Her2 low quantification method

Abstract

Compositions, methods, and systems for detecting human epidermal growth factor receptor 2 (HER2)-low expression levels in a biological sample are disclosed. Also disclosed are compositions, methods, and systems for distinguishing HER2-low expression from HER2-negative or HER2-positive expression. Compositions for improved RNA recovery during sample preparation steps and detection of nucleic acids in the methods herein are also disclosed.

Description

HER2 LOW QUANTIFICATION METHODCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 597,284, filed November 8, 2023, which is incorporated by reference herein in its entirety.FIELD

[0002] The present invention relates generally to the area of detecting human epidermal growth factor receptor 2 (HER2) expression and distinguishing expression levels of HER2, particularly distinguishing HER2-low expression from HER2-negative or HER2- positive expression.BACKGROUND

[0003] HER2 testing is a standard procedure for all new breast cancer diagnoses, as well as in case of tumor progression and / or residual tumor after neoadjuvant treatment. This analysis relies on a combination of immunohistochemistry (IHC) and in situ hybridization (ISH). IHC detects the expression and intensity of HER2 protein on the cell membrane by a three-tier scoring system (from score 0 to score 3+), while ISH detects the presence of gene amplification using HER2 and CEP17 probes. To date, HER2+ breast cancer patients are defined as IHC score 3+ or score 2+ with a positive ISH (ERBB2 gene amplified), and subsequently qualified for anti-HER2 targeted therapy. Only about 15 percent of women diagnosed with breast cancer are HER2+ and eligible for anti-HER2 therapy.

[0004] The standard of HER2 amplification tightly linked to therapy efficacy is changing. Activity of trastuzumab deruxtecan (T-DXd), a novel HER2-targeted antibody drug conjugate (ADC), is seen in patients with HER2 IHC scores of 1+ and 2+ (unamplified). However, due to the design of currently used HER2 assays (designed to distinguish HER2 amplified from unamplified tumors) the scoring accuracy for HER2 IHC in the low range (0 and 1+) has been shown to be poor, with less than 70% interrater agreement on 15 of 80 College of American Pathologists Proficiency Testing survey cases. This is most likely due to the very weak staining of tumors with HER2 protein in the low expression range of normal breast epithelial cells. HER2 has a broad range of expression inzumors from around 1000 molecules to over 1,000,000 molecules / cell in some studies. Very few assays have sufficient dynamic range to span this 3-log difference in expression level. For example, chromogenic IHC generally has less than one log of linear dynamic range. Thus, to accurately assess HER2 protein expression in breast cancer requires either more than one assay with the use of more than one antibody concentration or an assay with 3 log dynamic range.

[0005] The methods, compositions, and devices presented herein achieve rapid, sensitive, qualitative, and quantitative detection of HER2-expression from a single sample, in some embodiments, using a closed and affordable instrument.SUMMARY

[0006] Conventional methods used for identification of low HER2 expression levels is not a trivial task because it relies on multiple methodological and analytical variables. These variables might trouble the testing sensitivity and reproducibility, particularly for the discrimination between HER2-low score 1+ and “HER2-zero” (i.e., IHC score 0), which comprises also the subset of “HER2 ultra-low” (i.e., score 0 with incomplete and faint staining in <10% of tumor cells). The tissue sample handling and processing remains a crucial task, mainly subjected to the “garbage-in-garbage-out” paradigm. Variables such as fixation, antigen retrieval, antibody clones, reaction time, temperature, and substrate concentration can all influence the IHC staining intensity. Additionally, the choice of staining methodology, particularly antigen retrieval, and the availability of different antibody clones with varying specificity can also impact the accuracy and reproducibility of results. Disclosed herein are compositions, methods, and devices to detect and identify HER2 expression, and more particularly, distinguish HER2-low from HER2-positive and HER2-negative, as well as discrimination between HER2-low levels of expression.

[0007] Various embodiments contemplated herein may include, but need not be limited to, one or more of the following:

[0008] A method for detecting, identifying, and / or discriminating between human epidermal growth factor receptor 2 (HER2)-low expression levels in a biological sample from a subject with cancer, the method comprising: incubating the biological sample in a lysis reagent and recovering nucleic acid from said lysis reagent, contacting the nucleic acidwith a set of primers and probes for detecting the presence of ERBB2 biomarker; subjecting the nucleic acid, primers, and probes to amplification conditions; detecting expression levels of ERBB2, expressed as a Ct value or a ACt value, wherein ACt is the Ct value for a reference gene minus the Ct value for the ERBB2 biomarker; comparing the Ct value or the ACt value to a range of Ct values or ACt values, and determining that the subject has HER2- low expression when the Ct value or the ACt value for ERBB2 is within a first range of Ct values or ACt values. HER2-low expression levels as described herein encompasses HER2- ultralow expression levels. Accordingly, the methods disclosed herein can discriminate between HER2-ultralow expression levels and other levels of HER2-low expression. The methods disclosed herein can further comprise distinguishing HER2-low expression from HER2-negative or HER2-positive expression in the biological sample, and determining: that the subject is HER2 -negative when the Ct value or the ACt value for ERBB2 is within a second range of Ct values or ACt values; and / or that the subject is HER2 -positive when the Ct value or the ACt value for ERBB2 is within a third range of Ct values or ACt values. The biological sample can be a formalin fixed paraffin-embedded (FFPE) sample, an in vitro transcribed (IVT) RNA, a tissue sample, cells, a biopsy, or combination thereof. The subject can be diagnosed with a cancer selected from breast cancer, gastric cancer, lung cancer, esophageal carcinoma, bladder cancer, colon cancer, or a combination thereof.

[0009] In specific aspects, HER2-low expression can be determined if (i) the Ct value or the ACt value for ERBB2 expression level corresponds to a peptide quantities between about 2 and about 20 attomole / mm2, (ii) the Ct value or the ACt value for ERBB2 expression level corresponds to a FISH score of less than about 6 signals / cell based on HER2 copy number, (iii) the Ct value for ERBB2 expression level is about 23 to about 30, or (iv) the ACt value for ERBB2 expression level is about -5.0 to about -0.25, preferably about -3.0 to about -0.25, or more preferably about -2.5 to about -0.25. In some examples, HER2-low expression can be determined if the Ct value for ERBB2 is about 23 to about 30 or the ACt value for ERBB2 is less than about -0.25 to about -2.5. In other examples, HER2-low expression can be determined if the Ct value for ERBB2 is about 23 to about 30 and the ACt value for ERBB2 is less than about -0.25 to about -2.5. In further examples, HER2-low expression can be determined if the ACt value for ERBB2 is about -0.25 to about -2.5.

[0010] As described herein, the method includes incubating the biological sample in a lysis reagent and recovering nucleic acid from said lysis reagent. Incubating the biological sample and recovering nucleic acid can comprise adding an FFPE lysis buffer and a proteolytic enzyme to the biological sample to form a mixture, heating the mixture to a temperature ranging from about 50°C to about 100°C to form a lysed mixture, optionally contacting the lysed mixture with an alcohol, placing the lysed mixture in a cartridge, the cartridge comprising a cartridge body having a plurality of chambers in fluidic communication, a reaction vessel having one or more reaction chambers and configured for amplification and detection of the nucleic acid, a fluidic path between the plurality of chambers and the reaction vessel, and a filter in the fluidic path, and capturing nucleic from the lysed mixture onto the filter in the cartridge. In other embodiments, incubating the biological sample and recovering nucleic acid can comprise placing the biological sample in a cartridge, the cartridge comprising a cartridge body having a plurality of chambers in fluidic communication, a reaction vessel having one or more reaction chambers and configured for amplification and detection of the nucleic acid, a fluidic path between the plurality of chambers and the reaction vessel, and a filter in the fluidic path; adding an FFPE lysis buffer and a proteolytic enzyme to the biological sample to form a mixture, optionally heating the mixture to a temperature ranging from about 50°C to about 100°C to form a lysed mixture, optionally contacting the lysed mixture with an alcohol, and capturing nucleic acids from the lysed mixture onto the filter in the cartridge. In some embodiments, the method comprises heating the mixture at a temperature in the range of 50-85°C. The lysis mixture can be incubated for at least 10 minutes, preferably for at least 30 minutes, more preferably for about 30 to about 120 minutes.

[0011] The methods described herein can comprise contacting the lysed mixture with an alcohol selected from ethanol, PEG, or a combination thereof. The alcohol can be in an amount of from 30% to 65% by volume of the mixture, preferably in an amount of from 45% to 55% by volume of the mixture. In some embodiments, the methods include contacting the lysed mixture with an oil, bovine serum albumin (BSA), or a combination thereof. The oil can be selected from mineral oil, paraffin oil, or a combination thereof. In some aspects of the methods herein, the oil or BSA can be in an amount of from 0.01% to 5% by volume, preferably from 0.01% to 3% by volume, more preferably from 0.01% to 1% by volume, of the lysed mixture.

[0012] The FFPE lysis buffer can comprise a salt, an antioxidant or a chelating agent, a buffer, a detergent, an antifoam, and an antimicrobial agent. The FFPE lysis buffer can be present in an amount of from 30% to 65% by volume of the mixture. The buffer can comprise Tris, phosphate buffer, PBS, citrate buffer, TAPS, Bicine, Tricine, TAPSO, HEPES, TES, MOPS, PIPES, Cacodylate, SSC, MES, or combinations thereof. The antioxidant and / or chelating agent can comprise N-acetyl-L-cysteine, ethylenediaminetetraacetic acid (EDTA), diethylene triamine pentaacetic acid (DTP A), ethylenediamine-N,N'-disuccinic acid (EDDS), l,2-bis(o- aminophenoxy)ethane-N,N,N',N'- tetraacetic acid (BAPTA), a phosphonate chelating agent, or combinations thereof. The proteolytic enzyme can comprise a protease (e.g., proteinase K, trypsin, chymotrypsin, papain, or pepsin), a guanidinium compound, formamide, lithium perchlorate, magnesium chloride, urea, thiourea, or combinations thereof.

[0013] The method can further comprise detecting expression levels of ESRI, PGR, and MKi67 genes in the sample. Each of the primers and probes for amplifying and detecting ERBB2 can comprise at least 15 contiguous nucleotides having a sequence that is at least 85% identical to at least 15 contiguous nucleotides of the ERBB2 gene at exons 15, 16, and / or 17, and when present, the primers and probes for amplifying and detecting ESRI can comprise at least 15 contiguous nucleotides having a sequence that is at least 85% identical to at least 15 contiguous nucleotides of the ESRI gene at exons 5 and / or 6, the primers and probes for amplifying and detecting PGR can comprise at least 15 contiguous nucleotides having a sequence that is at least 85% identical to at least 15 contiguous nucleotides of the PGR gene at exons 2, 3, 4, 5, 6, and / or 7, and the primers and probes for amplifying and detecting MKi67 can comprise at least 15 contiguous nucleotides having a sequence that is at least 85% identical to at least 15 contiguous nucleotides of the MKi67 gene at exons 2 and / or 3. The primers and / or probes comprise a detectable label.

[0014] The methods described herein for distinguishing HER2-low from HER2- negative or HER2 -positive expression does not include immunofluorescence & image analysis, AQUA (Automated quantitative analysis) score, or LCMS quantification.

[0015] The method of detecting and distinguishing HER2-low expression from HER2-negative or HER2-positive expression as described herein can be performed at the same facility where the biological sample is collected from a subject. In some aspects, the method can be performed within 48 hours, preferably within 24 hours of obtaining thebiological sample from the subject. In other embodiments, the method can be performed within 3 hours, preferably within 2 hours from step a) incubating the biological sample in a lysis reagent. After detection and identification, the methods can further comprise administering a treatment regimen to the subject based on the determined expression level. For example, the subject can be treated with a HER2 -targeted drug therapy, preferably a HER2-low targeted drug therapy. The HER2-targeted drug therapy can be an antibody-drug conjugate (ADC) such as, trastuzumab deruxtecan (T-DXd), trastuzumab, pertuzumab, trastuzumab emtansine (T-DMI), or combinations thereof.

[0016] Cartridges for detecting, identifying, and distinguishing HER2-low expression from HER2-negative or HER2-positive expression in a biological sample from a subject, according to the methods herein are also disclosed. The cartridge can comprise a cartridge body comprising a plurality of chambers therein, wherein the plurality of chambers includes: a sample chamber having at least a fluid outlet in fluid communication with another chamber of the plurality; and an optional lysis chamber in fluidic communication with the sample chamber, optionally wherein the sample chamber and lysis chamber are the same. The cartridge also comprises a reaction vessel fluidically coupled to the plurality of chambers of the cartridge body and configured for: i) amplification of nucleic acid and ii) detection and identification of a plurality of amplification products via real-time PCR; a filter disposed in a fluidic path between the lysis chamber, if present, or the sample chamber, and the reaction vessel, and a set of primers and probes for detecting ERBB2. The set of primers and probe for detecting ERBB2 can be selected from: (i) a forward primer, a reverse primer, and a probe each comprising at least 15 contiguous nucleotides having a sequence that is at least 85% identical to at least 15 contiguous nucleotides of the ERBB2 gene; or (ii) a forward primer comprising at least 15 contiguous nucleotides having a sequence that is at least 85% identical to at least 15 contiguous nucleotides of the ERBB2 gene at exons 15, 16, and / or 17, and at least one reverse primer comprising at least 15 contiguous nucleotides having a sequence that is at least 85% identical to at least 15 contiguous nucleotides of the ERBB2 gene at exons 15, 16, and / or 17, or (iii) a forward primer comprising at least 15 contiguous nucleotides having a sequence that is at least 85% identical to at least 15 contiguous nucleotides of SEQ ID NO: 3 or 6, and at least one reverse primer comprising at least 15 contiguous nucleotides having a sequence that is at least 85% identical to at least 15 contiguous nucleotides of SEQ IDNO: 4 or 6. The cartridge can further comprise a set of primers and probe for detecting ESRI gene, PGR gene, MKi67 gene, or a combination thereof. The cartridge is configured to carry out non-isothermal amplification, optionally by thermal cycling or temperature oscillation.

[0017] Kits for detecting, identifying, and distinguishing HER2-low expression from HER2-negative or HER2-positive expression in a biological sample from a subject, according to the methods herein are also disclosed. The kit can comprise a set of primers and probe for detecting ERBB2, wherein the set of primers and probe for detecting ERBB2 and optionally, a set of primers and optional probe for detecting ESRI, PGR, or MKi67 mRNA biomarker, or a combination thereof. The kit can further comprise one or more lysis reagents for releasing nucleic acid from the biological sample. The one or more lysis reagents can comprise an FFPE lysis buffer, a proteolytic enzyme, optionally an alcohol, and optionally an oil or bovine serum albumin.

[0018] Systems for detecting, identifying, and distinguishing human epidermal growth factor receptor 2 (HER2)-low expression from HER2-negative or HER2 -positive expression in a biological sample from a subject, according to the methods herein are disclosed. The system comprises a module having a receiving bay for receiving a cartridge as disclosed herein, wherein the module includes one or more mechansims within the receiving bay for manipulating a fluid sample within the cartridge, and an instrument that interfaces with the reaction vessel: and a memory having programmable instructions recorded thereon, that are specially configured to operate the module of a cancer assay protocol to determine expression levels of nucleic acid sequence characteristics of HER2- low. The module and / or system can further comprise a scanner or reader configured to read an identifier on the cartridge; wherein the instructions are configured to determine an applicable protocol based on reading or scanning of the identifier; and wherein the system operates the module of the applicable protocol based on an input from the scanner or reader. The module and / or system further comprises an enclosure; and a plurality of modules that includes said module, wherein modules are substantially identical and configured to concurrently perform assays on cartridges received therein.BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIGS. 1A-1C show an overview of a sample cartridge with a valve assembly configured for performing differing sample processes, including chemical lysing of targets, which is configured for PCR and integrated nucleic acid analysis of the HER2 biomarker panel in accordance with some embodiments of the invention. FIG. 1A shows the sample cartridge body with reaction vessel, FIG. IB shows an exploded view of the sample cartridge, and FIG. 1C shows components of the valve assembly, in accordance with some embodiments.

[0020] FIG. 2 illustrates various valve assemblies A, B, C, D, each suited for one or more types of target lysing, any of which may be used in a respective sample cartridge.

[0021] FIG. 3 is a flow diagram illustrating the ACSO guidelines for classifying HER2 (ERBB2) levels.

[0022] FIG. 4 illustrates HER2-low assay calibration in cell micro array, adapted from Moutafi M. et al., Lab Investigation 2022, 102:1101-1108.

[0023] FIG. 5 illustrates AQUA scoring on patient samples identifies HER2-low cases.

[0024] FIG. 6 shows the linearity of HER2 copy number with Cts and dCts in molecular RT-qPCR test.

[0025] FIG. 7 shows the linearity of HER2 in molecular RT-qPCR test with cell lines. Lysate of 300 cells gives ERBB2 Cts in range of 23-30 for HER2 low expressing cell lines. LCMS MS quantification of HER2 is per mm2. 1 mm2section will have about 67 cells (based on cell diameter of 15-16 micron). 10 mm2section will have approximately 1000 cells (approximate tissue area input).

[0026] FIG. 8 shows refined HER2 scale. The current cutoff for ERBB2 / HER2 positive is -1.0 in molecular RT-qPCR assays. The new cutoff for ERBB2 / HER2 positive based on Metadata is -0.25. A new HER2-low cutoff is -0.25 to -2.5 / -2.0, corresponding to 6.7 to 5 attomole.

[0027] FIG. 9 shows comparison of human epidermal growth factor receptor 2 determined by either RT-qPCR or by immunohistochemistry with FISH assessment of IHC2+ (adapted from Wu et al., Breast Cancer Research and Treatment, 2018, 172:32-338).The figure shows a graph of ERBB2 dCt values by IHC plus FISH where FISH was used to resolve the IHC 2+ equivocals into HER2-positive or HER2-negative status. Fig. 9 further shows cutoffs in Metadata, based on FIG. 8, with ERBB2 ACt cutoff of -0.25. The line with the cut off of -2.5 classifies samples from IHC 1+ and IHC2+ in the HER2-low category.There is overall concordance with the cut offs for HER2-low.

[0028] FIG. 10 illustrates clinical overview of HER2-low status. FIG. 10 (top) illustrates DESTINY04 trial with Trastuzumab Deruxtecan with HER2 low patients. Modi et al., NEJM, 2022. The Ab:Drug ratio is 1:8 delivering a high payload. FIG. 10 (bottom) illustrates large retrospective study showing minimal prognostic differences between HER2- low and HER2 negative. Peiffer et al., JAMA Oncology, 2023.DETAILED DESCRIPTION

[0029] The present disclosure describes methods, compositions, devices, and systems that facilitate the rapid detection and distinction of human epidermal growth factor receptor 2 (HER2)-low expression from HER2-negative and HER2-positive expression in a biological sample from a subject. The subject may or may not be diagnosed with cancer, prior to performing the disclosed methods. The methods, compositions, devices, and systems are readily automated and can be employed in point-of-care devices, enabling detection and discrimination between HER2 expression levels in the subject, so that the appropriate treatment can be administered in a timely manner. In some instances, the mRNA assay may take about 75 minutes or less to amplify a portion of the ERBB2 mRNA and reference mRNA within the sample and to generate the test results. The approach relies on nucleic acid amplification to measure HER2 expression, which entails incubating the biological sample in a lysis reagent and recovering nucleic acid from said lysis reagent, contacting the nucleic acid with a set of primers and probes that target the ERBB2 gene biomarker, subjecting the nucleic acid, primers, and probes to amplification conditions, measuring the expression levels of ERBB2, expressed as a Ct value or a ACt value, wherein ACt is the Ct value for a reference gene minus the Ct value for the ERBB2 biomarker, comparing the Ct value and / or the ACt value to a range of Ct values and / or ACt values, and determining if the subject is HER2 -positive, HER2-negative, or HER2-low, based on the ACt and / or ACt values.Definitions

[0030] Terms used in the claims and specification are defined as set forth below unless otherwise specified.

[0031] The term “nucleic acid” refers to a nucleotide polymer, and unless otherwise limited, includes analogs of natural nucleotides that can function in a similar manner (e.g., hybridize) to naturally occurring nucleotides.

[0032] The term nucleic acid includes any form of DNA or RNA, including, for example, genomic DNA; complementary DNA (cDNA), which is a DNA representation of mRNA, usually obtained by reverse transcription of messenger RNA (mRNA) or by amplification; DNA molecules produced synthetically or by amplification; mRNA; and non-coding RNA.

[0033] The term nucleic acid encompasses double- or triple-stranded nucleic acid complexes, as well as single- stranded molecules. In double- or triple- stranded nucleic acid complexes, the nucleic acid strands need not be coextensive (i.e, a double- stranded nucleic acid need not be double-stranded along the entire length of both strands).

[0034] The term nucleic acid also encompasses any modifications thereof, such as by methylation and / or by capping. Nucleic acid modifications can include addition of chemical groups that incorporate additional charge, polarizability, hydrogen bonding, electrostatic interaction, and functionality to the individual nucleic acid bases or to the nucleic acid as a whole. Such modifications may include base modifications such as 2’- position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at cytosine exocyclic amines, substitutions of 5-bromo-uracil, sugar-phosphate backbone modifications, unusual base pairing combinations such as the isobases isocytidine and isoguanidine, and the like.

[0035] More particularly, in some embodiments, nucleic acids, can include polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), and any other type of nucleic acid that is an N- or C-glycoside of a purine or pyrimidine base, as well as other polymers containing nonnucleotidic backbones, for example, polyamide (e.g., peptide nucleic acids (PNAs)) and polymorpholino polymers (see, e.g., Summerton and Weller (1997) “Morpholino Antisense Oligomers: Design, Preparation, and Properties,” Antisense & Nucleic Acid Drug Dev. 7: 1817-195; Okamoto etal. (20020) “Development of electrochemically gene- analyzing method using DNA- modified electrodes,” Nucleic Acids Res. Supplement No. 2:171-172), and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA. The term nucleic acid also encompasses locked nucleic acids (LNAs), which are described in U.S. Patent Nos. 6,794,499, 6,670,461, 6,262,490, and 6,770,748, which are incorporated herein by reference in their entirety for their disclosure of LNAs.

[0036] The nucleic acid(s) can be derived from a completely chemical synthesis process, such as a solid phase-mediated chemical synthesis, from a biological source, such as through isolation from any species that produces nucleic acid, or from processes that involve the manipulation of nucleic acids by molecular biology tools, such as DNA replication, PCR amplification, reverse transcription, or from a combination of those processes.

[0037] As used herein, the term “gene” encompasses coding sequences, introns, and any associated control sequences that participate in the expression of the coding sequences.

[0038] As used herein, the term “complementary” refers to the capacity for precise pairing between two nucleotides; i.e., if a nucleotide at a given position of a nucleic acid is capable of hydrogen bonding with a nucleotide of another nucleic acid to form a canonical base pair, then the two nucleic acids are considered to be complementary to one another at that position. Complementarity between two single-stranded nucleic acid molecules may be “partial,” in which only some of the nucleotides bind, or it may be complete when total complementarity exists between the single-stranded molecules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.

[0039] The term “corresponds to,” as used herein, is defined as related to or associated with, e.g., be similar, analogous, or equivalent to. For example, a mold may correspond to an anatomical structure if the cavity in the mold is shaped to resemble the anatomical structure. In the context of, for example, a Ct value or a ACt value for ERBB2 expression level corresponds to a peptide quantity, is intended to identify the ERBB2 expression level (expressed as Ct value or ACt value) that is correspondingly equivalentrelative to a peptide quantity at a defined HER2 score (e.g., HER2-negative, HER2- ultralow, HER2-low, or HER2-positive).

[0040] “Selective hybridization” or “selective annealing” refers to the binding of a nucleic acid to a target nucleic acid in the absence of substantial binding to other nucleic acids present in the hybridization mixture under defined stringency conditions. Those of skill in the art recognize that relaxing the stringency of the hybridization conditions allows sequence mismatches to be tolerated.

[0041] In some embodiments, hybridizations are carried out under stringent hybridization conditions. The phrase “stringent hybridization conditions” generally refers to a temperature in a range from about 5 °C to about 20°C or 25 °C below than the melting temperature (Tm) for a specific sequence at a defined ionic strength and pH. As used herein, the Tmis the temperature at which a population of double-stranded nucleic acid molecules becomes half-dissociated into single strands. Methods for calculating the Tmof nucleic acids are well known in the art (see, e.g., Berger and Kimmel (1987) METHODS IN ENZYMOLOGY, VOL.152: GUIDE TO MOLECULAR CLONING TECHNIQUES, San Diego: Academic Press, Inc. and Sambrook et al. (1989) MOLECULAR CLONING: A LABORATORY MANUAL, 2ND ED., VOLS. 1-3, Cold Spring Harbor Laboratory), both incorporated herein by reference for their descriptions of stringent hybridization conditions). As indicated by standard references, a simple estimate of the Tmvalue may be calculated by the equation: Tm=81.5+0.41(% G+C), when a nucleic acid is in aqueous solution at 1 M NaCl (see, e.g., Anderson and Young, Quantitative Filter Hybridization in NUCLEIC ACID HYBRIDIZATION (1985)). The melting temperature of a hybrid (and thus the conditions for stringent hybridization) is affected by various factors such as the length and nature (DNA, RNA, base composition) of the primer or probe and nature of the target nucleic acid (DNA, RNA, base composition, present in solution or immobilized, and the like), as well as the concentration of salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol). The effects of these factors are well known and are discussed in standard references in the art. Illustrative stringent conditions suitable for achieving specific hybridization of most sequences are: a temperature of at least about 60°C and a salt concentration of about 0.2 molar at pH7. Tmcalculation for oligonuclotide sequences based on nearest-neighbors thermodynamics can carried out as described in “A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics” John SantaLucia, Jr., PNAS February 17, 1998 vol. 95 no. 4 1460-1465 (which is incorporated by reference herein for this description).

[0042] The term “oligonucleotide” is used to refer to a nucleic acid that is relatively short, generally shorter than 200 nucleotides, more particularly, shorter than 100 nucleotides, most particularly, shorter than 50 nucleotides. Typically, oligonucleotides are single-stranded DNA molecules.

[0043] The term “primer” refers to an oligonucleotide that is capable of hybridizing (also termed “annealing”) with a nucleic acid and serving as an initiation site for nucleotide (RNA or DNA) polymerization under appropriate conditions (i.e., in the presence of four different nucleoside triphosphates and an agent for polymerization, such as DNA or RNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature. The appropriate length of a primer depends on the intended use of the primer, but primers are typically at least 7 nucleotides long and, in some embodiments, range from 10 to 30 nucleotides, or, in some embodiments, from 10 to 60 nucleotides, in length. In some embodiments, primers can be, e.g., 15 to 50 nucleotides long. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. A primer need not reflect the exact sequence of the template but must be sufficiently complementary to hybridize with a template.

[0044] A primer is said to “anneal to” or “hybridize to” another nucleic acid if the primer, or a portion thereof, hybridizes to a nucleotide sequence within the nucleic acid. The statement that a primer hybridizes to a particular nucleotide sequence is not intended to imply that the primer hybridizes either completely or exclusively to that nucleotide sequence. For example, in some embodiments, amplification primers used herein are said to “anneal to” or be “specific for” a nucleotide sequence.” This description encompasses primers that anneal wholly to the nucleotide sequence, as well as primers that anneal partially to the nucleotide sequence.

[0045] The term “primer pair” refers to a set of primers including a 5’ “upstream primer” or “forward primer” that hybridizes with the complement of the 5’ end of the DNA sequence to be amplified and a 3’ “downstream primer” or “reverse primer” that hybridizes with the 3’ end of the sequence to be amplified. As will be recognized by those of skill inthe art, the terms “upstream” and “downstream” or “forward” and “reverse” are not intended to be limiting, but rather provide illustrative orientations in some embodiments.

[0046] A “probe” is a nucleic acid capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, generally through complementary base pairing, usually through hydrogen bond formation, thus forming a duplex structure. The probe can be labeled with a detectable moiety to permit facile detection of the probe, particularly once the probe has hybridized to its complementary target. Alternatively, however, the probe may be unlabeled, but may be detectable by specific binding with a ligand that is labeled, either directly or indirectly. Probes can vary significantly in size.

[0047] As used herein with reference to a portion of a primer or a nucleotide sequence within the primer, the term “specific for” a nucleic acid, refers to a primer or nucleotide sequence that can specifically anneal to the target nucleic acid under suitable annealing conditions.

[0048] The term “target” is used herein with reference to “target nucleic acids,” as well as “target organisms.” The former refers to nucleic acids to be detected, and the latter refers to organisms to be detected. The term, “target nucleic acid” is generally used herein to refer to a segment of nucleic acid that is defined by a primer pair and that gives rise to an amplicon produced in an amplification reaction; the term “amplification target” is also used herein to refer to this type of target nucleic acid. Primers and probes are also said to “target” nucleic acid sequences, and so these sequences can also be understood as “target nucleic acids.” Additionally, primers and probes are said to “target” or “be specific for” genes. In this usage, the primers and probes can be used to detect the presence of a particular gene by specifically hybridizing to a portion of the gene that indicates its presence. The meaning of “target” and “target nucleic acids” will be clear to one of skill in the art from the context in which the term is employed. In some embodiments, multiple target nucleic acids can be detected to detect a single target organism. In some embodiments, a single target nucleic acid can be detected to detect a single target organism. In some embodiments, an assay can employ multiple target nucleic acids for one or more target organisms and single target nucleic acids for one or more different target organisms.

[0049] Amplification according to the present teachings encompasses any means by which at least a part of at least one target nucleic acid is reproduced, typically in a templatedependent manner, including without limitation, a broad range of techniques for amplifying nucleic acid sequences, either linearly or exponentially. Illustrative means for performing an amplifying step include PCR, nucleic acid strand-based amplification (NASBA), two- step multiplexed amplifications, rolling circle amplification (RCA), and the like, including multiplex versions and combinations thereof, for example but not limited to, OLA / PCR, PCR / OLA, LDR / PCR, PCR / PCR / LDR, PCR / LDR, LCR / PCR, PCR / LCR (also known as combined chain reaction-CCR), helicase-dependent amplification (HDA), and the like. Descriptions of such techniques can be found in, among other sources, Ausubel et al.; PCR Primer: A Laboratory Manual, Diffenbach, Ed., Cold Spring Harbor Press (1995); The Electronic Protocol Book, Chang Bioscience (2002); Msuih et al., J. Clin. Micro. 34:501-07 (1996); The Nucleic Acid Protocols Handbook, R. Rapley, ed., Humana Press, Totowa, NJ. (2002); Abramson et al., Curr Opin Biotechnol. 1993 Feb.;4(l):41-7, U.S. Pat. No. 6,027,998; U.S. Pat. No. 6,605,451, Barany et al., PCT Publication No. WO 97 / 31256; Wenz et al., PCT Publication No. WO 01 / 92579; Day et al., Genomics, 29(1): 152-162 (1995), Ehrlich et ah, Science 252:1643-50 (1991); Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press (1990); Favis et al., Nature Biotechnology 18:561-64 (2000); and Rabenau et al., Infection 28:97-102 (2000); Belgrader, Barany, and Lubin, Development of a Multiplex Ligation Detection Reaction DNA Typing Assay, Sixth International Symposium on Human Identification, 1995 (available on the world wide web at: promega.com / geneticidproc / ussymp6proc / blegrad.html- ); LCR Kit Instruction Manual, Cat. #200520, Rev. #050002, Stratagene, 2002; Barany, Proc. Natl. Acad. Sci. USA 88:188- 93 (1991); Bi and Sambrook, Nucl. Acids Res. 25:2924-2951 (1997); Zirvi et al., Nucl. Acid Res. 27:e40i-viii (1999); Dean et al., Proc Natl Acad Sci USA 99:5261-66 (2002); Barany and Gelfand, Gene 109:1-11 (1991); Walker et al., Nucl. Acid Res. 20:1691-96 (1992); Polstra et al., BMC Inf. Dis. 2: 18- (2002); Lage et al., Genome Res. 2003 Feb.;13(2):294-307, and Landegren et al., Science 241:1077-80 (1988), Demidov, V., Expert Rev Mol Diagn. 2002 Nov.;2(6):542-8., Cook et al., J Microbiol Methods. 2003 May;53(2): 165-74, Schweitzer et al., Curr Opin Biotechnol. 2001 Feb.;12(l):21-7, U.S. Pat. No. 5,830,711, U.S. Pat. No. 6,027,889, U.S. Pat. No. 5,686,243, PCT PublicationNo. WO0056927A3, and PCT Publication No. WO9803673A1.

[0050] In some embodiments, amplification comprises at least one cycle of the sequential procedures of: annealing at least one primer with complementary or substantially complementary sequences in at least one target nucleic acid; synthesizing at least one strand of nucleotides in a template-dependent manner using a polymerase; and denaturing the newly-formed nucleic acid duplex to separate the strands. The cycle may or may not be repeated. Amplification can comprise thermocycling or can be performed isothermally.

[0051] As used herein, the term “amplification conditions” refers to conditions that promote amplification of a target nucleic acid in the presence of suitable primers.

[0052] As used herein, “in solution” means not immobilized on a substrate of any kind, for example, a bead or a surface in a cassette, such as a chamber wall.

[0053] A “multiplex amplification reaction” is one in which two or more nucleic acids distinguishable by sequence are amplified simultaneously.

[0054] The term “qPCR” is used herein to refer to quantitative real-time polymerase chain reaction (PCR), which is also known as “real-time PCR” or “kinetic polymerase chain reaction;” all terms refer to PCR with real-time signal detection.

[0055] The term “melt curve analysis” refers to the use of the dissociation characteristics of a segment of double-stranded nucleic during heating. Originally, strand dissociation was observed using UV absorbance measurements, but techniques based on fluorescence measurements are now the most common approach. The temperaturedependent dissociation between two DNA-strands can be measured in a “melt assay,” for example, using a DNA-intercalating fluorophore, such as SYBR green or EvaGreen, or fluorophore-labelled DNA probes. In the case of SYBR green (which fluoresces 1000-fold more intensely while intercalated in the minor groove of two strands of DNA), the dissociation of the DNA during heating is measurable by the large reduction in fluorescence that results. Alternatively, juxtapositioned probes (one featuring a fluorophore and the other, a suitable quencher) can be used to determine the complementarity of the probe to the target nucleic acid sequence.

[0056] A “reagent” refers broadly to any agent used in a reaction, other than the analyte (e.g., nucleic acid being analyzed). Illustrative reagents for a nucleic acid amplification reaction include, but are not limited to, buffer, metal ions, polymerase, reversetranscriptase, primers, template nucleic acid, nucleotides, labels, dyes, nucleases, dNTPs, and the like. Reagents for enzyme reactions include, for example, substrates, cofactors, buffer, metal ions, inhibitors, and activators.

[0057] The term “label,” as used herein, refers to any atom or molecule that can be used to provide a detectable and / or quantifiable signal. In particular, the label can be attached, directly or indirectly, to a nucleic acid or protein. Suitable labels that can be attached to probes include, but are not limited to, radioisotopes, fluorophores, chromophores, mass labels, electron dense particles, magnetic particles, spin labels, molecules that emit chemiluminescence, electrochemically active molecules, enzymes, cofactors, and enzyme substrates.

[0058] The term “dye,” as used herein, generally refers to any organic or inorganic molecule that absorbs electromagnetic radiation and produces a detectable signal (e.g., a fluorescent signal).

[0059] The term “quencher,” as used herein generally refers to any organic or inorganic molecule that reduces the level of a detectable signal.

[0060] As used herein, the term “detecting” refers to “determining the presence of’ an item, such as a nucleic acid sequence, e.g., one that is indicative of the presence of a HER2 expression. Detection can include the determination of the presence of a HER2 expression, without definitive identification of the level of expression (such as HER2-low or HER2-positive).

[0061] The term “identifying,” as used herein, refers to the action of recognizing a sample as having a certain expression level of HER2, e.g. HER2-negative, HER2-ultralow, HER2-low, or HER2-positive.

[0062] As used herein, the term “treatment regimen” refers to any medical intervention intended to mitigate the symptoms and / or the pathology of a disorder. The treatment regimen can include one or more actions (e.g., bed rest, increasing fluid intake), non-prescription or prescription medications, supplements, foods, drinks, or the use of medical devices (e.g., a respirator).

[0063] As used herein, “Clinical Laboratory Improvement Amendments (CLIA)” refers to The Clinical Laboratory Improvement Amendments of 1988 (CLIA) regulations ineffect as of the original filing date of the present application. The CLIA regulations include federal standards applicable to all U.S. facilities or sites that test human specimens for health assessment or to diagnose, prevent, or treat disease. A “CLIA-compliant” test is one that complies with these regulations. “CLIA-waived” tests include tests that does not comply with all of these regulations. For example, CLIA-waived tests include test systems cleared by the U.S. Food and Drug Administration for home use and those tests approved for waiver under the CLIA criteria.

[0064] An “endogenous control,” as used herein refers to a moiety that is naturally present in the sample to be used for detection. In some embodiments, an endogenous control is a “sample adequacy control” (SAC), which may be used to determine whether there was sufficient sample used in the assay, or whether the sample comprised sufficient biological material, such as cells. In some embodiments, an endogenous control is an RNA (such as an mRNA, tRNA, ribosomal RNA, etc.), such as a human RNA for a human sample. Nonlimiting exemplary endogenous controls include ABL mRNA, GUSB mRNA, GAPDH mRNA, TUBE mRNA, and UP Kia mRNA. In some embodiments, an endogenous control, such as an SAC, is selected that can be detected in the same manner as the target nucleic acid (e.g., RNA) is detected and, in some embodiments, simultaneously with the target nucleic acid (e.g., RNA).

[0065] An “exogenous control,” as used herein, refers to a moiety that is added to a sample or to an assay, such as a “sample processing control” (SPC). In some embodiments, an exogenous control is included with the assay reagents. An exogenous control is typically selected that is not expected to be present in the sample to be used for detection, or is present at very low levels in the sample such that the amount of the moiety naturally present in the sample is either undetectable or is detectable at a much lower level than the amount added to the sample as an exogenous control. I n some embodiments, an exogenous control comprises a nucleotide sequence that is not expected to be present in the sample type used for detection of the target nucleic acid (e.g., RNA). In some embodiments, an exogenous control comprises a nucleotide sequence that is not known to be present in the species from whom the sample is taken. In some embodiments, an exogenous control comprises a nucleotide sequence from a different species than the subject from whom the sample was taken. In some embodiments, an exogenous control comprises a nucleotide sequence that is not known to be present in any species. In some embodiments, an exogenous control isselected that can be detected in the same manner as the target nucleic acid (e.g., RNA) is detected and, in some embodiments, simultaneously with the target nucleic acid (e.g., RNA). In some embodiments, the exogenous control is an RNA. In some such embodiments, the exogenous control is an Armored RNA®, which comprises RNA packaged in a bacteriophage protective coat. See, e.g., WalkerPeach et al, Clin. Chem. 45: 12: 2079-2085 (1999).

[0066] The term “cancer” is intended to include any member of a class of diseases characterized by the uncontrolled growth of aberrant cells. The term includes all known cancers and neoplastic conditions, whether characterized as malignant, benign, soft tissue, or solid, and cancers of all stages and grades including pre- and post-metastatic cancers. Examples of different types of cancer include, but are not limited to, breast cancer; lung cancer (e.g., non-small cell lung cancer); digestive and gastrointestinal cancers such as colorectal cancer, gastrointestinal stromal tumors, gastrointestinal carcinoid tumors, colon cancer, rectal cancer, anal cancer, bile duct cancer, small intestine cancer, and stomach(gastric) cancer; esophageal cancer; gallbladder cancer; liver cancer; pancreatic cancer; appendix cancer; ovarian cancer; renal cancer (e.g., renal cell carcinoma); cancer of the central nervous system; skin cancer; lymphomas; choriocarcinomas; head and neck cancers; osteogenic sarcomas; and blood cancers. As used herein, a “tumor” comprises one or more cancerous cells. In one embodiment, the breast tumor is derived from a subject with an invasive or in situ form of ductal carcinoma or lobular carcinoma. In another embodiment, the breast tumor is derived from a subject with recurrent or metastatic breast cancer.

[0067] In the present disclosure, the phrase “level of expression” refers to expression of either mRNA or protein whose abundance is measured quantitatively.

[0068] The term “HER2-low” as used in clinical practice relies on the standard IHC and ISH approach; thus, tumors with low level of HER2 expression (defined as a HER2 IHC score of 1+ or 2+) and no detectable ERBB2 amplification fall into this category. As a standard or routinely, the HER- 2 status is, accordingly, performed by immunohistochemistry with one of two FDA-approved commercial kits available; namely the Dako Herceptest™ and the Ventana Pathway™, respectively. These are semi-quantitative assays which stratify expression levels into 0 (<20,000 receptors per cell, no expression visible by IHC staining), 1+ (-100,000 receptors per cell, partial membrane staining, <10% of cellsoverexpressing HER2), 2+ (-500,000 receptors per cell, light to moderate complete membrane staining, >10% of cells overexpressing HER2), and 3+ (-2,000,000 receptors per cell, strong complete membrane staining, >10% of cells overexpressing HER2).

[0069] A “biomarker” in the context of the present disclosure refers to a biological compound, such as a polynucleotide or polypeptide which is differentially expressed in a sample taken from patients having cancer as compared to a comparable sample taken from control subjects (e.g., a person with a negative diagnosis or normal or healthy subject) or differentially expressed in a sample from a patient having cancer as compared to a sample from a patient who does not have cancer. The biomarker can be a nucleic acid, a fragment of a nucleic acid, a polynucleotide, or an oligonucleotide that can be detected and / or quantified.

[0070] The term “biological sample” is intended any sampling of cells, tissues, or bodily fluids in which expression of an intrinsic gene can be detected. Examples of such biological samples include, but are not limited to, biopsies and smears. Bodily fluids useful in the present invention include blood, lymph, urine, saliva, nipple aspirates, fine needle aspirates (ENA), gynecological fluids, or any other bodily secretion or derivative thereof. Blood can include whole blood, plasma, serum, or any derivative of blood. In some embodiments, the biological sample includes breast cells, particularly breast tissue from a biopsy, such as a breast tumor tissue sample. Biological samples may be obtained from a subject by a variety of techniques including, for example, by scraping or swabbing an area, by using a needle to aspirate cells or bodily fluids, or by removing a tissue sample (i.e., biopsy). Methods for collecting various biological samples are well known in the art. In some embodiments, a tissue sample is obtained by, for example, fine needle aspiration biopsy, core needle biopsy, or excisional biopsy. Fixative and staining solutions may be applied to the cells or tissues for preserving the specimen and for facilitating examination. Biological samples, particularly tissue samples, may be transferred to a glass slide for viewing under magnification. In one embodiment, the biological sample is a formalin-fixed, paraffin-embedded breast tissue sample, particularly a primary breast tumor sample.

[0071] In particular embodiments, the “sample” as used herein includes any biological specimen obtained from a patient. Samples include, without limitation, whole blood, plasma, serum, red blood cells, white blood cells (e.g., peripheral blood mononuclearcells), ductal lavage fluid, nipple aspirate, lymph (e.g., disseminated tumor cells of the lymph node), bone marrow aspirate, saliva, urine, stool (i.e., feces), sputum, bronchial lavage fluid, tears, fine needle aspirate (e.g., harvested by random periareolar fine needle aspiration), any other bodily fluid, a tissue sample (e.g., tumor tissue) such as a biopsy of a tumor (e.g., needle biopsy) or a lymph node (e.g., sentinel lymph node biopsy), and cellular extracts thereof. In some embodiments, the sample is whole blood or a fractional component thereof such as plasma, serum, or a cell pellet. In preferred embodiments, the sample is obtained by isolating circulating cells of a solid tumor from whole blood or a cellular fraction thereof using any technique known in the art. In other embodiments, the sample is a formalin fixed paraffin embedded (FFPE) tumor tissue sample, e.g., from a solid tumor of the breast.HER2 Expression

[0072] The human epidermal growth factor receptor- 2 (HER2) is a receptor tyrosine kinase, belonging to the ErbB family (EGFR / HER1, HER2, HER3, and HER4), involved in signal transduction pathways that mediate key cellular processes including cell proliferation, differentiation, and survival. Overexpression of the HER2 protein or amplification of the HER2 gene (also known as ERBB2) is an important predictive biomarker for identifying patients with breast and other cancers, who may benefit from HER2-targeted therapy. The mechanisms of HER2 activation include not only the overexpression of the HER2 protein and amplification of the HER2 but also somatic mutations in HER2, leading to activation of the HER2 gene.

[0073] Historically, ERBB2 gene amplification and protein overexpression in tumor biopsy material, as measured by in situ hybridization (ISH) or immunohistochemistry (IHC), has been used to select patients most likely to benefit from HER2-based therapeutic strategies. Although for therapeutic decisions HER2 status is commonly dichotomized in positive and negative according to immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH), HER2 expression is a continuum. The levels of ERBB2 mRNA progressively increase across samples classified as IHC score 0, IHC score 1+, IHC score 2+ non amplified by FISH, IHC score 2+ amplified by FISH, and IHC score 3+.

[0074] HER2 protein expression spans about 3 orders of magnitude. Previous studies have estimated a functional range for conventional chromogenic stain-based assaysbetween 1 and 1.5 orders of magnitude. The functional range for fluorescence-based assays extends to 2+ orders of magnitude and LC-MS / MS assays to 3.5 log. Although the current assay combination of IHC and ISH can separate amplified from unamplified cases (with sensitivity as high as 95%), it is insufficient to identify low levels of HER2 expression. This is because the assay is designed to distinguish gene amplified levels of HER2 from gene unamplified levels. To span the full dynamic range of cancer HER2 expression more than one assay is needed. Here, the inventors have constructed and validated a new assay for determination of HER2 in the critical range where the HER2 targeting antibody drug conjugate (ADC) drugs will be effective, but also the range that lacks reproducibility with current assays. More specifically, the inventors have developed an assay that is a standalone or complementary to the conventional assay with sensitivity and linearity in the range of HER2 that is largely below the conventional assay usage threshold. This assay can be incorporated in the clinical setting for these low HER2 unamplified cases allowing more accurate patient stratification for potential benefit from ADC therapy.

[0075] Methods for detecting and discriminating between human epidermal growth factor receptor 2 (HER2)-low expression levels in a biological sample of a subject are disclosed herein. Methods for detecting and distinguishing HER2-low expression from HER2-negative or HER2-positive expression in a biological sample of a subject are also disclosed herein. For the purpose of the disclosure, the term subject, or subject sample, refers to an individual regardless of health and / or disease status. A subject can be a subject, a study participant, a control subject, a screening subject, or any other class of individual from whom a sample is obtained and assessed in the context of the disclosure. Accordingly, a subject can be diagnosed with cancer, such as breast cancer, can present with one or more symptoms of cancer, or a predisposing factor, such as a family (genetic) or medical history (medical) factor, for cancer, can be undergoing treatment or therapy for cancer, or the like. Alternatively, a subject can be healthy with respect to any of the aforementioned factors or criteria. It will be appreciated that the term “healthy” as used herein, is relative to breast cancer status, as the term “healthy” cannot be defined to correspond to any absolute evaluation or status. Thus, an individual defined as healthy with reference to any specified disease or disease criterion, can in fact be diagnosed with any other one or more diseases, or exhibit any other one or more disease criterion, including one or more cancers other than breast cancer. However, the healthy controls are preferably free of any cancer. In variousembodiments, the subject can include non-human animals, e.g., canines, felines, equines, primates, and other non-human mammals, as well as humans.

[0076] The biological sample can be any sample as described herein. For example, the biological sample can be a formalin fixed paraffin-embedded (FFPE) sample, an in vitro transcribed (IVT) RNA, a tissue sample, a blood sample, cells, a biopsy sample (e.g., fine needle aspirate), or combination thereof. In some examples, the biological sample can be a fixed paraffin-embedded tissue (e.g., FFPET) sample. While histological samples are typically fixed with an aldehyde fixative such as formalin (formaldehyde) and glutaraldehyde, it is believed the methods described herein additionally work with tissues fixed using other fixation techniques such as alcohol immersion, and the like. Illustrative samples include, but are not limited to, FFPET samples from human tissues, laboratory animal tissues, companion animal tissues, or livestock animal tissues. Thus, for example, the samples include tissue samples from humans including, but not limited to samples from healthy humans (e.g., healthy human tissue samples), samples from a diseased subject and / or diseased tissue, samples used for diagnostic and / or prognostic assays and the like. Suitable samples also include samples from non-human animals. Biological samples such as FFPET samples from, for example, a non-human primate, such as a chimpanzee, gorilla, orangutan, gibbon, monkey, macaque, baboon, mangabey, colobus, langur, marmoset, lemur, a mouse, rat, rabbit, guinea pig, hamster, cat dog, ferret, fish, cow, pig, sheep, goat, horse, donkey, chicken, goose, duck, turkey, amphibian, or reptile can be used in the methods described herein. In some embodiments, the sample to be tested is obtained from an individual who has one or more symptoms of cancer.

[0077] In addition, biological samples such as FFPET samples of any age can be used with the methods described herein including, but not limited to, samples that are fresh, less than one week old, less than two weeks old, less than one month old, less than two months old, less than three months old, less than six months old, less than 9 months old, less than one year old, at least one year old, at least two years old, at least three years old, at least four years old, at least five years old, at least six years old, at least seven years old, at least eight years old, at least nine years old, at least ten years old, at least fifteen years old, at least twenty years old, or older.

[0078] In some embodiments, the methods described herein are performed on one or more sections taken from a fixed, embedded tissue sample (e.g., an FFPET sample). The sections can be of any desired thickness. Thus, in some embodiments, both thin sections or thick sections are contemplated, including, but not limited to, sections that are less than 1 micron thick, about 1 micron thick, about 2 microns thick, about 3 microns thick, about 4 microns thick, about 5 microns thick, about 6 microns thick, about 7 microns thick, about 8 microns thick, about 9 microns thick, about 10 microns thick, about 15 microns thick, or about 20 microns thick, depending upon the desired application. In certain applications, the sections can be, for example, up to about 1 micron thick, up to about 2 microns thick, up to about 3 microns thick, up to about 4 microns thick, up to about 5 microns thick, up to about 6 microns thick, up to about 7 microns thick, up to about 8 microns thick, up to about 9 microns thick, up to about 10 microns thick, up to about 15 microns thick, up to about 20 microns thick, or up to about 25 or 30 microns thick. In some embodiments, the sections can be defined by a range of sizes, including, but not limited to, between about 1 and about 5 microns thick, between about 1 and about 20 microns thick, between about 1 and about 10 microns thick, or between about 5 and about 10 microns thick.

[0079] In many cases, the fixed embedded tissue samples (e.g., FFPET samples) comprise an area of diseased tissue, for example a tumor or other cancerous tissue. While such FFPET samples find utility in the methods described herein, FFPET samples that do not comprise an area of diseased tissue, for example FFPET samples from normal, untreated, placebo-treated, or healthy tissues, also can be used in the methods described herein. In some embodiments of the methods described herein, a desired diseased area or tissue, or an area containing a particular region, feature or structure within a particular tissue, is identified in a FFPET sample, or a section or sections thereof, prior to isolation of nucleic acids as described herein, in order to increase the percentage of nucleic acids obtained from the desired region. Such regions or areas can be identified using any method known to those of skill in the art, including, but not limited to, visual identification, staining, for example hematoxylin and eosin staining, immunohistochemical labeling, and the like. In any event, in some embodiments, the desired area of the tissue sample, or sections thereof, can be dissected, either by macrodissection or microdissection, to obtain the starting material for the isolation of a nucleic acid sample using the methods described herein.

[0080] In certain illustrative, but non-limiting embodiments, the sample comprises a diseased area or tissue comprising cells from a cancer. In some embodiments the cancer comprises a cancer selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), Adrenocortical carcinoma, AIDS -related cancers (e.g., kaposi sarcoma, lymphoma), anal cancer, appendix cancer, astrocytomas, atypical teratoid / rhabdoid tumor, bile duct cancer, extrahepatic cancer, bladder cancer, bone cancer (e.g., Ewing sarcoma, osteosarcoma, malignant fibrous histiocytoma), brain stem glioma, brain tumors (e.g., astrocytomas, brain and spinal cord tumors, brain stem glioma, central nervous system atypical teratoid / rhabdoid tumor, central nervous system embryonal tumors, central nervous system germ cell tumors, craniopharyngioma, ependymoma, breast cancer, bronchial tumors, burkitt lymphoma, carcinoid tumors (e.g., childhood, gastrointestinal), cardiac tumors, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative disorders, colon cancer, colorectal cancer, craniopharyngioma, cutaneous t-cell lymphoma, duct cancers e.g. (bile, extrahepatic), ductal carcinoma in situ (DCIS), embryonal tumors, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer (e.g., intraocular melanoma, retinoblastoma), fibrous histiocytoma of bone, malignant, and osteosarcoma, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumors (GIST), germ cell tumors (e.g., ovarian cancer, testicular cancer, extracranial cancers, extragonadal cancers, central nervous system), gestational trophoblastic tumor, brain stem cancer, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, histiocytosis, langerhans cell cancer, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumors, pancreatic neuroendocrine tumors, kaposi sarcoma, kidney cancer (e.g., renal cell, Wilm's tumor, and other kidney tumors), langerhans cell histiocytosis, laryngeal cancer, leukemia, acute lymphoblastic (ALL), acute myeloid (AML), chronic lymphocytic (CLL), chronic myelogenous (CML), hairy cell, lip and oral cavity cancer, liver cancer (primary), lobular carcinoma in situ (LCIS), lung cancer (e.g., childhood, non-small cell, small cell), lymphoma (e.g., AIDS-related, Burkitt (e.g., non-Hodgkin lymphoma), cutaneous T-Cell (e.g., mycosis fungoides, Sezary syndrome), Hodgkin, non-Hodgkin, primary central nervous system (CNS)), macroglobulinemia, Waldenstrom, male breast cancer, malignantfibrous histiocytoma of bone and osteosarcoma, melanoma (e.g., childhood, intraocular (eye)), merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer, midline tract carcinoma, mouth cancer, multiple endocrine neoplasia syndromes, multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndromes, Myelogenous Leukemia, Chronic (CML), multiple myeloma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity cancer, lip and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic neuroendocrine tumors (islet cell tumors), papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, plasma cell neoplasm, pleuropulmonary blastoma, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, renal pelvis and ureter, transitional cell cancer, rhabdomyosarcoma, salivary gland cancer, sarcoma (e.g., Ewing, Kaposi, osteosarcoma, rhadomyosarcoma, soft tissue, uterine), Sezary syndrome, skin cancer (e.g., melanoma, merkel cell carcinoma, basal cell carcinoma, nonmelanoma), small intestine cancer, squamous cell carcinoma, squamous neck cancer with occult primary, stomach (gastric) cancer, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, trophoblastic tumor, ureter and renal pelvis cancer, urethral cancer, uterine cancer, endometrial cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, Wilm’s tumor, and the like.

[0081] In some embodiments, the biological sample can be a cell (e.g., the test cell from which a cellular extract is produced). The cell can be a tumor cell such as a breast cancer cell, a gastric cancer cell, and / or a HER2-expressing tumor cell. In certain instances, the tumor cell is a circulating tumor cell, or a fine needle aspirate (ENA) cell obtained from a tumor. In other embodiments, the cell (e.g., the test cell from which a cellular extract is produced) is isolated from a sample that is obtained, e.g., from a breast or gastric cancer patient. Non-limiting examples of samples include bodily fluid samples such as, for example, a whole blood, serum, plasma, ductal lavage fluid, nipple aspirate, lymph, bone marrow aspirate, urine, saliva, and / or fine needle aspirate (ENA) sample. In particular embodiments, the sample comprises a whole blood, serum, plasma, and / or tumor tissue sample such as breast or gastric tumor tissue or HER2-expressing tumor tissue.

[0082] As described herein, the methods for detecting and discriminating betweenHER2-low expression levels, and distinguishing HER2-low expression from HER2-negative or HER2 -positive expression can include incubating the biological sample in a lysis reagent and recovering nucleic acid from said lysis reagent. The lysis reagent generally comprises a buffer sufficient to maintain the pH of the solution at a pH ranging from about pH 3 or about pH 4 to about pH 6, or about pH 7, or about pH 8, or about pH 9. In some embodiments the lysis reagent additionally comprises one or more proteolytic enzyme(s) / protease, one or more chaotropic agent(s), one or more chelating agents, one or more detergents, one or more salts, one or more antioxidants, or a combination thereof. In certain embodiments the lysis reagent additionally contains an antifoaming agent, and / or a preservative / biocide, and / or one or more second chaotrope / denaturing agent, and / or a second detergent, and / or calcium chloride or equivalent salt. In certain embodiments the lysis reagent omits the protease which can then be added immediately prior to use.

[0083] In some embodiments, the buffer in the lysis reagent buffers the solution at a pH ranging from about pH 6.5 up to about pH 7.5. In some embodiments, the buffer buffers the solution at a pH ranging from about pH 6.6, or about pH 6.7, or about pH 6.8 up to about pH 7.5 or up to about pH 7.4, or up to about pH 7.3, or up to about pH 7.2. In certain embodiments the pH is buffered at pH 7.05 (+ / - 0.1).

[0084] In certain embodiments, the concentration of the buffer ranges from about 10 mM up to about 100 mM, or from about 20 mM up to about 50 mM, or is about 50 mM.

[0085] Any of a number of buffers used in biology are suitable for use in the lysis reagent. Such include, but are not limited to buffers such as citrate buffer (sodium citrate), Tris (tris(hydroxymethyl)methylamine), phosphate, PBS, citrate, TAPS (1 3- { [tris(hydroxymethyl)methyl]amino}propanesulfonic acid), Bicine (N,N-bis(2- hydroxy ethyl) glycine), Tricine (N-tris(hydroxymethyl)methylglycine), TAPSO (3-[N- Tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic Acid), HEPES (4-2- hydroxyethyl-1 -piperazineethanesulfonic acid), TES (2- { [tris(hydroxymethyl)methyl]amino] ethanesulfonic acid), MOPS (3-(N- morpholino)propanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)), Cacodylate (dimethylarsinic acid), SSC (saline sodium citrate), MES (2-(N- morpholino)ethanesulfonic acid), and the like.

[0086] In some embodiments, the lysis reagent additionally includes one or more salts, such as calcium salt, sodium salt, magnesium salt, or equivalent salts. When present,the salt can be in the lysis reagent at a concentration ranging from about 1 mM up to about 500 mM, from about 300 mM to about 500 mM, from about 350 mM up to about 450 mM, from about 2 mM up to about 200 mM, or up to about 100 mM. In some embodiments, the salt is calcium chloride and is present at a concentration ranging from about 1 mM, or about 2 mM or about 5 mM, or about 8 mM up to about 50 mM, or up to about 40 mM, or up to about 30 mM, or up to about 20 mM, or up to about 15 mM, or up to about 10 mM. In certain embodiments the lysis reagent contains a magnesium salt such as MgCh. In certain embodiments the concentration of the magnesium salt in the lysis reagent ranges from about 2 mM up to about 20 mM, from about 5 mM up to about 15 mM, or is about 10 mM.

[0087] As indicated herein, the lysis reagent can comprise one or more antioxidant and / or chelating agents. Antioxidant and / or chelating agents are known to those of skill in the art and include, but are not limited to N-acetyl-L-cysteine, ethylenediaminetetraacetic acid (EDTA), diethylene triamine pentaacetic acid (DTP A), ethylenediamine-N,N'- disuccinic acid (EDDS), l,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), and phosphonate chelating agents (e.g., including, but not limited to nitrilotris(methylene)phosphonic acid (NTMP), ethylenediamine tetra(methylene phosphonic acid) (EDTMP), diethylenetriamine penta(methylene phosphonic acid (DTPMP), 1 -hydroxy ethylidene-l,l-diphosphonic acid (HEDP), and the like). In some embodiments the chelating agent comprises EDTA, or DTAP. In some embodiments, the chelating agent comprises EDTA. In some embodiments, the chelating agent comprises N- acetyl-L-cysteine.

[0088] When present, the antioxidant and / or chelating agent can be present in the lysis reagent at a concentration ranging from about 5 mM up to about 200 mM, or from about 10 mM up to about 100 mM. In some embodiments, the antioxidant and / or chelating agent is present at a concentration ranging from about 10 mM, or from about 20 mM, or from about 30 mM, or from about 40 mM up to about 60 mM, or up to about 70 mM, or up to about 80 mM, or up to about 90 mM, or up to about 100 mM. In some embodiments, the antioxidant and / or chelating agent is present at a concentration of about 50 mM. In some embodiments, the antioxidant and / or chelating agent comprises 0.5% to about 5% of the lysis reagent. In some embodiments, the antioxidant and / or chelating agent comprises 0.5% to about 3%, or to about 2%, or to about 1.5% of the lysis reagent. In some embodiments, the antioxidant and / or chelating agent comprises 1% of the lysis reagent.

[0089] In some examples, the chelating agent comprises EDTA or DTAP at a concentration of about 4 M. In some examples, the chelating agent comprises EDTA at a concentration of about 4 M. In some examples the chelating agent comprises EDTA or DTAP at a concentration of about 35 mM. In some examples, the chelating agent comprises EDTA at a concentration of about 35 mM. In some examples, the antioxidant and / or chelating agent is N-acetyl-L-cysteine comprising 0.5% to about 3%, or to about 2%, or to about 1.5% of the lysis reagent. In some examples, the antioxidant and / or chelating agent is N-acetyl-L-cysteine comprising about 1% of the lysis reagent. In some examples, the lysis reagents in the methods described herein need not be limited to the use of the chelating agents described above. Using the teaching and examples provided herein, other chelating agents will be available to one of skill in the art.

[0090] As indicated herein, the lysis reagent can comprise one or more detergents. In some embodiments, the detergent comprises an ionic detergent or a non-ionic detergent. Illustrative, but non-limiting detergents for use in some embodiments of the lysis reagent includes benzethonium chloride (cationic), BRU® 35 (nonionic), BRU® 58 (nonionic), cetylpyridinium chloride monohydrate (cationic), cetyltrimethylammonium bromide (cationic), CHAPS (zwitterionic), CHAPSO (zwitterionic), 1 -decanesulfonic acid sodium salt (anionic), n-decyl-P-D-glucopyranoside (nonionic), n-decyl-P-D-maltoside (nonionic), deoxy-BIGCHAP (nonionic), digitonin (nonionic), 1 -dodecanesulfonic acid sodium salt (anionic), n-dodecyl-P-D-glucopyranoside (nonionic), dodecyl-P-D-maltoside (nonionic), dodecyltrimethylammonium bromide (cationic), HECAMEG (nonionic), 1 -heptanesulfonic acid sodium salt anhydrous (anionic), 1 -heptanesulfonic acid sodium salt monohydrate (anionic), 1 -hexanesulfonic acid sodium salt anhydrous (anionic), 1 -hexanesulfonic acid sodium salt monohydrate (anionic), n-lauroylsarcosine sodium salt (anionic), lithium dodecylsulfate (LiDS) (anionic), MEGA-8 (nonionic), MEGA-9 (nonionic), 1- nonanesulfonic acid sodium salt (anionic), n-nonyl-P-D-glucopyranoside (nonionic), n- nonyl-P-D-maltoside (nonionic), 1-octanesulfonic acid sodium salt (anionic), n-octyl-P-D- glucopyranoside (nonionic), n-octyl-P-D-thioglucopyranoside (nonionic), octyl- D- glucopyranoside (nonionic), 1 -pentanesulfonic acid sodium salt anhydrous (anionic), 1- pentanesulfonic acid sodium salt monohydrate (anionic), PLURONIC® F-68 (nonionic), saponin (nonionic), SDS (sodium dodecylsulfate) (anionic), sodium cholate (anionic), sodium deoxycholate (anionic), sucrose monolaurate (nonionic), sulfobetaine SB 12(zwitterionic), sulfobetaine SB 14 (zwitterionic), n-tetradecyl-β-D-maltoside (nonionic), n- tridecyl-β-D-maltoside (nonionic), TRITON® X-100 (nonionic), TRITON® X-114 (nonionic), TWEEN®20 (nonionic), TWEEN® 80 (nonionic), n-undecyl-β-D-maltoside (nonionic), N-lauroylsarcosine (anionic), or a combination thereof.

[0091] When present, the detergent can be present in the lysis reagent at a concentration ranging from about 5 mM up to about 200 mM, or from about 10 mM up to about 100 mM, or from about 20 mM up to about 50 mM, or from about 30 mM up to about 40 mM. In some embodiments the detergent ranges from about 5 mM, or from about 10 mM, or from about 15 mM or from about 20 mM or from about 25 mM up to about 200 mM or up to about 150 mM, or up to about 100 mM, or up to about 75 mM, or up to about 50 mM, or up to about 40 mM. In some embodiments, the detergent is present at a concentration of about 35 mM. In some embodiments, the detergent is present at a percentage ranging from about 0.5% (v / v) up to about 30% (v / v), or from about 1% (v / v) up to about 20% (v / v) or from about 5% up to about 15% (v / v). In some embodiments the detergent comprises about 0.1% to about 2% of said solution, or about 0.5% to about 1.5% of said solution, or about 1% of the lysis reagent. In some embodiments the detergent comprises from about 0.1%, or from about 0.2% up to about 3% or up to about 2%, or up to about 1% of the lysis reagent, by volume. In certain embodiments the detergent comprises about 0.2% to about 0.4% of the lysis reagent, by volume.

[0092] In some embodiments, the detergents used in the lysis reagents described herein need not be limited to the detergents described above. Using the teaching and examples provided herein, other detergents will be available to one of skill in the art.

[0093] As indicated herein, the lysis reagent can comprise one or more chaotropes (chaotropic agent(s)). Chaotropic agents are well known to those of skill in the art and include, but are not limited to, 1 -octanesulfonic acid sodium salt, ammonium sulfate, butanol, dithiothreitol, ethanol, guanidinium hydrochloride, guanidinium thiocyanate, lithium chloride, lithium perchlorate, magnesium chloride, phenol, propanol, sodium dodecyl sulfate, thiourea, tributyl phosphate, urea, and the like. In some embodiments the chaotropic agent comprises a chaotropic salt (e.g., lithium perchlorate, magnesium chloride, guanidinium salts, lithium chloride, and the like). In some embodiments, the chaotropic agent comprises a guanidinium compound. In some embodiments, the guanidiniumcompound comprises guanidinium hydrochloride and / or guanidinium thiocyanate. In some embodiments the chaotropic agent(s) do not include an alcohol and / or an organic solvent. In some embodiments, the chaotropic agent(s) do not include organic solvents that dissolve / solubilize paraffin.

[0094] In some embodiments, when present the chaotropic agent(s) are present in the lysis reagent at a concentration ranging from about 1 M up to about 10 M, or from about 2 M, or from about 2.5 M, or from about 3 M, up to about 7 M, or up to about 8 M or up to about 9 M. In some embodiments, the chaotropic agents are present at a concentration of about 1 M, or about 1.5 M, or about 2 M, or about 2.5 M, or about 3 M, or about 3.5 M, or about 4 M, or about 4.5 M, or about 5 M, or about 5.5 M, or about 6 M, or about 6.5 M, or about 7 M, or about 7.5 M, or about 8 M, or about 8.5 M, or about 9 M, or about 9.5 M, or about 10 M, or in some embodiments at even higher concentrations. In some embodiments the chaotropic agent is present at a concentration of about 4 M, or about 4.5 M. In some embodiments, the chaotropic agent is present in the lysis reagent at a concentration of about 7 M.

[0095] In some embodiments, the lysis reagents in the methods described herein need not be limited to the use of the chaotropic agents described above. Using the teaching and examples provided herein, other chaotropic agents will be available to one of skill in the art.

[0096] As indicated herein, the lysis reagent can additionally comprise a second detergent (different than the first detergent(s)). In some embodiments, the second detergent comprises an ionic detergent or a non-ionic detergent. In some embodiments, the second detergent comprises TWEEN® 20. In some embodiments, when present, the second detergent is present in the lysis reagent at a concentration ranging from about 5 mM up to about 200 mM, or from about 10 mM up to about 100 mM, or from about 20 mM up to about 50 mM, or from about 30 mM up to about 40 mM. In some embodiments the detergent ranges from about 5 mM, or from about 10 mM, or from about 15 mM or from about 20 mM or from about 25 mM up to about 200 mM or up to about 150 mM, or up to about 100 mM, or up to about 75 mM, or up to about 50 mM, or up to about 40 mM. In some embodiments, the second detergent is present at a concentration of about 34 mM. In some embodiments, the detergent is present at a percentage ranging from about 0.5% (v / v)up to about 30% (v / v), or from about 1% (v / v) up to about 20% (v / v) or from about 5% up to about 15% (v / v). In some embodiments the detergent is present at about 10% (v / v). In some embodiments, the second detergent comprises TWEEN® 20 at about 10% (v / v) in the lysis reagent.

[0097] In some embodiments, the lysis reagent can additionally comprise a second chaotrope different than the first chaotrope. Suitable second chaotropes include, but are not limited to, 1 -octanesulfonic acid sodium salt, ammonium sulfate, butanol, dithiothreitol, ethanol, guanidinium hydrochloride, guanidinium thiocyanate, lithium chloride, lithium perchlorate, magnesium chloride, phenol, propanol, sodium dodecyl sulfate, thiourea, tributyl phosphate, urea, and the like. In some embodiments the chaotropic agent comprises a chaotropic salt (lithium perchlorate, magnesium chloride, guanidinium salts, lithium chloride, and the like). In some embodiments, the chaotropic agent comprises a guanidinium compound. In some embodiments, the guanidinium compound comprises guanidinium hydrochloride and / or guanidinium thiocyanate. In some embodiments the chaotropic agent(s) do not include an alcohol and / or an organic solvent. In some embodiments, the chaotropic agent(s) do not include organic solvents that dissolve / solubilize paraffin. In some embodiments, the second chaotrope comprises urea or thiourea. In some embodiments, the second chaotrope comprises urea. In some embodiments, when present the second chaotropic agent(s) is present in the lysis reagent at a concentration ranging from about 1 M up to about 10 M, or from about 2 M or from about 3 M up to about 7 M, or up to about 8 M or up to about 9 M. In some embodiments, the chaotropic agents are present at a concentration of about 1 M, or about 2 M, or about 3 M, or about 4 M, or about 5 M, or about 6 M, or about 7 M, or about 8 M, or about 9 M, or about 10 M, or in some embodiments at even higher concentrations. In some embodiments the second chaotropic agent is present at a concentration of about 6 M.

[0098] In some embodiments the lysis reagent additionally includes one or more proteolytic enzymes (proteases). Suitable proteases include, but are not limited to serine proteases, threonine proteases, cysteine proteases, aspartate proteases, metalloproteases, glutamic acid proteases, metalloproteases, and combinations thereof. Illustrative suitable proteases include but are not limited to proteinase k (a broad-spectrum serine protease), subtilysin trypsin, chymotrypsin, pepsin, papain, a guanidinium compound, formamide, lithium perchlorate, magnesium chloride, urea, thiourea, or combinations thereof.

[0099] When present in the lysis reagent, the protease can be present at an amount that provides an activity that ranges from 1 U / ml up to about 200 U / ml of lysis reagent. In some embodiments, the amount provides an activity ranging from about 1 U / ml, or from about 5 U / ml, or from about 10 U / ml, or from about 15 U / ml, up to about 200 U / ml, or up to about 100 U / ml, or up to about 80 U / ml, or up to about 60 U / ml, or up to about 40 U / ml, or up to about 30 U / ml of lysis reagent. In some embodiments, the amount of protease ranges from about 0.05 to about 5 mg / ml. In some embodiments, the amount of protease ranges from about 0.1 mg / ml, or about 0.2 mg / ml, or about 0.3 mg / ml, or about 0.4 mg / ml, or about 0.5 mg / ml, or about 0.6 mg / ml, or about 0.7 mg / ml, or about 0.8 mg / ml up to about 5 mg / ml, or up to about 4 mg / ml, or up to about 3 mg / ml, or up about 2 mg / ml, or up to about 1 mg / ml.

[0100] In some embodiments, the lysis reagents in the methods described herein need not be limited to the use of the proteases described above. Using the teaching and examples provided herein, other proteases will be available to one of skill in the art.

[0101] In some embodiments, the lysis reagent additionally comprises an oil. The oil can be selected from mineral oil, liquid paraffin oil, or combinations thereof. When present, the oil can be present in the lysis reagent at a concentration ranging from about 0.01% up to about 5% by volume, from about 0.01% up to about 4% by volume, from about 0.01% up to about 3% by volume, from about 0.01% up to about 2% by volume, from about 0.01% up to about 1.8% by volume, from about 0.01% up to about 1.5% by volume, from about 0.01% up to about 1% by volume, from about 0.1% up to about 2% by volume, from about 0.1% up to about 1.5% by volume, from about 0.2% up to about 4% by volume, from about 0.2% up to about 3% by volume, from about 2% up to about 1% by volume, from about 0.2% up to about 1% by volume, or from 0.01% to 1% by volume.

[0102] In some embodiments, the lysis reagent additionally comprises bovine serum albumin (BSA), or another serum albumin. When present, BSA can be in the lysis reagent at a concentration ranging from about 0.01% up to about 5% by volume, from about 0.01% up to about 4% by volume, from about 0.01% up to about 3% by volume, from about 0.01% up to about 2% by volume, from about 0.01% up to about 1.8% by volume, from about 0.01% up to about 1.5% by volume, from about 0.01% up to about 1% by volume, from about 0.1% up to about 2% by volume, from about 0.1% up to about 1.5% by volume, from about0.2% up to about 4% by volume, from about 0.2% up to about 3% by volume, from about 2% up to about 1% by volume, from about 0.2% up to about 1% by volume, or from 0.01% to 1% by volume.

[0103] Various studies have shown there is ~2-3 logs of RNA lost during the sample preparation steps (compared to control that bypasses sample preparation steps), leading to delayed cycle thresholds and increased assay variation. It was a surprising discovery that samples processed in accordance with the methods using the materials described herein, particularly using the lysis reagent(s) described herein (especially an oil or BSA,) give earlier Ct results, sometimes better than 2 Cts, or better than 3 Cts, or better than 4 Cts, as compared to various commercial lysis systems.

[0104] The methods and lysis reagents described herein are well suited for either RNA or DNA extraction. Accordingly, in such embodiments a single lysis reagent and extraction method can be used to extract essentially any nucleic acid (e.g., DNA, mRNA, non-coding RNA, and the like).

[0105] As described herein, the methods include incubating the biological sample (e.g., one or more sections of a fixed, paraffin-embedded, tissue sample) in a lysis reagent at an elevated temperature, ranging from about 40°C to about 110°C. In certain embodiments the lysis reagent lacks a protease, however, more typically a protease (e.g., proteinase K) is included.

[0106] The sample can be generally heated in the lysis reagent at a temperature of about 40°C up to about 110°C. In some embodiments the sample is heated at a temperature ranging from about 40°C, or from about 45°C, or from about 50°C, or from about 55°C, or from about 60°C, or from about 65°C, or from about 70°C, or from about 74°C up to about 80°C, or up to about 85°C, or up to about 90°C, or up to about 95 °C, or up to about 100°C, or up to about 105°C, or up to about 110°C, or up to about 115°C. In some embodiments, the sample is heated at a temperature ranging from about 80°C to about 90°C.

[0107] When the biological sample includes tissue sections, it is noted that where thinner sections are used it is possible and can be desirable to utilize a plurality of sections (e.g., at least 2 sections, or at least 3 sections, or at least 4 sections, or at least 5 sections, or at least 6 sections, or at least 7 sections, or at least 8 sections, or at least 9 sections, or atleast 10 sections). Particularly where the section is 5 pm thick or smaller multiple sections can be desirable.

[0108] The incubation time ranges from about 10 minutes up to about 4 hours. In some embodiments, the incubation time ranges from about 10 minutes, or from about 15 minutes, or from about 20 minutes, or from about 25 minutes, or from about 30 minutes up to about 24 hours, or up to about 12 hours, or up to about 6 hours, or up to about 4 hours, or up to about 3.5 hours, or up to about 3 hours, or up to about 2.5 hours, or up to about 2 hours, or up to about 1.5 hours, or up to about 1 hour. In some embodiments, the incubation time ranges from about 30 minutes up to about 1 hour.

[0109] In some embodiments, the step of incubating the biological sample in a lysis reagent and recovering nucleic acid from said lysis reagent can be carried out in an automated cartridge. For example, the method can comprise placing the biological sample in a cartridge, the cartridge comprising a cartridge body having a plurality of chambers in fluidic communication, a reaction vessel having one or more reaction chambers and configured for amplification and detection of the nucleic acid, a fluidic path between the plurality of chambers and the reaction vessel, and a filter in the fluidic path; adding a lysis reagent to the biological sample to form a mixture, and heating the mixture to a temperature ranging from about 50°C to about 100°C to form a lysed mixture. In other embodiments, the method can be semi-automated and includes incubating the biological sample in a lysis reagent and / or recovering nucleic acid from said lysis reagent manually. One or more of the remaining method steps can be automated in a cartridge body.

[0110] After the biological sample is heated in the lysis reagent the extracted nucleic acid (e.g., DNA, RNA) is recovered. Numerous methods for DNA and / or RNA recovery are known to those of skill in the art. In some embodiments, the nucleic acid is precipitated and / or bound to a solid substrate. Precipitation and / or binding to a substrate is readily accomplished by use of an alcohol, for example a lower alcohol (e.g., a Ci-Ce alcohol). Other precipitating agents are provided in US Patent Publication No. 2021 / 0324372, which is incorporated herein by reference in its entirety. For example, precipitation and / or binding to a substrate can be accomplished by use of a polysaccharide comprising one or more uronic acid units, such as a modified pectin. The nucleic acids can be recovered from the lysis reagent, e.g., using an alcohol extraction (e.g., an alcohol precipitation). In someembodiments the alcohol is ethanol or isopropanol. In some embodiment the alcohol is ethanol. It will be recognized that in some embodiments, dry alcohols can be used. The alcohol or precipitating agent can be present in an amount of from 5% to 75% by volume of the mixture, such as from 10% to 75% by volume, from 10% to 65% by volume, from 20% to 75% by volume, from 20% to 65% by volume, from 30% to 75% by volume, from 30% to 65% by volume, from 30% to 60% by volume, from 35% to 55% by volume, from 40% to 75% by volume, from 40% to 65% by volume, from 45% to 60% by volume, or from 45% to 55% by volume.

[0111] In certain embodiments, an alcohol such as polyethylene glycol (e.g., PEG 200), methanol, propanol, or ethanol can be added to the lysis reagent before exposure to the tissue sections or after exposure to the tissue sections. In certain embodiments the lysis reagent containing extracted nucleic acid(s), and optionally PEG, can be stored, and / or repeatedly used, e.g., for up to about 4 hrs, or up to about 8 hours, or up to about 1 day, or up to about 2 days, or up to about 3 days, or up to about 4 days, or up to about 1 week, or up to about 2 weeks, or up to about one month, or up to about two months, or up to about 3 months, or up to about 6 months, or up to about one year, or up to about 2 years, or up to about 3 years, or up to about 4 years, or up to about 5 years, or longer.

[0112] In some embodiments the alcohol is used to simply precipitate the nucleic acid(s). In some embodiments, the alcohol is used to precipitate the nucleic acids in the presence of compatible solid phase that results in binding of the nucleic acid to that solid phase. For example, in some embodiments, the alcohol treatment is performed in the present of a glass or cellulose substrate resulting in the binding of the nuclei acid(s) to that substrate. Remaining contaminants can be washed away while retaining the recovered nucleic acids that are then ready for amplification or other uses.

[0113] In some embodiments the solid phase comprises glass, silica, or cellulose.The solid phase can be provided by the walls of a container, as a fiber (e.g., glass fiber), as a membrane (e.g., cellulose membrane), in the form of beads (e.g., microparticles, or nanoparticles, etc.), and the like. In certain embodiments, the nucleic acid recovery can be performed in a GENEXPERT® cartridge, e.g., as described below. According, the method is rapid, simple, and easily amenable to automation and high throughput methodologies.Illustrative, but non-limiting methods for recovery of the nucleic acids are illustrated herein in the Examples.

[0114] The nucleic acids (e.g., DNA, RNA) extracted using the methods and reagents described herein are of good and sufficient quality and can readily be amplified to detect and / or quantify one or more target nucleic acid sequences in the sample. The nucleic acids are particular well suited to PCR amplification, detection, and / or quantification reactions including, but not limited to RT-PCR. While the extracted nucleic acids are well suited for use in any RT-PCR systems, in some embodiments, as illustrated herein in the Examples, the nucleic acids are particularly well suited for use in the GENEXPERT® systems (Cepheid Systems Inc.). In these embodiments, the method can include capturing nucleic acids from the lysed mixture onto the filter in the GENEXPERT® cartridge.

[0115] The GENEXPERT® System is a closed, self-contained, fully-integrated and automated platform that represents a paradigm shift in the automation of molecular analysis, producing accurate results in a timely manner with minimal risk of contamination. The GENEXPERT® System combines on-board sample preparation with real-time PCR (polymerase chain reaction) amplification and detection functions for fully integrated and automated nucleic acid analysis. The system is designed to purify, concentrate, detect and identify targeted nucleic acid sequences thereby delivering answers directly from unprocessed samples.

[0116] Accordingly, in some embodiments, methods are provided for identification and / or quantitative measurement of a target nucleic acid sequence in a biological sample. In some embodiments the methods comprise extracting a nucleic acid (e.g., a DNA, an RNA) from a fixed paraffin embedded biological tissue sample according any of the extraction methods described herein, subjecting the extracted nucleic acid to amplification using a pair of oligonucleotide primers capable of amplifying a region of a target nucleic acid, to obtain an amplified sample; and determining the presence and / or quantity of the target nucleic acid. In some embodiments, the target nucleic acid is a DNA (e.g., a gene). In some embodiments, the target nucleic acid is an RNA (e.g., an mRNA, a non-coding RNA, and the like).

[0117] In some embodiments, the nucleic acids extracted using the methods described herein are well suited for use in detection methods, diagnostic methods,prognostic methods, methods of monitoring treatments (e.g., cancer treatment), and the like. Accordingly, in some illustrative, but non-limiting embodiments, the nucleic acids extracted from fixed paraffin-embedded samples (e.g., from FFPET samples) can be used to identify the presence and / or the expression level of a gene, and / or the mutational status of a gene.

[0118] Such methods are particular well suited to identification of the presence, and / or expression level, and / or mutational status of one or more cancer markers. Accordingly, in some embodiments, the nucleic acids extracted using the methods described herein are utilized to detect the presence, and / or copy number, and / or expression level, and / or mutational status of one or more cancer markers.

[0119] In some embodiments, a normal level (a “control”) for each target marker (e.g., RNA) can be determined as an average (or median) level or range that is characteristic of normal cells or other reference material, against which the level measured in the sample can be compared. The determined average (or median) or range of target marker (e.g., RNA) in normal subjects can be used as a benchmark for detecting above-normal levels of target RNA indicative of a disease state (e.g., HER2 -positive, HER2-low, HER2-ultra low). In some embodiments, normal levels of target nucleic acid can be determined using individual or pooled RNA-containing samples from one or more individuals.

[0120] In some embodiments, determining a normal level of expression of a target nucleic acid marker (e.g., RNA) comprises detecting a complex comprising a probe hybridized to a nucleic acid selected from a target RNA, a DNA amplicon of the target RNA, and a complement of the target RNA. That is, in some embodiments, a normal level of expression can be determined by detecting a DNA amplicon of the target RNA, or a complement of the target RNA rather than the target RNA itself. In some embodiments, a normal level of such a complex is determined and used as a control (or “reference”). The normal level of the complex, in some embodiments, correlates to the normal level of the target RNA.

[0121] In some embodiments, a control comprises RNA from cells of a single individual, cells known to be healthy from the same subject. In some embodiments, a control comprises RNA from a pool of cells from multiple individuals. In some embodiments, a control is drawn from anatomically and / or cytologically normal areas of the of the individual from whom the test sample was obtained. In some embodiments, a controlcomprises commercially-available human RNA. In some embodiments, a normal level or normal range has already been predetermined prior to testing a sample for an elevated level.

[0122] In some embodiments, the normal level of target RNA can be determined from one or more continuous cell lines, typically cell lines previously shown to have expression levels of the at least one target RNA that approximate the level of expression in normal cells.

[0123] As described herein, the methods for detecting and discriminating between HER2-low expression levels, and distinguishing HER2-low expression from HER2- negative or HER2 -positive expression in a biological sample comprises detecting the level of expression of at least one target RNA, more particularly of the ERBB2 gene. The method can further comprises comparing the level of expression of the at least one target RNA to a control (or reference) level of expression of the at least one target RNA. A control level of expression of the at least one target RNA is, in some embodiments, the level of expression of the at least one target RNA in a normal cell. In some such embodiments, a control level may be referred to as a normal level.

[0124] In some embodiments, the level of expression of the at least one target RNA is compared to a reference level of expression, e.g., from a confirmed HER2-low expression, HER2 -positive expression, or HER2-negative. In some such embodiments, a similar level of expression of the at least one target RNA relative to the reference sample indicates HER2-low expression, HER2 -positive expression, or HER2-negative.

[0125] In some embodiments, a control level of expression of a target RNA is determined contemporaneously, such as in the same assay or batch of assays, as the level of expression of the target RNA in a sample. In some embodiments, a control level of expression of a target RNA is not determined contemporaneously as the level of expression of the target RNA in a sample. In some such embodiments, the control level of expression has been determined previously.HER2 Primers and Probes

[0126] As indicated herein, HER2-expression can be detected and determined by nucleic acid amplification. Particularly, the method for detecting and discriminating between HER2-low expression levels, and distinguishing HER2-low expression fromHER2-negative or HER2-positive expression can include contacting the nucleic acid from the biological sample with a set of primers and probes for detecting the presence of ERBB2 gene, subjecting the nucleic acid, primers, and probes to amplification conditions, and determining expression levels of ERBB2. The amplification reactions can be designed to detect 1, 2, 3, 4, 5, up to 30 or more target nucleic acids (including one or more target nucleic acid of the ERBB2 gene, and optionally one or more target nucleic acid of an additional gene(s)) per amplification reaction mixture.

[0127] The considerations for primers and probes for detecting HER2 expression are described in more detail below. The reference genomes and position of primers and probes for the gene(s) being targeted in an illustrative test are provided below in Table 1. Primers and probes that can be used in the methods disclosed herein are specific for measuring mRNA expression levels of Human Epidermal Growth Factor Receptor 2 (ERBB2) and a control gene. In some embodiments, one or more additional primers and probes that can be used in the methods disclosed herein are specific for measuring mRNA expression levels of Estrogen Receptor 1 (ESR), Progesterone Receptor 1 (PGR), cell proliferative antigen identified by Monoclonal Antibody Ki-67 (MKi67), Cytoplasmic FMRI -Interacting Protein 1 (CYFIP1), or a combination thereof. Particular embodiments of a HER2 expression test may include one amplification target per gene target to be detected or more than one amplification targets. For example, 1, 2, 3, 4, or all of the target genes listed in Table 1 can in various embodiments be detected using 2, 3, or more amplification targets per gene.Table 1: Reference genome positions

[0128] The compositions and methods disclosed herein can utilize automated reverse transcription cDNA synthesis from mRNA and real-time quantitative polymerase chain reaction (RT-PCR) for gene specific amplification. Detection of the amplified DNA isachieved by fluorogenic target-specific probe hybridization followed by 5’- exonuclease cleavage of the probe to release fluorophore. As described herein, the primers and probes in the test can be designed to amplify and detect the ERBB2 gene sequence and optionally one or more, two or more, three or more, four or more additional gene sequences (e.g., ESR, PGR, MKi67, and one “reference” CYFIP1) from the biological sample. The assay may then calculate delta Ct (ACt) between each target and reference for determining the positive and negative presence of target gene using quantitative algorithm derived from clinical standards.

[0129] Messenger RNA (mRNA) provides genetic information from DNA to the ribosome by coding specific amino acid sequence that gives rise to the protein products via gene expression. A newly transcribed pre-mRNA from DNA needs to be modified and spliced by removing various non-coding sequences known as introns in order to give rise to a matured mRNA containing protein coding sequences (exons). The methods described herein aim at detecting the mRNA targets from the biological sample, e.g., the formalin fixed and paraffin embedded tissue samples (FFPE). The RNA from the FFPE tissue samples is known to be fragmented during the tissue preparation processing that affects the RNA quality necessary for detection. The strategy to detect mature mRNA is to utilize a forward and a reverse primer targeting two or more adjacent exons with a sizable intron (e.g., >1000 nt length) in between. A <100 nt amplicon size from short RT-PCR amplification cycle ensures specific mature mRNA amplifications instead of non-specific DNA or unspliced pre-mRNA sequences while compensating for an efficient detection of short RNA fragments from the FFPE tissue samples.

[0130] The methods and systems may utilize up to 10 optical channels to individually detect four target genes, ESR, PGR, ERBB2, and MKi67, one CYFIP1 reference gene, and an Internal Control gene. One forward and one reverse primer can be used for RT-PCR amplifications while one or more probes are used for qPCR detection. For some targets, such as PGR, three primer and probe sets covering three distinct exon / exon junctions can be designed to improve PGR coverages and PGR mRNA detectability. The primer and probe designs are evaluated based on their maximum sequence homology to specific target genes, SNP frequencies, annealing temperatures (Tm), secondary structures (primer dimer and hairpin free energy), detectability (Ct, EPF), and background fluorescence signal.Controls

[0131] In some embodiments, an assay described herein for detecting and distinguishing HER2 expression in a biological sample as described above comprises at least one endogenous control. In some embodiments, the endogenous control is a sample adequacy control (SAC). In some such embodiments, if no HER2 biomarker is detected in a sample, and the SAC is also not detected in the sample, the assay result is considered “invalid” because the sample may have been insufficient. While not intending to be bound by any particular theory, an insufficient sample may be too dilute, contain too little cellular material, or contain an assay inhibitor, etc. In some embodiments, the failure to detect a SAC may indicate that the assay reaction failed. In some embodiments, an endogenous control is an RNA (such as an mRNA, tRNA, ribosomal RNA, etc.). Nonlimiting exemplary endogenous controls include ABL mRNA, GUSB mRNA, GAPDH mRNA, TUBB mRNA, and UPKla mRNA.

[0132] In some embodiments, an assay described herein for detecting and distinguishing HER2 expression in a biological sample as described above comprises at least one exogenous control. In some embodiments, the exogenous control is a sample processing control (SPC). In some such embodiments, if no HER2 biomarker described above is detected in a sample, and the SPC is also not detected in the sample, the assay result is considered “invalid” because there may have been an error in sample processing, including but not limited to, failure of the assay. Nonlimiting exemplary errors in sample processing include, inadequate sample processing, the presence of an assay inhibitor, the presence of a nuclease (such as an RNase), or compromised reagents, etc. In some embodiments, an exogenous control (such as an SPC) is added to a sample. In some embodiments, an exogenous control (such as an SPC) is added during performance of an assay, such as with one or more buffers or reagents. In some embodiments, when a GeneXpert® system is to be used, the SPC is included in the GeneXpert® cartridge. In some embodiments, an exogenous control (such as an SPC) is an Armored RNA®, which is protected by a bacteriophage coat.

[0133] In some embodiments, an endogenous control and / or an exogenous control is / are detected contemporaneously, such as in the same test, as detection of the HER2 expression biomarkers. In some embodiments, a test comprises reagents for detectingHER2 expression biomarkers described above, and a SAC and / or an exogenous control, simultaneously in the same assay reaction mixture. In some such embodiments, for example, an assay reaction mixture comprises primer sets for amplifying the HER2 expression biomarkers described above, a primer set for amplifying a SAC and / or a primer set for amplifying an exogenous control, as well as optional labeled probes for detecting the amplification products (such as, for example, TaqMan® probes).

[0134] In some examples, the control is an encapsidated RNA pseudovirus in the form of a dry bead in the cartridge. The control is mixed with RT-PCR reaction mixture to monitor the assay integrity and potential inhibition that may compromise assay efficiency. The control sequence can be made up of recombinant fragments derived from Yersinia enterocolitica, Tritrichomonas foetus and the human genome, and no sequence homology to the remaining target and reference genes.

[0135] In some aspects of the methods, CYFIP1 (Cytoplasmic FMRI -Interacting Protein 1) can be used as a reference (also referred to herein as a positive control biomarker) that is indicative of the quality of the sample. Without wishing to be bound by theory, the use of CYFIP1 as a positive control means that when CYFIP1 expression is detected at a sufficient level, the sample is deemed to be of high enough quality to continue with analysis, and / or, when CYFIP1 expression is not detected at a sufficient level, the sample is deemed to be of low quality and further analysis is not performed using that sample. Accordingly, in some aspects, the cutoff value can be a threshold value of measured CYFIP1 expression that indicates that CYFIP1 is present in the biological sample. As would be appreciated by the skilled artisan, said threshold value can be derived by the user performing the preceding methods based on the experimental conditions being used to measure the expression levels of the recited biomarkers.Polynucleotides

[0136] Polynucleotides are provided for detecting the biomarkers described above.In some embodiments, synthetic polynucleotides are provided. Synthetic polynucleotides, as used herein, refer to polynucleotides that have been synthesized in vitro either chemically or enzymatically. Chemical synthesis of polynucleotides includes, but is not limited to, synthesis using polynucleotide synthesizers, such as OligoPilot™ (GE Healthcare), ABI 3900 DNA Synthesizer (Applied Biosystems), and the like. Enzymatic synthesis includes,but is not limited, to producing polynucleotides by enzymatic amplification, e.g., PCR. A polynucleotide may comprise one or more analog of the canonical nucleotides (e.g., modified nucleotides).

[0137] In some embodiments, a polynucleotide is provided that comprises a region that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to, or at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to, at least 6, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 contiguous nucleotides of the respiratory pathogen targets, and / or exemplary controls discussed above.

[0138] In various embodiments, an exemplary polynucleotide comprises at least: 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In various embodiments, a polynucleotide comprises fewer than: 200, 150, 100, 50, 40, 30, or 20 nucleotides. In various embodiments, an exemplary polynucleotide is between 6 and 200, between 8 and 200, between 8 and 150, between 8 and 100, between 8 and 75, between 8 and 50, between 8 and 40, between 8 and 30, between 15 and 100, between 15 and 75, between 15 and 50, between 15 and 40, or between 15 and 30 nucleotides long.

[0139] In some embodiments, detection of each target nucleic acid can be carried out using a single labeled primer or probe, specific for each target nucleic acid. Different primers and / or probes can have the same label. By using primers or probes labeled with different detectable moieties (e.g., different fluorescent reporter dyes), numerous target nucleic acids can be detected simultaneously in a single reaction mixture. In some embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more different labels can be used in a single reaction mixture or a plurality of reaction mixtures. Each target nucleic acid can be independently monitored using such multiplexing technology. In some embodiments, detection of a plurality of target nucleic acids can be carried out using a single labeled primer or probe. A melt curve may be generated in orderto distinguish two or more target nucleic acids that each use the same label, but such analysis may not be necessarily required.Polynucleotide Modifications

[0140] In some embodiments, the methods of detecting at least one target nucleic acid described herein employ one or more polynucleotides that have been modified, such as polynucleotides comprising one or more affinity-enhancing nucleotide analogs. Modified polynucleotides useful in the methods described herein include primers for reverse transcription, PCR amplification primers, and probes. In some embodiments, the incorporation of affinity-enhancing nucleotides increases the binding affinity and specificity of a polynucleotide for its target nucleic acid as compared to polynucleotides that contain only the canonical deoxy ribonucleotides, which allows for the use of shorter polynucleotides or for shorter regions of complementarity between the polynucleotide and the target nucleic acid.

[0141] In some embodiments, affinity-enhancing nucleotide analogs include nucleotides comprising one or more base modifications, sugar modifications, and / or backbone modifications. In some embodiments, modified bases for use in affinityenhancing nucleotide analogs include 5 -methylcytosine, isocytosine, pseudoisocytosine, 5- bromouracil, 5-propynyluracil, 6- aminopurine, 2-aminopurine, inosine, diaminopurine, 2- chloro-6-aminopurine, xanthine and hypoxanthine. In some embodiments, affinityenhancing nucleotide analogs include nucleotides having modified sugars such as 2'- substituted sugars, such as 2'-O-alkyl-ribose sugars, 2'-amino-deoxyribose sugars, 2'-fluoro- deoxyribose sugars, 2'-fluoro-arabinose sugars, and 2'-O-methoxyethyl-ribose (2'MOE) sugars. In some embodiments, modified sugars are arabinose sugars, or d-arabino-hexitol sugars.

[0142] In some embodiments, affinity-enhancing nucleotide analogs include backbone modifications such as the use of peptide nucleic acids (PNA; e.g., an oligomer including nucleobases linked together by an amino acid backbone). Other backbone modifications include phosphorothioate linkages, phosphodiester- modified nucleic acids, combinations of phosphodiester and phosphorothioate nucleic acid, methylphosphonate, alkylphosphonates, phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates, carbonates, phosphate triesters, acetamidates, carboxymethyl esters,methylphosphorothioate, phosphorodithioate, p-ethoxy modifications, and combinations thereof.

[0143] In some embodiments, a polynucleotide includes at least one affinityenhancing nucleotide analog that has a modified base, at least nucleotide (which may be the same nucleotide) that has a modified sugar, and / or at least one intemucleotide linkage that is non-naturally occurring.

[0144] In some embodiments, an affinity-enhancing nucleotide analog contains a locked nucleic acid (“LNA”) sugar, which is a bicyclic sugar. In some embodiments, a polynucleotide for use in the methods described herein comprises one or more nucleotides having an LNA sugar. In some embodiments, a polynucleotide contains one or more regions consisting of nucleotides with LNA sugars. In other embodiments, a polynucleotide contains nucleotides with LNA sugars interspersed with deoxyribonucleotides. See, e.g., Frieden, M. et al. (2008) Curr. Pharm. Des. 14(11): 1138-1142.Primers

[0145] In some embodiments, the polynucleotide is a primer. Primers useful in the methods described herein are generally capable of selectively hybridizing to: genomic DNA, a target RNA (genomic or transcript), a cDNA reverse transcribed from the target RNA, and / or an amplicon that has been amplified from genomic DNA, a target RNA, or a cDNA (collectively referred to as “template”), and, in the presence of the template, a polymerase and suitable buffers and reagents, can be extended to form a primer extension product. Primers are generally of a sufficient length to ensure selective hybridization to their target nucleic acids. Generally, primers of at least 15 nucleotides in length hybridize specifically in most contexts, and this length can be reduced, e.g., by including of affinityenhancing modifications, such as those discussed above. Primers can but need not be exactly complementary to their target nucleic acids. Primers can have any degree of complementarity described above for exemplary polynucleotides. In illustrative embodiments, primers can be 8 to 40 nucleotides in length and at least 90% complementary to their target nucleic acids; 8 to 40 nucleotides in length and at least 95% complementary to their target nucleic acids; 8 to 40 nucleotides in length and at least 99% complementary to their target nucleic acids; 8 to 30 nucleotides in length and at least 90% complementary to their target nucleic acids; 8 to 30 nucleotides in length and at least 95% complementaryto their target nucleic acids; 8 to 30 nucleotides in length and at least 99% complementary to their target nucleic acids. In embodiments wherein a primer is less than 100% complementary to it target nucleic acid, having the 3’ nucleotide in the primer be complementary to its target nucleic acid facilitates the production of an extension product.

[0146] In some embodiments, a primer that selectively hybridizes to its target nucleic acid hybridizes to its target nucleic acid with at least 5-fold greater affinity than to non-target nucleic acid under the same assay conditions. In some embodiments, a primer that selectively hybridizes to its target nucleic acid hybridizes to its target nucleic acid with at least 10-fold greater affinity than to non-target nucleic acid under the same assay conditions.

[0147] In some embodiments, a primer pair is designed to produce an amplicon that is 50 to 1500 nucleotides long, 50 to 1000 nucleotides long, 50 to 750 nucleotides long, 50 to 500 nucleotides long, 50 to 400 nucleotides long, 50 to 300 nucleotides long, 50 to 200 nucleotides long, 50 to 150 nucleotides long, 100 to 300 nucleotides long, 100 to 200 nucleotides long, or 100 to 150 nucleotides long.

[0148] In some embodiments, the primer is labeled with a detectable moiety. In some embodiments, a primer is not labeled.Probes

[0149] In some embodiments, the polynucleotide is a probe. Probes useful in the methods described herein are generally capable of selectively hybridizing to: genomic DNA, a target RNA (genomic or transcript), a cDNA reverse transcribed from the target RNA, and / or an amplicon that has been amplified from genomic DNA, a target RNA, or a cDNA (collectively referred to as “template”). Generally, probes of at least 15 nucleotides in length hybridize specifically in most contexts, and this length can be reduced, e.g., by including of affinity-enhancing modifications, such as those discussed above. Probes can but need not be exactly complementary to their target nucleic acids. For example, probes can deliberately include “mismatches” to adjust the Tm of a melt probe. Probes can have any degree of complementarity described above for exemplary polynucleotides. In illustrative embodiments, probes can be 8 to 40 nucleotides in length and at least 70% complementary to their target nucleic acids; 8 to 40 nucleotides in length and at least 75%complementary to their target nucleic acids; 8 to 40 nucleotides in length and at least 80% complementary to their target nucleic acids; 8 to 40 nucleotides in length and at least 85% complementary to their target nucleic acids; 8 to 40 nucleotides in length and at least 90% complementary to their target nucleic acids; 8 to 40 nucleotides in length and at least 95% complementary to their target nucleic acids; 8 to 40 nucleotides in length and at least 99% complementary to their target nucleic acids; 8 to 30 nucleotides in length and at least 90% complementary to their target nucleic acids; 8 to 30 nucleotides in length and at least 95% complementary to their target nucleic acids; 8 to 30 nucleotides in length and at least 99% complementary to their target nucleic acids. In embodiments wherein a probe is less than 100% complementary to a target nucleic acid, any points or regions of non-complementarity are typically located so as not to disrupt the ability of the probe to selectively hybridize to its target nucleic acid.

[0150] In some embodiments, a probe that selectively hybridizes to its target nucleic acid hybridizes to its target nucleic acid with at least 5 -fold greater affinity than to nontarget nucleic acid under the same assay conditions. In some embodiments, a probe that selectively hybridizes to its target nucleic acid hybridizes to its target nucleic acid with at least 10-fold greater affinity than to non-target nucleic acid under the same assay conditions.

[0151] In some embodiments, the compositions and methods disclosed herein can include primers and probe for the ERBB2 biomarker, comprising an oligonucleotide complementary to a region within exons 15, 16, and / or 17 of the nucleotide sequence of ERBB2 published in the NCBI database under the accession number NC_000017.11. For example, the primers and probes for amplifying and detecting ERBB2 can comprise at least 15 contiguous nucleotides having a sequence that is at least 85% identical to at least 15 contiguous nucleotides of the ERBB2 gene at exons 15, 16, and / or 17. In specific examples, primers and probe for the ERBB2 biomarker can include an oligonucleotide 15 to 30, 15 to 25, or 16 to 22 nucleotides long and complementary to a region within the amplicon produced from its primer pair described herein.

[0152] In some examples, the set of primers and probe for detecting ERBB2 is selected from: (i) a forward primer, a reverse primer, and a probe each comprising at least 15 contiguous nucleotides having a sequence that is at least 85% identical to at least 15contiguous nucleotides of the ERBB2 gene; or (ii) a forward primer comprising at least 15 contiguous nucleotides having a sequence that is at least 85% identical to at least 15 contiguous nucleotides of the ERBB2 gene at exons 15, 16 and / or 17, at least one reverse primer comprising at least 15 contiguous nucleotides having a sequence that is at least 85% identical to at least 15 contiguous nucleotides of the ERBB2 gene at exons 15, 16 and / or 17, and a probe comprising at least 15 contiguous nucleotides having a sequence that is at least 85% identical to at least 15 contiguous nucleotides of the ERBB2 gene at exons 15, 16 and / or 17, or (iii) a forward primer comprising at least 15 contiguous nucleotides having a sequence that is at least 85% identical to at least 15 contiguous nucleotides of SEQ ID NO:3 or 6, at least one reverse primer comprising at least 15 contiguous nucleotides having a sequence that is at least 85% identical to at least 15 contiguous nucleotides of SEQ ID NO:4 or 6, and a probe comprising at least 15 contiguous nucleotides having a sequence that is at least 85% identical to at least 15 contiguous nucleotides of SEQ ID NO: 5 or 6.

[0153] In some embodiments, the compositions and methods disclosed herein can include primers and probe for the ESRI biomarker, comprising an oligonucleotide complementary to a region within exons 5 and / or 6 of the nucleotide sequence of ESRI published in the NCBI database under the accession number NG_008493.1. For example, the primers and probes for amplifying and detecting ESRI comprise at least 15 contiguous nucleotides having a sequence that is at least 85% identical to at least 15 contiguous nucleotides of the ESRI gene at exons 5 and / or 6. In specific examples, primers and probe for the ESRI biomarker can include an oligonucleotide 10 to 30, 12 to 25, or 16 to 22 nucleotides long and complementary to a region within the amplicon produced from its primer pair described herein.

[0154] In some embodiments, the compositions and methods disclosed herein can include primers and probe for the PGR biomarker, comprising an oligonucleotide complementary to a region within exons 2, 3, 4, 5, 6, and / or 7 of the nucleotide sequence of PGR published in the NCBI database under the accession number NG_016475.1. For example, the primers and probes for amplifying and detecting PGR comprise at least 15 contiguous nucleotides having a sequence that is at least 85% identical to at least 15 contiguous nucleotides of the PGR gene at exons 2, 3, 4, 5, 6, and / or 7. In specific examples, primers and probe for the PGR biomarker can include an oligonucleotide 10 to30, 12 to 25, or 16 to 22 nucleotides long and complementary to a region within the amplicon produced from its primer pair described herein.

[0155] In some embodiments, the compositions and methods disclosed herein can include primers and probe for the MKi67 biomarker, comprising an oligonucleotide complementary to a region within exons 2 and / or 3 of the nucleotide sequence of MKi67 published in the NCBI database under the accession number NC_000010.11. For example, the primers and probes for amplifying and detecting MKi67 comprise at least 15 contiguous nucleotides having a sequence that is at least 85% identical to at least 15 contiguous nucleotides of the MKi67 gene at exons 2 and / or 3. In specific examples, primers and probe for the MKi67 biomarker can include an oligonucleotide 10 to 30, 12 to 25, or 16 to 22 nucleotides long and complementary to a region within the amplicon produced from its primer pair described herein.

[0156] In some embodiments, the compositions and methods disclosed herein can include primers and probe for the CYFIP biomarker, comprising an oligonucleotide complementary to a region within exons 20 and / or 21 of the nucleotide sequence of CYFIP published in the NCBI database under the accession number NC_000015.10. For example, the primers and probes for amplifying and detecting CYFIP comprise at least 15 contiguous nucleotides having a sequence that is at least 85% identical to at least 15 contiguous nucleotides of the CYFIP gene at exons 20 and / or 21. In specific examples, primers and probe for the CYFIP biomarker can include an oligonucleotide 10 to 30, 12 to 25, or 16 to 22 nucleotides long and complementary to a region within the amplicon produced from its primer pair described herein.Polynucleotide Labels

[0157] In some embodiments, the primer or probe is labeled with a detectable moiety. Detectable moieties include directly detectable moieties, such as fluorescent dyes, and indirectly detectable moieties, such as members of binding pairs. When the detectable moiety is a member of a binding pair, in some embodiments, the probe can be detectable by incubating the probe with a detectable label bound to the second member of the binding pair. In some embodiments, a primer or probe is not labeled, such as when a primer or probe is immobilized, e.g., on a microarray or bead. A labeled primer is extendable, e.g., by a polymerase. In some embodiments, a probe is extendable. In other embodiments, a probeis not extendable. The following discussion centers on probes, as these are more typically employed for detecting in the methods described here, but those of skill in the art appreciate that the polynucleotide labeling strategies described below apply equally to the labeling of primers.

[0158] In some embodiments, the probe is a FRET probe that, in some embodiments, is labeled at the 5'-end with a fluorescent dye (donor) and at the 3'-end with a quencher (acceptor), a chemical group that absorbs (i.e., suppresses) fluorescence emission from the dye when the groups are in close proximity (e.g., attached to the same probe). Thus, in some embodiments, the emission spectrum of the dye should overlap considerably with the absorption spectrum of the quencher. In other embodiments, the dye and quencher are not at the ends of the FRET probe.

[0159] Illustrative FRET probes, which include, but are not limited to, a TaqMan® probe, a Molecular beacon probe and a Scorpion probe. A TaqMan® probe is a linear probe that typically has a fluorescent dye covalently bound at one end of the DNA and a quencher molecule covalently bound elsewhere, such as at the other end of the DNA. The FRET probe comprises a sequence that is complementary to a region of the cDNA or amplicon such that, when the FRET probe is hybridized to the cDNA or amplicon, the dye fluorescence is increased due to increased distance between dye and quencher; when the FRET probe is non-hybridized, the dye fluorescence is quenched; and when the probe is digested during amplification of the cDNA or amplicon, the dye is released from the probe and produces a fluorescence signal. In some embodiments, the amount of target nucleic in the sample is proportional to the amount of fluorescence measured during amplification.

[0160] Like TaqMan® probes, Molecular Beacons use FRET to detect a PCR product via a probe having a fluorescent dye and a quencher attached at the ends of the probe. Unlike TaqMan® probes, Molecular Beacons remain intact during the PCR cycles. Molecular Beacon probes form a stem-loop structure when free in solution, thereby allowing the dye and quencher to be in close enough proximity to cause fluorescence quenching. When the Molecular Beacon hybridizes to a target nucleic acid, the stem-loop structure is abolished so that the dye and the quencher become separated in space and the dye fluoresces. Molecular Beacons are available, e.g., from Gene Link™ (see www.genelink.com / newsite / products / mbintro.asp).

[0161] In some embodiments, Scorpion probes can be used as sequence-specific primers and for PCR product detection. Like Molecular Beacons, Scorpion probes form a stem- loop structure when not hybridized to a target nucleic acid. However, unlike Molecular Beacons, a Scorpion probe achieves both sequence-specific priming and PCR product detection. A fluorescent dye molecule is attached to the 5 ’-end of the Scorpion probe, and a quencher is attached elsewhere, such as to the 3 ’-end. The 3’ portion of the probe is complementary to the extension product of the PCR primer, and this complementary portion is linked to the 5 ‘-end of the probe by a non-amplifiable moiety. After the Scorpion primer is extended, the target-specific sequence of the probe binds to its complement within the extended amplicon, thus opening up the stem-loop structure and allowing the dye on the 5 ‘-end to fluoresce and generate a signal. Scorpion probes are available from, e.g., Premier Biosoft International (see www.premierbiosoft.com / tech_notes / Scorpion.html).

[0162] In some embodiments, labels that can be used on the FRET probes include colorimetric and fluorescent dyes, such as Alexa Fluor dyes; BODIPY dyes, such as BODIPY FL, Cascade Blue, and Cascade Yellow; coumarin and its derivatives, such as 7- amino-4-methylcoumarin, aminocoumarin and hydroxycoumarin; cyanine dyes, such as Cy3 and Cy5; eosins and erythrosins; fluorescein and its derivatives, such as fluorescein isothiocyanate; macrocyclic chelates of lanthanide ions, such as Quantum Dye™; Marina Blue; Oregon Green; rhodamine dyes, such as rhodamine red, tetramethylrhodamine and rhodamine 6G; Texas Red; fluorescent energy transfer dyes, such as thiazole orange- ethidium heterodimer; and TOT AB.

[0163] Specific examples of dyes include, but are not limited to, those identified above and the following: Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500. Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, and, Alexa Fluor 750; aminereactive BODIPY dyes, such as B0DIPY 493 / 503, B0DIPY 530 / 550, B0DIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, BODIPY650 / 655, BODIPY FL, BODIPY R6G, BODIPY TMR, and, BODIPY-TR; Cy3, Cy5, 6- FAM, Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500,Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, SYPRO, TAMRA, 2’, 4’, 5 ’,7’ -Tetrabromosulfonefluorescein, and TET.

[0164] Examples of dye / quencher pairs (i.e., donor / acceptor pairs) include, but are not limited to, fluorescein / tetramethylrhodamine; lAEDANS / fluorescein; EDANS / dabcyl; fluorescein / fluorescein; BODIPY FL / BODIPY FL; and fluorescein / QSY 7 or QSY 9 dyes. When the donor and acceptor are the same, FRET may be detected, in some embodiments, by fluorescence depolarization. Certain specific examples of dye / quencher pairs (i.e., donor / acceptor pairs) include, but are not limited to, Alexa Fluor 350 / Alexa Fluor488; Alexa Fluor 488 / Alexa Fluor 546; Alexa Fluor 488 / Alexa Fluor 555; Alexa Fluor488 / Alexa Fluor 568; Alexa Fluor 488 / Alexa Fluor 594; Alexa Fluor 488 / Alexa Fluor 647;Alexa Fluor 546 / Alexa Fluor 568; Alexa Fluor 546 / Alexa Fluor 594; Alexa Fluor546 / Alexa Fluor 647; Alexa Fluor 555 / Alexa Fluor 594; Alexa Fluor 555 / Alexa Fluor 647;Alexa Fluor 568 / Alexa Fluor 647; Alexa Fluor 594 / Alexa Fluor 647; Alexa Fluor350 / QSY35; Alexa Fluor 350 / dabcyl; Alexa Fluor 488 / QSY 35; Alexa Fluor 488 / dabcyl;Alexa Fluor 488 / QSY 7 or QSY 9; Alexa Fluor 555 / QSY 7 or QSY9; Alexa Fluor 568 / QSY7 or QSY 9; Alexa Fluor 568 / QSY 21; Alexa Fluor 594 / QSY 21; and Alexa Fluor 647 / QSY21. In some instances, the same quencher may be used for multiple dyes, for example, a broad spectrum quencher, such as an Iowa Black® quencher (Integrated DNA Technologies, Coralville, IA) or a Black Hole Quencher™ (BHQ™; Sigma-Aldrich, St. Louis, MO).

[0165] Specific examples of fluorescently labeled ribonucleotides useful in the preparation of probes for use in some embodiments of the methods described herein are available from Molecular Probes (Invitrogen), and these include, Alexa Fluor 488-5-UTP, Fluorescein- 12-UTP, BODIPY FL-14-UTP, BODIPY TMR-14-UTP,Tetramethylrhodamine-6-UTP, Alexa Fluor 546-14-UTP, Texas Red-5-UTP, and BODIPYTR-14-UTP. Other fluorescent ribonucleotides are available from Amersham Biosciences(GE Healthcare), such as Cy3-UTP and Cy5-UTP.

[0166] Specific examples of fluorescently labeled deoxyribonucleotides useful in the preparation of probes for use in the methods described herein include Dinitrophenyl (DNP)-l ‘-dUTP, Cascade Blue-7-dUTP, Alexa Fluor 488-5-dUTP, Fluorescein- 12-dUTP,Oregon Green 488-5-dUTP, BODIPY FL-14-dUTP, Rhodamine Green-5-dUTP, AlexaFluor 532-5-dUTP, BODIPY TMR- 14-dUTP, Tetramethylrhodamine-6-dUTP, Alexa Fluor 546-14-dUTP, Alexa Fluor 568-5-dUTP, Texas Red-12-dUTP, Texas Red-5-dUTP,BODIPY TR- 14-dUTP, Alexa Fluor 594-5-dUTP, BODIPY 630 / 650- 14-dUTP, BODIPY650 / 665- 14-dUTP; Alexa Fluor 488-7-OBEA-dCTP, Alexa Fluor 546-16-OBEA-dCTP,Alexa Fluor 594-7-OBEA-dCTP, and Alexa Fluor 647-12-OBEA-dCTP. Fluorescently labeled nucleotides are commercially available and can be purchased from, e.g., Invitrogen.

[0167] As noted above, exemplary detectable moieties also include members of binding pairs. Exemplary binding pairs include, but are not limited to, biotin and streptavidin, antibodies and antigens, etc.Assay Methods

[0168] The methods described herein can utilize RNA targets for detection by direct hybridization or, more easily, by reverse transcribing a target RNA to produce a cDNA that is complementary to the target RNA. This cDNA can be directly detected by direct hybridization or by amplification of the cDNA template.

[0169] Nucleic acid amplification provides rapid, sensitive, and specific detection of nucleic acid targets, and has been employed in a wide variety of assay formats to detect nucleic acid targets. Those of skill in the art can, following the guidance herein, carry out the methods described herein in any number of different nucleic acid amplification-based assays, using, for example, any of the nucleic acid amplification methods discussed above. Such methods can entail thermocycling, but need not do so, as in the case of isothermal amplification. Exemplary methods include, but are not limited to, isothermal amplification, real time RT-PCR, endpoint RT-PCR, and amplification using T7 polymerase from a T7 promoter annealed to a DNA, such as provided by the SenseAmp Plus™ Kit available at Implen, Germany. Amplification and detection can be carried out in solution or can make use of a solid support (e.g., a biochip). Nucleic acid amplification-based assays can employ a single reaction chamber or multiple reaction chambers. Amplification can be nested or non-nested. In some embodiments, detection includes electrochemical detection.

[0170] In some embodiments of amplification by polymerase chain reaction (PCR), an exemplary cycle comprises an initial denaturation at 90°C to 100°C for 20 seconds to 5 minutes, followed by cycling that comprises denaturation at 90°C to 100°C for 1 to 10seconds, followed by annealing and amplification at 60°C to 75°C for 10 to 40 seconds. A further exemplary cycle comprises 20 seconds at 94°C, followed by up to 3 cycles of 1 second at 95°C, 35 seconds at 62°C, 20 cycles of 1 second at 95°C, 20 seconds at 62°C, and 14 cycles of 1 second at 95°C, 35 seconds at 62°C. In some embodiments, for the first cycle following the initial denaturation step, the cycle denaturation step is omitted. In some embodiments, Taq polymerase is used for amplification. In some embodiments, the cycle is carried out at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, or at least 45 times. In some embodiments, Taq is used with a hot-start function. In some embodiments, detection of the target nucleic acids occurs in less than 3 hours, less than 2.5 hours, less than 2 hours, less than 1 hour, or less than 30 minutes from initial denaturation through the last extension. In some embodiments, target nucleic acids are detected by a method that includes real-time quantitative PCR, e.g., using FRET probes, such as those described above.

[0171] In particular embodiments, target nucleic acids (such as the ERBB2 gene target) and / or optional controls, can be detected by (a) incubating the biological sample in a lysis reagent and recovering nucleic acid from said lysis reagent, (b) contacting the nucleic acid with a set of primers and probes for detecting the presence of the target nucleic acid (such as ERBB2); (c) subjecting the nucleic acid, primers, and probes to amplification conditions; (d) determining expression levels of the target nucleic acid (such as ERBB2), expressed as a Ct value or a ACt value, wherein ACt is the Ct value for a reference gene minus the Ct value for the target nucleic acid (such as ERBB2); (e) comparing the Ct value or the ACt value to a range of Ct values or ACt values, and (f) determining that the subject has HER2-low expression when the Ct value or the ACt value for the target nucleic acid (such as ERBB2) is within a first range of Ct values or ACt values. The method can further comprise discrimination various levels of HER2-low expression, for example, determining that the subject has HER2-ultralow expression when the Ct value or the ACt value for the target nucleic acid is within a second range of Ct values or ACt values. The method can also comprise determining that the subject is HER2-negative when the Ct value or the ACt value for the target nucleic acid (such as ERBB2) is within a third range of Ct values or ACt values, or that the subject is HER2 -positive when the Ct value or the ACt value for the target nucleic acid (such as ERBB2) is within a fourth range of Ct values or ACt values.

[0172] The American Society of Clinical Oncology ( ASCO) / College of American Pathologists (CAP) Expert. Panel has provided guidelines on HER2 interpretation in breast cancer, including algorithms for defining positive, equivocal, and negative values for both HER2 protein expression and gene amplification. The current IHC algorithm with the expanded spectrum of HER2 reporting category and the reporting results for ISH assays are shown in Tables 2 and 3, respectively (Source: Breast Biomarker Reporting, CAP CancerProtocol Templates, vl.4.1.1, November 2021 update). In some embodiments of the methods disclosed herein, the range of values for HER2-low expression can be within a Ct range and / or ACt range corresponding to a FISH score of less than about 6 signals / cell based on HER2 copy number.Table 2: Reporting results of HER2 testing by immunohistochemistry and the corresponding expanded spectrum of positivity (ASCO / CAP 2021 )Table 3: Reporting results of HER'2 testing by in situ hybridization (ASCO / CAP 2021)CN: Average HER2 copy number ( signal s / cell); IHC: immunohistochemical score; ratio: HER2 / CEP17 ratio.

[0173] Moutafi M. et al., in Laboratory Investigation 2022, 102: 1101-1108 describe quantitative measurement of HER2 expression to subclassify ERBB2 unamplified breast cancer. Particularly, Moutafi uses a chromogenic assay (quantitative immunofluorescence coupled with a mass spectrometry standardized HER2 array) to generate an assay that can measure absolute amounts of HER2 protein in attomol / mm2on conventional histology sections. The expression is measured in attomols / ug total protein in the cell lines, then converted to attomols / mm2by image analysis and determination of area, supported by the AQUA method of analysis which allows assessment of signal per area rather than per cell. Table 4 details protein concentrations (pg), HER2 concentration (amol) for amplified and non-amplified cell lines and their corresponding cell numbers. The resultant assay is linear between 2 and 20 attomols / mm2, which is below the levels seen in amplified cell lines or tumors, but within the range of expression seen in normal breast epithelium. In some embodiments of the methods disclosed herein, the range of values for HER2-low expression can be within a Ct range and / or ACt range corresponding to HER2 peptide quantities between about 2 and about 20 attomole / mm2.Table 4: Concentration of HER2 for amplified and non-amplified cell lines and their corresponding cell numbers.

[0174] In more particular embodiments, HER2-low expression can be identified if the Ct value for ERBB2 expression level is at least about 23 up to about 33. In some aspects, the Ct value for ERBB2 representing HER2-low expression can be between about23 to about 30, or about 24 to about 30, or about 25 to about 30, or about 23 to about 29, or about 24 to about 29, or about 25 to about 29, or about 23 to about 28, or about 24 to about28, or about 25 to about 28, or about 23 to about 27, or about 24 to about 27, or about 25 to about 27. In some aspects, the Ct value for ERBB2 representing HER2-low expression can be about 23, or about 24, or about 24, or about 26, or about 27, or about 28, or about 29, or about 30. In some aspects, the Ct value for ERBB2 representing HER2-low expression can be at least about 23, or at least about 24, or at least about 25, or at least about 26, or at least about 27, or at least about 28, or at least about 29, or at least about 30. In some aspects, theCt value for ERBB2 representing HER2-low expression can be less than about 35.

[0175] In some embodiments, HER2-low expression can be identified if the ACt value for ERBB2 expression level is at least about -6.0 to about -0.1. In some aspects, theACt value for ERBB2 representing HER2-low expression can be between about -5.5 to about -0.1, or about -5.5 to about -0.15, or about -5.5 to about -0.20, or about -5.5 to about -0.25, about -5.0 to about -0.1, or about -5.0 to about -0.15, or about -5.0 to about -0.20, or about -5.0 to about -0.25, about -4.7 to about -0.1, or about -4.7 to about -0.15, or about -4.7 to about -0.20, or about -4.7 to about -0.25, about -4.5 to about -0.1, or about -4.5 to about -0.15, or about -4.5 to about -0.20, or about -4.5 to about -0.25, about -4.0 to about -0.1, or about -4.0 to about -0.15, or about -4.0 to about -0.20, or about -4.0 to about -0.25, about -3.9 to about -0.1, or about -3.9 to about -0.15, or about -3.9 to about -0.20, or about -3.9 to about -0.25. In some aspects, the ACt value for ERBB2 representing HER2-low expression can be less than about -0.1, or less than about -0.15, or less than about -0.20, or less than about -0.25, or less than about -0.30, or less than about -0.40, or less than about -0.50, or less than about -0.60, or less than about -0.70, or less than about -0.80, or less than about -0.9, or less than about -1.0, or less than about -1.1, or less than about -1.2, less than about -1.5, or less than about -1.7, or less than about -2. 0, or less than about -2.25, or less than about -2.5, or less than about -2.7, or less than about -3. 0, or less than about -3.2, or less than about -3.5, or less than about -3.7, or less than about -4.0, or less than about -4.2. In some aspects, the ACt value for ERBB2 representing HER2-low expression can be at least about -6.5, or at least about -6.25, or at least about -6.1, or at least about -6.0, or at leastabout -5.9, or at least about -5.8, or at least about -5.7, or at least about -5.6, or at least about-5.5, or at least about -5.4, or at least about -5.3, or at least about -5.2, or at least about -5.1, or at least about -5.0, or at least about -4.9, or at least about -4.8, or at least about -4.7, or at least about -4.6, or at least about -4.5 or at least about -4.4, or at least about -4.3, or at least about -4.2, or at least about -4.1, or at least about -4.0, or at least about -3.9, or at least about-3.8, or at least about -3.7, or at least about -3.6, or at least about -3.5, or at least about -3.4, or at least about -3.2, or at least about -3.1, or at least about -3.0. In some aspects, the ACt value for ERBB2 representing HER2-low expression can be about -5.0 to about -0.25, preferably about -3.0 to about -0.25, or more preferably about -2.5 to about -0.25. In some examples, HER2-low expression is identified if the ACt value for ERBB2 is less than about-0.25 to about -2.5. The ACt value can be determined by comparing the Ct value of the biomarker to the Ct of a control or reference marker.

[0176] In some embodiments, HER2-low expression can be identified by both Ct and ACt values. For example, HER2-low expression can be identified when the Ct value forERBB2 expression level is between about 23 to about 30, or about 24 to about 30, or about25 to about 30, or about 23 to about 29, or about 24 to about 29, or about 25 to about 29, or about 23 to about 28, or about 24 to about 28, or about 25 to about 28, or about 23 to about27, or about 24 to about 27, or about 25 to about 27 and the ACt value for ERBB2 expression level is between about -6.0 to about -0.1. In some examples, HER2-low expression can be identified when the Ct value for ERBB2 expression level is at least about23, or at least about 24, or at least about 25, or at least about 26, or at least about 27, or at least about 28, or at least about 29, or at least about 30, the ACt value for ERBB2 representing HER2-low expression is less than about -0.1, or less than about -0.15, or less than about -0.20, or less than about -0.25, or less than about -0.30, or less than about -0.40, or less than about -0.50, or less than about -0.60, or less than about -0.70, or less than about-0.80, or less than about -0.9, or less than about -1.0, or less than about -1.1, or less than about -1.2. In some examples, HER2-low expression is identified if the Ct value for ERBB2 is about 23 to about 30 and the ACt value for ERBB2 is less than about -0.25 to about -2.5.

[0177] In some embodiments, HER2 -positive expression can be identified if the ACt value for ERBB2 expression level is at least about -0.25, such as at least about -0.20, or at least about -0.15, or at least about -0.10, or at least about -0.05, or at least about 0.05, or at least about 0.10, or at least about 0.20, or at least about 0.30, or at least about 0.40, or atleast about 0.50. In some aspects, the ACt value for ERBB2 representing HER2-positive expression can be greater than about -0.25 to about 5.0, or greater than about -0.25 to about -2.5.

[0178] In some embodiments, HER2-negative expression can be identified if the ACt value for ERBB2 expression level is less than about -5.0, such as less than about -5.5, or less than about -5.75, or less than about -6.0, or less than about -6.25, or less than about -6.50, or less than about -6.75, or less than about -7.0, or less than about -7.25, or less than about -7.50, or less than about -7.75. In some aspects, the ACt value for ERBB2 representing HER2-negative expression can be less than about -5.0 to about -10.0, or less than about -5.0 to about -7.5.

[0179] In some embodiments, quantitation of the results of real-time PCR assays is done by constructing a standard curve from a nucleic acid of known concentration and then extrapolating quantitative information for target nucleic acids of unknown concentration. In some embodiments, the nucleic acid used for generating a standard curve is a DNA (for example, an endogenous control, or an exogenous control). In some embodiments, the nucleic acid used for generating a standard curve is a purified double- stranded plasmid DNA or a single- stranded DNA generated in vitro.

[0180] In some embodiments, in order for an assay to indicate that a given target nucleic acid is not present in a sample, the Ct values for an endogenous control (such as an SAC) and / or an exogenous control (such as an SPC) must be within previously-determined valid ranges. For example, in some embodiments, the absence of a particular target nucleic acid cannot be confirmed unless the controls are detected, indicating that the assay was successful.

[0181] In some embodiments, a threshold or range of Ct (or a “cutoff Ct”) values for a target nucleic acid (including an endogenous control and / or exogenous control), below which the gene is considered to be detected, has previously been determined. In some embodiments, a threshold Ct is determined using substantially the same assay conditions and system (such as a GeneXpert®) on which the samples will be tested.

[0182] Real-time PCR is performed using any PCR instrumentation available in the art. Typically, instrumentation used in real-time PCR data collection and analysiscomprises a thermal cycler, optics for fluorescence excitation and emission collection, and optionally a computer and data acquisition and analysis software.

[0183] In some embodiments, the number of target nucleic acids in an assay exceeds the number of labels that can be detected, e.g., in particular instrument. Therefore, the PCR amplification can be followed by a melt analysis to increase the number of possible reported results.

[0184] Another approach to detect target nucleic acids can include high-resolution melt. Target nucleic acids can also be detected by real-time PCR but in more than one reaction chambers. Examples of other approaches that can be employed in the methods describe herein include bead-based flow cytometric assay. See Lu J. et al. (2005) Nature 435:834-838, which is incorporated herein by reference for this description. In some embodiments, the approach for detecting a target nucleic acid does not include bead-based flow cytometric assay, microfluidic devices and single-molecule detection, simple gel electrophoresis, use of a capture probe attached to a solid-support, separation of reaction mixture into multiple reaction chambers, array-based detection, nested amplification, electrochemical detection, high resolution melt only, or a combination thereof.

[0185] Preferably, determining expression levels of the target nucleic acid (such as ERBB2) in the methods for discriminating between HER2-low expression levels, and distinguishing HER2-low expression from HER2-negative or HER2-positive expression does not include immunofluorescence & image analysis, AQUA (Automated quantitative analysis) score, or LCMS quantification.Semi- Automated and Automated Assay Methods

[0186] Readily automated approaches are of great interest in performing the methods for discriminating between HER2-low expression levels, and distinguishing HER2-low expression from HER2-negative or HER2-positive expression. The methods described herein can be carried out in a semi-automated or substantially automated manner using a commercially available nucleic acid amplification system. Exemplary nonlimiting nucleic acid amplification systems that can be used to carry out the methods include the GENEXPERT® system, a GENEXPERT® Infinity system, and GENEXPERT® Xpress System (Cepheid, Sunnyvale, Calif.). In some embodiments, the amplification system maybe available at the same location as the individual to be tested, such as a health care provider’s office, a clinic, or a community hospital, so processing is not delayed by transporting the sample to another facility. Assays according to the method described herein can be completed in under 3 hours, in some embodiments, under 2 hours, in some embodiments, under 1 hour, using an automated system, for example, the GENEXPERT® system. The GENEXPERT® utilizes a self-contained, single-use cartridge. Sample extraction, amplification, and detection may all carried out within this self-contained sample cartridge as further described below.

[0187] As described herein, the methods for discriminating between HER2-low expression levels, and distinguishing HER2-low expression from HER2-negative or HER2- positive expression can include incubating the biological sample in a lysis reagent and recovering nucleic acid from said lysis reagent. This step can be automated, for example, in the GENEXPERT® system. Accordingly, the method can include (a) placing the biological sample in a cartridge; (b) adding an FFPE lysis buffer and a proteolytic enzyme io the biological sample to form a mixture, (c) optionally heating the mixture to a temperature ranging from about 50°C to about 100°C to form a lysed mixture, (d) optionally contacting the lysed mixture with an alcohol, and (e) capturing nucleic acids from the lysed mixture onto the filter in the cartridge.

[0188] In other embodiments, the step of incubating the biological sample in a lysis reagent and recovering nucleic acid from said lysis reagent is semi-automated and can include (a) adding an FFPE lysis buffer and a proteolytic enzyme to the biological sample to form a mixture, (b) optionally heating the mixture to a temperature ranging from about 50°C to about 100°C to form a lysed mixture, (c) optionally contacting the lysed mixture with an alcohol, (d) placing the lysed mixture in a cartridge; and (e) capturing nucleic from the lysed mixture onto the filter in the cartridge.

[0189] After the sample is added to the cartridge, released nucleic acid (NA) can be bound to a NA-binding substrate, such as a silica or glass substrate. The sample supernatant is then removed, and the NA eluted in an elution buffer, such as a Tris / EDTA buffer. The eluate may then be processed in the cartridge to detect target nucleic acids as described herein. In some embodiments, the eluate is used to reconstitute at least some of the PCR reagents, which can be present in the cartridge as lyophilized particles.

[0190] A cartridge having a plurality of chambers can have the set of primers and probes described herein, or a subset thereof, disposed in a chamber. In some embodiments, the set of primers and probes described herein, or a subset thereof, are disposed in more than one of the plurality of chambers.

[0191] In some embodiments, RT-PCR is used to amplify and analyze the presence of the target nucleic acids. In some embodiments, the reverse transcription uses MMLV and / or CAT-A RT enzyme and an incubation of 5 to 20 minutes at 40°C to 50°C. In some embodiments, the PCR uses Taq polymerase with hot-start function, such as AptaTaq (Roche). In some embodiments, the initial denaturation is at 90 °C to 100 °C for 20 seconds to 5 minutes; the cycling denaturation temperature is 90°C to 100°C for 1 to 10 seconds; the cycling anneal and amplification temperature is 60 °C to 75 °C for 10 to 40 seconds; and up to 50 cycles are performed.

[0192] In some embodiments, a double-denature method is used to amplify low- copy number target nucleic acids. A double-denature method comprises, in some embodiments, a first denaturation step followed by addition of primers and / or probes for detecting target nucleic acids. All or a substantial portion of the nucleic acid-containing sample (such as a DNA eluate) is then denatured a second time before, in some instances, a portion of the sample is aliquoted for cycling and detection of the target nucleic acids.While not intending to be bound by any particular theory, the double-denature protocol may increase the chances that a low-copy number target nucleic acid (or its complement) will be present in the aliquot selected for cycling and detection because the second denaturation effectively doubles the number of target nucleic acids (i.e., it separates the target nucleic acid and its complement into two separate templates) before an aliquot is selected for cycling. In some embodiments, the first denaturation step comprises heating to a temperature of 90°C to 100°C for a total time of 30 seconds to 5 minutes. In some embodiments, the second denaturation step comprises heating to a temperature of 90°C to 100°C for a total time of 5 seconds to 3 minutes. In some embodiments, the first denaturation step and / or the second denaturation step is carried out by heating aliquots of the sample separately. In some embodiments, each aliquot may be heated for the times listed above. As a non-limiting example, a first denaturation step for an NA-containing sample (such as a DNA eluate) may comprise heating at least one, at least two, at least three, or at least four aliquots of the sample separately (either sequentially orsimultaneously) to a temperature of 90°C to 100°C for 60 seconds each. As a non-limiting example, a second denaturation step for a NA-containing sample (such as a DNA eluate) containing enzyme, primers, and probes may comprise heating at least one, at least two, at least three, or at least four aliquots of the eluate separately (either sequentially or simultaneously) to a temperature of 90°C to 100°C for 5 seconds each. In some embodiments, an aliquot is the entire NA-containing sample (such as a DNA eluate). In some embodiments, an aliquot is less than the entire NA-containing sample (such as a DNA eluate).

[0193] In some embodiments, an off-line centrifugation is used, for example, with samples with low cellular content. The sample, with or without a buffer added, is centrifuged and the supernatant removed. The pellet is then resuspended in a smaller volume of either supernatant or the buffer. The resuspended pellet is then analyzed as described herein.Exemplary Automation and Systems

[0194] Many existing fully integrated nucleic acid amplification and test systems capable of sample preparation are normally quite complicated and costly. The nucleic acid amplification and test systems provided herein perform rapid, simple, convenient, and affordable nucleic acid analysis.System Overview

[0195] In one aspect, the disclosure pertains to a sample cartridge that utilizes a valve body platform that allows for detection of enveloped and free target nucleic acids. In some embodiments, the valve body includes a sample processing region or lysing chamber that provides for either or both mechanical and chemical lysis. This allows a single cartridge to provide lysing for a multitude of differing types of targets. In some embodiments, the sample cartridge can perform processing and detection of targets requiring mechanical lysing and chemical lysing.

[0196] The sample cartridge device can be any device configured to perform one or more process steps relating to preparation and / or analysis of a biological fluid sample according to any of the methods described herein. In some embodiments, the sample cartridge device is configured to perform at least sample preparation. The sample cartridgecan further be configured to perform additional processes, such as detection of a target nucleic acid in a nucleic acid amplification test (NAAT), e.g., Polymerase Chain Reaction (PCR) assay, by use of a reaction vessel attached to the sample cartridge. In some embodiments, the reaction vessel extends from the body of the cartridge. Preparation of a fluid sample generally involves a series of processing steps, which can include chemical, electrical, mechanical, thermal, optical or acoustical processing steps according to a specific protocol. Such steps can be used to perform various sample preparation functions, such as cell capture, cell lysis, binding of analyte, and binding of unwanted material.

[0197] A sample cartridge suitable for use with the composition and methods disclosed herein, includes one or more transfer ports through which the prepared fluid sample can be transported into an attached reaction vessel for analysis. FIG. 1A illustrates an exemplary assay cartridge 100 suitable for sample preparation and analytics testing by PCR when received in an instrument module in accordance with some embodiments. The sample cartridge is attached with a reaction vessel 116 (also referred to as a “reaction tube” or “PCR tube”) adapted for analysis of a fluid sample processed within the sample cartridge 100. In some embodiments the reaction vessel extends from the cartridge body. Such a sample cartridge 100 includes various components including a main housing 102 having one or more chambers 108 for processing of the fluid sample, which typically include sample preparation before analysis. In these embodiments, the sample cartridge can be a fully integrated nucleic acid amplification and test system combining sample preparation, amplification, and detection together. The instrument module facilitates the processing steps needed to perform sample preparation and the prepared sample is transported through one of a pair of transfer ports into fluid conduit of the reaction vessel 116 attached to the housing of the sample cartridge 100. The prepared biological fluid sample is then transported into a reaction chamber of the reaction vessel where the biological fluid sample undergoes nucleic acid amplification. In some embodiments, the amplification is a polymerase chain reaction. In some embodiments, concurrent with the amplification of the biological fluid sample, an excitation means, and an optical detection means of the module is used to detect optical emissions that indicate the presence or absence of a target nucleic acid analyte of interest. It is appreciated that such a reaction vessel could include various differing chambers, conduits, or micro-well arrays for use in detecting the target analyte. The sample cartridge can be provided with means to perform preparation of the biologicalfluid sample before transport into the reaction vessel. Any chemical reagent required for cell lysis or means for binding or detecting an analyte of interest (e.g., reagent beads) can be contained within one or more chambers of the sample cartridge, and as such can be used for sample preparation.

[0198] An exemplary use of a reaction vessel for analyzing a biological fluid sample is described in commonly assigned U.S. Patent No. 6,818,185, entitled “Cartridge for Conducting a Chemical Reaction,” filed May 30, 2000, the entire contents of which are incorporated herein by reference for all purposes. Examples of the sample cartridge and associated modules are shown and described in U.S. Patent No. 6,374,684, entitled “FluidControl and Processing System” filed August 25, 2000, and U.S. Patent No, 8,048,386, entitled “Fluid Processing and Control,” filed February 25, 2002, U.S. Patent Application No. 63 / 217,672 entitled “Universal Assay Cartridge and Methods of Use” filed July 1, 2021; U.S. Provisional Application No. 63 / 319,993 entitled “Unitary Cartridge Body and Associated Components and Methods of Manufacture” filed March 15, 2022; and U.S. Patent No. 10,562,030 entitled “Molecular Diagnostic Assay System” filed July 22, 2016; the entire contents of which are incorporated herein by reference in their entirety for all purposes.

[0199] Various aspects of the sample cartridge 100 can be further understood by referring to U.S. Patent No. 6,374,684 (“the ‘684 patent”), which described certain aspects of a sample cartridge in greater detail. Such sample cartridges can include a fluid control mechanism, such as a rotary fluid control valve assembly, that is fluidically connected to the chambers of the sample cartridge. The term “chamber” can be used interchangeably with the terms “well”, “tube”, and the like. Rotation of the rotary fluid control valve permits fluidic communication between chambers and the valve so as to control flow of a biological fluid sample deposited in the cartridge into different chambers in which various reagents can be provided according to a particular protocol as needed to prepare the biological fluid sample for analysis. To operate the rotary valve, the cartridge processing module comprises a motor such as a stepper motor that is typically coupled to a drive train that engages with a feature of the valve in the sample cartridge to control movement of the valve in coordination with movement of the syringe, thereby resulting movement of the fluid sample according to the desired sample preparation protocol. The fluid metering and distribution function of therotary valve according to a particular sample preparation protocol is demonstrated in the ‘684 patent.Exemplary Assay Cartridge and Valve AssembliesOverview

[0200] As shown in FIG. 1A, the test cartridge 100 comprises a cartridge body 102 containing a plurality of chambers 108 for reagents or buffers and sample processing. The chambers are disposed around a central syringe barrel 106 that is in fluid communication with a valve body 110 (see FIGS. IB and 1C) and that is sealed with a gasket 104. The valve body 110 can include a cap 112 and the entire cartridge body can be supported on a cartridge base 101. The valve body typically contains one or channels or cavities (chamber(s) 114) that can contain a filter as described herein that can function to bind and elute a nucleic acid. In some embodiments the cartridge further comprises one or more temperature-controlled channels or chambers that can, in certain embodiments, function as thermocycling chambers. A “plunger” not shown can be operated to draw fluid into the syringe barrel 106 and rotation of the valve body 110 provides selective fluid communication between the various reagent chambers 108 and channels, reaction chamber(s), mixing chambers, and optionally, any temperature controlled regions. Thus, the various reagent chambers 108, reaction chambers, filter material(s), and temperature- controlled chambers or channels are selectively in fluid communication by rotation of the plunger and reagent movement (e.g., chamber loading or unloading) is operated by the “syringe” action of the plunger within the valve assembly. In other embodiments, the various reagent chambers, reaction chambers, filter material, and temperature-controlled chambers or channels are selectively in fluid communication by linear progression (e.g., by forced movement) of the reagents and sample from one chamber to the next.Reaction Modules

[0201] In certain embodiments the cartridge is configured for insertion into a reaction module. The module is configured to receive the cartridge therein. In certain embodiments the reaction module provides heating plates to heat the temperature controlled chamber or channel. The module can optionally additionally include a fan to providecooling where the temperature controlled channel or chamber is a thermocycling channel or chamber. Electronic circuitry can be provided to pass information (e.g., optical information) top a computer for analysis. In certain embodiments the module can contain optical blocks to provide excitation and / or detection of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) optical signals representing, e.g., signal DNAs amplified for various PCR targets. In various embodiments an electrical connector can be provided for interfacing the module with a system e.g. system controller or with a discrete analysis / controller unit. In certain embodiments, the module also contains a controller that operates a plunger in the syringe barrel and the rotation of the valve body.Analytical System

[0202] In certain embodiments a system (e.g., a processing unit) is provided. The system includes an enclosure that is configured to support and power multiple sample processing modules, where each processing module is configured to hold and operate a removable cartridge. In some embodiments, the system is configured to operate the sample processing modules to perform a PCR assay for one or more target nucleic acid analytes and optionally to determine the level of one or more target RNA / DNA sequences within a corresponding removable sample cartridge. Typically, the processing on a sample within the corresponding removable sample cartridge involves operating the cartridge to perform a method as described herein. In certain embodiments the system is configured to contain one sample processing module. In certain embodiments the system is configured to contain at least two or more sample processing modules (e.g., at least 4, 8, 12, 16, 20, 24, 28, 32, 64, 128 or more) sample processing modules. In some embodiments, the system provides a user interface that allows the user input operational instructions and / or to monitor operation of the cartridges to determine the presence and / or quantity of one or more nucleic acids.

[0203] While the methods described herein are described primarily with reference to the GENEXPERT® cartridge by Cepheid Inc. (Sunnyvale, Calif.) (see, e.g., FIG. 1A), it will be recognized, that in view of the teachings provided herein the methods can be implemented on other cartridge / microfluidic systems, including alternative cartridge designs having valve assemblies that involve multiple interfacing components, as well as cartridge body defined by multiple interfacing components to form the multiple chambers of the cartridges, for example, those described in Korean Application No. 102293717B1 andKR102362853B1, cartridges that utilizes ultrasonic waves to lyse cells in a biological sample, for example, those described in International Application No. WO2021 / 245390A1, cartridges and systems that utilizes an electrowetting grid for microdroplet manipulation and electrosensor arrays configured to detect analytes of interest, for example, those described in International Application No. WO2016 / 077341A2, cartridges that facilitate movement of nucleic acid from one chamber to the next chamber by opening a vent pocket, for example, those described in International Application No. WO2012 / 145730A2, multiplexed assay systems comprising a plurality of thermocycling units such that individual chambers can be heated, cooled, and / or compressed to mix fluid within the chamber or to propel fluid in the chamber into another chamber, for example, those described in International Application No. WO2Q15 / 138343A1, and as well as systems for rapid amplification of nucleic acids facilitated by flexible portions of the sample cartridge aligned to accomplish temperature cycling for nucleic acid amplification, for example, those described in International Application No.WO2017 / 147085Al. Such cartridge / microfluidic systems can include, for example microfluidic systems implemented using soft lithography, micro / nano-fabricated microfluidic systems implemented using hard lithography, and the like.

[0204] In certain embodiments, the cartridge for detecting and discriminating between HER2-low expression levels, and distinguishing HER2-low expression from HER2-negative or HER2-positive expression in a biological sample from a subject with cancer can comprise (a) a cartridge body comprising a plurality of chambers therein, wherein the plurality of chambers includes: (i) a sample chamber having at least a fluid outlet in fluid communication with another chamber of the plurality; and (ii) an optional lysis chamber in fluidic communication with the sample chamber, optionally wherein the sample chamber and lysis chamber are the same; (b) a reaction vessel fluidically coupled to the plurality of chambers of the cartridge body and configured for: i) amplification of nucleic acid and ii) detection and identification of a plurality of amplification products via real-time PCR; (c) a filter disposed in a fluidic path between the lysis chamber, if present, or the sample chamber, and the reaction vessel, and (d) a set of primers and probes for detecting the target nucleic acid (such as ERBB2).

[0205] In certain embodiments of the cartridge described herein, the lysis chamber comprises one or more lysis reagents for releasing nucleic acid. The reaction vessel can comprise one or more reaction chambers for amplification and detection of theamplification products. In some examples, the reaction vessel comprises one reaction chamber for amplification and detection of the amplification products. Each reaction chamber is configured to detect a single amplification product and / or a plurality of amplification products. In some embodiments, the cartridge is a Clinical Laboratory Improvement Amendments (CLIA)-compliant cartridge.

[0206] Additionally, it is appreciated that the assay methods described herein can further be realized in entirely different systems, including: isothermal nucleic acid amplification systems, digital RT-PCR, electrochemical PCR, lateral flow testing cartridges, electrochemical sensors, nucleic acid sequencing, CRISPR / Cas based technologies, chemiluminescence, and nanoparticle-based colorimetric detection.

[0207] In various embodiments, the signal DNA(s) from PCR (nucleic acid amplification) reactions are amplified for detection and quantification. In certain embodiments, the amplification comprise any of a number of methods including, but not limited to polymerase chain reaction (PCR), ligase chain reaction (LCR), ligase detection reaction (LDR), multiplex ligation-dependent probe amplification (MLP A), ligation followed by Q-replicase amplification, primer extension, strand displacement amplification (SDA), hyperbranched strand displacement amplification, multiple displacement amplification (MDA), nucleic acid strand-based amplification (NASBA), rolling circle amplification (RCA), and the like.

[0208] In illustrative, but non-limiting embodiments, the amplification reaction may produce an optical signal that is proportional to the amount of amplified target nucleic acid (e.g., signal DNA). Illustrative optical signals include, but are not limited to a fluorescent signal, a chemiluminescent signal, an electrochemiluminescent signal, a colorimetric signal, and the like. In certain embodiments the optical signal is a fluorescent optical signal generated by a fluorescent indicator. In certain embodiments the fluorescent indicator is a non-specific intercalating dye that binds to double-stranded DNA products, while in certain other embodiments, the fluorescent indicator comprises a target sequence- specific probe (e.g., a TAQMAN® probe, a SCORPION® probe, a MOLECULAR BEACON®, and the like).

[0209] Single PCR reactions (nucleic acid amplification), or multiple PCR reactions (nucleic acid amplifications) run sequentially (or simultaneously in separate temperaturecontrolled channels or chambers) can also use the same detectable label since sequentially run PCR signal DNAs are analyzed sequentially and the simultaneous PCR signal DNAs are distinguished by the occurrence in different temperature controlled channels or chambers. The signal produced by this amplification can be distinguished from other amplification products because it is not run at the same time and / or because it is run in a different reaction channel / chamber. However, where multiple nucleic acid amplifications are run simultaneously in the same chamber the reaction products of for each analysis are typically detected and / or quantified by the use of different and distinguishable labels.

[0210] In certain embodiments, amplification products (amplified nucleic acid from nucleic acid analysis) can be detected using methods well known to those of skill in the art. In certain embodiments the amplification is a straightforward simple PCR amplification reaction. In certain embodiments, however, a nested PCR reaction is used to amplify the nucleic acid from the nucleic acid analysis. In various embodiments, multiplexed PCR assays are contemplated, particularly where it is desired to analyze multiple products of the nucleic acid analysis in the same amplification reaction. In certain embodiments in such multiplexed amplification reactions, each probe (e.g., for each specific analyte) has its own specific dye / fluor so that it is detectable independently of the other probes. In certain embodiments, typically, for signal generation, the probes used in various amplification reactions utilize a change in the fluorescence of a fluorophore due to a change in its interaction with another molecule or moiety brought about by changing the distance between the fluorophore and the interacting molecule or moiety for detection and / or quantification of the amplified product. Alternatively, other methods of detecting a polynucleotide in a sample, including, but not limited to, the use of radioactively labeled probes, are contemplated.Exemplary Assay ConfigurationsReagents for Biomarker Panel Assay

[0211] As described herein, the nucleic acid can be bound to a nucleic acid-binding substrate, also referred to herein as a filter. In some examples, the filter comprises glass fibers and optionally a polymeric binder. The glass fibers may be modified with a nucleic acid binding ligand such as an alkylamine, a cycloalkylamine, an alkyloxy amine, apolyamine moiety, an arylamine, an intercalating agent, a DNA groove binder, a peptide, an amino acid, a protein, or a combination thereof. In some examples, the filter comprises a 500 micron to 2000 microns thick glass fiber disk having a pore size of 0.2 microns to 1 micron. In some aspects, the sample can be contacted with a binding reagent, wash reagent, or a combination during or after lysis. The binding reagent can promote binding of nucleic acids to the filter, facilitating the removal of non-target material. In some embodiments, the binding reagent can include a binding polymer such as polyacrylic acid (PAA), polyacrylamide (PAM), polyethylene glycol (PEG), poly(sulfobetaine), or a salt, or combinations thereof. In some embodiments, the filtering reagent and / or the washing reagent can include the binding reagent. For example, the binding reagent, the filtering reagent, and / or the washing reagent can include a binding polymer (e.g., PEG 200), buffer, inorganic salt(s), antioxidant and / or chelating agent, antifoam SE15, sodium azide, disaccharide or disaccharide derivative, earner protein, a chaotropic agent (such as guanidium hydrochloride) detergent, DMSO, or a combination thereof. The binding polymer can be present in an amount of at least 10% v / v, at least 20% v / v, at least 30% v / v, and / or less than 60% v / v, less than 40% v / v, less than 30% v / v, less than 20% v / v, or less than 10% v / v or can fall within any range bounded by any of these values, e.g., from 10% to 60% v / v, of the binding reagent, filtering reagent, and / or the washing reagent. The buffer can be selected from the group consisting of Tris, 2-amino-2-hydroxymethyl-l,3- propanediol, HEPES, phosphate buffer, PBS, citrate buffer, TAPS, Bicine, Tricine, TAPSO, HEPES, TES, MOPS, PIPES, Cacodylate, SSC, and MBS. The concentration of the buffer can range from about 5 mM to about 100 mM, such as from about 5 mM to about 50 mM. The salt, such as NaCl, KC1, or MgCh, can be present at a concentration from about 0.05 M to about 1 M, such as from about 0.1 M to about 0.5 M. The antioxidant and / or chelating agent comprises an agent selected from the group consisting of N-acetyl-L-cysteine, ethylenediaminetetraacetic acid (EDTA), diethylene triamine pentaacetic acid (DTP A), ethylenediamine-N,N'-disuccinic acid (EDDS), l,2-bis(o-aminophenoxy)ethane-N,N,N',N'- tetraacetic acid (BAPTA), and a phosphonate chelating agent. In some embodiments the antioxidant and / or chelating agent comprises EDTA. In certain embodiments the antioxidant and / or chelating agent comprise 0.2% to about 5%, about 0.2% to about 3%, or about 0.5% to about 2%, or about 0.5% of the binding reagent, filtering reagent, and / or the washing reagent. In some embodiments the concentration of the antioxidant and / or chelating agent inthe binding reagent, filtering reagent, or the washing reagent ranges from about 2 mM to about 50 mM or about 5 mM to about 20 mM. In some embodiments, the detergent is an ionic detergent or a non-ionic detergent. The detergent can be selected from an ionic detergent or a non-ionic detergent. In some examples, the detergent comprises a detergent selected from the group consisting of N-lauroylsarcosine, sodium dodecyl sulfate (SDS), cetyl methyl ammonium bromide (CT AB), TRITON®-X-100, n-octyl-|3-D- glucopyranoside, CHAPS, n-octanoylsucrose, n-octyl-P-D-maltopyranoside, n-octyl-P-D- thioglucopyranoside, PLURONIC® F-127, TWEEN® 20, Brij-35, and n-heptyl-P-D- glucopyranoside. The detergent can comprise about 0.1% to about 2% of the binding reagent, filtering reagent, and / or the washing reagent, and / or ranges from about 10 mM up to about 100 mM. The binding reagent, filtering reagent and / or the washing reagent can have a pH ranging from about pH 6.0 to about pH 8.0 (such as from about 6.5 to about 7.5).

[0212] The sample supernatant is then removed and the nucleic acid is eluted in an elution buffer such as a Tris / EDTA buffer. The elution buffer can comprise ammonia or an alkali metal hydroxide. In general, the elution buffer has a pH above about 9, above about 10, or above about 11. The elution buffer can further comprise a polyanion, optionally a carrageenan, a earner nucleic acid, or i-carrageenan and KOH. The eluate may then be processed in the cartridge to detect target genes as described herein. In some embodiments, the eluate is used to reconstitute at least some of the PCR reagents, which are present in the cartridge as lyophilized particles. Particularly, the lyophilized particles can be in the form of beads and comprise primers, probes, a salt, dNTPs, a thermostable polymerase, a reverse transcriptase, or a combination thereof. The lyophilized can be present in the reaction vessel of the cartridge.

[0213] As would be appreciated by the skilled artisan, a Ct value is the number of cycles in a quantitative PCR experiment that are required for the fluorescent signal associated with the amplification of a specific target nucleic acid to exceed a predetermined threshold value. As would be appreciated by the skilled artisan, this threshold value can be the background fluorescence levels measured in the experiment.

[0214] The methods described herein can be carried out at the same facility where the biological sample was collected from a subject. For example, the method can be a point- of-care method. In other instances, the method can be carried out in a hospital, an urgent care center, an emergency room, a physician’s office, a health clinic, or a home. In furtherinstances, the method is a Clinical Laboratory Improvement Amendments (CLIA)-waived test. In some embodiments, information concerning the diagnosis of HER2 expression level in the subject is communicated to a medical practitioner. A “medical practitioner,” as used herein, refers to an individual or entity that diagnoses and / or treats patients, such as a hospital, a clinic, a physician’s office, a physician, a nurse, or an agent of any of the aforementioned entities and individuals. In some embodiments, the methods are carried out at a laboratory that has received the subject’s sample from the medical practitioner or agent of the medical practitioner. The laboratory carries out the detection by any method, including those described herein, and then communicates the results to the medical practitioner. A result is “communicated,” as used herein, when it is provided by any means to the medical practitioner. In some embodiments, such communication may be oral or written, may be by telephone, in person, by e-mail, by mail or other courier, or may be made by directly depositing the information into, e.g., a database accessible by the medical practitioner, including databases not controlled by the medical practitioner. In some embodiments the result of the assay is combined with clinical parameters, data, or information about other risk factors to make a diagnosis. In some embodiments, the information is maintained in electronic form. In some embodiments, the information can be stored in a memory or other computer readable medium, such as RAM, ROM, EEPROM, flash memory, computer chips, digital video discs (DVD), compact discs (CDs), hard disk drives (HDD), magnetic tape, etc. The results may also be provided using a web-based application that may be provided to the health care practitioner or to the patient on a smart phone or other mobile device. In some aspects, results may be provided to the patient via a mobile device.

[0215] In some embodiments, the method further comprises receiving a communication from the laboratory that indicates the diagnosis in the sample. A “laboratory,” as used herein, is any facility that detects the target gene in a sample by any method, including the methods described herein, and communicates the result to a medical practitioner. In some embodiments, a laboratory is under the control of a medical practitioner. In some embodiments, a laboratory is not under the control of the medical practitioner.Exemplary Detection Methods, Results, and Handling of Results

[0216] HER2-low determination can be performed as an mRNA assay on Cepheid’s GENEXPERT® systems. Whole tissue sections can be used for the method. A single 4-pm FFPE section can be placed into a 1.5-mL tube as a scroll or scraped with a scalpel blade from an unstained section placed on a glass microscope slide. FFPE lysis reagent and proteinase K can be added to the sample. The sample is then mixed and incubated (for e.g., at 80°C for 30 minutes). Ethanol can be optionally added to the sample. Following each lysate preparation method, an aliquot of the FFPE lysate can be added to the cartridge (if lysate was prepared off-board), and the cartridge placed in a GeneXpert module. Each single-use, disposable cartridge can be preloaded with all necessary RT-PCR assay reagents, including wash buffers, elution buffers, and lyophilized beads containing primers, enzymes, and nucleotides, including primers and probes for quantitative real-time PCR measurement of the ERBB2 mRNA marker and one reference gene (for e.g., CYFIP1). The cartridge integrates sample purification, nucleic acid amplification, and detection of the target sequence using real-time RT-PCR and real-time PCR assays. The mRNA assay may take about 75 minutes or less to amplify a portion of the ERBB2 mRNA and reference mRNA within the sample and to generate the test results. The GX system includes the GeneXpert instrument, barcode reader, a computer, and preloaded software for running tests and viewing the results. For each sample, cycle threshold (Ct) value for the target gene can be obtained, along with simultaneously measured Ct values for the reference gene and an internal control gene (CIC). The delta Ct value for each target is calculated by reference gene Ct - target gene and is HER2 -positive, HER2 -negative, HER2-low, HER2-ultralow, or indeterminate for expression of ERBB2, based on validated assay cutoffs and sample adequacy specifications. In some cases, final positive, negative, or low results can be analyzed for concordance (OPA, positive percent agreement, and negative percent agreement) vs IHC and / or FISH measurements for the same marker.

[0217] Before the start of the PCR reaction, the GENEXPERT® System measures the fluorescence signal from the probes to monitor bead rehydration, reaction tube filling, probe integrity, and dye stability. This Probe Check Control (PCC) passes if it meets validated acceptance criteria.

[0218] In some embodiments, a computer-based analysis program is used to translate the raw data generated by the detection assay into data of predictive value for a clinician. The clinician can access the predictive data using any suitable means. Thus, in some embodiments, the present invention provides the further benefit that the clinician, who is not likely to be trained in genetics or molecular biology, need not understand the raw data. The data is presented directly to the clinician in its most useful form. The clinician is then able to immediately utilize the information in order to optimize the care of the subject.

[0219] When the GENEXPERT® System is used, the results are interpreted automatically and are shown in a “View Results” window.

[0220] The present disclosure contemplates any method capable of receiving, processing, and transmitting the information to and from laboratories conducting the assays, information provides, medical personal, and subjects. For example, in some embodiments of the present invention, a sample is obtained from a subject and submitted to a testing service (e.g., clinical lab at a medical facility, genomic profiling business, etc.), located in any part of the world (e.g., in a country different than the country where the subject resides or where the information is ultimately used) to generate raw data. Where the sample comprises a tissue or other biological sample, the subject may visit a medical center to have the sample collected and sent to the testing service, or subjects may collect the sample themselves and directly send it to a testing service. Where the sample includes previously determined biological information, the information may be directly sent to the testing service by the subject (e.g., an information card containing the information may be scanned by a computer and the data transmitted to a computer of the profiling center using an electronic communication systems). Once received by the testing service, the sample is processed and a set of test results is produced, specific for the diagnostic or prognostic information desired for the subject.

[0221] The test results can be prepared in a format suitable for interpretation by a treating clinician. For example, rather than providing raw expression data, the prepared format may represent a diagnosis or risk assessment for the subject, with or without recommendations for particular treatment options. The test results may be displayed to the clinician by any suitable method. For example, in some embodiments, the testing servicegenerates a report that can be printed for the clinician (e.g., at the point of care) or displayed to the clinician on a computer monitor.

[0222] In some embodiments, the information is first analyzed at the point of care or at a regional facility. The raw data is then sent to a central processing facility for further analysis and / or to convert the raw data to information useful for a clinician or patient. The central processing facility provides the advantage of privacy (all data is stored in a central facility with uniform security protocols), speed, and uniformity of data analysis. The central processing facility can then control the fate of the data following treatment of the subject. For example, using an electronic communication system, the central facility can provide data to the clinician, the subject, or researchers.

[0223] In some embodiments, the subject is able to directly access the data using the electronic communication system. The subject may choose further intervention or counseling based on the results. In some embodiments, the data is used for research use. For example, the data may be used to further optimize the inclusion or elimination of markers as useful indicators of a particular condition or stage of disease or as a companion diagnostic to determine a treatment course of action.

[0224] In some embodiments, the methods disclosed herein comprise administering a treatment regimen to the subject based on the determined expression level. The subject can be treated with a HER2-targeted drug therapy, preferably a HER2-low targeted drug therapy. For example, the HER2-targeted drug therapy is an antibody-drug conjugate (ADC) such as, trastuzumab deruxtecan (T-DXd), trastuzumab, pertuzumab, trastuzumab emtansine (T-DMI), or combinations thereof. The specific treatment can depend on the specific type of cancer present in the subject. In some examples, the subject is diagnosed with a cancer selected from breast cancer, gastric cancer, lung cancer, esophageal carcinoma, bladder cancer, colon cancer, or a combination thereof.Kits0225] Also contemplated is a kit for carrying out the methods described herein. Such kits include one or more reagents useful for practicing any of these methods. A kit generally includes a package with one or more containers holding the reagents, as one or more separate compositions or, optionally, as an admixture where the compatibility of thereagents will allow. The kit can also include other material(s) that may be desirable from a user standpoint, such as a buffer(s), a diluent(s), a standard(s), and / or any other material useful in sample processing, washing, or conducting any other step of the assay.

[0226] Kits preferably include instructions for carrying out one or more of the screening methods described herein. Instructions included in kits can be affixed to packaging material or can be included as a package insert. While the instructions are typically written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user can be employed. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. As used herein, the term “instructions” can include the address of an internet site that provides the instructions.

[0227] In some embodiments, a kit includes primer pairs for amplifying and / or detecting the above-described biomarker targets, optionally with probes specific for these targets. In some embodiments, these kits can include primers pairs and optional probes for detecting one or more of the above-described controls.

[0228] In some embodiments, the kit can include any of the reagents described above provided with or in one or more GENEXPERT® cartridge(s). See e.g., US Patents 5,958,349; 6,403,037; 6,440,725; 6,783,736; 6,818, 185; each of which is herein incorporated by reference for this description).

[0229] In certain embodiments, the kit for detecting and distinguishing HER2-low expression from HER2-negative or HER2-positive expression can comprise a set of primers and probe for detecting ERBB2 as described herein. The kit can further comprise a set of primers and optional probe for detecting ESRI, PGR, or MKi67 mRNA biomarker, or a combination thereof. The kit may further comprise one or more lysis reagents for releasing nucleic acid from the biological sample. For example, the kit can comprise one or more lysis reagents which comprise an FFPE lysis buffer, a proteolytic enzyme, optionally an alcohol, and optionally an oil or bovine serum albumin.

[0230] In certain embodiments, the kits are provided for the extraction of a nucleic acid from a cell and / or tissue sample. In certain embodiments the kit will typically comprises a container containing a lysis reagent as described herein. In certain embodiments the kit further comprises a container containing a protease (e.g., proteinase K, trypsin,chymotrypsin, papain, etc.). In certain embodiments the protease and the lysis reagent are mixed together. In certain embodiments the protease and the lysis reagent are provided in separate containers.

[0231] In certain embodiments the kit can further comprise a device for the collection of a cell or tissue sample. Illustrative devices include, but are not limited to a device selected from the group consisting of a device or device tip for performing a scrape, a wipe, a device or device tip for obtaining an aspirate, a punch biopsy device, and a blade for obtaining a skin biopsy. For example, in certain embodiments, the kit comprises a device or device tip for obtaining a fine needle aspirate and / or for obtaining a vacuum assisted aspirate. In certain embodiments the kit comprises a device for performing a buccal scrape, or a gynecological scrape. Illustrative devices include, but are not limited to a multispatula, an extended tip spatula, a cytobrush, a cytopick, a cervexbrush, swab, a baynebrush, a profilebrush, a bulb aspirator, an Ayre spatula, an Aylesbury device, and the like. In typical embodiments the device for collection of a cell or tissue sample is provided in packaging that preserves sterility of the sample collecting device before use.

[0232] In certain embodiments, the kit can comprise a container configured to receive a cell or tissue sample and to store that sample in said lysis reagent or in a buffer. In certain embodiments the container configured to receive a cell or tissue sample is configured for storage and / or shipping. Thus, in certain embodiments, the container configured to receive a cell or tissue sample, is provided with a label to identify the sample, and, in certain embodiments sealable packaging to hold the container during storage and / or shipping and / or a shipping container.

[0233] In certain embodiments, the kit can optionally further include a sterile swab (e.g., an alcohol swab) for cleaning the sample site, and / or a drying pad (e.g., a gauze pad) for drying the site, and / or a dressing (e.g. bandage) for dressing the site after obtaining the sample.

[0234] In certain embodiments, the components for a single collection operation are packaged together in a packet. Such packets can include, for example, a single use disposable sample device, optionally a sterile swab, optionally a drying pad, and optionally a dressing. In certain embodiments the kit includes at least 2 packets, or at least 3 packets,or at least 4 packets, or at least 5 packets, or at least 6 packets, or at least 7 packets, or at least 8 packets.

[0235] In certain embodiments, the kit can further contain instructional materials teaching collection methods utilizing the kit components and, optionally, providing guidance to overcome problems that may occur during collection. The instructional materials can also include information and / or instructions regarding the use of the lysis reagent and / or instructions for the collection, and / or storage, and / or shipping of a cell or tissue sample. In certain embodiments the kits additionally contain reagents and / or instructions teaching the use of the lysis buffer for isolation and recovery of a nucleic acid.

[0236] Often and typically the instructional materials are provided in written form and can be printed on the kit components themselves (e.g. on the cover of a box, container, or on an envelope), or can be provided as an insert / instructional page or booklet. While the instructional materials typically comprise written or printed materials, they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials.EXAMPLESExample 1:

[0237] Human epidermal growth factor receptor 2 (ERBB2 / HER2) is an important predictive biomarker for oncology therapy decisions. With the advent of a new class of HER2-targeted therapies called HER2- Antibody Drug Conjugates (HER2-ADC), the assessment of HER2 status has changed, requiring pathologists to not only reliably differentiate HER2 positive vs HER2 negative breast cancer, but to now identify “HER2- low” tumors that may be eligible for treatment with a HER2-ADC. HER2-low tumors in this new paradigm are identified as being HER2 IHC 1+ or 2+ / FISH-negative.

[0238] Exemplified herein is a method for detecting and distinguishing human epidermal growth factor receptor 2 (HER2)-low expression from HER2-negative or HER2- positive expression in a biological sample. For IVDx tests and assays using nucleic acidextraction methods from different specimens as a pre-step to amplification and detection of targets (using PCR, RT-PCR in this case), there can be significant losses of the small nucleic acids during sample preparation steps, particularly in cases where the free nucleic acids are spiked into the test to serve as assay controls, for verification studies and for analytical validation of assay limits, reproducibility and precision. The examples provided herein demonstrated improved RNA recovery during sample preparation steps. Particularly, various additives were used in the lysis method to improve free RNA recovery in the cartridge. Firstly, melted paraffin sections were used to act as potential carrier or blanking agent in a proprietary reagent mixture, which enabled improved recovery and precision in detection of small nucleic acids.

[0239] RNA recovery and detection : RNA was spiked into 650uL of paraffin lysate background matrix (blank paraffin and inactivated Proteinase K in a mixture of 1:1 parts PEPE Lysis Reagent and pure ethanol) and added to the sample chamber of the GENEXPERT® cartridge. The entire 650uL contents was passed through the sample preparation steps, including passing through the cartridge filter. Various studies have shown there is -2-3 logs of RNA lost during the sample preparation steps (compared to control that bypasses sample preparation steps), leading to delayed cycle thresholds and increased assay variation. The results below demonstrated improve RNA recovery during sample preparation steps using the paraffin lysate mixture described herein.

[0240] Contrived matrix additives: Described below are additives that were used to formulate a stable, clear contrived matrix that aid in improved recovery and detection of nucleic acids. Tables 5 and 6 show results of Xpert® prototype test target Ct values in mock lysate spiked with IVT.1) Mineral oil as an additive in Mock Lysate at 0.05-0.07% resulted in 0.4-1.0 cycle Ct gain in open carts. There was no penalty in the probe check and EPF values at these concentrations of mineral oil.2) Liquid paraffin at 0.05-0.07% along with proteinase Kinase enzyme (range 0.09- 0.1%) also improved the Cts (1.2- 0.4 cycle lower) when tested in open carts.3) BSA (Bovine Serum Albumin) at a concentration range of 0.1 to 0.04% as an additive improved the recovery and stability of IVTs spiked in Mock Lysate. BSA at a concentration of 0.083% aided in gaining 0.6- 1.2 cycles for all targets when the IVT panel 3 spiked mock lysate was stored at RT for up to 5 days.Table 5: Xpert® prototype test target Ct values in mock lysate spiked with IVT.p ; ; pTable 6: Xpert® prototype test target Ct values in mock lysate spiked with IVT, stores and tested at specific temperature and time points.

[0241] Detection of HER2 low samples: Xpert® prototype test detects and reports presence or absence of ERBB2 (HER2) gene based on the dCt values, where in values lower than -1.0 indicates HER2 -ve and values greater than -1.0 are classified as HER2 4-ve sample. ATCC cancer cell lines with quantified HER2 protein levels were used to prepare a standard curve with dCt values in prototype assay. HER2 expression was precisely detected in low and very low HER2 cell lines using prototype test as illustrated in Table 7. Table 8 shows a comparison of ERBB2 copy number to corresponding Ct values for HER2-low cell lines.Table 7: dCt values for HER2-low cell lines in Xpert® prototype testTable 8: Comparison of ERBB2 copy number to corresponding Ct values for HER2-low cell lines.

Claims

CLAIMS1. A method for identifying human epidermal growth factor receptor 2 (HER2) low expression in a biological sample from a subject, the method comprising: a) incubating the biological sample in a lysis reagent and recovering nucleic acid from said lysis reagent, b) contacting the nucleic acid with a set of primers and probes for detecting the presence of an ERBB2 biomarker; c) subjecting the nucleic acid, primers, and probes to amplification conditions; d) detecting ERBB2, expressed as a Ct value or a ACt value, wherein ACt is theCt value for a reference gene minus the Ct value for the ERBB2 biomarker; e) comparing the Ct value or the ACt value to a range of Ct values or ACt values, and f) identifying HER2-low expression when the Ct value or the ACt value for ERBB2 is within a first range of Ct values or ACt values.

2. The method of claim 1, comprising identifying HER2-ultralow expression in the biological sample.

3. The method of claim 1 or 2, further comprising distinguishing HER2-low expression from HER2-negative or HER2-positive expression in the biological sample by identifying:HER2-negative expression when the Ct value or the ACt value for ERBB2 is within a second range of Ct values or ACt values; orHER2-positive expression when the Ct value or the ACt value for ERBB2 is within a third range of Ct values or ACt values.

4. A method for identifying human epidermal growth factor receptor 2 (HER2) expression and distinguishing HER2-low expression, HER2-negative expression, and HER2-positive expression in a biological sample from a subject, the method comprising: a) incubating the biological sample in a lysis reagent and recovering nucleic acid from said lysis reagent,b) contacting the nucleic acid with a set of primers and probes for detecting the presence of an ERBB2 biomarker; c) subjecting the nucleic acid, primers, and probes to amplification conditions; d) detecting ERBB2, expressed as a Ct value or a ACt value, wherein ACt is theCt value for a reference gene minus the Ct value for the ERBB2 biomarker; e) comparing the Ct value or the ACt value to a range of Ct values or ACt values, and f) identifying a. HER2-low expression when the Ct value or the ACt value for ERBB2 is within a first range of Ct values or ACt values, b. HER2-negative when the Ct value or the ACt value for ERBB2 is within a second range of Ct values or ACt values; or c. HER2 -positive expression when the Ct value or the ACt value for ERBB2 is within a third range of Ct values or ACt values.

5. The method of any one of claims 1-4, wherein the biological sample is a formalin fixed paraffin-embedded (FFPE) sample, an in vitro transcribed (IVT) RNA, a tissue sample, cells, a biopsy, or combination thereof.

6. The method of any one of claims 1-5, wherein HER2-low expression is identified if(i) the Ct value or the ACt value for ERBB2 expression level corresponds to a peptide quantity between about 2 and about 20 attomole / mm2,(ii) the Ct value or the ACt value for ERBB2 expression level corresponds to a FISH score of less than about 6 signals / cell based on HER2 copy number,(iii) the Ct value for ERBB2 expression level is about 23 to about 30, or(iv) the ACt value for ERBB2 expression level is about -5.0 to about -0.25, about-3.0 to about -0.25, or about -2.5 to about -0.25.

7. The method of claim 6, wherein HER2-low expression is identified if the Ct value for ERBB2 is about 23 to about 30 or the ACt value for ERBB2 is less than about -0.25 to about -2.5.

8. The method of claim 6 or 7, wherein HER2-low expression is identified if the Ct value for ERBB2 is about 23 to about 30 and the ACt value for ERBB2 is less than about -0.25 to about -2.5.

9. The method of claim 7 or 8, wherein HER2-low expression is identified if the ACt value for ERBB2 is about -0.25 to about -2.5.

10. The method of any one of claims 1-9, wherein incubating the biological sample in a lysis reagent and recovering nucleic acid from said lysis reagent comprises: a) adding an FFPE lysis buffer and a proteolytic enzyme to the biological sample to form a mixture, b) heating the mixture to a temperature ranging from about 50°C to about 100°C to form a lysed mixture, c) optionally contacting the lysed mixture with an alcohol, d) placing the lysed mixture in a cartridge, the cartridge comprising a cartridge body having a plurality of chambers in fluidic communication, a reaction vessel having one or more reaction chambers and configured for amplification and detection of the nucleic acid, a fluidic path between the plurality of chambers and the reaction vessel, and a filter in the fluidic path; and e) capturing nucleic from the lysed mixture onto the filter in the cartridge.

11. The method of any one of claims 1-10, wherein incubating the biological sample in a lysis reagent and recovering nucleic acid from said lysis reagent comprises: a) placing the biological sample in a cartridge, the cartridge comprising a cartridge body having a plurality of chambers in fluidic communication, a reaction vessel having one or more reaction chambers and configured for amplification and detection of the nucleic acid, a fluidic path between the plurality of chambers and the reaction vessel, and a filter in the fluidic path; b) adding an FFPE lysis buffer and a proteolytic enzyme to the biological sample to form a mixture, c) optionally healing the mixture to a temperature ranging from about 50°C to about 100°C to form a lysed mixture, d) optionally contacting the lysed mixture with an alcohol, ande) capturing nucleic acids from the lysed mixture onto the filter in the cartridge.

12. The method of claim 10 or 11, where the method comprises: contacting the lysed mixture with an alcohol selected from ethanol, PEG, or a combination thereof.

13. The method of claim 12, wherein the alcohol is in an amount of from 30% to65% by volume of the mixture, or in an amount of from 45% to 55% by volume of the mixture.

14. The method of any one of claims 10-13, wherein incubating the biological sample in a lysis reagent and recovering nucleic acid from said lysis reagent further comprises: contacting the lysed mixture with an oil, bovine serum albumin (BSA), or a combination thereof, wherein the oil or BSA is in an amount of from 0.01% to 5% by volume, from 0.01% to 3% by volume, or from 0.01% to 1% by volume, of the lysed mixture.

15. The method of claim 13, wherein the oil is selected from mineral oil, paraffin oil, or combinations thereof.

16. The method of any one of claims 10-15, wherein the FFPE lysis buffer comprises a salt, an antioxidant, a chelating agent, a buffer, a detergent, an antifoam agent, an antimicrobial agent, or a combination thereof, and wherein the FFPE lysis buffer is present in an amount of from 30% to 65% by volume of the mixture.

17. The method of claim 16, wherein the buffer comprises Tris, phosphate buffer, PBS, citrate buffer, TAPS, Bicine, Tricine, TAPSO, HEPES, TES, MOPS, PIPES,Cacodylate, SSC, MES, or combinations thereof.

18. The method of any one of claims 16-17, wherein the antioxidant and / or chelating agent comprise N-acetyl-L-cysteine, ethylenediaminetetraacetic acid (EDTA),diethylene triamine pentaacetic acid (DTP A), ethylenediamine-N,N'-disuccinic acid (EDDS), l,2-bis(o- aminophenoxy)ethane-N,N,N',N’-tetraacetic acid (BAPTA), a phosphonate chelating agent, or combinations thereof.

19. The method of any one of claims 10-18, wherein said proteolytic enzyme comprises a protease (e.g., proteinase K, trypsin, chymotrypsin, papain, or pepsin), a guanidinium compound, formamide, lithium perchlorate, magnesium chloride, urea, thiourea, or combinations thereof.

20. The method of any one of claims 10-19, wherein heating the mixture is performed at a temperature in the range of 50-85°C.

21. The method of any one of claims 10-20, wherein the lysis mixture is incubated for at least 10 minutes, for at least 30 minutes, or for about 30 to about 120 minutes.

22. The method of any one of claims 1-21, wherein the method of identifying HER2-low expression or distinguishing HER2-low expression, HER2-negative expression, and HER2 -positive expression is performed at the same facility where the biological sample is collected from a subject.

23. The method of any one of claims 1-22, wherein the method of identifying HER2-low expression or distinguishing HER2-low expression, HER2-negative expression, and HER2 -positive expression is performed within 48 hours, or within 24 hours of obtaining the biological sample from the subject.

24. The method of any one of claims 1-23, wherein the method of identifying HER2-low expression or distinguishing HER2-low expression, HER2-negative expression, and HER2 -positive expression is performed within 3 hours, or within 2 hours from step a) incubating the biological sample in a lysis reagent.

25. The method of any one of claims 1-24, wherein method further comprises identifying expression of the ESRI, PGR, and MKi67 genes in the sample.

26. The method of any one of claims 1-25, wherein each of the primers and probes for amplifying and detecting ERBB2 comprise at least 15 contiguous nucleotides having a sequence that is at least 85% identical to at least 15 contiguous nucleotides of the ERBB2 gene at exons 15, 16, and / or 17, and when present, the primers and probes for amplifying and detecting ESRI comprise at least 15 contiguous nucleotides having a sequence that is at least 85% identical to at least 15 contiguous nucleotides of the ESRI gene at exons 5 and / or 6, the primers and probes for amplifying and detecting PGR comprise at least 15 contiguous nucleotides having a sequence that is at least 85% identical to at least 15 contiguous nucleotides of the PGR gene at exons 2, 3, 4, 5, 6, and / or 7, and the primers and probes for amplifying and detecting MKi67 comprise at least 15 contiguous nucleotides having a sequence that is at least 85% identical to at least 15 contiguous nucleotides of the MKi67 gene at exons 2 and / or 3.

27. The method of any one of claims 1-26, wherein the primers and / or probes comprise a detectable label.

28. The method of any one of claims 1-27, wherein the method of identifying HER2-low expression or distinguishing HER2-low expression, HER2-negative expression, and HER2 -positive expression does not include immunofluorescence and image analysis, Automated quantitative analysis (AQUA) score, or LCMS quantification.

29. The method of any one of claims 1-28, further comprising administering a treatment regimen to the subject based on the determined expression.

30. The method of any one of claims 1-29, further comprising treating the subject with a HER2-targeted drug therapy, preferably a HER2-low targeted drug therapy.

31. The method of claim 30, wherein the HER2-targeted drug therapy is an antibody-drug conjugate (ADC), optionally trastuzumab deruxtecan (T-DXd), trastuzumab, pertuzumab, trastuzumab emtansine (T-DMI), or combinations thereof.

32. The method of any one of claims 1-31, wherein the subject is diagnosed with a cancer selected from breast cancer, gastric cancer, lung cancer, esophageal carcinoma, bladder cancer, colon cancer, or a combination thereof, or wherein the subject is diagnosed with a HER-negative cancer, or wherein the subject is diagnosed with a HER-positive cancer, prior to identifying HER2-low expression or distinguishing HER2-low expression, HER2-negative expression, and HER2-positive expression.

33. A cartridge for identifying HER2-low expression and / or distinguishing HER2-low expression, HER2-negative expression, and HER2-positive expression in a biological sample from a subject, the cartridge comprising: a cartridge body comprising a plurality of chambers therein, wherein the plurality of chambers includes: a sample chamber having at least a fluid outlet in fluid communication with another chamber of the plurality; and an optional lysis chamber in fluidic communication with the sample chamber, optionally wherein the sample chamber and lysis chamber are the same; a reaction vessel fluidically coupled to the plurality of chambers of the cartridge body and configured for: i) amplification of nucleic acid and ii) detection and identification of a plurality of amplification products via real-time PCR; a filter disposed in a fluidic path between the lysis chamber, if present, or the sample chamber, and the reaction vessel, and a set of primers and probes for detecting ERBB2.

34. The cartridge of claim 33, wherein the set of primers and probe for detectingERBB2 is selected from:(i) a forward primer, a reverse primer, and a probe each comprising at least 15 contiguous nucleotides having a sequence that is at least 85% identical to at least 15 contiguous nucleotides of the ERBB2 gene; or(ii) a forward primer comprising at least 15 contiguous nucleotides having a sequence that is at least 85% identical to at least 15 contiguous nucleotides of the ERBB2 gene at exons 15, 16, and / or 17, and at least one reverse primer comprising at least 15contiguous nucleotides having a sequence that is at least 85% identical to at least 15 contiguous nucleotides of the ERBB2 gene at exons 15, 16, and / or 17, or(iii) a forward primer comprising at least 15 contiguous nucleotides having a sequence that is at least 85% identical to at least 15 contiguous nucleotides of SEQ ID NO: 3 or 6, and at least one reverse primer comprising at least 15 contiguous nucleotides having a sequence that is at least 85% identical to at least 15 contiguous nucleotides of SEQ ID NO: 4 or 6.

35. The cartridge of claims 33-34, further comprising a set of primers and probe for detecting ESRI gene, PGR gene, MKi67 gene, or a combination thereof.

36. The cartridge of any one of claims 33-35, comprising a lysis chamber, wherein the lysis chamber comprises one or more lysis reagents for releasing nucleic acid.

37. The cartridge of any one of claims 33-36, wherein the reaction vessel comprises one or more reaction chambers for amplification and detection of the amplification products.

38. The cartridge of any one of claims 33-37, wherein the reaction vessel comprises one reaction chamber for amplification and detection of the amplification products.

39. The cartridge of any one of claims 37-38, wherein each reaction chamber is configured to detect a single amplification product.

40. The cartridge of any one of claims 37-39, wherein each reaction chamber is configured to detect a plurality of amplification products.

41. The cartridge of any one of claims 33-40, wherein the cartridge is a Clinical Laboratory Improvement Amendments (CLIA)-compliant cartridge.

42. The cartridge of any one of claims 33-41, wherein the cartridge is configured to carry out non-isothermal amplification, optionally by thermal cycling or temperature oscillation.

43. A kit for identifying HER2-low expression and / or distinguishing HER2-low expression, HER2-negative expression, and HER2-positive expression in a biological sample from a subject, the kit comprising: a set of primers and probe for detecting ERBB2, wherein the set of primers and probe for detecting ERBB2 is selected from:(i) a forward primer, a reverse primer, and a probe, each comprising at least 15 contiguous nucleotides having a sequence that is at least 85% identical to at least 15 contiguous nucleotides of the ERBB2 gene; or(ii) a forward primer comprising at least 15 contiguous nucleotides having a sequence that is at least 85% identical to at least 15 contiguous nucleotides of the ERBB2 gene at exons 15, 16, and / or 17, and at least one reverse primer comprising at least 15 contiguous nucleotides having a sequence that is at least 85% identical to at least 15 contiguous nucleotides of the ERBB2 gene at exons 15, 16, and / or 17, or(iii) a forward primer comprising at least 15 contiguous nucleotides having a sequence that is at least 85% identical to at least 15 contiguous nucleotides of SEQ ID NO: 3 or 6, and at least one reverse primer comprising at least 15 contiguous nucleotides having a sequence that is at least 85% identical to at least 15 contiguous nucleotides of SEQ ID NO: 4 or 6.

44. The kit of claim 43, further comprising a set of primers and optional probe for detecting ESRI, PGR, or MKi67 mRNA biomarker, or a combination thereof.

45. The kit of claim 43 or 44, further comprising one or more lysis reagents for releasing nucleic acid from the biological sample.

46. The kit of claim 45, wherein the one or more lysis reagents comprise: an FFPE lysis buffer, a proteolytic enzyme, optionally an alcohol, and optionally an oil or bovine serum albumin.

47. The kit of claim 46, wherein the alcohol is selected from ethanol, PEG, or a combination thereof.

48. The kit of claim 46 or 47, wherein the oil is selected from mineral oil, paraffin oil, or combinations thereof.

49. The kit of any one of claims 46-48, wherein the FFPE lysis buffer comprises a salt, an antioxidant or a chelating agent, a buffer, a detergent, an antifoam, and an antimicrobial agent.

50. The kit of claim 49, wherein the buffer comprises Tris, phosphate buffer,PBS, citrate buffer, TAPS, Bicine, Tricine, TAPSO, HEPES, TES, MOPS, PIPES,Cacodylate, SSC, and MES.

51. The kit of claim 49 or 50, wherein the antioxidant and / or chelating agent comprise N-acetyl-L-cysteine, ethylenediaminetetraacetic acid (EDTA), diethylene triamine pentaacetic acid (DTP A), ethylenediamine-N,N'-disuccinic acid (EDDS), l,2-bis(o- aminophenoxy)ethane-N,N,N',N’-tetraacetic acid (BAPTA), a phosphonate chelating agent, or combinations thereof.

52. The kit of any one of claims 46-51, wherein the proteolytic enzyme comprises a protease (e.g., proteinase K, trypsin, chymotrypsin, or papain), a guanidinium compound, formamide, lithium perchlorate, magnesium chloride, urea, thiourea, and a combination thereof.

53. The method, cartridge, or kit of any one of claims 1-52, wherein at least one of the primers or probes comprises a detectable label.

54. The method, cartridge, or kit of any one of claims 1-53, wherein each probe comprises a fluorescent dye and a quencher molecule.

55. The method, cartridge, or kit of any one of claims 1-54, wherein the kit or cartridge further comprises, or the method further employs, a primer pair specific for an exogenous control and / or an endogenous control, wherein the exogenous control is a sample processing control, and wherein the endogenous control is a sample adequacy control.

56. The method, cartridge, or kit of any one of claims 1-55, wherein the cartridge or kit facilitates and / or the method comprises identifying HER2-low expression and / or distinguishing HER2-low expression, HER2-negative expression, and HER2-postive expression within the biological sample within 2 hours of obtaining the biological sample.

57. The cartridge or method of any one of claims 1-56, wherein at least one of the plurality of chambers comprises one or more lyophilized reagents.

58. The cartridge or method of claim 57, wherein the one or more lyophilized reagents is / are in the form of one or more beads.

59. The cartridge or method of claim 57 or 58, wherein the one or more lyophilized reagents are selected from primers, probes, a salt, dNTPs, a thermostable polymerase, a reverse transcriptase, or a combination thereof.

60. The cartridge or method of any one of claims 57-59, wherein the one or more lyophilized reagents comprise lyophilized primers and probes.

61. The cartridge or method of any one of claims 57-60, wherein reagents and components in the reaction vessel are in solution.

62. The cartridge or method of any one of claims 1-61, wherein the filter is configured to bind the nucleic acid to be analyzed.

63. The cartridge or method of claim 62, wherein the filter comprises glass fibers and optionally a polymeric binder, or the glass fibers are optionally modified with a DNA binding ligand such as an alkylamine, a cycloalkylamine, an alkyloxy amine, a polyaminemoiety, an arylamine, an intercalating agent, a DNA groove binder, a peptide, an amino acid, a protein, or a combination thereof.

64. The cartridge or method of claim 62 or 63, wherein the filter comprises a 500 micron to 2000 microns thick glass fiber disk having a pore size of 0.2 microns to 1 micron.

65. The cartridge or method of any one of claims 1-64, wherein the filter is configured to bind unwanted material and allow the nucleic acid to pass through.

66. The cartridge or method of any one of claims 1-65, wherein the cartridge further comprises a binding reagent, wash reagent, eluting reagent, or a combination thereof.

67. A system for identifying HER2-low expression and / or distinguishing HER2- low expression, HER2-negative expression, and HER2 -positive expression in a biological sample from a subject, the system comprising: a module having a receiving bay for receiving the cartridge of any one of claims 34- 43, wherein the module includes one or more mechansims within the receiving bay for manipulating a fluid sample within the cartridge, and an instrument that interfaces with the reaction vessel; and a memory having programmable instructions recorded thereon, that are specially configured to operate the module of an assay protocol to identify expression of nucleic acid sequences characteristic of HER2-low.

68. The system of claim 67, wherein the module and / or system further comprises: a scanner or reader configured to read an identifier on the cartridge; wherein the instructions are configured to determine an applicable protocol based on reading or scanning of the identifier; and wherein the system operates the module of the applicable protocol based on an input from the scanner or reader.

69. The system of claim 67 or 68, wherein the module and / or system further comprises: an enclosure; a plurality of modules that includes said module, wherein modules are substantially identical and configured to concurrently perform assays on cartridges received therein.

70. The system of any one of claims 68-69, further comprising a networking platform for transmitting results derived from module operation.

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