Immune checkpoint blockade to prevent malignancy in individuals at high-risk for hereditary mismatch repair deficient tumors

Abstract

The present disclosure provides prophylactic methods for using an immune checkpoint inhibitor to prevent neoplasia in a Lynch Syndrome subject that lacks detectable tumors and has not received a prior anti-cancer therapy such as immunotherapy. In some embodiments, the Lynch Syndrome subject does not harbor germline mutations in POLE or POLDI and / or comprises a hereditary mismatch repair deficient (dMMR) germline mutation.

Description

Atty. Dkt. No.: 115872-3061 IMMUNE CHECKPOINT BLOCKADE TO PREVENT MALIGNANCY IN INDIVIDUALS AT HIGH-RISK FOR HEREDITARY MISMATCH REPAIR DEFICIENT TUMORS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No.63 / 578,057 filed August 22, 2023, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0002] The present disclosure relates to prophylactic methods for using an immune checkpoint inhibitor to prevent neoplasia in a Lynch Syndrome subject that lacks detectable tumors and has not received a prior anti-cancer therapy such as immunotherapy. In some embodiments, the Lynch Syndrome subject does not harbor germline mutations in POLE or POLD1 and / or comprises a hereditary mismatch repair deficient (dMMR) germline mutation. GOVERNMENT SUPPORT

[0003] This invention was made with government support under CA008748 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND

[0004] The following description of the background of the present technology is provided simply as an aid in understanding the present technology and is not admitted to describe or constitute prior art to the present technology.

[0005] Lynch syndrome (LS), a pan-cancer predisposition syndrome, is defined by inactivating pathogenic germline alterations in the DNA mismatch repair (MMR) genes (MLH1, MSH2, MSH6, PMS2, 3’ EPCAM terminal deletions). Importantly, patients with LS are at elevated risk for both synchronous and metachronous cancers. Inactivation of the second wild-type allele leads to impaired genome maintenance and renders cells unable to repair certain classes of mutations occurring during DNA replication. Over time this results in high tumor mutation burden (TMB) and enrichment of mutations in microsatellites, termed microsatellite instability (MSI). This high TMB and MSI leads to enrichment for mutations encoding proteins that are immunogenic, and are thought to sensitize mismatch repair deficient (dMMR) tumors to immune checkpoint blockade (ICB). -1- 4857-4385-9418.1Atty. Dkt. No.: 115872-3061

[0006] In a report of >46,000 solid tumor patients uninformed by germline analysis, it was found that ICB-containing treatment of the first cancer was associated with decreased incidence of second primary cancers (Heudel, Pierre, et al. ESMO open 6.1 (2021): 100044). However, the ability of ICB to prevent dMMR malignant or pre-malignant neoplasia development in Lynch syndrome (LS) patients is unknown. SUMMARY OF THE PRESENT TECHNOLOGY

[0007] In one aspect, the present disclosure provides a method for preventing neoplasia in a Lynch Syndrome patient comprising administering to the patient an effective amount of at least one immune checkpoint inhibitor (ICI), wherein the patient lacks detectable tumors and has not received a prior immunotherapy. In some embodiments, the patient does not harbor a germline mutation in POLE and / or POLD1. Additionally or alternatively, in certain embodiments, the patient comprises an inactivating pathogenic or likely pathogenic germline mutation in at least one DNA mismatch repair (MMR) gene. The at least one MMR gene may be selected from the group consisting of MLH1, MSH2, MSH6, PMS2 or EPCAM 3’ terminal region. In any of the preceding embodiments of the methods disclosed herein, the at least one ICI targets PD-1, PD-L1 or CTLA4. Examples of the at least one ICI comprises ipilimumab, pembrolizumab, nivolumab, dostarlimab, avelumab, atezolizumab, and durvalumab.

[0008] Additionally or alternatively, in some embodiments, the Lynch Syndrome patient has not received a prior cancer diagnosis. In other embodiments, the Lynch Syndrome patient has received at least one prior cancer diagnosis. In certain embodiments, the at least one prior cancer diagnosis is a cancer selected from among carcinoma, sarcoma, melanoma, hematopoietic cancer, adrenal cancers, bladder cancers, blood cancers, bone cancers, brain cancers, breast cancers, carcinoma, cervical cancers, colon cancers, colorectal cancers, corpus uterine cancers, ear, nose and throat (ENT) cancers, endometrial cancers, esophageal cancers, gastrointestinal cancers, head and neck cancers, Hodgkin's disease, intestinal cancers, kidney cancers, larynx cancers, leukemias, liver cancers, lymph node cancers, lymphomas, lung cancers, melanomas, mesothelioma, myelomas, nasopharynx cancers, neuroblastomas, non-Hodgkin's lymphoma, oral cancers, ovarian cancers, pancreatic cancers, penile cancers, pharynx cancers, prostate cancers, rectal cancers, sarcoma, seminomas, skin cancers, stomach cancers, teratomas, testicular cancers, thyroid cancers, uterine cancers, vaginal cancers, vascular tumors, biliary cancer, urothelial cancer, non-melanoma skin cancer, and germ cell tumor. -2- 4857-4385-9418.1Atty. Dkt. No.: 115872-3061

[0009] In another aspect, the present disclosure provides a method for preventing new primary visceral tumors in a Lynch Syndrome patient comprising administering to the patient an effective amount of at least one immune checkpoint inhibitor (ICI), wherein the patient has received at least one previous cancer diagnosis prior to administration of the at least one ICI. In some embodiments, the new primary visceral tumors comprise tumors of an internal organ or system of the body selected from among the gastrointestinal organs and tract inclusive of peritoneal and / or abdominal cavity, gynecological organs, genitourinary organs, respiratory organs among other internal systems and organs. Additionally or alternatively, in some embodiments, the patient comprises a germline mutation in POLE and / or POLD1. In certain embodiments, the patient comprises an inactivating pathogenic or likely pathogenic germline mutation in at least one DNA mismatch repair (MMR) gene. The at least one MMR gene may be selected from the group consisting of MLH1, MSH2, MSH6, PMS2 or EPCAM 3’ terminal region. In certain embodiments, the at least one previous cancer diagnosis is a cancer selected from among carcinoma, sarcoma, melanoma, hematopoietic cancer, adrenal cancers, bladder cancers, blood cancers, bone cancers, brain cancers, breast cancers, carcinoma, cervical cancers, colon cancers, colorectal cancers, corpus uterine cancers, ear, nose and throat (ENT) cancers, endometrial cancers, esophageal cancers, gastrointestinal cancers, head and neck cancers, Hodgkin's disease, intestinal cancers, kidney cancers, larynx cancers, leukemias, liver cancers, lymph node cancers, lymphomas, lung cancers, melanomas, mesothelioma, myelomas, nasopharynx cancers, neuroblastomas, non-Hodgkin's lymphoma, oral cancers, ovarian cancers, pancreatic cancers, penile cancers, pharynx cancers, prostate cancers, rectal cancers, sarcoma, seminomas, skin cancers, stomach cancers, teratomas, testicular cancers, thyroid cancers, uterine cancers, vaginal cancers, vascular tumors, biliary cancer, urothelial cancer, non- melanoma skin cancer, and germ cell tumor.

[0010] In any of the preceding embodiments of the methods disclosed herein, the at least one ICI targets PD-1, PD-L1 or CTLA4. Examples of the at least one ICI comprises ipilimumab, pembrolizumab, nivolumab, dostarlimab, avelumab, atezolizumab, and durvalumab. In some embodiments of the methods disclosed herein, the patient has a high or low tumor mutation burden (TMB). Additionally or alternatively, in some embodiments, the patient comprises tumors having a TMB ranging from of 30-95 mutations per Megabase. In certain embodiments, the TMB is about 30-35, about 35-40, about 40-45, about 45-50, about 50-55, about 55-60, about 60-65, about 65-70, about 70-75, about 75-80, about 80-85, about 85-90, or about 90-95 mutations per Megabase. -3- 4857-4385-9418.1Atty. Dkt. No.: 115872-3061

[0011] In any and all embodiments of the methods described herein, the patient has been classified as microsatellite stable (MSS), or indeterminate Microsatellite unstable (MSI-I), or MSI.

[0012] Additionally or alternatively, in some embodiments of the methods disclosed herein, the patient has an increased number of mutation associated neoantigens (MANA) or frameshift variants relative to a healthy control subject or predetermined threshold. In some embodiments, the number of mutation associated neoantigens (MANA) or frameshift variants is increased by at least 0.5 fold, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35- fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 55-fold, at least 60-fold, at least 65-fold, at least 70-fold, at least 75-fold, at least 80-fold, at least 85-fold, at least 90- fold, at least 95-fold, or at least 100-fold relative to a healthy control subject or predetermined threshold. In any and all embodiments of the methods disclosed herein, the patient has previously received surgery, radiation, or chemotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIGs.1A-1F: Patient case example, clinical timeline, and tumor characteristics of Lynch syndrome patients with subsequent primary malignancies (SPM) after immune checkpoint blockade (ICB). FIG.1A, Case example of patient with multiple primary malignancies pre-ICB exposure. (Patient 7 from FIG.1B; FIG.9) Timeline of cancer diagnoses and sequential local and systemic interventions including combined androgen blockade (CAB), androgen receptor antagonist (ARA) and ICB. Targeted next-generation sequencing (MSK-IMPACT) comparing pre-ICB prostate cancer (PCA) and post-ICB small bowel cancer (SBA) by tumor mutational burden, somatic mutational profile, and copy-number alterations. Single shared somatic mutation, PTPRT at chromosome 20. FIG.1B: Timeline for SPM development post-ICB exposure (n=21 patients) inclusive of: SBA: Small bowel, CRC: Colorectal, GCA: Gastric, PCA: Prostate, SN: Sebaceous neoplasm, HCC: Hepatocellular, UCC: Urothelial, SCC: Squamous cell, CHO: Cholangiocarcinoma, ECA: Esophageal, BRC: Breast, LYM: Lymphoma, UCE: Uterine, PDA: Pancreatic, VUV: Vulvar. FIGs.1C-1F: MSISensor score (FIG.1C), tumor mutational burden (FIG.1D), frameshift mutations (FIG.1E), and comparative analysis of strong binders to HLA Class 1 MHC (FIG.1F) between pre- and post-ICB tumors. P- values are derived from pairwise t-testing for the paired pre and post tumor from each -4- 4857-4385-9418.1Atty. Dkt. No.: 115872-3061 patient without replicates for each separate analysis. Analyses included nine patients with 9 pre and 9 post tumors except analysis c (MSISensor score) which included 8 patients with 8 pre and 8 post tumors due to an MSI score not available for patient 6. *MSI status on 1 sample with inadequate tumor purity was confirmed via miMSI.

[0014] FIGs.2A-2F: Incidence of subsequent primary malignancies (SPM) and site of origin in the study cohort and comparator Lynch syndrome (LS) cohorts. FIG.2A, Rate of SPM per observation year in the ICB treated cohort (pink) and comparator cohorts inclusive of internal non-immune checkpoint blockade (non-ICB) exposed patients with LS (light blue) and external multi-center patients with LS undergoing prospective surveillance after the first cancer diagnosis (dark blue)15. FIG.2B, Overall SPM incidence at 3-years matched follow-up in the ICB treated (pre- and post-ICB) and non-ICB treated cohort (pre- and post-chemotherapy). The number of matched patient pre / post ICB or pre / post chemotherapy is reported below the figure. The 3 year incidence of 2ndneoplasm for each clinical condition is reported above the box plot representing the mean and standard deviation (SEM). Pre and post 3 year incidence were compared using paired t test. ns: non significant, P>0.05. FIG.2C, Incidence of cutaneous and visceral neoplasms at 3-years matched follow-up in patients pre- and post-ICB, and pre- and post-chemotherapy. The numbers of matched patient pre / post ICB or pre / post chemotherapy are respectively 65 and 95 patients. The 3 year incidence of 2ndneoplasm category (skin or visceral) for each clinical condition is reported above the box plot representing the mean and standard deviation (SEM). Pre and post 3 year incidence were compared using repeated measure one way ANOVA test corrected for multiple comparisons comparing 3 year incidences of de novo cutaneous or visceral neoplasms. *: P≤0.05 and P>0.01, ns: non significant, P>0.05. FIG.2D, Time to first cutaneous or visceral neoplasm post-ICB exposure. The number of patients is reported on the figure, each patient being accounted only once based on first 2ndneoplasm type (skin or visceral). The median time to first 2ndneoplasm post ICB is reported. Survival curves were compared using log rank test and the p-value is reported on the figure. The dashed lines represent the upper and lower limit of the confidence interval at 95% for each conditions with matched colors. FIGs.2E-2F: Cumulative incidence of cutaneous (FIG.2E) versus visceral (FIG.2F) post-ICB SPM. Patients with a matched follow up pre- and post-ICB of 36 months were selected and all non-index cancers occurring during the window of observation were accounted for. Cumulative incidence is reported as the cumulative sum of non-index cancers occurring during the period of -5- 4857-4385-9418.1Atty. Dkt. No.: 115872-3061 observation. Number of patients and cumulative observation time in months is reported. Fisher’s exact test comparing the cumulative observation time without a neoplasm event to the number of months with an event was used for statistical analysis. ****: P<0.0001 ; **: P≤0.01 and >0.001.

[0015] FIG.3: Study cohort of Lynch syndrome patients. Segregated by microsatellite status as designated by MSIsensor14. MSI: Microsatellite unstable; MSS: Microsatellite stable.

[0016] FIGs.4A-4D: Histological evaluation of patient 7’s sequential tumors demonstrating distinct histological and immunohistochemical features (Patient example FIG.1A, patient 7 FIG.2B, FIGs.10, 13). FIG.4A, Tumor 3: Gastric adenocarcinoma. FIG.4B, Tumor 4: Colorectal adenocarcinoma. FIG.4C, Tumor 5: Prostate adenocarcinoma. FIG.4D, Tumor 6: Small bowel adenocarcinoma.

[0017] FIGs.5A-5B: Paired analysis of pre- and post-immune checkpoint blockade (ICB) exposed tumors using MSK-IMPACT next-generation sequencing derived data28. Nonsynonymous somatic mutations from the MSK IMPACT variant call format (VCF) output from each sequenced patient tumor were input into the SnpEff program (https: / / pcingola.github.io / SnpEff / ) to predict translated amino acid sequences for each respective mutation. The number of unique mutation associated neoantigens (MANA) was determined by taking the defined peptide sequence and evaluating all potential contiguous 9’mer variations using a sliding reading frame. FIG.5A: Comparison of total missense mutations in pre- and post-ICB tumors. FIG.5B: Comparison of mutation associated neoantigens between pre- and post-ICB tumors.

[0018] FIGs.6A-6H: Sensitivity analyses of pre- and post-immune checkpoint blockade (ICB) exposed tumors using MSK-IMPACT next generation sequencing derived data28. FIGs.6A-6D: MSIsensor score, tumor mutational burden (TMB), frameshift mutation rates and number of mutation associated neoantigens (MANA) between pre- and post-ICB exposed tumors when grouped by gastrointestinal cancer only. FIGs. 6E-6H: MSIsensor score, tumor mutational burden (TMB), frameshift mutation rates and number of mutation associated neoantigens (MANA) between pre- and post-ICB exposed tumors when grouped by urothelial cancers only.

[0019] FIGs.7A-7B: Immunohistochemical analysis of immune cell infiltration of pre-immune checkpoint blockade (ICB) and post-ICB tumors. FIG.7A: Intrapatient -6- 4857-4385-9418.1Atty. Dkt. No.: 115872-3061 comparison of paired pre- and post-ICB tumors. CD8 tumor infiltrating lymphocytes (CD8 TILS) in the non-ICB exposed urothelial cancer and the post-ICB exposure cutaneous squamous cell carcinoma. FIG.7B: Interpatient comparions of CD8 TILS, programmed cell death 1 protein (PD1) positive TILS and programmed cell death ligand 1 (PDL1) combined positive score (PDL1 CPS) in tumors arising prior to ICB exposure, tumors arising whilst patients were on ICB and tumors arising post completion of ICB.

[0020] FIGs.8A-8D: Incidence of subsequent primary malignancies (SPM) in the immune checkpoint blockade (ICB) treated and non-ICB (chemotherapy) treated Lynch syndrome patients. FIG.8A: SPM incidence at 5-years matched follow-up in the ICB treated (pre- and post-ICB) and non-ICB treated cohort (pre- and post-chemotherapy). FIG.8B: SPM incidence in patients defined as high risk (HR). HR-patients were defined as patients presenting with multiple non concomitant cancers (>=2) in a pre-ICB delay matched with post-ICB follow up.3-year and 5-year incidence calculation was performed excluding the index cancer and concomitant cancers (<1 month between diagnoses of cancers). Incidence of pre-ICB and post-ICB cancers on matched follow up in HR-patients was compated using a paired t test and the same methodology was used for patients not exposed to ICB, using initiation of chemotherapy as a reference for matched pre- and post- chemotherapy with 3-year and 5-year incidence calculation. There was a statistically significant reduction in post-ICB and post-chemotherapy cancers in both the matched 3- and matched 5- year cohorts. FIG.8C: Swimmers plot demonstration cancer occurrence pre- and post-ICB exposure in the HR patient cohort. FIG.8D: Cumulative post-ICB cancer incidence in the ICB-exposed HR-patient cohort.

[0021] FIG.9: Clinicodemographic characteristics of Lynch syndrome patients treated with immune checkpoint blockade. FPM: First Primary Malignancy, MSI: Microsatellite Instability, MSS: Microsatellite Stable, MS-I: Microsatellite indeterminate, PD-1i: Programmed Cell Death Protein 1 Inhibitor, PD-L1i: Programmed Cell Death Ligand1 Inhibitor, CTLA4i: Cytotoxic T Lymphocyte protein inhibitor.

[0022] FIG.10: Clinical and treatment characteristics of immune checkpoint blockade (ICB) exposed patients with and without development of a subsequent primary malignancy (SPM). Given risk of multiple primary cancers in Lynch syndrome, the tumor targeted by the ICB was ranked as to the order in which it had occurred. IQR: Interquartile range. -7- 4857-4385-9418.1Atty. Dkt. No.: 115872-3061

[0023] FIG.11: Malignant precursor lesions post-immune checkpoint blockade (ICB) exposure. TT: target tumor; TTP: time to polyp; TA: tubular adenoma; TVA: tubulo-villous adenoma; SA: serrated adenoma; IHC: immunohistochemistry; dMMR: deficient DNA mismatch repair; pMMR: proficient DNA mismatch repair. *Incomplete baseline colonoscopy pre-ICB exposure. NA: Not available

[0024] FIG.12: Analysis of shared mutations and mutation associated noeantigens (MANA) between pre- and post-immune checkpoint blockade exposed tumors. Tumor pairs with shared neoantigen number are aligned with the corresponding shared mutation(s) for that specific pair.

[0025] FIG.13: Characteristics of tumors targeted (TT) by immune checkpoint blockade (ICB) and the subsequent primary malignancies (SPM) post-ICB. SPM: Second primary malignancy; SNV: Short nucleotide variant; ICB: Immune checkpoint blockade; ADT: Androgen deprivation therapy; CR: Complete response; SD: Stable disease; POD: Progressions of disease; PSA: Prostate Specific Antigen; TVA: Tubulovillous adenoma; SCC: Squamous cell carcinoma of skin; HCC: Hepatocellular carcinoma; DCIS: Ductal carcinoma in situ; *Not present radiologically on baseline imaging. DETAILED DESCRIPTION

[0026] It is to be appreciated that certain aspects, modes, embodiments, variations and features of the present methods are described below in various levels of detail in order to provide a substantial understanding of the present technology.

[0027] In practicing the present methods, many conventional techniques in molecular biology, protein biochemistry, cell biology, immunology, microbiology and recombinant DNA are used. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Patent No.4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription -8- 4857-4385-9418.1Atty. Dkt. No.: 115872-3061 and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al. eds (1996) Weir’s Handbook of Experimental Immunology. Methods to detect and measure levels of polypeptide gene expression products (i.e., gene translation level) are well-known in the art and include the use of polypeptide detection methods such as antibody detection and quantification techniques. (See also, Strachan & Read, Human Molecular Genetics, Second Edition. (John Wiley and Sons, Inc., NY, 1999)).

[0028] The Examples described herein are distinguishable from the data presented by Harrold E et al., the American Society of Clinical Oncology (ASCO) Annual meeting (2022) (https: / / ascopubs.org / doi / abs / 10.1200 / JCO.2022.40.16_suppl.10505), which represented preliminary and inaccurate evaluation of a subset of a Lynch Syndrome patient cohort. The present Examples focus on the rates of new primary malignancy in high-risk Lynch syndrome patients receiving immune checkpoint inhibitors (ICI) compared to the new primary malignancy rates in control patients with Lynch syndrome who did not receive ICI. Prior studies have concluded that in LS-patients in receipt of ICI for cancer treatment, the risk of a second primary malignancy and polyps remain high following treatment with ICI. In contrast, the Examples described herein demonstrate that the risk of developing new primary malignancy rates was significantly preempted in Lynch syndrome patients receiving ICI relative to Lynch syndrome patients that did not receive ICI. Moreover, as demonstrated herein, ICI treatment in high-risk LS patients unexpectedly prevented the formation of new primary malignancies that were visceral neoplasms as opposed to cutaneous neoplasms. Definitions

[0029] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. For example, reference to “a cell” includes a combination of two or more cells, and the like. Generally, the nomenclature used herein and the laboratory procedures in cell -9- 4857-4385-9418.1Atty. Dkt. No.: 115872-3061 culture, molecular genetics, organic chemistry, analytical chemistry and nucleic acid chemistry and hybridization described below are those well-known and commonly employed in the art.

[0030] As used herein, the term “about” in reference to a number is generally taken to include numbers that fall within a range of 1%, 5%, or 10% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value).

[0031] As used herein, the “administration” of an agent or drug to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function. Administration can be carried out by any suitable route, including but not limited to, orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intrathecally, or topically. Administration includes self- administration and the administration by another.

[0032] As used herein, the term “antibody” collectively refers to immunoglobulins or immunoglobulin-like molecules including by way of example and without limitation, IgA, IgD, IgE, IgG and IgM, combinations thereof, and similar molecules produced during an immune response in any vertebrate, for example, in mammals such as humans, goats, rabbits and mice, as well as non-mammalian species, such as shark immunoglobulins. As used herein, “antibodies” (includes intact immunoglobulins) and “antigen binding fragments” specifically bind to a molecule of interest (or a group of highly similar molecules of interest) to the substantial exclusion of binding to other molecules (for example, antibodies and antibody fragments that have a binding constant for the molecule of interest that is at least 103M-1greater, at least 104M-1greater or at least 105M-1greater than a binding constant for other molecules in a biological sample). The term “antibody” also includes genetically engineered forms such as chimeric antibodies (for example, humanized murine antibodies), heteroconjugate antibodies (such as, bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J., Immunology, 3rd Ed., W.H. Freeman & Co., New York, 1997.

[0033] More particularly, antibody refers to a polypeptide ligand comprising at least a light chain immunoglobulin variable region or heavy chain immunoglobulin variable region which specifically recognizes and binds an epitope of an antigen. Antibodies are composed of a heavy and a light chain, each of which has a variable region, termed the variable heavy -10- 4857-4385-9418.1Atty. Dkt. No.: 115872-3061 (VH) region and the variable light (VL) region. Together, the VHregion and the VLregion are responsible for binding the antigen recognized by the antibody. Typically, an immunoglobulin has heavy (H) chains and light (L) chains interconnected by disulfide bonds. There are two types of light chain, lambda (λ) and kappa (κ). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each heavy and light chain contains a constant region and a variable region, (the regions are also known as “domains”). In combination, the heavy and the light chain variable regions specifically bind the antigen. Light and heavy chain variable regions contain a “framework” region interrupted by three hypervariable regions, also called “complementarity-determining regions” or “CDRs”. The extent of the framework region and CDRs have been defined (see, Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991, which is hereby incorporated by reference). The Kabat database is now maintained online. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, largely adopt a β-sheet conformation and the CDRs form loops which connect, and in some cases form part of, the β-sheet structure. Thus, framework regions act to form a scaffold that provides for positioning the CDRs in correct orientation by inter-chain, non-covalent interactions.

[0034] The CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located. Thus, a VH CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, whereas a VLCDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found. An antibody that binds PD-1, PD-L1 or CTLA4 protein will have a specific VHregion and the VLregion sequence, and thus specific CDR sequences. Antibodies with different specificities (i.e. different combining sites for different antigens) have different CDRs. Although it is the CDRs that vary from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within the CDRs are called specificity determining residues (SDRs). “Immunoglobulin-related compositions” as used herein, refers to antibodies (including monoclonal antibodies, polyclonal antibodies, humanized antibodies, chimeric antibodies, recombinant antibodies, -11- 4857-4385-9418.1Atty. Dkt. No.: 115872-3061 multi-specific antibodies, bispecific antibodies, etc.,) as well as antibody fragments. An antibody or antigen binding fragment thereof specifically binds to an antigen.

[0035] As used herein, the term “antibody-related polypeptide” means antigen-binding antibody fragments, including single-chain antibodies, that can comprise the variable region(s) alone, or in combination, with all or part of the following polypeptide elements: hinge region, CH1, CH2, and CH3domains of an antibody molecule. Also included in the technology are any combinations of variable region(s) and hinge region, CH1, CH2, and CH3domains. Antibody-related molecules useful in the present methods, e.g., but are not limited to, Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VLor VHdomain. Examples include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CLand CH1domains; (ii) a F(ab′)2fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VHand CH1domains; (iv) a Fv fragment consisting of the VLand VHdomains of a single arm of an antibody, (v) a dAb fragment (Ward et al., Nature 341: 544-546, 1989), which consists of a VHdomain; and (vi) an isolated complementarity determining region (CDR). As such “antibody fragments” or “antigen binding fragments” can comprise a portion of a full length antibody, generally the antigen binding or variable region thereof. Examples of antibody fragments or antigen binding fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multi-specific antibodies formed from antibody fragments.

[0036] As used herein, the term “diabodies” refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VHVL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen binding sites. Diabodies are described more fully in, e.g., EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).

[0037] As used herein, the terms “single-chain antibodies” or “single-chain Fv (scFv)” refer to an antibody fusion molecule of the two domains of the Fv fragment, VLand VH. Single-chain antibody molecules may comprise a polymer with a number of individual molecules, for example, dimer, trimer or other polymers. Furthermore, although the two domains of the Fvfragment, VLand VH, are coded for by separate genes, they can be joined, -12- 4857-4385-9418.1Atty. Dkt. No.: 115872-3061 using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VLand VHregions pair to form monovalent molecules (known as single-chain Fv(scFv)). Bird et al. (1988) Science 242:423-426 and Huston et al. (1988) Proc. Natl. Acad Sci. USA 85:5879-5883. Such single-chain antibodies can be prepared by recombinant techniques or enzymatic or chemical cleavage of intact antibodies.

[0038] Any of the above-noted antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for binding specificity and neutralization activity in the same manner as are intact antibodies.

[0039] The term “antigen binding fragment” refers to a fragment of the whole immunoglobulin structure which possesses a part of a polypeptide responsible for binding to antigen. Examples of the antigen binding fragment useful in the present technology include scFv, (scFv)2, scFvFc, Fab, Fab′ and F(ab′)2, but are not limited thereto. Any of the above- noted antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for binding specificity and neutralization activity in the same manner as are intact antibodies.

[0040] As used herein, an “antigen” refers to a molecule to which an antibody (or antigen binding fragment thereof) can selectively bind. The target antigen may be a protein, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. In some embodiments, the target antigen may be a polypeptide (e.g., a PD-1, PD-L1 or CTLA4 polypeptide). An antigen may also be administered to an animal to generate an immune response in the animal.

[0041] As used herein, the term “biological sample” means sample material derived from living cells. Biological samples may include tissues, cells, protein or membrane extracts of cells, and biological fluids (e.g., ascites fluid or cerebrospinal fluid (CSF)) isolated from a subject, as well as tissues, cells and fluids present within a subject. Biological samples of the present technology include, but are not limited to, samples taken from eye, breast tissue, renal tissue, the uterine cervix, the endometrium, the head or neck, the gallbladder, parotid tissue, the prostate, the brain, the pituitary gland, kidney tissue, muscle, the esophagus, the stomach, the small intestine, the colon, the liver, the spleen, the pancreas, thyroid tissue, heart tissue, lung tissue, the bladder, adipose tissue, lymph node tissue, the uterus, ovarian tissue, adrenal tissue, testis tissue, the tonsils, thymus, blood, hair, buccal, skin, serum, plasma, CSF, semen, prostate fluid, seminal fluid, urine, feces, sweat, -13- 4857-4385-9418.1Atty. Dkt. No.: 115872-3061 saliva, sputum, mucus, bone marrow, lymph, and tears. Biological samples can also be obtained from biopsies of internal organs. Biological samples can be obtained from subjects for diagnosis or research or can be obtained from non-diseased individuals, as controls or for basic research. Samples may be obtained by standard methods including, e.g., venous puncture and surgical biopsy. In certain embodiments, the biological sample is a tissue sample obtained by needle biopsy.

[0042] As used herein, the term “CDR-grafted antibody” means an antibody in which at least one CDR of an “acceptor” antibody is replaced by a CDR “graft” from a “donor” antibody possessing a desirable antigen specificity.

[0043] As used herein, the term “chimeric antibody” means an antibody in which the Fc constant region of a monoclonal antibody from one species (e.g., a mouse Fc constant region) is replaced, using recombinant DNA techniques, with an Fc constant region from an antibody of another species (e.g., a human Fc constant region). See generally, Robinson et al., PCT / US86 / 02269; Akira et al., European Patent Application 184,187; Taniguchi, European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., WO 86 / 01533; Cabilly et al. U.S. Patent No.4,816,567; Cabilly et al., European Patent Application 0125,023; Better et al., Science 240: 1041-1043, 1988; Liu et al., Proc. Natl. Acad. Sci. USA 84: 3439-3443, 1987; Liu et al., J. Immunol 139: 3521-3526, 1987; Sun et al., Proc. Natl. Acad. Sci. USA 84: 214-218, 1987; Nishimura et al., Cancer Res 47: 999-1005, 1987; Wood et al., Nature 314: 446-449, 1885; and Shaw et al., J. Natl. Cancer Inst.80: 1553-1559, 1988.

[0044] As used herein, a “control” is an alternative sample used in an experiment for comparison purpose. A control can be “positive” or “negative.” For example, where the purpose of the experiment is to determine a correlation of the efficacy of a therapeutic agent for the treatment for a particular type of disease, a positive control (a compound or composition known to exhibit the desired therapeutic effect) and a negative control (a subject or a sample that does not receive the therapy or receives a placebo) are typically employed.

[0045] As used herein, the term “effective amount” refers to a quantity sufficient to achieve a desired therapeutic and / or prophylactic effect, e.g., an amount which results in the prevention of, or a decrease in a disease or condition described herein or one or more signs or symptoms associated with a disease or condition described herein. In the context of -14- 4857-4385-9418.1Atty. Dkt. No.: 115872-3061 therapeutic or prophylactic applications, the amount of a composition administered to the subject will vary depending on the composition, the degree, type, and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. The compositions can also be administered in combination with one or more additional therapeutic compounds. In the methods described herein, the therapeutic compositions may be administered to a subject having one or more signs or symptoms of a disease or condition described herein. As used herein, a “therapeutically effective amount” of a composition refers to composition levels in which the physiological effects of a disease or condition are ameliorated or eliminated. A therapeutically effective amount can be given in one or more administrations.

[0046] As used herein, “expression” includes one or more of the following: transcription of the gene into precursor mRNA; splicing and other processing of the precursor mRNA to produce mature mRNA; mRNA stability; translation of the mature mRNA into protein (including codon usage and tRNA availability); and glycosylation and / or other modifications of the translation product, if required for proper expression and function.

[0047] As used herein, the term “gene” means a segment of DNA that contains all the information for the regulated biosynthesis of an RNA product, including promoters, exons, introns, and other untranslated regions that control expression.

[0048] As used herein, “humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some embodiments, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues which are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance such as binding affinity. Generally, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains (e.g., Fab, Fab′, F(ab′)2, or Fv), in which all or substantially all of the hypervariable loops correspond to those of a non-human -15- 4857-4385-9418.1Atty. Dkt. No.: 115872-3061 immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus FR sequence although the FR regions may include one or more amino acid substitutions that improve binding affinity. The number of these amino acid substitutions in the FR are typically no more than 6 in the H chain, and in the L chain, no more than 3. The humanized antibody optionally may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Reichmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.2:593-596 (1992). See e.g., Ahmed & Cheung, FEBS Letters 588(2):288-297 (2014).

[0049] As used herein, the term “hypervariable region” refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region generally comprises amino acid residues from a “complementarity determining region” or “CDR” (e.g., around about residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the VL, and around about 31-35B (H1), 50-65 (H2) and 95-102 (H3) in the VH (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or those residues from a “hypervariable loop” (e.g., residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the VL, and 26-32 (H1), 52A-55 (H2) and 96-101 (H3) in the VH (Chothia and Lesk J. Mol. Biol.196:901-917 (1987)).

[0050] As used herein, the terms “individual”, “patient”, or “subject” can be an individual organism, a vertebrate, a mammal, or a human. In some embodiments, the individual, patient or subject is a human.

[0051] As used herein, “Lynch syndrome” or “Hereditary nonpolyposis colon cancer (HNPCC)” is an inherited cancer syndrome caused by a germline mutation in a single allele of one of several genes involved in DNA mismatch repair (MMR), including MLH1, MSH2, MSH6, PMS2, and 3’EPCAM deletion. Lynch syndrome patients develop early-onset cancer, and frequently exhibit multiple tumors, including those of the colon, rectum, endometrium, stomach, ovary, pancreas, urinary tract, small intestine, and other sites. A germline mutation in a MMR gene may be “likely pathogenic” or “pathogenic” as per ACMG classification. Level 4 (likely pathogenic) and Level 5 (pathogenic) mutations in the MMR genes are both considered equally as being diagnostic of Lynch Syndrome. -16- 4857-4385-9418.1Atty. Dkt. No.: 115872-3061

[0052] As used herein, “microsatellites” are repeated DNA sequences that occur approximately every 50–100 Kb base pairs throughout the human genome. MSI is a hypermutable phenotype caused by the loss of DNA mismatch repair activity and is implicated in the development of CRC, endometrial cancer and multiple other cancer types. MSI is detected in about 15% of all CRCs; 3% are of which are associated with Lynch syndrome and the other 12% occur sporadically. In some cases, sporadic MSI is caused by acquired hypermethylation of the promoter of the MLH1 gene, which occurs in tumors with the CpG island methylator phenotype.

[0053] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. For example, a monoclonal antibody can be an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including, e.g., but not limited to, hybridoma, recombinant, and phage display technologies. For example, the monoclonal antibodies to be used in accordance with the present methods may be made by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or may be made by recombinant DNA methods (See, e.g., U.S. Patent No.4,816,567). The “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol.222:581-597 (1991), for example.

[0054] As used herein, the terms “new primary malignancy” or “subsequent primary malignancy,” “second primary malignancy” or “SPM” are interchangeable, and refer to any malignancy that demonstrates different histology and morphology from prior malignancy, or arises in a different location in the same or different organ as the prior malignancy. For -17- 4857-4385-9418.1Atty. Dkt. No.: 115872-3061 example, a Lynch syndrome patient may develop a first cancer in the uterus / endometrium and then subsequently a new primary malignancy in the colorectum. Furthermore, as Lynch syndrome patients are at high risk of multiple primary cancers, it is not unusual to develop more than one primary colorectal cancer in the different parts of colorectum. Beyond morphological and histological differences, such new primary malignancy may also be differentiated through genomic differences with respect to the nature of the acquired somatic genomic alterations harbored by the new primary malignancy in contrast to the prior malignancy or malignancies. New primary malignancies are entirely separate and distinct from recurrent malignancies (i.e., thereoccurrence of the prior malignancy).

[0055] As used herein, the term “pharmaceutically-acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal compounds, isotonic and absorption delaying compounds, and the like, compatible with pharmaceutical administration. Pharmaceutically-acceptable carriers and their formulations are known to one skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences (20th edition, ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, Pa.). Examples of pharmaceutically-acceptable carriers include a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, useful for introducing the active agent into the body.

[0056] As used herein, the term “polynucleotide” or “nucleic acid” means any RNA or DNA, which may be unmodified or modified RNA or DNA. Polynucleotides include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, RNA that is mixture of single- and double-stranded regions, and hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double- stranded regions. In addition, polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.

[0057] As used herein, the terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to mean a polymer comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. Polypeptide refers to both short chains, commonly referred to as peptides, glycopeptides or oligomers, -18- 4857-4385-9418.1Atty. Dkt. No.: 115872-3061 and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. Polypeptides include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques that are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature

[0058] As used herein, “prevention,” “prevent,” or “preventing” of a disorder or condition refers to one or more compounds that, in a statistical sample, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset of one or more symptoms of the disorder or condition relative to the untreated control sample.

[0059] As used herein, the term “separate” therapeutic use refers to an administration of at least two active ingredients at the same time or at substantially the same time by different routes.

[0060] As used herein, the term “sequential” therapeutic use refers to administration of at least two active ingredients at different times, the administration route being identical or different. More particularly, sequential use refers to the whole administration of one of the active ingredients before administration of the other or others commences. It is thus possible to administer one of the active ingredients over several minutes, hours, or days before administering the other active ingredient or ingredients. There is no simultaneous treatment in this case.

[0061] As used herein, “specifically binds” refers to a molecule (e.g., an antibody or antigen binding fragment thereof) which recognizes and binds another molecule (e.g., an antigen), but that does not substantially recognize and bind other molecules. The terms “specific binding,” “specifically binds to,” or is “specific for” a particular molecule (e.g., a polypeptide, or an epitope on a polypeptide), as used herein, can be exhibited, for example, by a molecule having a KD for the molecule to which it binds to of about 10−4 M, 10−5 M, 10−6 M, 10−7 M, 10−8 M, 10−9 M, 10−10 M, 10−11 M, or 10−12 M. The term “specifically binds” may also refer to binding where a molecule (e.g., an antibody or antigen binding fragment thereof) binds to a particular polypeptide (e.g., a PD-1, PD-L1 or CTLA4 polypeptide), or an epitope on a particular polypeptide, without substantially binding to any other polypeptide, or polypeptide epitope. -19- 4857-4385-9418.1Atty. Dkt. No.: 115872-3061

[0062] As used herein, the term “simultaneous” therapeutic use refers to the administration of at least two active ingredients by the same route and at the same time or at substantially the same time.

[0063] As used herein, the term “therapeutic agent” is intended to mean a compound that, when present in an effective amount, produces a desired therapeutic effect on a subject in need thereof.

[0064] As used herein, “visceral malignancy” is any neoplasm of an internal organ or system of the body including but not limited to the gastrointestinal organs and tract inclusive of peritoneal and / or abdominal cavity, gynecological organs, genitourinary organs, respiratory organs among other internal systems and organs, and excludes neoplasms of the skin organ which are cutaneous neoplasms including, for example, cutaneous melanoma, sebaceous neoplasms, cutaneous basal cell carcinoma and cutaneous squamous cell carcinoma.

[0065] Methods for Preventing Neoplasia and New Primary Visceral Tumors in Lynch Syndrome

[0066] In one aspect, the present disclosure provides a method for preventing neoplasia in a Lynch Syndrome patient comprising administering to the patient an effective amount of at least one immune checkpoint inhibitor (ICI), wherein the patient lacks detectable tumors and has not received a prior immunotherapy. In some embodiments, the patient does not harbor a germline mutation in POLE and / or POLD1. Additionally or alternatively, in certain embodiments, the patient comprises an inactivating pathogenic or likely pathogenic germline mutation in at least one DNA mismatch repair (MMR) gene. The at least one MMR gene may be selected from the group consisting of MLH1, MSH2, MSH6, PMS2 or EPCAM 3’ terminal region. In any of the preceding embodiments of the methods disclosed herein, the at least one ICI targets PD-1, PD-L1 or CTLA4. Examples of the at least one ICI comprises ipilimumab, pembrolizumab, nivolumab, dostarlimab, avelumab, atezolizumab, and durvalumab.

[0067] Additionally or alternatively, in some embodiments, the Lynch Syndrome patient has not received a prior cancer diagnosis. In other embodiments, the Lynch Syndrome patient has received at least one prior cancer diagnosis. In certain embodiments, the at least one prior cancer diagnosis is a cancer selected from among carcinoma, sarcoma, melanoma, hematopoietic cancer, adrenal cancers, bladder cancers, blood cancers, bone -20- 4857-4385-9418.1Atty. Dkt. No.: 115872-3061 cancers, brain cancers, breast cancers, carcinoma, cervical cancers, colon cancers, colorectal cancers, corpus uterine cancers, ear, nose and throat (ENT) cancers, endometrial cancers, esophageal cancers, gastrointestinal cancers, head and neck cancers, Hodgkin's disease, intestinal cancers, kidney cancers, larynx cancers, leukemias, liver cancers, lymph node cancers, lymphomas, lung cancers, melanomas, mesothelioma, myelomas, nasopharynx cancers, neuroblastomas, non-Hodgkin's lymphoma, oral cancers, ovarian cancers, pancreatic cancers, penile cancers, pharynx cancers, prostate cancers, rectal cancers, sarcoma, seminomas, skin cancers, stomach cancers, teratomas, testicular cancers, thyroid cancers, uterine cancers, vaginal cancers, vascular tumors, biliary cancer, urothelial cancer, non-melanoma skin cancer, and germ cell tumor.

[0068] In another aspect, the present disclosure provides a method for preventing new primary visceral tumors in a Lynch Syndrome patient comprising administering to the patient an effective amount of at least one immune checkpoint inhibitor (ICI), wherein the patient has received at least one previous cancer diagnosis prior to administration of the at least one ICI. In some embodiments, the new primary visceral tumors comprise tumors of an internal organ or system of the body selected from among the gastrointestinal organs and tract inclusive of peritoneal and / or abdominal cavity, gynecological organs, genitourinary organs, respiratory organs among other internal systems and organs. Additionally or alternatively, in some embodiments, the patient comprises a germline mutation in POLE and / or POLD1. In certain embodiments, the patient comprises an inactivating pathogenic or likely pathogenic germline mutation in at least one DNA mismatch repair (MMR) gene. The at least one MMR gene may be selected from the group consisting of MLH1, MSH2, MSH6, PMS2 or EPCAM 3’ terminal region. In certain embodiments, the at least one previous cancer diagnosis is a cancer selected from among carcinoma, sarcoma, melanoma, hematopoietic cancer, adrenal cancers, bladder cancers, blood cancers, bone cancers, brain cancers, breast cancers, carcinoma, cervical cancers, colon cancers, colorectal cancers, corpus uterine cancers, ear, nose and throat (ENT) cancers, endometrial cancers, esophageal cancers, gastrointestinal cancers, head and neck cancers, Hodgkin's disease, intestinal cancers, kidney cancers, larynx cancers, leukemias, liver cancers, lymph node cancers, lymphomas, lung cancers, melanomas, mesothelioma, myelomas, nasopharynx cancers, neuroblastomas, non-Hodgkin's lymphoma, oral cancers, ovarian cancers, pancreatic cancers, penile cancers, pharynx cancers, prostate cancers, rectal cancers, sarcoma, seminomas, skin cancers, stomach cancers, teratomas, testicular cancers, thyroid cancers, -21- 4857-4385-9418.1Atty. Dkt. No.: 115872-3061 uterine cancers, vaginal cancers, vascular tumors, biliary cancer, urothelial cancer, non- melanoma skin cancer, and germ cell tumor.

[0069] In any of the preceding embodiments of the methods disclosed herein, the at least one ICI targets PD-1, PD-L1 or CTLA4. Examples of the at least one ICI comprises ipilimumab, pembrolizumab, nivolumab, dostarlimab, avelumab, atezolizumab, and durvalumab. In some embodiments of the methods disclosed herein, the patient has a high or low tumor mutation burden (TMB). Additionally or alternatively, in some embodiments, the patient comprises tumors having a TMB ranging from of 30-95 mutations per Megabase. In certain embodiments, the TMB is about 30-35, about 35-40, about 40-45, about 45-50, about 50-55, about 55-60, about 60-65, about 65-70, about 70-75, about 75-80, about 80-85, about 85-90, or about 90-95 mutations per Megabase.

[0070] In any and all embodiments of the methods described herein, the patient has been classified as microsatellite stable (MSS), or indeterminate Microsatellite unstable (MSI-I), or MSI.

[0071] Additionally or alternatively, in some embodiments of the methods disclosed herein, the patient has an increased number of mutation associated neoantigens (MANA) or frameshift variants relative to a healthy control subject or predetermined threshold. In some embodiments, the number of mutation associated neoantigens (MANA) or frameshift variants is increased by at least 0.5 fold, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35- fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 55-fold, at least 60-fold, at least 65-fold, at least 70-fold, at least 75-fold, at least 80-fold, at least 85-fold, at least 90- fold, at least 95-fold, or at least 100-fold relative to a healthy control subject or predetermined threshold. In any and all embodiments of the methods disclosed herein, the patient has previously received surgery, radiation, or chemotherapy.

[0072] Additionally or alternatively, in some embodiments, the ICI is administered intravenously, intramuscularly, intraperitoneally, subcutaneously, rectally, parenterally, or intradermally. In any of the preceding embodiments of the methods disclosed herein, the ICI is administered once per every two weeks, once per every three weeks, or once a month. In certain embodiments, the ICI is administered for at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at -22- 4857-4385-9418.1Atty. Dkt. No.: 115872-3061 least 9 months, at least 10 months, at least 11 months, at least 1 year, or at least 2 or more years.

[0073] The ICI compositions of the present technology may optionally be administered as a single bolus to a subject in need thereof. Administration can be carried out by any suitable route, including orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intracranially, intratumorally, intrathecally, or topically. Administration includes self-administration and the administration by another. It is also to be appreciated that the various modes of treatment of medical conditions as described are intended to mean “substantial”, which includes total but also less than total treatment, and wherein some biologically or medically relevant result is achieved.

[0074] In some embodiments, the ICIs comprise pharmaceutical formulations which may be administered to subjects in need thereof in one or more doses (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or more doses). Dosage regimens can be adjusted to provide the desired response.

[0075] Typically, an effective amount of the ICI compositions of the present technology, sufficient for achieving a prophylactic effect, range from about 0.000001 mg per kilogram body weight per day to about 10,000 mg per kilogram body weight per day. Typically, the dosage ranges are from about 0.0001 mg per kilogram body weight per day to about 100 mg per kilogram body weight per day. For administration of ICIs, the dosage ranges from about 0.0001 to 100 mg / kg, and more usually 0.01 to 5 mg / kg every week, every two weeks or every three weeks, of the subject body weight. For example, dosages can be 1 mg / kg body weight or 10 mg / kg body weight every week, every two weeks or every three weeks or within the range of 1-10 mg / kg every week, every two weeks or every three weeks. In one embodiment, a single dosage of ICI ranges from 0.1-10,000 micrograms per kg body weight. In one embodiment, ICI concentrations in a carrier range from 0.2 to 2000 micrograms per delivered milliliter. An exemplary treatment regime entails administration once per every two-three weeks or once a month or once every 3 to 6 months. The ICIs may be administered on multiple occasions. Intervals between single dosages can be hourly, daily, weekly, monthly or yearly. Intervals can also be irregular as indicated by measuring blood levels of the ICI in the subject. In some methods, dosage is adjusted to achieve a serum ICI concentration in the subject of from about 75 μg / mL to about 125 μg / mL, 100 μg / mL to about 150 μg / mL, from about 125 μg / mL to about 175 μg / mL, or from about 150 μg / mL to about 200 μg / mL. Alternatively, the ICIs can be administered as a sustained release formulation, in which case less frequent administration -23- 4857-4385-9418.1Atty. Dkt. No.: 115872-3061 is required. Dosage and frequency vary depending on the half-life of the ICI in the subject. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, or until the subject shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.

[0076] Toxicity. Optimally, an effective amount (e.g., dose) of an ICI described herein will provide therapeutic benefit without causing substantial toxicity to the subject. Toxicity of the ICIs described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD50 (the dose lethal to 50% of the population) or the LD100 (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effect is the therapeutic index. The data obtained from these cell culture assays and animal studies can be used in formulating a dosage range that is not toxic for use in human. The dosage of the ICIs described herein lies within a range of circulating concentrations that include the effective dose with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the subject’s condition. See, e.g., Fingl et al., In: The Pharmacological Basis of Therapeutics, Ch.1 (1975).

[0077] Formulations of Pharmaceutical Compositions. According to the methods of the present technology, the ICIs can be incorporated into pharmaceutical compositions suitable for administration. The pharmaceutical compositions generally comprise recombinant or substantially purified ICI and a pharmaceutically-acceptable carrier in a form suitable for administration to a subject. Pharmaceutically-acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions for administering the ICI compositions (See, e.g., Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, PA 18th ed., 1990). The pharmaceutical compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration. -24- 4857-4385-9418.1Atty. Dkt. No.: 115872-3061

[0078] The terms “pharmaceutically-acceptable,” “physiologically-tolerable,” and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a subject without the production of undesirable physiological effects to a degree that would prohibit administration of the composition. For example, “pharmaceutically- acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients can be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous. “Pharmaceutically-acceptable salts and esters” means salts and esters that are pharmaceutically-acceptable and have the desired pharmacological properties. Such salts include salts that can be formed where acidic protons present in the composition are capable of reacting with inorganic or organic bases. Suitable inorganic salts include those formed with the alkali metals, e.g., sodium and potassium, magnesium, calcium, and aluminum. Suitable organic salts include those formed with organic bases such as the amine bases, e.g., ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. Such salts also include acid addition salts formed with inorganic acids (e.g., hydrochloric and hydrobromic acids) and organic acids (e.g., acetic acid, citric acid, maleic acid, and the alkane- and arene-sulfonic acids such as methanesulfonic acid and benzenesulfonic acid). Pharmaceutically-acceptable esters include esters formed from carboxy, sulfonyloxy, and phosphonoxy groups present in the ICIs, e.g., C1-6 alkyl esters. When there are two acidic groups present, a pharmaceutically-acceptable salt or ester can be a mono-acid-mono-salt or ester or a di-salt or ester; and similarly where there are more than two acidic groups present, some or all of such groups can be salified or esterified. An ICI described herein can be present in unsalified or unesterified form, or in salified and / or esterified form, and the naming of such ICI is intended to include both the original (unsalified and unesterified) compound and its pharmaceutically-acceptable salts and esters. Also, certain embodiments of the present technology can be present in more than one stereoisomeric form, and the naming of such ICI is intended to include all single stereoisomers and all mixtures (whether racemic or otherwise) of such stereoisomers. A person of ordinary skill in the art, would have no difficulty determining the appropriate timing, sequence and dosages of administration for particular drugs and compositions of the present technology. -25- 4857-4385-9418.1Atty. Dkt. No.: 115872-3061

[0079] Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and compounds for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or compound is incompatible with the ICI, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0080] A pharmaceutical composition of the present technology is formulated to be compatible with its intended route of administration. The ICI compositions of the present technology can be administered by parenteral, topical, intravenous, oral, subcutaneous, intraarterial, intradermal, transdermal, rectal, intracranial, intrathecal, intraperitoneal, intranasal; or intramuscular routes, or as inhalants.

[0081] Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating compounds such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and compounds for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0082] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, e.g., water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, e.g., by the use of a coating such as lecithin, -26- 4857-4385-9418.1Atty. Dkt. No.: 115872-3061 by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal compounds, e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be desirable to include isotonic compounds, e.g., sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition a compound which delays absorption, e.g., aluminum monostearate and gelatin.

[0083] Sterile injectable solutions can be prepared by incorporating an ICI of the present technology in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the ICI into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof. The ICIs of the present technology can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained or pulsatile release of the active ingredient.

[0084] Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the ICI can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding compounds, and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating compound such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening compound such as sucrose or saccharin; or a flavoring compound such as peppermint, methyl salicylate, or orange flavoring. -27- 4857-4385-9418.1Atty. Dkt. No.: 115872-3061

[0085] For administration by inhalation, the ICI is delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0086] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, e.g., for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the ICI is formulated into ointments, salves, gels, or creams as generally known in the art.

[0087] The ICI can also be prepared as pharmaceutical compositions in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0088] In one embodiment, the ICI is prepared with carriers that will protect the ICI against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically-acceptable carriers. These can be prepared according to methods known to those skilled in the art, e.g., as described in U.S. Pat. No.4,522,811.

[0089] Kits of the Present Technology

[0090] The present technology provides kits comprising at least one immune checkpoint inhibitor (ICI) and instructions for using the same in the prevention of neoplasia and / or new primary visceral tumors in Lynch Syndrome patients. In certain embodiments, the Lynch Syndrome patients lack detectable tumors and have not received a prior immunotherapy. The components of the kits of the present technology are packed in suitable containers and may be stored in unit or multi-dose containers, for example, sealed ampoules, vials, bottles, syringes, and test tubes, as an aqueous, preferably sterile, solution or as a lyophilized, preferably sterile, formulation for reconstitution. The kit may further comprise a second -28- 4857-4385-9418.1Atty. Dkt. No.: 115872-3061 container which holds a diluent suitable for diluting the pharmaceutical composition towards a higher volume. Suitable diluents include, but are not limited to, the pharmaceutically acceptable excipient of the pharmaceutical composition and a saline solution. Furthermore, the kit may comprise instructions for diluting the pharmaceutical composition and / or instructions for administering the pharmaceutical composition, whether diluted or not. The containers may be formed from a variety of materials such as glass or plastic and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper which may be pierced by a hypodermic injection needle). The kit may further comprise more containers comprising a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and the like. The kits may optionally include instructions customarily included in commercial packages of therapeutic or diagnostic products, that contain information about, for example, the indications, usage, dosage, manufacture, administration, contraindications and / or warnings concerning the use of such therapeutic or diagnostic products.

[0091] The kit can also comprise, e.g., a buffering agent, a preservative or a protein- stabilizing agent. Each component of the kit can be enclosed within an individual container and all of the various containers can be within a single package, along with instructions for using the kit. The kits of the present technology may contain a written product on or in the kit container. The written product describes how to use the reagents contained in the kit, e.g., for prevention of neoplasia and / or new primary visceral tumors in Lynch Syndrome patients that lack detectable tumors and have not received a prior immunotherapy. In certain embodiments, the use of the reagents can be according to the methods of the present technology.

[0092] EXAMPLES

[0093] The present technology is further illustrated by the following Examples, which should not be construed as limiting in any way.

[0094] Example 1: Materials and Methods

[0095] Study Population

[0096] The Memorial Sloan Kettering LS database was queried for all cancer affected patients with pathogenic or likely pathogenic germline variants in MLH1, MSH2 (including -29- 4857-4385-9418.1Atty. Dkt. No.: 115872-3061 EPCAM terminal deletions with epigenetic silencing of MSH2), MSH6, or PMS2 diagnostic of LS (n=837). Patients with LS who received ≥1 cycle of ICB between 03 / 2014, and 03 / 2023 (n=172) were then identified with annotation from the electronic medical record for baseline demographics, tumor stage and pathology, cancer treatment and response, and endoscopic surveillance. Patients with congenital mismatch repair deficiency were excluded. ICB included the CTLA4 inhibitor ipilimumab, PD-1 inhibitors: pembrolizumab, nivolumab, dostarlimab and PD-L1 inhibitors: avelumab, atezolizumab, durvalumab. All patients with further tumor profiling provided written consent for an institutional review board–approved prospective protocol (NCT01775072) for tumor and matched normal DNA sequencing via MSK-IMPACT (MSK–Integrated Mutation Profiling of Actionable Cancer Targets), a clinical NGS platform FDA authorized to identify genetic variants in up to 505 cancer-related genes as well as MSI status 27, 28.

[0097] We additionally assessed the non-ICB treated cohort (n=665). Patients whose last date of follow-up was 2019 or earlier or without documentation of death were excluded from the analysis (n=207) as we could not reliably exclude the possibility of additional tumors. The remaining comparator cohort of non-ICB exposed patients (n=458) was annotated for baseline demographics, all tumors inclusive of cutaneous malignancies and cancer treatment. For further comparisons with ICB exposure, we considered a subgroup of patients who received chemotherapy (N=136).

[0098] Post-ICB pre-neoplasia rate: We previously reported the adenoma rate in a LS prospective endoscopic surveillance cohort, without ICB exposure12. In a recently published LS cohort (n=163)13, inclusive of 60% with CRC history, Del Carmen et al calculated the mean number of adenomas per procedure (MAP) over the number of all colonoscopies performed reporting a MAP of 0.54 with a mean duration of follow up of 8.7 years. We replicated this analysis in our internal cohort of patients who underwent colonoscopies post-ICB exposure (n=61) and we report the mean number of adenomas per procedure over all post-ICB colonoscopies performed.

[0099] Tumor Assessment for dMMR and MSI

[0100] Tumors were designated as dMMR or MSI utilizing one or more methods of analysis. Immunohistochemical (IHC) staining for the MMR proteins was performed as previously described29 with a tumor being designated as dMMR if IHC staining demonstrated loss of protein expression in one or more of the 4 MMR proteins -30- 4857-4385-9418.1Atty. Dkt. No.: 115872-3061 corresponding to the underlying germline alteration known to be present in the patient. When sufficient tissue was available, IHC was also performed on colonic adenomas from colonoscopy surveillance post-ICB exposure. For MMR ICH we used the following antibodies: MLH1 (clone G168-728, diluted 1:250; BD PharMingen or clone M1, ready-to- use, Ventana), MSH2 (clone G219.1129, ready-to-use, Cell Marque), MSH6 (clone 44, ready-to-use, Ventana), and PMS2 (clone A16.4, diluted 1:100, BD Biosciences).

[0101] For MSI, the MSIsensor algorithm was applied as previously described14. MSIsensor scores ≥ 10 defined MSI status, scores ≥ 3 to < 10 an indeterminate (MSI-I) status, and scores < 3 microsatellite stable (MSS) status. If discordance existed between IHC and MSIsensor, miMSI, an alternative classifier-based bioinformatics tool which has a better sensitivity on low tumor samples, was used to further assess MSI30. Tumor mutation burden (TMB) was calculated by dividing the total number of reported non-synonymous mutations by the genomic size where mutations were reported (Mut / MB) for each sample.

[0102] Neoantigen Prediction and HLA binding affinity analysis

[0103] Computational predictions of MHC class I-binding affinity as a surrogate for immunogenicity are routinely used in epitope identification and vaccine discovery studies.

[0104] Patients in the cohort with at least one sample before and after ICB treatment (N=9) were evaluated for similarity in tumor molecular features. Nonsynonymous somatic mutations from the MSK IMPACT variant call format (VCF) output from each sequenced patient tumor were input into the SnpEff program (https: / / pcingola.github.io / SnpEff / ) to predict translated amino acid sequences for each respective mutation. For missense mutations, 9’mer peptides were predicted with a sliding reading frame that was centered around the mutation site (to account for the different possibilities of the mutated amino acid in the 1-9 position). For frameshift mutations, 9’mers were defined starting 8 amino acids upstream of the insertion or deletion event and possible 9’mers were consecutively defined continuing along the mutated peptide sequence. The number of unique MANAs was determined by taking the defined peptide sequence and evaluating all potential contiguous 9’mer variations using a sliding reading frame. Patient-specific Human Leukocyte Antigen (HLA) Class I (HLA-A, HLA-B, HLA-C) subtypes was determined from MSK-IMPACT. The patient-specific HLA class-I binding affinity of all possible unique 9’mer sequences was determined using NetMHCpan version 4.0 (DTU Health); a widely-used artificial neural network-based method31, 32 trained on quantitative binding data and mass- -31- 4857-4385-9418.1Atty. Dkt. No.: 115872-3061 spectrometry derived MHC ligands to predict MHC peptide binding affinity. Strong HLA- binding NAs for each patient were identified if the NA sequence was predicted to have a binding threshold of less than or equal to 1%. Patient pre and post samples were paired and assessed with paired t-testing. If a patient had more than one tumor either before or after ICB, the median value of the respective dependent variable was determined for that patient to produce a single data point pre and post ICB for each patient. Patient 19's pre-ICB urothelial carcinoma was not included due to inadequate sample coverage of MSK IMPACT (57X). Sensitivity analyses were performed in mismatch-repair deficient gastrointestinal cancers only (FIG.6A) and mismatch-repair deficient urothelial cancers only (FIG.6B). For these analyses, Student's t-tests were used without pairing.

[0105] Pre- and post-ICB SPM incidence calculation

[0106] To assess the 3-year or 5-year incidence of de novo neoplasms pre- and post- ICB, we selected patients with at least 36 months or 60 months of post-ICB follow up, respectively. After exclusion of the index cancer for which the patient received ICB and concomitant cancers (< 1 months between diagnoses), we assessed the total number of non- index cancers occurring during the defined observation time of 3-year or 5-year incidence pre- and post-ICB. Comparison of the incidence of pre-ICB and post-ICB on matched follow up was performed using a paired t test (FIG.2B, FIG.8). The same methodology was used for patients not exposed to ICB, using initiation of chemotherapy as a reference for matched pre- and post-chemotherapy 3-year and 5-year incidence calculation.

[0107] Post-ICB SPM were further subdivided by visceral versus cutaneous site of origin; we compared incidence of cutaneous tumors, and incidence of visceral tumors, pre- and post-ICB exposure versus pre- and post- chemotherapy exposure (FIG.2C). We subsequently compared time to first neoplasm post ICB exposure (FIG.2D) and incidence of cutaneous versus visceral tumors pre- and post-ICB (FIGs.2E-2F). The sebaceous neoplasms in our study have been labelled as such as a consequence of the inherent pathological challenges of categorizing sebaceous adenomas and carcinomas due to the considerable overlap of histologic criteria for sebaceous adenoma, sebaceoma and sebaceous carcinoma. This is particularly true when tumor content is small as in a biopsy or when a carcinoma is low grade or well differentiated.

[0108] High risk patients were defined as all cumulative patients presenting with multiple non concomitant cancers (>=2) in a pre-ICB delay matched with post-ICB follow -32- 4857-4385-9418.1Atty. Dkt. No.: 115872-3061 up. To allow enough post-ICB follow-up, patients with a post-ICB follow up of less than 18 months, corresponding to the median ICB duration +6 months, were excluded from the analysis. In case of other cancers occurring outside of the matched pre-ICB post -CB follow-up window, these extra cancers were not considered for the definition of high risk. As for the whole population, the 3-year and 5-year incidence calculation was performed excluding the index cancer and concomitant cancers (<1 month between diagnoses of cancers). Comparison of the incidence of pre-ICB and post-ICB on matched follow up in high risk patients was performed using a paired t test (FIG.8). The same methodology was used for patients not exposed to ICB, using initiation of chemotherapy as a reference for matched pre- and post-chemotherapy 3-year and 5-year incidence calculation.

[0109] For cumulative incidence calculation in high risk patients of de novo cancers (FIG.8), we selected patients with a matched follow up pre- and post-ICB of 36 months and accounted for all non-index cancers occurring during the window of observation. Cumulative incidence is reported as the cumulative sum of non-index cancers occurring during the period of observation. Number of patients and cumulative observation time in months is reported. Fisher’s exact test comparing the cumulative observation time without a metastatic event to the number of months with an event was used for statistical analysis.

[0110] CD8, PD-1, PDL1 Immunohistochemistry and Analysis

[0111] IHC analysis of 15 tumors (18 samples overall, 5 matched pre and post ICB) for CD8, PD1 and PDL1 expression was possible. Antibodies used include: CD8 (clone C8 / 144B, diluted 1:1000, Dako), PD-1 (clone NAT105, ready to use, Cell Marque) and PDL1 (clone E1L3N, diluted 1:100, Cell signalling). The regions with best representation of tumor, away from edges or other artifacts were chosen for analysis. The relevant cells were counted in three 40x High Power Fields (HPF); the final result is expressed as count / HPF. For CD8 and PD1, the counts include TIL (tumor-infiltrating cells), Stromal (within tumor area), and the Sum. For PDL1, the counts include the number of tumor cells staining, and immune cell staining. Additionally, for PDL1, an overall CPS score is provided.

[0112] Analyses of immune populations was performed by comparing quantification per high power field of CD8+, PD-1+ or PD-L1+ cells in 15 different MMRd tumors from 10 patients. Tumors were classified according to the timing of diagnosis compared to ICB exposure: pre-ICB, on ICB or post-ICB discontinuation and means compared using ordinary -33- 4857-4385-9418.1Atty. Dkt. No.: 115872-3061 one way ANOVA test corrected for multiple comparisons. A p-value <0.05 was considered as significant.

[0113] HLA Loss of Heterozygosity Analysis

[0114] HLA genotyping was performed by Polysolver33. The copy number profile for each tumor was found using FACETS (Fraction and Allele-Specific Copy Number Estimates from Tumor Sequencing) v0.5.1434 compared to its matched normal in a two- pass manner: an initial run for purity and ploidy estimation followed by a second run for focal event detection. Each fit was manually reviewed to evaluate the quality of the fit. Loss of heterozygosity over HLA-A, HLA-B, and HLA-C was identified using LOHHLA (Loss Of Heterozygosity in Human Leukocyte Antigen)35 with the purity and ploidy estimates from FACETS. A loss of heterozygosity event was defined as copy number of an alternate allele <0.5 and the p-value related to this allelic imbalance <0.001.

[0115] Statistical Analyses

[0116] According to parameters, categorical variables are expressed in the form of frequencies or / and percentages and are compared where appropriate using two-sided Fisher’s exact testing. Continuous variables are calculated as mean and / or median with variation expressed as the mean and standard deviation (SEM) and interquartile range, respectively. For continuous variables minimum and maximum values are reported when appropriate. Continuous variables are compared with two sided student t testing or two- sided paired t test when matched samples from the same patients where available. For multiple comparisons, we used two sided one-way ordinary ANOVA tests, corrected for multiplicity using the Dunnet test. In all cases, a p-value less than or equal to 0.05 was considered significant. Survival curve estimates calculated with the Kaplan Meier method and compared with log-rank test. Statistics for incidence and cumulative incidence calculation are reported in designated method section. Data originating from the whole cohort was used repeatedly in subgroup analyses, including for the high-risk patients analyses as defined in the related method section.

[0117] Data has been analyzed with GraphPad Prism V9 software (Dotmatics, MA, USA) and R (v4.0.0; https: / / www.R-project.org / ) software package. The level of significance displayed in all the figures across the article is the following: ns: non significant, P>0.05; *: P≤0.05 and P>0.01; **: P≤0.01 and >0.001; ***: P≤0.001 and P>0.0001; ****: P≤0.0001. -34- 4857-4385-9418.1Atty. Dkt. No.: 115872-3061

[0118] Code Availability Statement: Nonsynonymous somatic variants identified via MSK-IMPACT targeted next generation sequencing were assessed by the SnpEff program v5.1d (https: / / pcingola.github.io / SnpEff / ) to predict translated amino acid sequences for each respective mutation. The patient-specific HLA class-I binding affinity of all possible 9’mer sequences was determined using NET MHCpan version 4.0 (DTU Health): https: / / services.healthtech.dtu.dk / services / NetMHCpan-4.0 / . Data has been analyzed with GraphPad Prism V9 software (Dotmatics, MA, USA) and R (v4.0.0; https: / / www.R- project.org / ) software package.

[0119] Example 2: Immune Checkpoint Inhibitors Prevent Neoplasia in Lynch Syndrome Subjects

[0120] Herein, we quantify the enduring risk of pre-malignant and malignant neoplasia in patients with LS post-ICB. Among 837 cancer-affected patients with LS at our institution, we identified 172 who received ICB between 03 / 2014-03 / 2023 (FIG.3) with median follow-up time of 3.12 years (37 months, IQR 11.25-43). Over 60% harbored MSH2 or MLH1 pathogenic germline variants; colorectal cancer (CRC) was the first primary cancer diagnosis in 42% and endometrial cancer in 16%. Median age at first primary malignancy was 47 years (range, 10-81, IQR 39-56) with 51% having multiple malignancies (FIG.9).

[0121] In 63% of patients, the ICB was given for the patient’s first cancer. ICB with an anti-PD-1 antibody was used in 80% and an anti-PD-L1 antibody in 10% (FIG.9). ICB was predominantly administered in the metastatic disease setting (66%) and 82% of patients received ICB for a dMMR tumor. Median duration of treatment was 9 months (IQR 3.75- 22).

[0122] A subsequent primary malignancy (SPM) developed in 12% (21 / 172) of LS cancer patients on or following ICB exposure (FIGs.10, 13, FIGs.1A-1B). Median TMB in the initial tumors targeted with ICB was 42.3 (range 1- 93.9). 13 of 21 patients (62%) had regression in the initial tumor targeted by ICB, while 7 / 21 (33%) had tumor stability. Median time to development of a new malignancy post first ICB exposure was 21 months (IQR range, 6-38). Eight patients developed multiple malignancies, for a total of 40 pathologically confirmed post-ICB exposure tumors. All post-ICB malignancies were histologically distinct from the tumors for which the patients were receiving ICB. Using a next-generation sequencing panel of >500 genes (2MB) on available samples11, pre- and -35- 4857-4385-9418.1Atty. Dkt. No.: 115872-3061 post-ICB tumors shared 1 or fewer somatic mutations (FIG.1A, FIG.4, patient case demonstration). Of the new cancers, immunohistochemical staining (IHC) for MMR was available in 32 samples; 91% (29 / 32) were dMMR. Eight patients were diagnosed with SPM ≤8 months after first ICB exposure, these tumors were not present clinically or radiologically evident prior to ICB initiation.

[0123] We next evaluated colonic pre-neoplasia in patients with LS post-ICB exposure. Post-ICB completion, 61 patients with LS underwent surveillance colonoscopy with 39% (24 / 61) diagnosed with ≥1 pre-malignant polyp(s) (tubular, tubulovillous or serrated adenomas); 54% of this cohort (33 / 61) had a history of prior CRC (FIG.11). Median time to polyp development was 14 months (IQR, 10.5-22 months) from last colonoscopy and 11.5 months (IQR, 6.5-22.5 months) from last ICB dose. In 14 patients with colonic polyps, the original target tumor treated by ICB was CRC; 5 of these patients also developed post-ICB tumors (FIG.11, patients 1, 7, 11, 12 and 14). MMR IHC staining in 19 / 24 patients’ polyps, identified 6 (32%) patients with dMMR adenomas, including three ≤ 3mm in size, with protein loss pattern consistent with the underlying LS-variant.

[0124] We previously reported adenoma rate in a LS prospective endoscopic surveillance cohort, without ICB exposure12. Baseline colonoscopy identified adenoma(s) in 33% (33 / 100) of patients and 29% (28 / 89) had interval adenomas at 1-2 years of follow- up with half of this cohort having had a prior CRC. In a recently published LS cohort (n=163)13, inclusive of 60% with CRC history, baseline adenoma detection rate was 21%; the mean number of adenomas per colonoscopies performed in this cohort was 0.54 with a mean duration of follow up of 8.7 years. The mean number of post-ICB adenomas per colonoscopies performed in our cohort (n=61) was 0.57 with a mean duration of follow up of 2.14 years (range, 1-79 months) post-ICB completion.

[0125] We next performed targeted NGS analysis on paired pre-ICB and post-ICB tumors from 9 patients (FIGs.2C-2F). Post-ICB tumors exhibited fewer frameshift mutations although this did not reach statistical significance (p=0.07). TMB in 7 / 8 post- ICB dMMR tumors was >10 mt / MB (mean TMB in post-ICB dMMR tumors=41.5, range 7-98.9). MSISensor score, a measure of MSI level14, was significantly lower in post-ICB tumors (p=0.05). Post-ICB tumor MSISensor score was also lower than the reference average for MSI tumors defined for each cancer type from the broader MSK clinical sequencing cohort (FIG.13). -36- 4857-4385-9418.1Atty. Dkt. No.: 115872-3061

[0126] We further assessed predicted 9’mer mutation associated neoantigens (MANA) in paired tumors pre-ICB and post-ICB (FIGs.1E-1F, FIG.5). Post-ICB tumors exhibited significantly fewer (53, IQR 14-66) unique MANA predicted to strongly bind (threshold <=1%) to patient-specific Class I MHC compared to pre-ICB tumors (69, IQR 60-95). These trends were preserved in sensitivity analyses when assessing only dMMR gastrointestinal or only urothelial cancers (FIG.6). MANA peptides were compared across the 9 patients with sequenced pre-ICB and post-ICB tumors (FIG.12). Nearly all (98.8%) assessed MANA peptide sequences identified were unique to the individual tumor sample without overlap with the other tumor(s) from the same patient (FIG.12). We assessed for HLA loss of heterozygosity (LOH) as a potential mechanism of immune escape. No HLA LOH in any of the sequenced tumors that developed either on ICB or post-ICB, was observed.

[0127] We also evaluated expression of CD8, PD1 and PD-L1 IHC in pre-ICB and post- ICB exposed tumors (15 tumors) with a statistically significant reduction in CD8+TILs in tumors developing on ICB that was not seen in tumors that developed post-ICB completion. (FIGs.7A-7B).

[0128] To contextualise our finding of enduring neoplasia risk amongst patients with LS who had received ICB, we evaluated the rate of SPM both in our internal cohort of patients with LS without ICB exposure and in a prospective LS multicentre study15 (FIG.2A). The rate of SPM per mean observation years was comparable at 3.2%, 4.1%, and 4.9% in the non-ICB exposed internal cohort, the external cohort, and our ICB exposed cohort, respectively.

[0129] Given the limitations of historical cohort comparisons, we then assessed intra- patient tumor development rates in patients with matched duration of follow-up pre-ICB and post-ICB. Rate of tumor development in the pre-ICB and post-ICB period was equivalent with 3-years of follow-up (FIG.2B). We replicated this analysis in non-ICB exposed patients with LS from our institution and demonstrated unchanged rates of tumor development pre- and post-chemotherapy exposure at 3-years (n=95) of matched follow-up (FIG.2B). When extended to 5-years of follow-up, the results were consistent in both the ICB and the chemotherapy groups (FIG.8A). Interestingly, when separating SPM into cutaneous versus visceral malignancies, we identified a reduction in post-ICB visceral tumor rate whilst conversely, the rate of cutaneous neoplasm increased significantly (FIGs. 2C-2F). In our historical comparator cohort, the overall rate of cutaneous malignancies was -37- 4857-4385-9418.1Atty. Dkt. No.: 115872-3061 12%; in the post-ICB cohort with SPM, 48%, (10 / 21), of patients developed ≥1 cutaneous malignancy. On exploratory analysis, when restricted to high-risk patients with a history of 2 or more malignancies pre-ICB exposure, the rate of SPM was significantly lower at 3- years and 5-years of follow-up (FIGs.8B-8D).

[0130] Immunoprevention with ICB in patients with LS is an attractive hypothesis given the success of ICB in locally advanced and metastatic dMMR tumors 3-9. Our finding that 12% of ICB treated patients with LS developed a new cancer less than 2 years after first ICB exposure and that 39% harbored a pre-malignant colonic polyp provides initial evidence that ICB does not durably eliminate the risk of pre-malignant neoplasia or subsequent malignant neoplasia. This is even more surprising since the majority (91%) of new cancers and a subset of pre-malignant neoplasia identified post-ICB treatment were dMMR.

[0131] Subsequent primary tumors following exposure to ICB, in addition to being predominately dMMR, still harbored high TMB, but were found to be genetically ‘younger’ than the dMMR tumors targeted by ICB as evidenced by the lower MSI level and a trend towards lower number of frameshift mutations, which are known to increase in dMMR tumors over time and contribute proportionally to tumor immunogenicity16. Notably, despite the lower genomic measures of immunogenicity, including lower unique MANA, some SPM still responded to ICB, including three complete radiographic responses.

[0132] Our data suggest that there is a window of time between malignant precursor lesion formation and the development of an invasive carcinoma where these tumor precursors may not be susceptible to ICB. There are several potential interpretations, but dMMR alone may not be sufficient and stage of tumor development appears to be a factor. This has been modeled in prior studies16, 17 and may explain recent data wherein genetic ablation of MMR alone was insufficient in inducing tumor immunogenicity18. Mandal and Germano16,17 both show that conversion to a immunogenic phenotype in a cell line from the point of MMR ablation by CRISPR required months of passaging in tissue culture and that immunogenicity was proportional to time and to the accumulation of single nucleotide variants, frameshift mutations and elevated levels of MSI 3,16. It may be that during the earliest period of tumor development, dMMR tumors are not immunogenic and therefore resistant to the effect of ICB. -38- 4857-4385-9418.1Atty. Dkt. No.: 115872-3061

[0133] In line with these studies, the assumption of later acquisition of dMMR status in LS-associated polyps has been questioned by evidence of colonoscopically ‘invisible’ precursor lesions or alternative routes to LS-associated CRC development19. dMMR crypt foci have been identified in the normal mucosa of patients with LS 20, 21 and an alternative 2-in-1 model of shortcut carcinogenesis has recently been proposed in MLH1-associated LS22. Molecular analyses of LS-associated CRC suggests that dMMR can be an early event preceding adenoma formation23 and furthermore an immune profile independent of MMR or neoantigen formation has been demonstrated in LS-associated polyps24. Indeed in our study 6 post-ICB colonic polyps were dMMR, 3 of which were >8 mm concordant with prior studies25; notably 3 tubular adenomas <8 mm in size were also dMMR.

[0134] Of particular note are two significant observations that have potential clinical implications for surveillance and prevention. The first is the higher rates of cutaneous SPM and lower rates of visceral SPM post-ICB, suggesting that subsequent visceral SPMs may be decreased by ICB exposure or that there is a shift in the type of SPM that occurs. This may have an impact on survival since visceral tumors can be lethal if not successfully treated. Also, dermatological screening, even in patients with LS without a prior diagnosis of the Muir-Torre variant, should be considered given the high number of cutaneous SPM. Second, high risk individuals with multiple malignancies prior to ICB exposure demonstrated significantly reduced rates of SPM.

[0135] Additionally, we demonstrate a lack of shared MANA and lower number of MANA with strong binding to Class 1 HLA between pre- and post-ICB tumors. As exploiting high rates of shared frameshift MANA among dMMR tumors underpins frameshift peptide-based vaccination approaches in LS26, it remains to be elucidated whether such strategies will be as effective in patients who have already been subject to ICB exposure.

[0136] Surprisingly, we also observed that none of the responders comprise germline mutations in POLE or POLD1. Patients with deficiencies in Polymerase POLE or POLD1 generate a high % of +1 insertions within microsatellite loci with a length of 5-6 bp, and are indicated for PD-1 blockade, whereas PD-1 blockade is considered inappropriate for patients that are wildtype for POLE and POLD1. See WO 2022099004 (describing that high POLEness score predicts immunotherapy response in cancers, whereas dMMR and tumor mutation burden did not. POLE and POLD1 with defective proofreading are -39- 4857-4385-9418.1Atty. Dkt. No.: 115872-3061 associated with increased insertion events and can contribute to the overall total microsatellite indel (MS-indel) burden in human cancers.).

[0137] Our study highlights the unique immunobiology of dMMR tumors and demonstrates a persistent risk of both new SPM and pre-neoplastic lesions after ICB exposure in patients with LS. This has immediate clinical implications in that high-risk surveillance of patients with LS should be continued, if clinically appropriate, both throughout and after ICB treatment.

[0138] Example 3: Prophylactic Clinical Trial in Therapy-Naïve LS Patients

[0139] A prophylactic clinical trial will be set up with 1 arm including Lynch Syndrome patients that lack detectable tumors, comprise a pathogenic germline mutation in MLH1, MSH2, MSH6, PMS2, or EPCAM 3’ deletion and have not received a prior anti-cancer therapy such as immunotherapy. Lynch syndrome patients will consist of two different cohorts including i) individuals with Lynch syndrome without a prior cancer diagnosis and ii) individuals with Lynch syndrome who are deemed to be at an especially high-risk of cancer(s) based on harboring a specific MMR gene mutation or based on a personal history of 1 or more prior cancer diagnosis. Both cohorts will be treated with ICB therapy. At least some patients harboring either a germline POLE or POLD1 mutation may also be included in this study. ICB will include the CTLA4 inhibitor ipilimumab, PD-1 inhibitors: pembrolizumab, nivolumab, dostarlimab and PD-L1 inhibitors: avelumab, atezolizumab, durvalumab. Patients will be characterized using the same assays described in Example 1.

[0140] It is anticipated that ICB therapy will prevent tumor formation / neoplasia in at least some of the Lynch syndrome patients that lack detectable tumors prior to treatment.

[0141] EQUIVALENTS

[0142] The present technology is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the present technology. It is to be understood that this present technology is not limited to particular methods, reagents, -40- 4857-4385-9418.1Atty. Dkt. No.: 115872-3061 compounds compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0143] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0144] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non- limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 cells refers to groups having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.

[0145] All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification. REFERENCES 1. Win, Aung Ko, et al. Journal of clinical oncology 30.9 (2012): 958. 2. Latham, Alicia, et al. Journal of clinical oncology 37.4 (2019): 286. 3. Le, Dung T., et al. New England Journal of Medicine 372.26 (2015): 2509-2520. 4. Le, Dung T., et al Science 357.6349 (2017): 409-413. 5. Diaz, L., et al. Annals of Oncology 28 (2017): v128-v129. 6. Marabelle, Aurelien, et al. Journal of Clinical Oncology 38.1 (2020): 1. 7. Cercek, Andrea, et al. New England Journal of Medicine (2022). -41- 4857-4385-9418.1Atty. Dkt. No.: 115872-3061 8. Chalabi, Myriam, et al. Nature medicine 26.4 (2020): 566-576. 9. Ludford, Kaysia, et al. Journal of Clinical Oncology (2023): JCO-22. 10. Heudel, Pierre, et al. ESMO open 6.1 (2021): 100044. 11. Zehir, Ahmet et al. Nature Medicine 23.6 (2017): 703-173 12. Yan et al. Cell host and microbe 27.4 (2020): 585-600 13. Del Carmen et al. Frontiers in Oncology 13:(2023): 1710 14. Middha et al. JCO Precision Oncology 1(2017): 1-17 15. Moller, Pal et al. Gut 66.9 (2017): 1657-1664 16. Mandal, Rajarsi, et al. Science 364.6439 (2019): 485-491. 17. Amodio, Vito, et al. Cancer Cell (2022). 18. Westcott, Peter MK, et al. bioRxiv (2021). 19. Ahadova, Aysel, et al. International Journal of Cancer 148.4 (2021): 800-811. 20. Kloor, Matthias, et al The lancet oncology 13.6 (2012): 598-606. 21. Staffa, Laura et al. PloS one vol.10,3 e0121980.27 Mar.2015, 22. Ahadova, Aysel et al. Gastroenterology (2023) 23. Sekine, Shigeki, et al Modern Pathology 30.8 (2017): 1144-1151. 24. Chang, Kyle et al. JAMA Oncology 4.8 (2018): 1085-1092. 25. Yurgelun, Matthew B., et al. Cancer prevention research 5.4 (2012): 574-582.. 26. Roudko, Vladimir, et al Cell 183.6 (2020): 1634-1649. 27. Cheng DT, Prasad M, Chekaluk Y, et al.: Comprehensive detection of germline variants by MSK-IMPACT, a clinical diagnostic platform for solid tumor molecular oncology and concurrent cancer predisposition testing. BMC Med Genomics 10:33, 2017 28. Cheng DT, Mitchell TN, Zehir A, et al.: Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT): A hybridization capture-based next-generation sequencing clinical assay for solid tumor molecular oncology. J Mol Diagn 17:251-264, 2015 29. Shia, Jinru. "The diversity of tumours with microsatellite instability: molecular mechanisms and impact upon microsatellite instability testing and mismatch repair protein immunohistochemistry." Histopathology 78.4 (2021): 485-497. 30. Ziegler, John, et al. "MiMSI-a deep multiple instance learning framework improves microsatellite instability detection from tumor next-generation sequencing." bioRxiv (2020). -42- 4857-4385-9418.1Atty. Dkt. No.: 115872-3061 31. Jurtz, Vanessa, et al. "NetMHCpan-4.0: improved peptide–MHC class I interaction predictions integrating eluted ligand and peptide binding affinity data." The Journal of Immunology 199.9 (2017): 3360-3368. 32. Paul, Sinu, et al. "Benchmarking predictions of MHC class I restricted T cell epitopes in a comprehensively studied model system." PLoS computational biology 16.5 (2020): e1007757. 33. Shukla, S. A. et al. Comprehensive analysis of cancer-associated somatic mutations in class I HLA genes. Nat. Biotechnol.33, 1152–1158 (2015). 34. Shen, R. & Seshan, V. E. FACETS: allele-specific copy number and clonal heterogeneity analysis tool for high-throughput DNA sequencing. Nucleic Acids Res.44, e131 (2016). 35. McGranahan, N. et al. Allele-specific HLA loss and immune escape in lung cancer evolution. Cell 171, 1259–1271 (2017). -43- 4857-4385-9418.1

Claims

Atty. Dkt. No.: 115872-3061 WHAT IS CLAIMED IS 1. A method for preventing neoplasia in a Lynch Syndrome patient comprising administering to the patient an effective amount of at least one immune checkpoint inhibitor (ICI), wherein the patient lacks detectable tumors and has not received a prior immunotherapy.

2. The method of claim 1, wherein the patient does not harbor a germline mutation in POLE.

3. The method of claim 1 or 2, wherein the patient does not harbor a germline mutation in POLD1.

4. The method of any one of claims 1-3, wherein the patient comprises an inactivating pathogenic or likely pathogenic germline mutation in at least one DNA mismatch repair (MMR) gene.

5. The method of claim 4, wherein the at least one MMR gene is selected from the group consisting of MLH1, MSH2, MSH6, PMS2 or EPCAM 3’ terminal region.

6. The method of any one of claims 1-5, wherein the Lynch Syndrome patient has not received a prior cancer diagnosis.

7. The method of any one of claims 1-5, wherein the Lynch Syndrome patient has received at least one prior cancer diagnosis.

8. The method of any one of claims 1-7, wherein the at least one ICI targets PD-1, PD- L1 or CTLA4.

9. The method of any one of claims 1-8, wherein the at least one ICI is selected from among ipilimumab, pembrolizumab, nivolumab, dostarlimab, avelumab, atezolizumab, and durvalumab.

10. The method of any one of claims 7-9, wherein the at least one prior cancer diagnosis is a cancer selected from among carcinoma, sarcoma, melanoma, hematopoietic cancer, adrenal cancers, bladder cancers, blood cancers, bone cancers, brain cancers, breast cancers, carcinoma, cervical cancers, colon cancers, colorectal cancers, corpus uterine cancers, ear, nose and throat (ENT) cancers, endometrial cancers, esophageal cancers, gastrointestinal cancers, head and neck cancers, Hodgkin's -44- 4857-4385-9418.1Atty. Dkt. No.: 115872-3061 disease, intestinal cancers, kidney cancers, larynx cancers, leukemias, liver cancers, lymph node cancers, lymphomas, lung cancers, melanomas, mesothelioma, myelomas, nasopharynx cancers, neuroblastomas, non-Hodgkin's lymphoma, oral cancers, ovarian cancers, pancreatic cancers, penile cancers, pharynx cancers, prostate cancers, rectal cancers, sarcoma, seminomas, skin cancers, stomach cancers, teratomas, testicular cancers, thyroid cancers, uterine cancers, vaginal cancers, vascular tumors, biliary cancer, urothelial cancer, non-melanoma skin cancer, and germ cell tumor.

11. A method for preventing new primary visceral tumors in a Lynch Syndrome patient comprising administering to the patient an effective amount of at least one immune checkpoint inhibitor (ICI), wherein the patient has received at least one previous cancer diagnosis prior to administration of the at least one ICI.

12. The method of claim 11, wherein the patient comprises an inactivating pathogenic or likely pathogenic germline mutation in at least one DNA mismatch repair (MMR) gene.

13. The method of claim 12, wherein the at least one MMR gene is selected from the group consisting of MLH1, MSH2, MSH6, PMS2, or EPCAM 3’ terminal region.

14. The method of any one of claims 11-13, wherein the at least one ICI targets PD-1, PD-L1 or CTLA4 or wherein the at least one ICI is selected from among ipilimumab, pembrolizumab, nivolumab, dostarlimab, avelumab, atezolizumab, and durvalumab.

15. The method of any one of claims 11-14, wherein the at least one previous cancer diagnosis is a cancer selected from among carcinoma, sarcoma, melanoma, hematopoietic cancer, adrenal cancers, bladder cancers, blood cancers, bone cancers, brain cancers, breast cancers, carcinoma, cervical cancers, colon cancers, colorectal cancers, corpus uterine cancers, ear, nose and throat (ENT) cancers, endometrial cancers, esophageal cancers, gastrointestinal cancers, head and neck cancers, Hodgkin's disease, intestinal cancers, kidney cancers, larynx cancers, leukemias, liver cancers, lymph node cancers, lymphomas, lung cancers, melanomas, mesothelioma, myelomas, nasopharynx cancers, neuroblastomas, non-Hodgkin's lymphoma, oral cancers, ovarian cancers, pancreatic cancers, penile cancers, pharynx cancers, prostate cancers, rectal cancers, sarcoma, seminomas, skin cancers, -45- 4857-4385-9418.1Atty. Dkt. No.: 115872-3061 stomach cancers, teratomas, testicular cancers, thyroid cancers, uterine cancers, vaginal cancers, vascular tumors, biliary cancer, urothelial cancer, non-melanoma skin cancer, and germ cell tumor.

16. The method of any one of claims 11-15, wherein the patient comprises a germline mutation in POLE and / or POLD1.

17. The method of any one of claims 11-16, wherein the patient has a high or low tumor mutation burden.

18. The method of any one of claims 1-17, wherein the patient has been classified as microsatellite stable (MSS), or indeterminate Microsatellite unstable (MSI-I), or MSI.

19. The method of any one of claims 1-18, wherein the patient has an increased number of mutation associated neoantigens (MANA) or frameshift variants relative to a healthy control subject or predetermined threshold.

20. The method of any one of claims 1-19, wherein the patient has previously received surgery, radiation, or chemotherapy. -46- 4857-4385-9418.1

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