCR, one-sided nested PCR, hemi-nested PCR, hemi-nested PCR, triply hemi-nested PCR, semi-nested PCR, one sided semi-nested PCR, 54 EP 4 737 593 A2 5 10 15 20 25 30 35 40 45 50 55 reverse semi-nested PCR method, or one-sided PCR, which are described in US Application No. 13 / 683,604, filed Nov. 21, 2012, U.S. Publication No. 2013 / 0123120, U.S. Application No. 13 / 300,235, filed Nov. 18, 2011, U.S. Publication No 2012 / 0270212, and U.S. Serial No. 61 / 994,791, filed May 16, 2014, which are hereby incorporated by reference in their entirety. If desired, any of these methods can be used for mini-PCR.

[0385] If desired, the extension step of the PCR amplification may be limited from a time standpoint to reduce amplification from fragments longer than 200 nucleotides, 300 nucleotides, 400 nucleotides, 500 nucleotides or 1,000 nucleotides. Thismay result in the enrichment of fragmented or shorter DNA (such as fetal DNA or DNA from cancer cells that have undergone apoptosis or necrosis) and improvement of test performance.

[0386] In some embodiments, multiplex PCR is used. In some embodiments, the method of amplifying target loci in a nucleic acid sample involves (i) contacting thenucleic acid samplewith a library of primers that simultaneously hybridize to least 100; 200; 500; 750; 1,000; 2,000; 5,000; 7,500; 10,000; 20,000; 25,000; 30,000; 40,000; 50,000; 75,000; or 100,000 different target loci to produce a reaction mixture; and (ii) subjecting the reaction mixture to primer extension reaction conditions (such as PCR conditions) to produce amplified products that include target amplicons. In some embodiments, at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or 99.5%of the targeted loci are amplified. In various embodiments, less than 60, 50, 40, 30, 20, 10, 5, 4, 3, 2, 1, 0.5, 0.25, 0.1, or 0.05% of the amplified products are primer dimers. In some embodiments, the primers are in solution (suchas being dissolved in the liquid phase rather than in a solid phase). In some embodiments, the primers are in solution and are not immobilized on a solid support. In some embodiments, the primers are not part of a microarray. In some embodiments, the primers do not include molecular inversion probes (MIPs).

[0387] In some embodiments, two or more (such as 3 or 4) target amplicons (such as amplicons from the miniPCR methoddisclosedherein) are ligated togetherand then the ligatedproductsaresequenced.Combiningmultipleamplicons into a single ligation product increases theefficiency of the subsequent sequencing step. In someembodiments, the target ampliconsare less than150,100, 90,75, or50basepairs in lengthbefore theyare ligated.Theselectiveenrichmentand / or amplificationmay involve tagging each individual molecule with different tags, molecular barcodes, tags for amplification, and / or tags for sequencing. In some embodiments, the amplified products are analyzed by sequencing (such as by high throughput sequencing) or by hybridization to an array, such as a SNP array, the ILLUMINA INFINIUM array, or the AFFYMETRIX gene chip. In some embodiments, nanopore sequencing is used, such as the nanopore sequencing technology developed by ...

Claims

1. A method for amplifying and sequencing DNA in a plasma sample from a subject who has been diagnosed with bladder cancer or colorectal cancer, comprising: (a) sequencing cellular DNA extracted from a tumor biopsy sample of the subject to identify at least 16 cancer mutations; (b) collecting a blood sample from the subject after the subject has been treated with surgery or neoadjuvant therapy; (c) extracting cell-free DNA (cfDNA) from a plasma fraction of the blood sample; (d) performing a targeted multiplex amplification reaction on the extracted cfDNA or DNA derived therefrom with at least 16 different primer pairs to generate amplicons, wherein the primer pairs are each designed to amplify a subject-specific single nucleotide variant locus that encompasses at least one of the cancer mutations identified in the tumor biopsy sample of the subject; and (e) performing high-throughput sequencing on the amplicons to generate sequence reads, wherein the subject-specific single nucleotide variant loci in the amplicons are each sequenced with a depth of read of at least 100,000, and identifying at least two of the cancer mutations in the sequence reads.

2. The method of claim 1, wherein the bladder cancer is stage 1, lb, or 2a bladder cancer, muscle-invasive bladder cancer or locally advanced muscle-invasive bladder cancer.

3. The method of claim 1, wherein the colorectal cancer is stage I, stage II, Stage III or stage IV colorectal cancer.

4. The method of any of claims 1-3, wherein the subject has been treated with surgery, first-line chemotherapy, and / or adjuvant therapy before collecting the blood sample.

5. The method of any of claims 1-4, wherein step (a) comprises sequencing the cellular DNA extracted from the tumor biopsy sample to identify 50-2,000 different cancer mutations.

6. The method of any of claims 1-5, wherein step (a) comprises sequencing the cellular DNA extracted from the tumor biopsy sample to identify 100-1,000 different cancer mutations.

7. The method of any of claims 1-6, wherein step (a) comprises performing whole-exome sequencing on the cellular DNA extracted from the tumor biopsy sample.

8. The method of any of claims 1-7, wherein step (d) comprises performing the targeted multiplex amplification reaction with 50-2,000 different primer pairs to generate amplicons, wherein the primer pairs are each designed to amplify a subject-specific single nucleotide variant locus that encompasses at least one of the cancer mutations identified in the tumor biopsy sample.

9. The method of any of claims 1-8, wherein step (d) comprises performing the targeted multiplex amplification reaction with 100-1,000 different primer pairs to generate amplicons, wherein the primer pairs are each designed to amplify a subject-specific single nucleotide variant locus that encompasses at least one of the cancer mutations identified in the tumor biopsy sample.

10. The method of any of claims 1-9, wherein step (d) comprises identifying at least two of the cancer mutations in the sequence reads with a confidence threshold of at least 90%, preferably wherein the confidence threshold is at least 95%.

11. The method of claim 10, wherein the identifying at least two of the cancer mutations identifies early relapse or metastasis of cancer with a sensitivity of greater than 95% at 0.01% tumor fraction in the sample.

12. The method of claim 10, wherein the identifying at least two of the cancer mutations identifies early relapse or metastasis of cancer in the subject diagnosed with bladder cancer, with a mean lead time of at least 128 days prior to imaging-based clinical relapse or metastasis.

13. The method of claim 10, wherein the identifying at least two of the cancer mutations identifies early relapse or metastasis of cancer in the subject diagnosed with bladder cancer, with a lead time of 50-265 days prior to imaging-based clinical relapse or metastasis.

14. The method claim 10, wherein the identifying at least two of the cancer mutations identifies early relapse or metastasis of cancer in the subject diagnosed with colorectal cancer, with a lead time of at least 9.13 months prior to imaging-based clinical relapse or metastasis.

15. The method of claim 10, wherein the identifying at least two of the cancer mutations identifies early relapse or metastasis of cancer in the subject with colorectal cancer, with a lead time of at least 10.2 months prior to imaging-based clinical relapse or metastasis.

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