Cancer treatment methods

Abstract

To provide methods for treating cancer such as melanomas.SOLUTION: A method comprises: (a) acquiring a value of responder status of a patient to a therapy comprising an inhibitor of PD-1 or PD-L1, where the value of responder status comprises a measure of the tumor mutational burden (TMB) in a melanoma sample, or a sample derived from a melanoma, from the subject; and (b) in response to an increased value of responder status, e.g., compared to a reference value of responder status, administering the inhibitor of PD-1 or PD-L1 to the patient, thereby treating the patient.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Patent Application No. 62 / 301,510, filed February 29, 2016. The entire contents of the above application are incorporated herein by reference. Let's say.

[0002] FIELD OF THE INVENTION The present invention provides methods and methods for treating cancer (hereinafter referred to as cancer), such as melanoma. and compositions. [Background technology]

[0003] Programmed death-1 / ligand (PD-1 / PD Substances that target the L1 (L2) system can relieve the suppression of antitumor T cell responses, which are manifested in many cancers. It has shown remarkable clinical activity (Wolchok, Cell 162:937, 2015). Volumab and pembrolizumab are clinically effective in 25-45% of patients with advanced melanoma. It induces a response and is now widely used with regulatory approval (Topalian et al., N Engl J Med 366:2443-54,2012;Hamid et al.,NE ngl J Med 369:134-44,2013; Herbst et al., Nature 515:563-7,2014;Robert et al.,N Engl J Med,372 :2521-32,2015;Robert et al.,N Engl J Med,372:3 20-30,2014;Larkin et al.,N Engl J Med,373:1270 Despite this activity, there is limited evidence to predict response and guide treatment decision-making. Clinically available validated markers for HIV-1 infection remain elusive. Clonal proliferation of tumors and PD-L1 expression by tumor or immune cells are potential markers of treatment response. (Topalian et al., N Engl J Med 366: 2443-54, 2012; Herbst et al., Nature 515:563-7, 20 14;Tumeh et al., Nature 515:568-71, 2014;Weber et al., Lancet Oncol,16:375-84,2015).

[0004] Despite these findings, no studies have shown that PD-1 or PD-L1 inhibitors can be effectively used to treat PD-1- or PD-L1-inhibitory disorders. The impact of mutational burden and specific oncogenic mutations in leukemia has not been systematically investigated. Furthermore, translating genomic research into routine clinical practice remains problematic because: Whole exome sequencing (WES) is not widely available, is expensive, and is very time-consuming. This is because it is technically difficult.

[0005] Thus, immunotherapy and genomic therapy for treating and diagnosing cancer, such as melanoma, are becoming increasingly popular. Novel therapeutic and diagnostic approaches, including clinical testing, are needed. Summary of the Invention

[0006] Overview of the invention The present invention is based, at least in part, on the role of mutational load in the regulation of PD-1 or PD-2 expression in melanoma. is based on the finding that PD-L1 correlates with therapeutic benefit from therapy including inhibitors of PD-L1. In one embodiment, for example, hybrid capture-based next generation sequencing ( Next-Generation Scanning (NGS) platforms are used to analyze genome or exome sequences from patient samples. Partial profiling serves as a valid surrogate for analysis of the total mutation load. In another embodiment, a single gene (e.g., the NF1 or LRP1B gene) is Mutations (alterations) detected in genes are used to predict response to treatment. Serves as a surrogate or further surrogate for mutation load. Targeted to detect mutation load. Using methods involving NGS platforms has several advantages, including, for example, faster, e.g., more clinically relevant, compared to whole genome or whole exome sequencing Manageable turnaround time (approximately 2 weeks), standardized informatics pipeline (i information pipeline), and more manageable costs. The methods disclosed herein include, but are not limited to, measuring PD-L1 expression. have other advantages over markers such as β-glucan, ... provides an objective measure (e.g., mutation load) rather than a pathological scoring The methods disclosed herein provide a method for targeting available mutations and immunotherapies. These methods also allow for the simultaneous detection of the mutation load for advanced melanoma. Clinical benefit of response to anti-PD-1 and / or anti-PD-L1 therapy in patients with This may provide a useful predictor.

[0007] Thus, the present invention provides, at least in part, a method for targeting the PD-1 pathway and / or by administering to a subject an effective amount of an agent (e.g., a therapeutic agent) that inhibits cancer, e.g., Methods for treating a subject having or at risk for melanoma are provided. In some embodiments, the therapeutic agent is an inhibitor of PD-1 or PD-L1. In the method, a therapeutic agent, such as an inhibitor of PD-1 or PD-L1, is administered to a subject. In some embodiments, the response state is administered according to the value of the response state relative to the target substance. This includes a measure of mutation load in samples from subjects (e.g., tumor samples). In embodiments, the measure of mutational load is determined for a given set of genes disclosed herein. the level of somatic mutations in the patient, the presence of somatic mutations in the NF1 gene, the LRP1B gene, The number of somatic mutations in a gene or genes in a given set of genes disclosed herein The method includes determining one or more of the number of C to T transitions in a sequence, or any combination thereof.

[0008] Treatment method In one aspect, the present invention relates to a method of treating a subject with cancer, e.g., melanoma. The method comprises: (a) administering to a subject a therapy, e.g., a therapy comprising an inhibitor of PD-1 or PD-L1; and obtaining a response state value for the method, where the response state value is obtained by measuring a sample from the subject, e.g., a measure of mutational load in a melanoma sample or melanoma-derived sample, (b) an increase in the value of the response state, e.g., in response to an increase in the value of the response state compared to a baseline value of the response state; If necessary, administering a therapy to a subject, thereby treating the subject.

[0009] In certain embodiments, the reference response status is a response status relative to a non-responder to a therapy. is the value.

[0010] In certain embodiments, the measure of mutational load is the following in a sample from a subject: Things, namely, (i) somatic mutations in a given set of genes listed in Table 1 (e.g., one or more levels of somatic mutations in (ii) the presence of a somatic mutation in the NF1 gene (e.g., one or more somatic mutations) or non-existence, (iii) the number of somatic mutations (e.g., one or more somatic mutations) in the LRP1B gene; ,or (iv) C to T changes in a given set of genes listed in Table 1 (e.g., , the number of C to T changes of 1 or more This may include one, two, three or all of the following decisions:

[0011] In certain embodiments, the following in a sample from a subject: (i) somatic mutations in a given set of genes listed in Table 1 (e.g., one or more increased levels of somatic mutations in a given set of genes listed in Table 1, e.g. an increase in somatic mutations (e.g., one or more somatic mutations) in a patient compared to a baseline level , (ii) the presence of a somatic mutation (e.g., one or more somatic mutations) in the NF1 gene; (iii) the number of somatic mutations (e.g., one or more somatic mutations) in the LRP1B gene; an increase in, e.g., somatic mutations in the LRP1B gene (e.g., one or more somatic mutations) its increase compared to the baseline number of (iv) C to T changes in a given set of genes listed in Table 1 (e.g., , one or more C to T changes), e.g., an increase in the number of Compared to a reference number of C to T changes in the set (e.g., 1 or more C to T changes) The increase if Therapy is administered to the subject depending on one, two, three, or all of the above.

[0012] In another aspect, the present invention provides a method for treating a subject with cancer, e.g., melanoma. The method relates to a therapy, e.g., a therapy comprising an inhibitor of PD-1 or PD-L1. The method includes administering to a subject a therapeutically effective amount of a compound to treat the subject, where the subject is, for example, experiencing a response. or capable of exhibiting an increase in the value of a response state when compared to a baseline value of the state. The response status value is determined based on a sample from the subject, e.g., a melanoma sample or contains a measure of mutational load in melanoma-derived samples.

[0013] In certain embodiments, the measure of mutational load is the following in a sample from a subject: Things, namely, (i) somatic mutations in a given set of genes listed in Table 1 (e.g., one or more levels of somatic mutations in (ii) the presence of a somatic mutation in the NF1 gene (e.g., one or more somatic mutations) or non-existence, (iii) the number of somatic mutations (e.g., one or more somatic mutations) in the LRP1B gene; ,or (iv) C to T changes in a given set of genes listed in Table 1 (e.g., , the number of C to T changes of 1 or more This may include one, two, three or all of the following decisions:

[0014] In certain embodiments, the following in a sample from a subject: (i) somatic mutations in a given set of genes listed in Table 1 (e.g., one or more increased levels of somatic mutations in a given set of genes listed in Table 1, e.g. an increase in somatic mutations (e.g., one or more somatic mutations) in a patient compared to a baseline level , (ii) the presence of a somatic mutation (e.g., one or more somatic mutations) in the NF1 gene; (iii) the number of somatic mutations (e.g., one or more somatic mutations) in the LRP1B gene; an increase in, e.g., somatic mutations in the LRP1B gene (e.g., one or more somatic mutations) its increase compared to the baseline number of (iv) C to T changes in a given set of genes listed in Table 1 (e.g., , one or more C to T changes), e.g., an increase in the number of Compared to a reference number of C to T changes in the set (e.g., 1 or more C to T changes) The increase if Therapy is administered to the subject depending on one, two, three, or all of the above.

[0015] In yet another aspect, the present invention provides a method for treating a subject with cancer, e.g., melanoma. The method relates to a method comprising: (a) a sample from a subject, e.g., a melanoma sample or a melanoma-derived sample In the following, namely: (i) Somatic mutations in a given set of genes (e.g., as listed in Table 1) level of mutations (e.g., ≥1 somatic mutation), (ii) the presence of a somatic mutation in the NF1 gene (e.g., one or more somatic mutations); Or non-existence, (iii) a somatic mutation in the LRP1B gene (e.g., one or more somatic mutations); number of, or (iv) C to T changes in a given set of genes listed in Table 1 (e.g., For example, the number of C to T transitions of 1 or more Determine one or more of (b) The following: (i) somatic mutations in a given set of genes listed in Table 1 (e.g., 1 an increase in the level of somatic mutations (e.g., a given set of genes listed in Table 1) the level of somatic mutations (e.g., one or more somatic mutations) in a patient compared to a baseline level of somatic mutations in that patient increase, (ii) the presence of a somatic mutation in the NF1 gene (e.g., one or more somatic mutations); , (iii) a somatic mutation in the LRP1B gene (e.g., one or more somatic mutations); an increase in the number of somatic mutations in the LRP1B gene (e.g., one or more somatic mutations the increase compared to a baseline number of individuals with a different (iv) C to T changes in a given set of genes listed in Table 1 (e.g., an increase in the number of mutations (e.g., one or more C to T changes), e.g., in the genes listed in Table 1 the reference number of C to T changes in a given set (e.g., 1 or more C to T changes); The increase compared to therapy, e.g., therapy comprising an inhibitor of PD-1 or PD-L1, in response to one or more of the following: to a subject.

[0016] How to choose a therapy In one aspect, the present invention provides a method for treating a subject with cancer, e.g., melanoma, For example, the present invention relates to a method for selecting a therapy that includes an inhibitor of PD-1 or PD-L1. The method includes administering to the subject a therapy, e.g., a therapy comprising an inhibitor of PD-1 or PD-L1. and selecting a therapy accordingly, wherein the response state The values ​​are measured in a sample from the subject, e.g., a melanoma sample or a melanoma-derived sample. The increase in the value of the response state, e.g., compared to the baseline value of the response state, includes a measure of the mutation load in the target gene. An increase in the level of the IL-1 receptor agonist (IL-1) relative to the level of the IL-1 receptor agonist (IL-1) indicates that the subject is or is likely to be a responder to the therapy. Alternatively, it may indicate that the subject will or is likely to respond to the therapy.

[0017] In certain embodiments, the measure of mutational load is the following in a sample from a subject: Things, namely, (i) somatic mutations in a given set of genes listed in Table 1 (e.g., one or more levels of somatic mutations in (ii) the presence of a somatic mutation in the NF1 gene (e.g., one or more somatic mutations) or non-existence, (iii) the number of somatic mutations (e.g., one or more somatic mutations) in the LRP1B gene; ,or (iv) C to T changes in a given set of genes listed in Table 1 (e.g., , the number of C to T changes of 1 or more This may include one, two, three or all of the following decisions:

[0018] In certain embodiments, the following in a sample from a subject: (i) somatic mutations in a given set of genes listed in Table 1 (e.g., one or more increased levels of somatic mutations in a given set of genes listed in Table 1, e.g. an increase in somatic mutations (e.g., one or more somatic mutations) in a patient compared to a baseline level , (ii) the presence of a somatic mutation (e.g., one or more somatic mutations) in the NF1 gene; (iii) the number of somatic mutations (e.g., one or more somatic mutations) in the LRP1B gene; an increase in, e.g., somatic mutations in the LRP1B gene (e.g., one or more somatic mutations) its increase compared to the baseline number of (iv) C to T changes in a given set of genes listed in Table 1 (e.g., , one or more C to T changes), e.g., an increase in the number of Compared to a reference number of C to T changes in the set (e.g., 1 or more C to T changes) The increase if Therapy is administered to the subject depending on one, two, three, or all of the above.

[0019] Subject or cancer assessment method In yet another aspect, the present invention provides a method for evaluating a subject with cancer, e.g., melanoma. The method relates to a method comprising: (a) administering to a subject a therapy, e.g., a therapy comprising an inhibitor of PD-1 or PD-L1; and obtaining a response state value for the method, where the response state value is obtained by measuring a sample from the subject, e.g., a measure of mutational load in a melanoma sample or melanoma-derived sample, (b) determining whether a subject is a responder (e.g., a complete or partial responder) or non-responder to a therapy; including identifying the answer as Here, a response state value equal to or greater than the reference response state value indicates that the subject is responding to the therapy. or that the subject is or is likely to be a responder to the therapy. indicates high sexuality, or Here, a response state value less than the reference response state value indicates that the subject is a non-responder to the therapy. that a subject is or is likely to be unresponsive to therapy, or that a subject is or is likely to be unresponsive to therapy Shows.

[0020] In certain embodiments, the measure of mutational load is the following in a sample from a subject: Things, namely, (i) somatic mutations in a given set of genes listed in Table 1 (e.g., one or more levels of somatic mutations in (ii) the presence of a somatic mutation in the NF1 gene (e.g., one or more somatic mutations) or non-existence, (iii) the number of somatic mutations (e.g., one or more somatic mutations) in the LRP1B gene; ,or (iv) C to T changes in a given set of genes listed in Table 1 (e.g., , the number of C to T changes of 1 or more This may include one, two, three or all of the following decisions:

[0021] In certain embodiments, the following in a sample from a subject: (i) somatic mutations in a given set of genes listed in Table 1 (e.g., one or more increased levels of somatic mutations in a given set of genes listed in Table 1, e.g. an increase in somatic mutations (e.g., one or more somatic mutations) in a patient compared to a baseline level , (ii) the presence of a somatic mutation (e.g., one or more somatic mutations) in the NF1 gene; (iii) the number of somatic mutations (e.g., one or more somatic mutations) in the LRP1B gene; an increase in, e.g., somatic mutations in the LRP1B gene (e.g., one or more somatic mutations) its increase compared to the baseline number of (iv) C to T changes in a given set of genes listed in Table 1 (e.g., , one or more C to T changes), e.g., an increase in the number of Compared to a reference number of C to T changes in the set (e.g., 1 or more C to T changes) The increase if Depending on one, two, three, or all of the above, therapy, e.g., PD-1 or PD-L1 inhibitors, may be used. A therapy comprising an inhibitor is administered to the subject.

[0022] In certain embodiments, the method further comprises the step of encoding a T cell receptor (TCR) gene (e.g., In one embodiment, the method further comprises sequencing the TCR gene (or genes). This involves determining the clonality of the TCR genes.

[0023] In a related aspect, a method for assessing a patient or patient population is provided. identify, select, or enter information or information that a patient population is participating in a clinical trial and providing a therapy for the patient or patient population, e.g., an inhibitor of PD-1 or PD-L1. and obtaining a response status value to therapy including the vitamin, and administering the patient to a subject as described herein. A subject or patient population is classified as a responder (e.g., complete or partial responder) to therapy or Identifying a patient or patient population as a non-responder or determining the likelihood or likelihood that the patient or patient population will respond to therapy This includes identifying whether

[0024] In another aspect, the present invention provides a method for assessing cancer, e.g., melanoma, in a subject. The method comprises: (a) administering to a subject a therapy, e.g., a therapy comprising an inhibitor of PD-1 or PD-L1; and obtaining a response state value for the method, where the response state value is obtained by measuring a sample from the subject, e.g., a measure of mutational load in a melanoma sample or melanoma-derived sample, (b) determining the responsiveness of the cancer to the therapy; Here, a response status value that is equal to or greater than the reference response status value indicates that the cancer is responding to therapy. indicates a likely or likely response, or Here, a response status value less than the reference response status value indicates that the cancer is not responding to therapy or is not responding to the therapy. This indicates that there is a high possibility of

[0025] In certain embodiments, the measure of mutational load is the following in a sample from a subject: Things, namely, (i) somatic mutations in a given set of genes listed in Table 1 (e.g., one or more levels of somatic mutations in (ii) the presence of a somatic mutation in the NF1 gene (e.g., one or more somatic mutations) or non-existence, (iii) the number of somatic mutations (e.g., one or more somatic mutations) in the LRP1B gene; ,or (iv) C to T changes in a given set of genes listed in Table 1 (e.g., , the number of C to T changes of 1 or more This may include one, two, three or all of the following decisions:

[0026] In certain embodiments, the following in a sample from a subject: (i) somatic mutations in a given set of genes listed in Table 1 (e.g., one or more increased levels of somatic mutations in a given set of genes listed in Table 1, e.g. an increase in somatic mutations (e.g., one or more somatic mutations) in a patient compared to a baseline level , (ii) the presence of a somatic mutation (e.g., one or more somatic mutations) in the NF1 gene; (iii) the number of somatic mutations (e.g., one or more somatic mutations) in the LRP1B gene; an increase in, e.g., somatic mutations in the LRP1B gene (e.g., one or more somatic mutations) its increase compared to the baseline number of (iv) C to T changes in a given set of genes listed in Table 1 (e.g., , one or more C to T changes), e.g., an increase in the number of Compared to a reference number of C to T changes in the set (e.g., 1 or more C to T changes) The increase if Therapy is administered to the subject depending on one, two, three, or all of the above.

[0027] In certain embodiments, the method further comprises the step of encoding a T cell receptor (TCR) gene (e.g., In one embodiment, the method further comprises sequencing the TCR gene (or genes). This involves determining the clonality of the TCR genes.

[0028] Additional aspects or embodiments of the invention include one or more of the following.

[0029] Response state and mutation load The invention described herein can be used, for example, to treat a subject with cancer, e.g., melanoma. Response to a therapy that inhibits PD-1 or PD-L1, for example, This may include obtaining a value of the state.

[0030] In certain embodiments, an increase in the value of the response state, e.g., an increase in the value of the response state compared to a baseline value of the response state, In response to that increase in cases, a therapy is administered to or selected for the subject. In an embodiment, the reference value of the response status is the value of the response status for a non-responder to the therapy. do.

[0031] In certain embodiments, the value of the response state is determined from a sample, e.g., a melanoma sample or contains a measure of mutational load in melanoma-derived samples.

[0032] In certain embodiments, an increase in the level of mutation load in a sample from the subject; For example, therapies may be targeted depending on the increase in mutation load compared to baseline levels. In certain embodiments, a baseline level of mutation load is administered or selected for the subject. Bell is the level of mutation load in samples from non-responders to therapy.

[0033] In certain embodiments, a melanoma sample from a subject is: (i) Relative to baseline levels of somatic mutations in a given set of genes listed in Table 1 The level of somatic mutations in a given set of genes listed in Table 1 compared increase, (ii) the presence of a somatic mutation in the NF1 gene; (iii) LRP1 compared with the reference number of somatic mutations in the LRP1B gene an increased number of somatic mutations in the B gene, or (iv) the baseline number of C to T transitions in a given set of genes listed in Table 1 C to T changes in a given set of genes listed in Table 1 compared to An increase in the number of A subject is identified as a responder to therapy if they exhibit one, two, three, or all of the following: .

[0034] In certain embodiments, the sample from the subject comprises: (i) Relative to baseline levels of somatic mutations in a given set of genes listed in Table 1 somatic mutations in a given set of genes listed in Table 1 that are reduced or unchanged compared to Different levels, (ii) the absence of somatic mutations in the NF1 gene; (iii) Similar, identical, or similar numbers of somatic mutations in the LRP1B gene compared to the reference number. decreased the number of somatic mutations in the LRP1B gene, or (iv) the baseline number of C to T transitions in a given set of genes listed in Table 1 For a given set of genes listed in Table 1, the results were similar, the same, or decreased compared to Number of C to T transitions in A subject is identified as a non-responder to therapy if they exhibit one, two, three, or all of the following: do.

[0035] In one embodiment, the predetermined set of genes includes at least one of the genes listed in Table 1. At least about 50 pieces, about 100 pieces or more, about 150 pieces or more, about 200 pieces or more, about 250 pieces or more Above, about 300 or more or all included.

[0036] In one embodiment, somatic mutations in a predetermined set of genes listed in Table 1 Baseline levels of abnormalities are listed in Table 1 in samples from non-responders to therapy. The level of somatic mutations in a given set of genes is

[0037] In one embodiment, the baseline number of somatic mutations in the LRP1B gene is a measure of the response to therapy. is the number of somatic mutations in the LRP1B gene in samples from non-responders .

[0038] In one embodiment, C to T in a given set of genes listed in Table 1 The reference number of changes to C is the number of changes from C to T in samples from non-responders to therapy. is the number of

[0039] In one embodiment, somatic mutations in a predetermined set of genes listed in Table 1 The level of variation can be determined by sequencing a predetermined set of genes listed in Table 1, e.g., a method comprising sequencing the coding regions of a predetermined set of genes listed in Table 1; In one embodiment, the presence or absence of a somatic mutation in the NF1 gene is determined. The absence of NF1 can be determined by sequencing the NF1 gene, e.g., by sequencing the coding region of the NF1 gene. In one embodiment, the LRP1B gene is determined by a method comprising sequencing the gene. The number of somatic mutations in the offspring can be determined by sequencing the LRP1B gene, e.g., LRP1 In some embodiments, the coding region of the B gene is determined by a method comprising sequencing the coding region of the B gene. In the example, the number of C to T transitions in a given set of genes listed in Table 1 is determined by a method comprising sequencing a predetermined set of genes listed in Table 1 do.

[0040] In certain embodiments, the method further comprises: determining, in response to a measure of mutation load, one of the following: That is, (a) administering a modified dose of a therapy to a subject; (b) altering the schedule or time course of therapy for a subject; (c) administering an additional agent in combination with the therapy, e.g., to non-responders or partial responders; And, or (d) prognosticating the time course of cancer progression in a subject This includes doing one, two, three or all of the following:

[0041] In certain embodiments, the method further comprises: A sample or melanoma-derived sample may be obtained, for example, directly or indirectly, and used as described herein. The samples are evaluated for mutation load or mutations (alterations) as described. This includes:

[0042] Somatic mutations in a given set of genes Therapies described herein (e.g., inhibitors of PD-1 or PD-L1) Therapies that include, for example, somatic mutations in a given set of genes listed in Table 1 ( and the development of cancer (e.g., melanoma) in response to increasing levels of a gene encoding ... The therapeutic agent may be administered to or selected for a subject.

[0043] In one embodiment, somatic mutations in a predetermined set of genes listed in Table 1 The level of variability may be determined by determining the level of variability in at least about 25, e.g., at least about 50, of the genes listed in Table 1. , about 100 or more, about 150 or more, about 200 or more, about 250 or more, about 300 or more or determining the level of somatic mutations in all.

[0044] In one embodiment, somatic mutations in a predetermined set of genes listed in Table 1 The level of variation may be determined per preselected unit (e.g., the coding region of a given set of genes). in a region, e.g., a megabase in the coding region of a given set of sequenced genes This involves determining the number of somatic mutations (per site).

[0045] In one embodiment, somatic mutations in a predetermined set of genes listed in Table 1 The increase in activity in a given set of genes listed in Table 1 compared to baseline levels of the An increase in the level of cell mutation, e.g., at least about 2-fold, at least about 3-fold, at least about 5 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 3 times 0-fold, at least about 40-fold, or at least about 50-fold increase in To do or select for a subject.

[0046] In one embodiment, somatic mutations in a predetermined set of genes listed in Table 1 The number of somatic mutations per megabase in the coding region of a given set of genes is approximately 3.3 or more, for example, about 5 or more, about 10 or more, about 15 or more, about 20 or more, about 25 or more , about 30 or more, about 35 or more, about 40 or more, about 45 or more, or about 50 or more Depending on this determination, a therapy is administered to or selected for the subject.

[0047] In one embodiment, somatic mutations in a predetermined set of genes listed in Table 1 The number of somatic mutations per megabase in the coding region of a given set of genes is approximately 23. 1 or more, for example, about 25 or more, about 30 or more, about 35 or more, about 40 or more, about 45 In response to a determination that the number of subjects is greater than or equal to about 50, administering a therapy to the subject or administering a therapy to the subject to choose for.

[0048] In one embodiment, somatic mutations in a predetermined set of genes listed in Table 1 The number of somatic mutations per megabase in the coding region of a given set of genes is approximately 3.3 to about 23.1, for example, about 5 to about 23, about 10 to about 23, or about 15 to about 23 In response to the determination, a therapy is administered to or selected for the subject.

[0049] In one embodiment, somatic mutations in a predetermined set of genes listed in Table 1 The increase in activity in a given set of genes listed in Table 1 compared to baseline levels of the An increase in the level of cell mutation, e.g., at least about 2-fold, at least about 3-fold, at least about 5 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 3 times A 0-fold, at least about a 40-fold, or at least about a 50-fold increase indicates that the subject is responding to the therapy. that the subject is or is likely to be a patient or that the subject will or is likely to respond to the therapy Indicates that.

[0050] In one embodiment, somatic mutations in a predetermined set of genes listed in Table 1 The genes listed in Table 1 were similar, the same, or decreased compared to baseline levels of the different genes. The level of somatic mutations in a given set may indicate whether the subject is a non-responder to therapy or is a It indicates that there is a high probability that the subject will respond to the therapy, or that there is a high probability that the subject will not respond to the therapy.

[0051] In one embodiment, somatic mutations in a predetermined set of genes listed in Table 1 The number of variations per megabase in the coding region of a given set of genes listed in Table 1 or more than about 3.3 somatic mutations, for example, more than about 5, more than about 10, more than about 15, or more than about 2 0 or more, about 25 or more, about 30 or more, about 35 or more, about 40 or more, about 45 or more A determination that the subject has about 50 or more markers indicates that the subject is a responder to therapy, e.g., a complete responder or or that the subject is or is likely to be a partial or complete responder to the therapy. This indicates a high possibility.

[0052] In one embodiment, somatic mutations in a predetermined set of genes listed in Table 1 The number of variations per megabase in the coding region of a given set of genes listed in Table 1 A determination that the subject has less than about 3.3 somatic mutations or less than about 3.3 somatic mutations indicates that the subject is a non-responder to therapy. or is likely to be unresponsive to therapy, or indicates that the subject will not respond to therapy or is likely to be unresponsive to therapy. vinegar.

[0053] In one embodiment, somatic mutations in a predetermined set of genes listed in Table 1 The number of variations per megabase in the coding region of a given set of genes listed in Table 1 or about 23.1 or more somatic mutations, for example, about 25 or more, about 30 or more, about 35 or more, A determination of about 40 or more, about 45 or more, or about 50 or more may indicate that the subject is responsive to therapy. The subject may be or be likely to be a responder, e.g., a complete responder, or the subject may respond to the therapy. Indicates that there is or is a high possibility of this happening.

[0054] In one embodiment, somatic mutations in a predetermined set of genes listed in Table 1 The number of variations per megabase in the coding region of a given set of genes listed in Table 1 A determination that the subject has fewer than about 23.1 somatic mutations per 1000 mg / mL of the 23.1 or 23.1 somatic mutations indicates that the subject is a partial or complete responder to therapy. are or are likely to be (or are partially or non-responders) likely to respond, or likely to partially respond, or likely to not respond .

[0055] In one embodiment, somatic mutations in a predetermined set of genes listed in Table 1 The number of variations per megabase in the coding region of a given set of genes listed in Table 1 About 3.3 to about 23.1 somatic mutations per 1000, for example, about 5 to about 23, about 10 to about 23, or A determination that the subject has about 15 to about 23 nucleotides indicates that the subject is a partial responder or a partial responder to the therapy. likely to respond (or partially respond, or likely to partially respond) Indicates that.

[0056] Mutations in the NF1 gene Therapies described herein (e.g., inhibitors of PD-1 or PD-L1) Therapies that involve, for example, a mutation in the NF1 gene (e.g., the coding region of the NF1 gene) Depending on the determination of the number of cell mutations, it is administered to a subject with cancer (e.g., melanoma), and can be selected for the subject.

[0057] In certain embodiments, the somatic mutation is in the NF1 gene (e.g., the coding region of the NF1 gene). In response to a determination that the tumor is present in the tumor area, a therapy is administered to the subject or selected for the subject. do.

[0058] In one embodiment, the presence of a somatic mutation in the coding region of the NF1 gene is The patient is likely to be a responder to therapy.

[0059] In one embodiment, (a) the somatic mutation is present in the coding region of the NF1 gene; and (b) the level of somatic mutations in a given set of genes listed in Table 1 Somatic cells per megabase in the coding region of a given set of genes listed in Table 1 About 23.1 mutations or more, for example, 25 mutations or more, about 30 mutations or more, about 35 mutations or more, about 38.5 mutations or more 40 or more, about 45 or more, or about 50 or more, The method is administered to or selected for the subject.

[0060] In one embodiment, (a) the presence of a somatic mutation in the coding region of the NF1 gene , and (b) somatic mutations in the BRAF gene and somatic mutations in the NRAS gene or a sample containing triple WT (wild type) for the BRAF, NRAS, and NF1 genes Samples (e.g., melanoma samples or melanoma-derived samples) as described in Table 1 The levels of somatic mutations in a given set of genes listed in Table 1 an increase in the level of somatic mutations in a given set of genes listed, e.g., at least For determining an increase of about 2-fold, at least about 3-fold, at least about 5-fold, or at least about 10-fold Accordingly, a therapy is administered to or selected for the subject.

[0061] Mutations in the LRP1B gene Therapies described herein (e.g., inhibitors of PD-1 or PD-L1) Therapies that include, for example, a gene encoding the LRP1B gene (e.g., a coding region of the LRP1B gene) responsive to a determination of the presence of a somatic mutation in a subject having cancer (e.g., melanoma). can be selected for the subject.

[0062] In certain embodiments, the LRP1B gene (e.g., the coding region of the LRP1B gene) ) about 1 or more, about 2 or more, about 3 or more, about 4 or more, or about 5 or more somatic cells in Depending on the determination of the presence of the mutation, a therapy is administered to or selected for the subject.

[0063] In certain embodiments, the LRP1B gene (e.g., the coding region of the LRP1B gene) ) about 1 or more, about 2 or more, about 3 or more, about 4 or more, or about 5 or more somatic cells in The presence of the mutation indicates that the subject is or is likely to be a responder to the therapy, or that the subject indicates a high likelihood of responding to therapy.

[0064] In one embodiment, the reference number of somatic mutations in the coding region of the LRP1B gene Increased number of somatic mutations in the coding region of the LRP1B gene compared with For example, at least about 2 times, at least about 2.5 times, at least about 2.8 times, at least about 3 times fold, at least about 3.5-fold, at least about 4-fold, or at least about 5-fold increase Therapies are administered to or selected for the subject depending on the patient's condition.

[0065] In one embodiment, the reference number of somatic mutations in the coding region of the LRP1B gene is a sample from a non-responder to therapy (e.g., a melanoma sample or The number of somatic mutations in the coding region of the LRP1B gene in the 1000-kDa population (sample of origin).

[0066] In one embodiment, the reference number of mutations in the coding region of the LRP1B gene is There are 0 or 1 cell mutations.

[0067] Change from C to T Therapies described herein (e.g., inhibitors of PD-1 or PD-L1) Therapies that include, for example, a predetermined set of genes, e.g., a set of genes listed in Table 1 Depending on the determination of the number of C to T changes in a constant set, cancer (e.g., melanoma) may be identified. The therapeutic agent may be administered to or selected for a subject.

[0068] In one embodiment, about 20 of the predetermined set of genes listed in Table 1 Determination of the presence of more than about 30, more than about 40, or more than about 50 C to T changes Depending on the condition, a therapy is administered to or selected for the subject.

[0069] In one embodiment, about 20 of the predetermined set of genes listed in Table 1 or more, about 24 or more, about 30 or more, about 40 or more, or about 50 or more C to T transitions Determining the presence of the phenotype indicates that the subject will be or is likely to be a responder to the therapy.

[0070] In one embodiment, C to T in a given set of genes listed in Table 1 in a given set of genes listed in Table 1 when compared to a reference number of changes to an increase in the number of C to T changes, e.g., at least about 8-fold, at least about 10-fold, or Depending on the determination of at least about a 12-fold, at least about a 15-fold, or at least about a 20-fold increase, The method is administered to or selected for the subject.

[0071] In one embodiment, the reference number of C to T changes is the survival rate from non-responders to therapy. C to T in a sample (e.g., a melanoma sample or melanoma-derived sample) is the number of changes to

[0072] In one embodiment, the reference number of C to T transitions is a gene listed in Table 1. There are approximately two C to T changes in a given set of

[0073] Mutation Type As described herein, various types of mutations (alterations), e.g., somatic mutations, can occur. , can be used to measure the mutation load in a sample.

[0074] In some embodiments, the mutations include one or more of the following: silent mutations (e.g., somatic mutations not identified as being associated with a cancer phenotype; change mutations (e.g., mutations that have no detectable effect on the fitness of the clone), Variants of unknown significance (VUS) (e.g., mutations for which pathogenicity cannot be confirmed or excluded), Point mutations, coding short variants (e.g., nucleotide substitutions or indels), nonsynonymous single nucleotide variants (SNVs) or splice variants.

[0075] Alternatively, or in combination, in some embodiments, the mutation is one of the following: does not include: rearrangements (e.g., translocations) tion), functional or germline mutations.

[0076] In certain embodiments, the somatic mutation is a silent mutation, eg, a synonymous mutation. In certain embodiments, the somatic mutation has not been identified as being associated with a cancer phenotype. In some embodiments, the somatic mutation is a passenger mutation, e.g., a clonal adaptation. In certain embodiments, somatic mutations are mutations that have no detectable effect on the degree of are variants of uncertain significance (VUS), i.e., mutations for which pathogenicity cannot be confirmed or excluded In some embodiments, the somatic mutation is a point mutation. In some embodiments, the somatic mutation is other than a rearrangement, e.g., other than a translocation. In some embodiments, the somatic mutation is a coding short mutation, e.g., a base substitution. or an indel. In one embodiment, the somatic mutation is a nonsynonymous single nucleotide variant (S In some embodiments, the somatic mutation is a splice variant. In embodiments, the somatic mutation is not a functional mutation.

[0077] In some embodiments, the mutation is not a germline mutation. , somatic mutations are not identical to or similar to germline mutations (e.g., distinguish In one embodiment, the increased level of somatic mutations is one or more classes of or type of somatic mutation (e.g., translocation, point mutation, indel, or any of these) In one embodiment, the increased level of somatic mutations is , one class or type of somatic mutation (e.g., only rearrangements, only point mutations, or In one embodiment, the level of somatic mutations is increased. The increase may be due to a somatic mutation (e.g., a mutation described herein, e.g., a mutation at a preselected position) at the target gene. In one embodiment, the level of BRAF is increased. The increased level of somatic mutations can be attributed to the presence of somatic mutations at preselected positions (e.g., as described herein). Described mutations, such as the V600 mutation in BRAF (e.g., The main cause is increased levels of the V600K mutation.

[0078] Therapeutic Substances and Methods A subject having a cancer described herein may be receiving a therapy, e.g., immunotherapy, e.g., PD- These patients may be treated with therapies including inhibitors of PD-L1 or PD-L1.

[0079] In one embodiment, the inhibitor of PD-1 is an anti-PD-1 antibody. In this form, the PD-1 inhibitor is nivolumab (ON O-4538, BMS-936558 or MDX1106), pembrolizumab (pe mbrolizumab) (MK-3475 or lambrolizumab zumab)), pidilizumab (CT-011), MEDI 0680(AMP-514), PDR001, REGN2810, BGB-108, BG B-A317, SHR-1210 (HR-301210, SHR1210 or SHR- 1210), PF-06801591 or AMP-224.

[0080] In some embodiments, the inhibitor of PD-L1 is an anti-PD-L1 antibody. In an embodiment, the inhibitor of PD-L1 is atezolizumab umab) (MPDL3280A, RG7446 or RO5541267), YW24 3.55.S70, MDX-1105, durvalumab (ME DI4736) or avelumab (MSB0010718C) be selected.

[0081] In one embodiment, the inhibitor of PD-1 or PD-L1 inhibits the PD-1 receptor , e.g., PD-1 receptor Fc fusion, PD-L1 receptor, e.g., PD-L1 receptor Fc fusion It is a combination.

[0082] In one embodiment, the subject is receiving a therapy other than an inhibitor of PD-1 or PD-L1. Are undergoing or have undergone different therapies, including therapeutic substances or treatments.

[0083] In some embodiments, the following: (i) Relative to baseline levels of somatic mutations in a given set of genes listed in Table 1 The level of somatic mutations in a given set of genes listed in Table 1 compared increase, (ii) the presence of a somatic mutation in the NF1 gene; (iii) LRP1 compared with the reference number of somatic mutations in the LRP1B gene an increased number of somatic mutations in the B gene, or (iv) the baseline number of C to T transitions in a given set of genes listed in Table 1 C to T changes in a given set of genes listed in Table 1 compared to An increase in the number of In response to one, two, three or all of the determinations, the different therapies are discontinued.

[0084] In some embodiments, the therapy is administered after discontinuing the different therapy. In some embodiments, the therapy is administered in combination with a different therapy.

[0085] In certain embodiments, the different therapies are chemotherapy, radiation therapy, immunotherapy, radiotherapy, or immunotherapy. a therapeutic agent selected from radioimmunotherapy, oncolytic virus therapy, surgical procedure, or any combination thereof; can be.

[0086] In one embodiment, the different therapy is dacarbazine e), temozolomide, interleukin 2 (IL-2), Interferon, ipilimumab, BRAF inhibitors, M EK inhibitor, talimogene laherparepvec erparepvec), adoptive cell transfer, or any combination thereof.

[0087] In one embodiment, the interferon is recombinant interferon alpha 2b or pegylated interferon alpha 2b. In one embodiment, The inhibitors are vemurafenib or dabrafenib. In one embodiment, the MEK inhibitor is cobimetinib. cobimetinib or trametinib. In embodiments, adoptive cell transfer involves modified T cells or modified dendritic cells.

[0088] In certain embodiments, the subject is a patient receiving, for example, an inhibitor of PD-1 or PD-L1. In certain embodiments, the subject is undergoing or has undergone immunotherapy comprising have not received immunotherapy, e.g., including inhibitors of PD-1 or PD-L1, and I have never received it.

[0089] melanoma The invention described herein can be used to treat cancer, e.g., melanoma, or It can be used to assess subjects with cancer, such as melanoma.

[0090] Melanoma is a melanocarcinoma defined according to any suitable melanoma classification system known to those of skill in the art. The tumor may be of any stage or risk group.

[0091] In some embodiments, the melanoma is stage 0, stage IA, or stage IB. , stage IIA, stage IIB, stage IIC, stage III, or stage IV melanoma Some of these, such as the American Joint Committee on Cancer According to the American Joint Committee on Cancer (AJCC) melanoma staging and classification system, Stage 0, Stage IA, Stage IB, Stage IIA, Stage IIB, Stage IIC, Stage I In one embodiment, the tumor is either stage II or stage IV melanoma. Melanoma is staged or classified according to Tables 2A and 2B set forth herein. can be.

[0092] In some embodiments, melanoma is characterized by the phenotype of melanoma described, for example, in Balch et al., J Clin O in situ (in situ) in situ melanoma (e.g., stage 0 melanoma), localized melanoma (e.g., melanoma), locally metastatic melanoma (e.g., stage III melanoma), melanoma) or distant metastatic melanoma (e.g., stage IV melanoma) In some embodiments, the melanoma is an advanced melanoma, e.g., stage It is stage III or IV melanoma.

[0093] In other embodiments, the melanoma is at the level of Clark (Weedon, Ski n pathology.2nd Edition.2002.Sydney:Chur Level 1, Level 2, Level 3, Level 4 based on Chill-Livingstone Level 4 or Level 5 melanoma, e.g., Level 1 (e.g., epithelial-only) Localized melanoma (melanoma in situ), Level 2 (e.g., invasion into the papillary dermis) ), level 3 (e.g., invasion of the junction between the papillary and reticular dermis), level 4 (e.g., Level 5 (e.g., invasion into the reticular dermis) or level 5 (e.g., invasion into the subcutaneous fat) melanoma It is one of the following.

[0094] In some embodiments, the melanoma is at or above Breslow depth (Breslow (1970) Annals of Surgery 172(5):902-908 Based on stage I, stage II, stage III, stage IV or stage IV melanoma Stage I (e.g., less than 0.75 mm deep), Stage II (e.g., 0. 76 mm to 1.50 mm deep), stage III (e.g., 1.51 mm to 2.25 mm deep) depth), stage IV (e.g., 2.26 mm to 3.0 mm depth), or stage V (e.g., melanoma (thickness greater than 3.00 mm).

[0095] In some embodiments, the melanoma is an aggressive melanoma. In this setting, advanced melanoma is stage III or later according to the AJCC staging and classification system. In one embodiment, the advanced melanoma is stage IV or stage IV melanoma. Stage III or IV disease based on staging and classification described in 2A and 2B In one embodiment, the advanced melanoma is stage III melanoma. melanoma, such as stage III melanoma described herein. In embodiments, the advanced melanoma is stage IV melanoma, e.g., melanoma as described herein. It is stage IV melanoma.

[0096] In some embodiments, the melanoma is metastatic melanoma. In this study, metastatic melanoma was classified into stages based on, for example, the AJCC staging and classification system. In one embodiment, the melanoma is stage III or stage IV. The disease is stage III or IV according to the staging and classification described in Tables 2A and 2B. In one embodiment, the metastatic melanoma is stage II melanoma. Stage I melanoma, such as stage III melanoma, as described herein. In another embodiment, the metastatic melanoma is stage IV melanoma, e.g., stage IV melanoma. It is stage IV melanoma as described in the fine print.

[0097] In other embodiments, melanoma, e.g., advanced melanoma, is treated with any of the methods described herein. Mutations in one or more of the genes listed in Table 1 (e.g., mutations), such as those described herein, In one embodiment, the melanoma is characterized by NF1, LRP1B, BRA, F, NRAS, TP53, MYC, APC / CTNNB1, IGF1R / HGF, PTE N, CDKN2A, CDK4, CDK6 and / or RB1 It has been confirmed that the gene contains or has the above mutations.

[0098] In one embodiment, the melanoma contains or has a mutation in the NF1 gene. In one embodiment, melanoma is characterized by a gene encoding ... In one embodiment, the melanocorticoids are characterized as containing or having a mutation in the melanocorticoids. It has been determined that these tumors contain or have mutations in the BRAF gene. In embodiments, the melanoma contains or has a mutation in the NRAS gene. In one embodiment, the melanoma is a tumor caused by a mutation in the NF1 gene and It has been shown that some patients with rhesus macular degeneration (HMD) have mutations in the LRP1B and LRP1B genes. In an embodiment, the melanoma may contain or have a mutation in the NF1 gene. Mutations in the BRAF gene, NRAS gene, or other genes have been confirmed. In some embodiments, the compound does not contain or has not been identified as having both of these. Melanomas have been identified that contain or harbor mutations in the LRP1B gene However, these tumors contain mutations in the BRAF gene, mutations in the NRAS gene, or both. In some embodiments, the melanoma is a It has been identified as a heavy wild-type (WT) melanoma, e.g., melanoma with NF1 gene mutations in the BRF gene and mutations in the NRAS gene It does not contain or has not been confirmed to have any of the following:

[0099] In one embodiment, the mutation in the NF1 gene results in a decrease in the activity of NF1 compared to wild-type activity. This results in a decrease in the activity of the NF1 gene product (e.g., NF1 protein) when For example, the mutation may affect the GTPase activator activity of the NF1 protein, the phosphatidylcholinesterase activity, Changes in phosphorus-binding activity and / or phosphatidylethanolamine-binding activity (e.g., In one embodiment, the mutation in NF1 can result in a mutation (e.g., , somatic mutations), such as substitutions (e.g., base substitutions), insertions or deletions, or includes it.

[0100] In one embodiment, the mutation in the BRAF gene is a mutation that results in a BRAF gene that is mutated relative to wild-type activity of BRAF. resulting in decreased activity of the BRAF gene product (e.g., BRAF protein) compared to For example, the mutation may result in an alteration (e.g., an increase) in the kinase activity of the BRAF protein. In one embodiment, the BRAF mutation may be a mutation (e.g., a somatic mutations), such as substitutions (e.g., base substitutions), insertions or deletions, or In certain embodiments, the mutation is a base substitution.

[0101] In one embodiment, the mutation in BRAF is located at codon V600. In some embodiments, the mutation in BRAF is a V600E mutation. In one embodiment, the mutation in BRAF is a V600K mutation. In one embodiment, the mutation in BRAF is a V600R mutation. The difference is the V600D mutation.

[0102] In one embodiment, the mutation in BRAF is a mutation other than a V600 mutation. In some embodiments, the mutation in BRAF is a K601 mutation. In one embodiment, the mutation in BRAF is a K601E mutation. The mutation in BRAF is a G469 mutation. In one embodiment, the mutation in BRAF is a G469E mutation. In one embodiment, the mutation in BRAF is a D594G mutation. In one embodiment, the mutation in BRAF is the L597 mutation. In one embodiment, the mutation in BRAF is a L597S mutation. The mutation in RAF is an S467 mutation. The mutation is S467L.

[0103] subject In certain embodiments, the subject has melanoma, e.g., a progression as described herein. and / or a gene encoding a gene for a melanoma, wherein the melanoma is a gene encoding a gene for a gene listed in Table 1. In some embodiments, the mutations include one or more mutations described herein. In melanoma, NF1, LRP1B, BRAF, NRAS, TP53, MYC, APC / CTNNB1, IGF1R / HGF, PTEN, CDKN2A, CDK4, CD containing or having a mutation in one or more of the genes selected from K6 and / or RB1 In other embodiments, the subject has a C to T mutation. Known or previously known to have melanoma, e.g., advanced melanoma It has been recognized.

[0104] Melanoma can be at any stage, e.g., any stage described herein, e.g., Progressive, recurrent, relapsing or refractory disease (including but not limited to) Cancer staging systems for melanoma include, for example, the American Joint Committee on Cancer (AMC) The American Joint Committee on Cancer (AJCC) melanoma disease Staging and classification systems are included. For example, melanoma can be classified as stage 0, stage IA, stage IB, Any of stage IIA, stage IIB, stage IIC, stage III, or stage IV melanoma For example, Stage 0, Stage I, and Stage 2 based on Tables 2A and 2B described herein. A, Stage IB, Stage IIA, Stage IIB, Stage IIC, Stage III or Stage IV It can be any of the following:

[0105] In one embodiment, the subject is a human, e.g., a human subject described herein. The subject is a human patient with melanoma (e.g., advanced melanoma).

[0106] In one embodiment, the subject has a different (e.g., non-PD-1 and / or non-PD-2) D-L1) Are receiving or have received treatment with a therapeutic substance or method. In one embodiment, the different therapeutic agents or treatment methods are chemotherapy, radiation, or therapy, immunotherapy, radioimmunotherapy, oncolytic virus therapy, surgical procedure or any of these In one embodiment, the different therapeutic agents or treatments are selected from the following combinations: Treatment options include dacarbazine and temozolomide. mide), interleukin 2 (IL-2), interferon (e.g., recombinant interferon Interferon alfa 2b or peginterferon alfa 2b), ipilimumab ( ipilimumab), BRAF inhibitors [e.g., vemurafenib (vemur afenib or dabrafenib), a MEK inhibitor [For example, cobimetinib or trametinib inib)], talimogene laherparepvec parepvec), and / or adoptive cell transfer (e.g., modified T cells or modified dendritic cells) cells).

[0107] In one embodiment, the mutation load and / or or, depending on the determination of the presence of a mutation, a different (e.g., non-PD-1 and / or non-PD-1) mutation. The PD-L1 checkpoint therapeutic agent or method is discontinued. In this state, the subject is more likely to respond to the different therapeutic substance or treatment method. Or it has been confirmed that the possibility is low.

[0108] In one embodiment, the subject is diagnosed with a PD-1 or PD-L1 inhibitor. In one embodiment, the subject is a melanoma patient participating in a clinical trial. (e.g., non-PD-1 and / or non-PD-L1) therapeutic agents or methods of treatment The patient is a melanoma patient participating in a clinical trial.

[0109] In one embodiment, the subject is 60 years of age or older. In yet another embodiment, the subject is 45 to 60 years old. In yet another embodiment, the subject is under 30 years of age. In one embodiment, the subject is 45 years of age or older and male. The subject is 45 years of age or younger and female. In one embodiment, the subject is Caucasian (Caucasian). In one embodiment, the subject has a family history of melanoma.

[0110] system In one embodiment, the present invention provides a method for assessing a subject with cancer, e.g., melanoma. The system includes at least one processor operatively connected to a memory. and wherein the at least one processor, when executed, (a) administering to a subject a therapy (e.g., a therapy comprising an inhibitor of PD-1 or PD-L1) method), where the response state value is obtained by measuring the response state of a sample (e.g., , melanoma samples, or melanoma-derived samples) fruit, (b) determining whether a subject is a responder (e.g., a complete or partial responder) or non-responder to a therapy; It is designed to identify the answer, Here, a response state value equal to or greater than the reference response state value indicates that the subject is responding to the therapy. or that the subject is or is likely to be a responder to the therapy. indicates high sexuality, or Here, a response state value less than the reference response state value indicates that the subject is a non-responder to the therapy. that a subject is or is likely to be unresponsive to therapy, or that a subject is or is likely to be unresponsive to therapy Shows.

[0111] In certain embodiments, the measure of mutational load is the following in a sample from a subject: Things, namely, (i) somatic mutations in a given set of genes listed in Table 1 (e.g., one or more levels of somatic mutations in (ii) the presence of a somatic mutation (e.g., one or more somatic mutations) in the NF1 gene; (iii) the number of somatic mutations (e.g., one or more somatic mutations) in the LRP1B gene; ,or (iv) C to T changes in a given set of genes listed in Table 1 (e.g., , the number of C to T changes of 1 or more This may include one, two, three or all of the following decisions:

[0112] In one embodiment, the following in a melanoma sample from a subject: Chi, (i) somatic mutations in a given set of genes listed in Table 1 (e.g., one or more The pre-defined set of genes listed in Table 1 compared to the baseline level of somatic mutations in the an increased level of somatic mutations (e.g., one or more somatic mutations) in the patient; (ii) the presence of a somatic mutation (e.g., one or more somatic mutations) in the NF1 gene; (iii) a source of somatic mutations (e.g., one or more somatic mutations) in the LRP1B gene; somatic mutations in the LRP1B gene (e.g., one or more somatic mutations) compared to baseline levels an increase in the number of cellular mutations, or (iv) C to T changes in a given set of genes listed in Table 1 (e.g., The incidence of the genes listed in Table 1 compared to the baseline level of The number of C to T transitions (e.g., one or more C to T transitions) in a given set of genes Increase in Therapy is administered to or selected for the subject depending on one, two, three, or all of the following: Select.

[0113] Kits, nucleic acid preparations and reaction mixtures In one aspect, the present invention relates to a kit comprising: (a)(i) somatic mutations in a given set of genes listed in Table 1 (e.g., one or more somatic mutations), (ii) a somatic mutation in the NF1 gene (e.g., one or more somatic mutations); (iii) a somatic mutation in the LRP1B gene (e.g., one or more somatic mutations); , (iv) C to T changes (e.g., one or more C to T changes) one or more detection reagents capable of detecting one or more of the following: (b) in determining the mutation load in melanoma samples and / or Includes instructions for use in the treatment of melanoma in

[0114] In one embodiment, the kit further comprises (c) an antibody for PD-1 or PD-L1. The present invention includes an inhibitor or a composition thereof.

[0115] In another aspect, the present invention provides a method for detecting a melanoma in a sample, such as a melanoma sample or melanoma cell line. The present invention relates to a purified or isolated preparation of nucleic acid from a sample derived from a human ovarian cancer, the preparation comprising: (i) somatic mutations in a given set of genes listed in Table 1 (e.g., one or more somatic mutations), (ii) a somatic mutation in the NF1 gene (e.g., one or more somatic mutations); (iii) a somatic mutation in the LRP1B gene (e.g., one or more somatic mutations); (iv) C to T changes (e.g., one or more C to T changes) Contains one or more of the following.

[0116] In certain embodiments, the preparation is carried out in a sequencing instrument or in such an instrument. To determine the mutation load of melanoma samples placed in the sample holder used It is used for

[0117] In yet another aspect, the present invention relates to a reaction mixture comprising: (a)(i) somatic mutations in a given set of genes listed in Table 1 (e.g., one or more somatic mutations), (ii) a somatic mutation in the NF1 gene (e.g., one or more somatic mutations); (iii) a somatic mutation in the LRP1B gene (e.g., one or more somatic mutations); ,or (iv) C to T changes (e.g., one or more C to T changes) one or more detection reagents capable of detecting one or more of the following: (b)(i) somatic mutations in a given set of genes listed in Table 1 (e.g., one or more somatic mutations), (ii) a somatic mutation in the NF1 gene (e.g., one or more somatic mutations); (iii) a somatic mutation in the LRP1B gene (e.g., one or more somatic mutations); ,or (iv) C to T changes (e.g., one or more C to T changes) a sample containing one or more of the following (e.g., a melanoma sample or a melanoma-derived sample): and nucleic acids derived from

[0118] In certain embodiments, the reaction mixture is in a sequencing instrument or in such an instrument. To determine the mutation load of a sample placed in a sample holder used in Used for.

[0119] In a related aspect, the present invention relates to a method for preparing a reaction mixture, the method comprising: (i) somatic mutations in a given set of genes listed in Table 1 (e.g., one or more somatic mutations), (ii) a somatic mutation in the NF1 gene (e.g., one or more somatic mutations); (iii) a somatic mutation in the LRP1B gene (e.g., one or more somatic mutations), or or (iv) C to T changes (e.g., one or more C to T changes) one or more detection reagents capable of detecting one or more of the following: (i) somatic mutations in a given set of genes listed in Table 1 (e.g., one or more somatic mutations), (ii) a somatic mutation in the NF1 gene (e.g., one or more somatic mutations); (iii) a somatic mutation in the LRP1B gene (e.g., one or more somatic mutations), or or (iv) C to T changes (e.g., one or more C to T changes) The method includes combining nucleic acid from a melanoma sample containing one or more of:

[0120] Unless otherwise defined, all technical and scientific terms used herein are intended to be limiting of the present invention. It has the same meaning as commonly understood by those skilled in the art. Methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention. Suitable methods and materials are described below. All publications, patent applications, patents, and other references are incorporated herein by reference in their entirety. In case of conflict, the present specification, including definitions, will take precedence. The methods and examples are illustrative only and are not limiting.

[0121] The details of one or more embodiments of the subject matter set forth in the invention are set forth in the accompanying drawings and the description below. Other features, objects and advantages of the present invention will become apparent from the description and drawings. This will be apparent from the appended claims.

[0122] Detailed Description The present invention is based, at least in part, on, for example, hybrid capture-based NGS platforms. The number of mutations detected in hundreds of genes by the PD-1 or PD-L This is based on the finding that IL-1 correlates with therapeutic benefit from therapy including IL-1. Classification of patients into several groups, e.g., three groups, may provide a clearer picture of "high" and "low" for most patients. "Enabling accurate prediction of mutation burden in cohorts for advanced melanoma and other cancers." Clinically actionable models of response to anti-PD-1 and / or anti-PD-L1 therapy in In other embodiments, several genes (e.g., NF1, LR) Mutations (alterations) in P1B were also assessed by the overall mutation burden and anti-PD-1 or anti-PD-L1 therapy. correlated with benefits from the law.

[0123] Without being bound by theory, the potential to generate immunogenic tumor neoantigens is As spontaneous mutations occur, they increase in a stochastic manner, increasing the likelihood of immune recognition. (Gubin and Schreiber, Science 350:158- However, assessment of the total mutation burden requires whole exome sequencing (WES). This approach requires specialized tissue processing, comparable normal specimens, and is currently primarily used in research. It is technically and informatically difficult to perform WES in clinical settings. Given this, alternative methods for detecting mutation load are needed. There are several validated hybrid capture-based NGS platforms available. It has several practical advantages, including, for example, more clinically tractable turnaround times. Round time (approximately 2 weeks), standardized informatics pipeline s pipeline), and more manageable costs. This approach It has other advantages over markers such as PD-L1 expression because it is a subjective measure. provides an objective measure (mutation load) rather than a degree (e.g., immunohistochemical scoring) (Hansen and Siu, JAMA Oncol 2(1):15-6, 2016). Furthermore, this platform allows for simultaneous identification of available mutations suitable for targeted therapy. Makes detection easier.

[0124] How many accurate predictive biomarkers for anti-PD-1 or anti-PD-L1 therapy have been identified? In melanoma, patients with high mutation burden are e.g. For example, patients with moderate / low mutation burden can receive anti-PD-1 monotherapy, while Patients who do so may be more susceptible to the more active (but more toxic) forms of nivolumab and ipilimumab, for example. (Larkin et al., N Engl J Med , 2015). Other cancers (e.g., NSCLC) have a lower overall response to anti-PD-1. (Rizvi et al., Lancet Oncol 16:257-65, 201 5; Garon et al., N Engl J Med, 2015). In these diseases, This approach stratifies patients to receive anti-PD-1 and other active agents, such as cytotoxic chemotherapy. Furthermore, it may be possible to identify "outliers" (e.g., tumors with high mutational burden that do not respond). There is a great need to identify the microbial and immunological properties of these proteins.

[0125] Thus, the present invention provides, at least in part, a method for targeting the PD-1 pathway and / or by administering to a subject an effective amount of an agent (e.g., a therapeutic agent) that inhibits cancer, e.g., Methods for treating a subject having or at risk for melanoma are provided. In some embodiments, the therapeutic agent is an inhibitor of PD-1 or PD-L1. In some embodiments, the therapeutic agent, e.g., an inhibitor of PD-1 or PD-L1, is administered to the patient. In some embodiments, the therapeutic agent is administered in response to a response state. The values ​​are calculated for a cancer sample from the subject, e.g., a melanoma sample or a melanoma-derived sample. In certain embodiments, the measure of mutation load is The level of somatic mutations in a given set of genes disclosed herein, including the NF1 gene the presence of somatic mutations in the LRP1B gene, the number of somatic mutations in the LRP1B gene, or The number of C to T transitions in a given set of genes disclosed in the literature, or their Any combination of the results of the determination. Also disclosed are methods and systems, nucleic acid preparations, kits, reaction mixtures, and methods for producing reaction mixtures. do.

[0126] Certain terms are defined below and throughout the specification.

[0127] As used herein, the terms "a," "an," and "the" refer to one or more (e.g., at least one) Point.

[0128] The word "or" is used herein to mean "and" unless the context clearly contradicts otherwise. " / or" and is interchangeable with "and / or." The use of the word "and / or" in this document implies "or" unless the context clearly contradicts it. The use of the word "and / or" does not imply that it is not interchangeable with the word "and / or."

[0129] "About" and "approximately" generally refer to quantities measured, taking into account the nature or precision of the measurements. The typical degree of error is or within 20 percent (%) of a range of values, typically within 10%, more typically within 5 % or less.

[0130] As used herein, the terms "obtain" or "obtain" refer to the "direct acquisition" of a physical entity or value. "directly obtain" or "indirectly obtain" a physical entity or value, e.g., a numerical value. "Directly obtain" means to obtain a physical entity or value through a process. "Indirectly obtained" means to carry out a process (e.g., by carrying out a synthetic or analytical method). "to be acquired" means that the physical entity or value is acquired by another party or source (e.g., a third party from whom the physical entity or value was acquired directly). It means receiving a physical entity or value from a research institution. In particular, it is possible to carry out processes that involve physical changes in physical substances, e.g., starting materials. Typical transformations include producing a physical entity from two or more starting materials, To cut or fragment, to separate or purify substances, to mix two or more separate entities to combine substances, to carry out chemical reactions involving the breaking or forming of covalent or non-covalent bonds. Obtaining a value directly involves determining the physical properties of a sample or another substance. To carry out a process that involves a change, e.g., to change a substance (e.g., a sample, analyte, or specimen) Analytical processes involving physical changes in the drug (referred to herein as "physical analysis"). and analytical methods, such as methods that include one or more of the following: includes: the conversion of a substance, e.g., an analyte, or a fragment or other derivative thereof, from another substance To separate or purify an analyte or a fragment or other derivative thereof into another substance, e.g. For example, combining with a buffer, solvent or reactant, or with the analyte or its fragment The structure of the fragment or other derivative may be determined by, for example, a covalent bond between a first atom and a second atom of the analyte. To alter by breaking or forming bonds or non-covalent bonds, or by using reagents or The structure of the fragment or other derivative can be determined by, for example, a covalent bond between a first atom and a second atom of the reagent. To change by breaking or forming bonds or noncovalent bonds.

[0131] As used herein, the term "obtaining a sequence" refers to "directly obtaining" or "indirectly obtaining" a sequence. By "obtaining" it is meant obtaining possession of a nucleotide sequence or an amino acid sequence. "Directly obtaining a sequence" means performing a process to obtain the sequence (e.g., synthetically or or analytical methods), e.g., sequencing methods (e.g., next generation sequencing ( "Indirectly obtaining a sequence" means obtaining a sequence from another party or supplier. obtain sequence information or knowledge from a source (e.g., a third-party laboratory that obtained the sequence directly) The sequence obtained does not have to be a complete sequence, e.g. , sequencing of at least one nucleotide, or a mutation as disclosed herein Obtaining information or knowledge that confirms the presence of a gene in a subject is included in obtaining the sequence. It can be enjoyed.

[0132] Directly obtaining a sequence involves extracting a physical material, e.g., a starting material, e.g., a tissue sample, e.g., For example, biopsies or physical examination of isolated nucleic acid (e.g., DNA or RNA) samples. Typical transformations involve the production of a compound from two or more starting materials. Producing a physical entity, such as cutting or fragmenting a substance, e.g., genomic DNA fragments , separating or purifying a substance (e.g., isolating a nucleic acid sample from tissue); 2 Combining these separate entities into a mixture, breaking covalent or non-covalent bonds, or Directly obtaining a value involves performing a chemical reaction involving the formation of a sample or It involves carrying out a process that involves a physical change in another substance (as described above).

[0133] As used herein, the term "obtaining a sample" refers to "directly obtaining" or By "indirectly obtained" is meant possession of a sample, e.g., a tissue sample or nucleic acid sample. "Directly obtaining a sample" means obtaining a sample through a process means to perform a procedure (e.g., a physical procedure, e.g., surgery or extraction) "Indirectly obtaining a sample" means obtaining a sample directly from another party or source (e.g., This means receiving a sample directly from a third-party laboratory. To obtain a tissue sample, a physical material, e.g., a starting material, e.g., a tissue, e.g., a tissue in a human patient, performing a process involving a physical change in tissue, or tissue that has already been isolated from the patient Typical transformations include producing a physical entity from a starting material, dissecting tissue, to separate or collect material (e.g., a tissue sample or a nucleic acid sample) Purification refers to the process of combining two or more separate entities into a mixture, either covalently or non-covalently. This involves performing a chemical reaction that involves breaking or forming a bond. In particular, this involves a physical change in a sample or another substance (e.g., as described above). This involves carrying out a process.

[0134] Genes or gene products (e.g., genes or gene products listed in Table 1, N F1 gene or gene product, or LRP1B gene or gene product) As used herein, "mutation" refers to a change in a gene or gene sequence compared to the normal or wild-type gene. mutations in the gene or gene product, e.g., changes in the integrity, sequence, structure, etc. of the gene or gene product; This refers to the presence of a mutation that affects the structure, amount, or activity of a protein in healthy tissue or cells. the amount, structure and / or activity of the cancerous tissue as compared to that of the cells (e.g., a control). or in amount, structure and / or activity in cancer cells. , associated with a pathology, e.g., cancer. For example, associated with cancer or in response to anti-cancer therapy. A gene or gene product that predicts responsiveness is a gene or gene product that predicts the responsiveness of a cancer tissue or cell compared to a healthy tissue or cell. Variant nucleotide sequences (e.g., mutations), amino acid sequences, in tissues or cancer cells Chromosomal translocation, intrachromosomal inversion, copy number, expression level, protein level, protein activity or epigenetic modifications (e.g., methylation or acetylation status), or translation They may have or result from post-translational modifications. Typical mutations include point mutations (e.g. (e.g., silent, missense or nonsense), deletion, insertion, inversion, duplication, amplification, translocation These include, but are not limited to, inter-, inter- and intra-chromosomal rearrangements. The mutation may be in a coding or non-coding region of the gene. The mutations may be associated with a phenotype, e.g., a cancerous phenotype (e.g., cancer risk, cancer progression, cancer treatment, or cancer cure). In certain embodiments, the phenotype may be associated with (or not associated with) one or more of the following: resistance to a specific therapeutic agent; In this context, the terms "mutation" and "mutation" are used interchangeably herein.

[0135] As used herein, a "functional mutation (functional change)" refers to a change in a sequence that is different from a reference sequence, e.g., a wild-type or Affects cell division, growth or survival compared to the non-mutated sequence, e.g., cell division In some embodiments, functional mutations include mutations (alterations) that promote growth or survival. is a database of functional mutations, e.g., the COSMIC database (cancer.sa nger.ac.uk / cosmic;Forbes et al., Nucl.Acids Res .2015;43(D1):D805-D811), In certain embodiments, functional mutations are identified as being functional, e.g., by COSMI Mutations with known functional status found as known somatic mutations in the C database In other embodiments, functional mutations are mutations with possible functional status, e.g. , a truncation in a tumor suppressor gene. In certain embodiments, a functional mutation driver mutations, e.g., that enhance cell survival or regeneration, thereby contributing to the clone. In some embodiments, a mutation that confers a selective advantage in a microenvironment. The mutation may result in clonal expansion. In certain embodiments, the functional mutation is (a) (b) self-sufficiency in growth signals, (c) reduced anti-growth signals, e.g., insensitivity to them (c) reduced apoptosis; (d) enhanced replicative capacity; (e) sustained angiogenesis; or (f) tissue invasion or metastasis. In this case, functional mutations are not passenger mutations but rather contribute to the fitness of a clone of cells, for example. In other embodiments, functional mutations are mutations that have a detectable effect on the It is not a variant of unknown origin (VUS), i.e., the pathogenicity of the variant cannot be confirmed or excluded. None.

[0136] A "binding entity" is any molecule to which a molecular tag can be directly or indirectly bound, The term "binding entity" refers to an entity that can specifically bind to an analyte. In certain embodiments, the binding entity may be an avidin molecule or a hapten bond. Allows for the separation of nucleic acids from mixtures such as antibodies or antigen-binding fragments thereof Typical binding entities include biotin molecules, haptens, antibodies, and antibody-binding fragments. Examples of suitable polypeptides include, but are not limited to, peptides and proteins.

[0137] "Complementary" refers to sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. The adenine residues of the first nucleic acid region are thymine residues that are antiparallel to the first region. form specific hydrogen bonds ("base pairing") with residues of the second nucleic acid region that are cysteine ​​or uracil Similarly, cytosine residues in the first nucleic acid strand are antiparallel to the first strand. The first strand of nucleic acid is capable of base pairing with a residue in the second nucleic acid strand, which is guanine. A region is said to be complementary to a second region of the same or a different nucleic acid if they When the two regions are arranged in an antiparallel manner, at least one nucleotide of the first region In some embodiments, the first region is a base paired with a residue in the second region. The first region includes a first portion and the second region includes a second portion, wherein the first and second portions are reversed. When arranged in a parallel manner, at least about 50% of the nucleotide residues of the first portion At least about 75%, at least about 90%, or at least about 95% of the nucleic acids in the second portion are In other embodiments, all nucleotides of the first portion may be base-paired with the nucleotide residues. The nucleotide residues in the second portion are capable of base pairing with nucleotide residues in the second portion.

[0138] The terms "cancer" and "tumor" are used interchangeably herein. These terms refer to the properties typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, and rapid growth. It refers to the presence of cells with a specific length and rate of proliferation as well as certain characteristic morphological features.

[0139] The terms "neoplastic" or "neoplastic" cells include benign, premalignant, and malignant cells. abnormal proliferation in cells or tissues, e.g., hyperproliferation, which may include (cancer) or metastatic stages It means the reproductive stage.

[0140] Cancer is said to be "inhibited" if at least one symptom of the cancer is alleviated, prevented, or reduced. In the present specification, the term "reduced recurrence or metastasis of cancer" refers to a case where the recurrence or metastasis of cancer is accelerated or prevented. Cancer is also said to be "inhibited" if the progression of the disease is slowed, delayed, or prevented.

[0141] The terms "suppression" or "inhibitor" refer to the suppression of a given molecule, e.g., an immune checkpoint inhibitor. The reduction in a certain parameter of the inhibitor, e.g., activity, can be at least 5 %, 10%, 20%, 30%, 40%, 50%, 60% or more of the activity (e.g. The term encompasses inhibition of PD-L activity (PD-L, PD-L1, or PD-L1 activity). It does not have to be 100%. Inhibitors can bind to targets directly (e.g., inhibits the target directly (e.g., by inhibiting target-associated activity) or indirectly (e.g., by interfering with target-associated activity) Inhibitors may, for example, be multispecific (e.g., bispecific) and / or For example, two or more different targets may be inhibited (simultaneously or sequentially). The PD-1 or PD-L1 inhibitors used in this study are PD-1, PD-L1, or both. can be suppressed.

[0142] The term "Programmed Death 1" or "PD-1" isoforms, mammalian, e.g., human PD-1, species homologs of human PD-1, and PD-1, e.g., human PD-1, includes analogs that contain at least one shared epitope with PD-1. The amino acid sequence of human PD-1 is known in the art (see, e.g., Shinohara et al., et al. (1994) Genomics 23(3):704-6; Finger LR et al., G ene(1997)197(1-2):177-87).

[0143] The term "PD-ligand 1" or "PD-L1" refers to any isoform, mammalian, e.g. For example, human PD-1, species homologs of human PD-L1, and at least one homolog of PD-L1 The amino acid sequence of PD-L1, e.g., human PD-L1, includes analogs that contain a common epitope. is known in the art.

[0144] As used herein, the term "indel" refers to an alteration of one or more nucleotides in the nucleic acid of a cell. In certain embodiments, an indel refers to an insertion, a deletion, or both. and deletions occur in close proximity on the nucleic acid, resulting in an insertion of one or more nucleotides and In some embodiments, an indel is a net change in the total number of nucleotides. In one embodiment, an indel results in a net fragmentation of about 1 to about 50 nucleotides. Bring about change.

[0145] As used herein, a "chemotherapeutic agent" refers to a cellular agent used to treat a disease state, particularly cancer. It means a chemical substance such as a toxic or cytostatic agent.

[0146] As used herein, "cancer therapy" and "cancer treatment" are synonymous.

[0147] As used herein, "chemotherapy," "chemotherapeutic agent," and "chemotherapeutic substance" are synonyms. It is a word.

[0148] The terms "homology" and "identity," used interchangeably herein, refer to the degree of similarity between two polypeptides. It refers to sequence similarity between nucleotide sequences or between two polypeptide sequences, rather than identity. "Percentage of identity or homology" and "Identity or homology (% The term "match" is used in the comparison of two or more polynucleotide sequences or two or more polypeptide sequences. "Sequence similarity" means the percentage of sequence similarity found between two or more polynucleotides. The percentage of similarity in base pair sequence (determined by any suitable method) between the octide sequences An array of 2 or more represents any integer value between 0 and 100% or between them. The identity or similarity may be aligned for comparison purposes. The positions in the compared sequences can be determined by comparing the positions in each sequence. If the position is occupied by a nucleotide base or an amino acid, the molecule will The degree of similarity or identity between polynucleotide sequences is determined by the degree of similarity between those polynucleotides. The number of identical or matching nucleotides at positions shared by the nucleotide sequences The degree of identity of polypeptide sequences is a function of the number of amino acids shared by those polypeptide sequences. Homology or similarity of polypeptide sequences is a function of the number of identical amino acids at positions where the sequence is identical. The degree of similarity is a function of the number of amino acids at positions shared by the polypeptide sequences. As used herein, the term "substantially identical" means at least 75%, at least At least 80%, at least 85%, at least 90%, 91%, 92%, 93%, 94%, 9 5%, 96%, 97%, 98%, 99% or higher.

[0149] As used herein, "more likely" or "increased likelihood" refers to an item, object, thing, or This means that there is a high probability (possibility) that a person or entity will cause an event. In these cases, patients respond to treatment with PD-1 or PD-L1 inhibitors, alone or in combination. Subjects likely to respond to PD-1 or PD-L1 inhibition, alone or in combination, are more likely to respond to treatment with the target than a reference subject or group of controls.

[0150] "Unlikely" refers to the probability (likelihood) that an event, item, object, thing, or person will cause the event. Therefore, the likelihood of a PD-1 response, whether alone or in combination, is low compared to the standard. Subjects who are unlikely to respond to treatment with an inhibitor of PD-L1 or PD-L1 alone or The likelihood of responding to treatment with the combined kinase inhibitors is assessed by the reference subject or subject group. is lower than

[0151] "Sequencing" a nucleic acid molecule involves determining the sequence of at least one nucleotide in the molecule. In some embodiments, the identity of more than all of the nucleotides in the molecule is determined. In other embodiments, the identity of a smaller number of nucleotides in the molecule is determined. Or all identities are determined.

[0152] As used herein, "next generation sequencing" or "NGS" or "NGS" refers to a method for sequencing a sequence of ... "G sequencing" refers to the process of sequencing individual nucleic acid molecules (e.g., in single molecule sequencing) or individual Nucleotide sequencing of the clonal expansion proxies of nucleic acid molecules in a high-throughput manner (e.g. , 10 5 refers to any sequencing method in which more than one molecule is sequenced simultaneously In one embodiment, the relative abundance of nucleic acid species in a library is determined by sequencing. The relative number of occurrences of their cognate sequences in the data obtained by the standard experiment Next generation sequencing methods are known in the art. , e.g. Metzker, M. (2010) Nature Biotechnology Reviews 11:31-46 (which is incorporated herein by reference). Next-generation sequencing is used to identify nucleic acids present in less than 5% of the samples. The present mutations can be detected.

[0153] "Sample", "tissue sample", "patient sample", "patient cell or tissue sample " or "specimen," respectively, refers to tissue, cells, e.g., circulating cells, obtained from a subject or patient. The origin of the tissue samples can be fresh, frozen and / or preserved organs, tissue samples , solid tissue from a biopsy or aspirate; blood or any blood component; body fluids, e.g., cerebrospinal fluid , amniotic fluid, peritoneal fluid, or interstitial fluid; or cells from any point in a subject's pregnancy or development. Tissue samples may contain compounds that are not naturally mixed with native tissue, such as preservatives, anticoagulants, etc. It may contain solidifying agents, buffers, fixatives, nutrients, antibiotics, etc. In one embodiment For this purpose, samples should be prepared as frozen samples or in formaldehyde or paraformaldehyde. Preserved as aldehyde-fixed paraffin-embedded (FFPE) tissue preparations. The sample may be embedded in a matrix (e.g., an FFPE block or frozen sample).

[0154] As used herein, "tumor nucleic acid sample" refers to a nucleic acid molecule from a tumor or cancer sample. Typically, it is DNA from a tumor or cancer sample, e.g., genomic DNA, or cDNA derived from RNA. In certain embodiments, the tumor nucleic acid sample is purified or isolated (eg, it is removed from its natural state).

[0155] As used herein, a "control" or "reference" or "nucleic acid sample" refers to a control or reference sample. Typically, it refers to a nucleic acid molecule derived from a gene or gene product. DNA that does not contain modifications or variations (e.g., does not contain mutations), e.g., genomic DNA A, or cDNA derived from RNA. In certain embodiments, the reference or control nucleic acid The sample is a wild-type or non-mutated sequence. In certain embodiments, the reference nucleic acid sample The protein is purified or isolated (e.g., it is removed from its native state) In other embodiments, the reference nucleic acid sample is a non-tumor sample, e.g., a blood control, positive control, or negative control. Neighboring tissue (NAT) or any other non-cancerous sample from the same or a different subject It is from Lu.

[0156] As used herein, "adjacent to an interrogation site" means that the site is sufficiently close. and a detection reagent complementary to the site is complementary to a reference sequence (e.g., a non-mutant sequence) in the target nucleic acid. or wild-type sequence) and a mutation (e.g., a mutation described herein) As used herein, the term "directly adjacent" means that the term "directly adjacent" can be used to refer to a region that is directly adjacent to the other region. This occurs when there is no intervening nucleotide between the two nucleotides.

[0157] As used herein, a "relevant mutation" refers to a defined mutation (e.g., a mutation described herein). a preselected mutant with respect to nucleotide or primary amino acid sequence In some embodiments, relevant mutations are mutations within n. where n is 2, 5, 10, 20, 30, 50, 100 or 200 nucleotides (n is the nucleotide number that defines the relevant mutation and the defining mutation). In some embodiments, the associated mutation is a translocation mutation.

[0158] As used herein, an "interrogation position" refers to a position at which a mutation of interest ( mutated in a gene (e.g., a mutation identified) or analyzed, e.g., by sequencing or is a mutated nucleotide (or amino acid) in the nucleic acid (or protein) to be recovered. at least one nucleotide (or, in the case of a polypeptide, , amino acid residues).

[0159] A "reference sequence" used herein, for example as a comparator for a mutant sequence, is a sequence that is A sequence that has a nucleotide or amino acid at a position different from the mutant being analyzed. In one embodiment, the reference sequence is wild-type with respect to at least the interrogation position. do.

[0160] Headings such as (a), (b), (i), etc., are merely for illustrative purposes only and are not intended to be limiting unless otherwise specified. The ranges are merely shown for ease of reading. The use of headings in this section does not imply that the steps or elements are in alphabetical or numerical order or that they are in any order. The steps do not necessarily have to be performed in the order listed.

[0161] Various aspects of the invention that are of interest are described in further detail below. Additional definitions are provided herein. This is described throughout the specification.

[0162] Therapeutic methods and substances "Treat," "treatment," and other forms of the term refer to the administration of a substance, e.g., a therapeutic substance, alone or in combination with another substance. or in combination with a second substance to prevent cancer growth, or by weight or volume to shrink cancer in a subject, or to improve a subject's expected survival or time to tumor progression. In these subjects, the administration of an amount effective to prolong the duration of the disease is intended to Treatment may include suppression of tumor growth, reduction of tumor burden, reduction in size or number of metastatic lesions, or the prevention of new metastases. Suppression of the occurrence of metastatic disease, prolongation of survival, prolongation of progression-free survival, prolongation of time to progression, and This may include, but is not limited to, improving quality of life. Treatment is said to have been successful if at least one symptom of cancer is alleviated, arrested, slowed, or prevented. This is the case when it is prevented.

[0163] Unless otherwise indicated, as used herein, "prevent," "preventing," and "prevention" The term "treatment" refers to actions taken before a subject begins to experience cancer regrowth and / or to measures of cancer severity. This includes the act of suppressing or reducing.

[0164] Unless otherwise indicated, as used herein, a "therapeutically effective amount" of a substance is an amount that is effective in treating cancer. or to provide a therapeutic benefit in the treatment or delay one or more symptoms associated with cancer. A therapeutically effective amount of a compound is an amount sufficient to prevent or minimize the progression of cancer. the amount of a therapeutic agent, alone or in combination with other therapeutic agents, that provides a therapeutic benefit in The term "therapeutically effective amount" means an amount that improves the overall treatment and alleviates the symptoms or causes of cancer. The therapeutic agent may comprise an amount that reduces or avoids or enhances the therapeutic efficacy of another therapeutic substance.

[0165] Unless otherwise indicated, as used herein, a "prophylactically effective amount" of a substance is an amount that is effective to prevent the recurrence of cancer. to prevent the progression of, or one or more symptoms associated with, cancer, or its recurrence A prophylactically effective amount of a substance is an amount sufficient to provide a prophylactic benefit in the prevention of cancer. "Prophylactically effective amount" means an amount of a substance, alone or in combination with other therapeutic substances, that results in a prophylactically effective amount. The term "prophylactic" includes an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic agent. Ugh.

[0166] The term "patient" or "subject" refers to a human (e.g., a male or female of any age group). women), e.g., pediatric patients (e.g., infants, children, adolescents), or adult patients (e.g., young adults In certain embodiments, the subject is a patient as described herein. Adult subjects (e.g., males or females) with or at risk for melanoma as described In certain embodiments, the subject is 10 years of age or older, 15 years of age or older, 20 years old and over, 25 years old and over, 30 years old and over, 35 years old and over, 40 years old and over, 45 years old and over, 50 years old and over Over 55, Over 60, Over 65, Over 70, Over 75, Over 80, Over 85 In some embodiments, the subject is 0-10 years old, 10-20 years old, or 90 years old or older. 20 years old, 20-30 years old, 30-40 years old, 40-50 years old, 50-60 years old, 60-70 years old, 7 In some embodiments, the subject is 25 to 80 years old or 80 to 90 years old. In other embodiments, the subject is 15 to 29 years old. In one embodiment, the subject is a woman and is between 15 and 29 years old. The elephant is over 65 years old.

[0167] The terms "patient" or "subject" are used in reference to the administration of a compound or drug. If so, the patient is the subject of treatment, observation and / or administration of a compound or drug.

[0168] The agents described herein, e.g., therapeutic agents, may be administered in combination with a second or different therapeutic agent. in combination with anti-cancer drugs and / or surgical procedures and / or radiation. It may be administered in combination with radiation therapy.

[0169] "In combination" means that the therapies or therapeutic agents must be administered simultaneously, and and / or must be formulated for delivery together. Although not intended, these delivery methods are within the scope of the present invention. The therapeutic agent may be administered simultaneously with, before, or after the administration of the additional therapeutic or therapeutic agent. The agent is administered at a dose and / or on a time schedule determined for that agent. Therapeutic agents used in combination can be administered together in a single composition or can be administered in different It is further understood that the individual compounds used in the regimen may be administered separately in a single composition. Each combination may be determined by the compatibility of the first therapeutic agent with the additional therapeutically active agent, and / or or taking into consideration the desired therapeutic effect to be achieved.

[0170] The term "responder" as used herein refers to a group of subjects who have a high risk of developing a disease, for example, as defined by the RECIST 1.1 criteria. A subject who responds to treatment and shows a decrease in tumor size or the extent of cancer in the body when Alternatively, the subject may exhibit disappearance of one or more (eg, most or all) symptoms of cancer.

[0171] As used herein, the term "partial responder" refers to a response defined by, for example, RECIST 1.1 criteria. A subject who, when treated according to the principles of the present invention, shows a decrease in tumor size or the extent of cancer in the body in response to treatment. Includes.

[0172] The term "complete responder" as used herein refers to a response defined by, for example, RECIST 1.1 criteria. This includes subjects who respond to treatment and show disappearance of most or all signs of cancer when treated according to the principles of Yes, the cancer does not have to be cured for a complete responder.

[0173] The term "non-responder" as used herein means a non-responder defined, for example, by the RECIST 1.1 criteria. This includes subjects who do not show a decrease in tumor size or the extent of cancer in their body when treated with the above method.

[0174] In some embodiments, the subject is a classical partial or complete responder. Definition (RECIST 1.1 criteria) or atypical immune-related reactions lasting at least 12 months Depending on the response, patients are classified as responders (e.g., complete or partial responders) or non-responders. In some embodiments, clinical deterioration (e.g., as determined by a clinician) may occur within 12 months. Subjects who demonstrate a mixed response that results in a response to a steroid or requires additional systemic therapy are considered non-responders. systemic therapy or clinical deterioration (e.g., before, at, or after additional systemic therapy or clinical deterioration) In some embodiments, the response to therapy is assessed by imaging and clinician-recorded clinical data. The RECIST 1.1 criteria are based on the Eisenhaue r et al., Eur J Cancer. 2009;45(2):228-247 There are.

[0175] In some embodiments, responders (e.g., complete or partial responders) are Preselection in a predetermined set of genes, e.g., in the coding regions of a predetermined set of genes per unit of the sample (e.g., about 3.3 somatic mutations per megabase) In some embodiments, the mutation load in a tumor sample is A responder (e.g., a complete responder) is a gene that expresses a specific gene in a given set of genes, e.g., a specific gene in a given set of genes. per preselected unit, e.g., per megabase, in the coding region of a A mutation load in a sample (e.g., a tumor sample) of approximately 23.1 or more mutations In some embodiments, responders (e.g., partial responders) are those that express a given set of genes. per preselected unit in the coding region of a given set of genes, for example For example, a sample with approximately 3.3 or more somatic mutations per megabase and less than 23.2 somatic mutations per megabase (e.g., tumor sample). , non-responders may be non-responsive to a given set of genes, e.g., a coding region of a given set of genes. less than about 3.3 somatic mutations per preselected unit, e.g., per megabase, in of the mutation load in a sample (e.g., a tumor sample).

[0176] In some embodiments, a responder (e.g., a complete or partial responder) may at least about 29% (e.g., at least about 35%, at least about 40%, at least about 5 0%, at least about 60%, at least about 70%, at least about 80%, at least about 8 have an objective response rate (ORR) of at least about 5%, at least about 90%, or at least about 95% In some embodiments, the patient is a member of a patient population (e.g., in a clinical trial). A responder (e.g., a complete responder) is one who has a response rate of at least about 85% (e.g., at least about 90%). or are part of a group of patients with an objective response rate (ORR) of at least about 95%. In some embodiments, responders (e.g., partial responders) are between about 29% and about 85% (e.g., For example, an objective response of about 30% to about 80%, about 40% to about 70%, or about 50% to about 60% In some embodiments, non-responders are members of a patient group that has an ORR. , an objective response rate (ORR) of less than about 25% (e.g., less than about 20% or less than about 10%) are part of a group of patients with

[0177] Exemplary Therapeutic Substances and Methods The substance, e.g., a therapeutic substance, can be a small molecule, a protein, a polypeptide, a peptide, an antibody molecule, or the like. , nucleic acids (e.g., siRNA, antisense, or microRNA), small molecules, or immunoglobulins It can be a cell therapy. Exemplary agents and agent classes are described herein.

[0178] In one embodiment, the agent, e.g., a therapeutic agent, binds to PD-1 or PD-L1. In one embodiment, the substance is an antibody molecule. The terms "antibody" and "antibody molecule," used interchangeably herein, refer to an immunoglobulin molecule. and the immunologically active portion of an immunoglobulin molecule, i.e., the portion that specifically binds to an antigen. The term "antigen binding site" refers to a molecule containing the antigen-binding site, such as a polypeptide of interest in the present invention. Molecules that specifically bind to a given polypeptide of interest in the present invention are those that specifically bind to the polypeptide. The polypeptide binds to the sample, but the sample naturally contains the polypeptide, e.g., a biological sample. A molecule that does not substantially bind to other molecules in a sample. Examples of therapeutically active moieties include those obtainable by treating antibodies with an enzyme, such as pepsin. F(ab) and F(ab')2 fragments are included. The present invention relates to polyclonal and As used herein, "monoclonal antibody" or " The term "monoclonal antibody composition" refers to a monoclonal antibody composition that contains an antigen-binding site capable of immunoreacting with a particular epitope. It refers to a population of antibody molecules containing only one species of PD-1 or PD-L1. Such antibodies are known in the art and target polypeptides such as PD-1 or PD-L1. Techniques for obtaining antibodies against are also known in the art.

[0179] Typical PD-1 inhibitors In one embodiment, the inhibitor of PD-1 is an anti-PD-1 antibody. In this form, the PD-1 inhibitor is nivolumab (ON O-4538, BMS-936558 or MDX1106), pembrolizumab (pe mbrolizumab) (MK-3475 or lambrolizumab zumab)), pidilizumab (CT-011), MEDI 0680(AMP-514), PDR001, REGN2810, BGB-108, BG B-A317, SHR-1210 (HR-301210, SHR1210 or SHR- 1210), PF-06801591 or AMP-224.

[0180] In other embodiments, the PD-1 inhibitor is nivolumab ), pembrolizumab or pidilizumab It is an anti-PD-1 antibody selected from the group consisting of izumab.

[0181] In some embodiments, the anti-PD-1 antibody is nivolumab ( CAS Registry Number: 946414-94-4). Other names for the drive include MDX-1106, MDX-1106-04, ONO-4538 or B Nivolumab is a fully human immunoglobulin G (IgE) antibody that specifically blocks PD1. G4 monoclonal antibody specific for nivolumab (clone 5C4) and PD1 Other human monoclonal antibodies that bind to In one embodiment, the inhibitor of PD-1 is disclosed in US Pat. Bitter is disclosed in US 8,008,449 and WO 2006 / 121168, respectively. The heavy and light chain amino acid sequences disclosed as SEQ ID NOs: 2 and 3 (or Substantially identical or similar amino acid sequences to, e.g., the specified amino acids sequences that are at least 85%, 90%, 95% or more identical to the sequence The drug that is being used is novolumab.

[0182] In some embodiments, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab (Lambrolizumab) b), MK-3475, MK03475, SCH-900475 or KEYTRUDA (Merck) is a humanized IgG4 monoclonal antibody that binds to PD-1. Pembrolizumab and other humanized anti-PD-1 antibodies are from Hamid , O. et al. (2013) New England Journal of Medicine e 369(2):134-44, US8,354,509 and WO2009 / 114 335. In one embodiment, the inhibitor of PD-1 is, for example, For example, the pembro Lizumab, as described in US8,354,509 and WO2009 / 114335 The heavy and light chain amino acid sequences disclosed as SEQ ID NOs: 4 and 5, respectively (or Substantially identical or similar amino acid sequences thereto, e.g., A sequence that is at least 85%, 90%, 95% or more identical to the amino acid sequence column).

[0183] In some embodiments, the anti-PD-1 antibody is pidilizumab b) Pidilizumab (CT-011; Cure Tech) binds to PD-1. It is a humanized IgG1k monoclonal antibody. Pidilizumab and other humanized anti-PD-1 antibodies The -1 monoclonal antibody is disclosed in WO2009 / 101611.

[0184] Other anti-PD-1 antibodies include, among others, AMP514 (Amplimune) mune), e.g., US8,609,089, US2010028330 and / or or the anti-PD1 antibodies disclosed in US20100114649.

[0185] Typical PD-L1 inhibitors In one embodiment, the inhibitor of PD-L1 is atezolizumab lizumab) (MPDL3280A, RG7446 or RO5541267), Y W243.55.S70, MDX-1105, durvalumab (MEDI4736) or avelumab (MSB0010718C ) is selected.

[0186] In some embodiments, the PD-L1 inhibitor is an antibody molecule. In embodiments, the anti-PD-L1 inhibitor is YW243.55.S70, MPDL 3280A, MEDI-4736, MSB-0010718C or MDX-1105 are selected.

[0187] In some embodiments, the anti-PD-L1 antibody is MSB0010718C. SB0010718C (also known as A09-246-2 (Merck Serono) ) is a monoclonal antibody that binds to PD-L1. MSB0010718C and Other humanized anti-PD-L1 antibodies are disclosed, for example, in WO2013 / 079174. Disclosed in WO2013 / 079174 as SEQ ID NOs: 24 and 25, respectively The heavy and light chain variable region amino acid sequences (or substantially identical or is a similar amino acid sequence, e.g., at least 85% similar to the specified amino acid sequence. %, 90%, 95% or more identical sequences).

[0188] In one embodiment, the PD-L1 inhibitor is YW243.55.S70. The YW243.55.S70 antibody was synthesized as described in WO2010 / 077634 (WO2010 / 0 The heavy and light chain variable region sequences of 77634 are shown in SEQ ID NOs: 20 and 21, respectively. and the anti-PD-L1 described in the sequence disclosed therein ( or amino acid sequences substantially identical or similar thereto, e.g., At least 85%, 90%, 95% or more identical to the amino acid sequence has a certain sequence).

[0189] In one embodiment, the PD-L1 inhibitor is MDX-1105. X-1105 is also known as BMS-936559 and is disclosed in WO2007 / 005874 and an anti-PD-L1 antibody as described in Substantially identical or similar amino acid sequences to these, e.g., the specified amino acids A sequence that is at least 85%, 90%, 95% or more identical to the amino acid sequence )

[0190] In one embodiment, the PD-L1 inhibitor is MDPL3280A (Gene MDPL3280A is a human F-cell PD-L1-binding antibody. c Optimized IgG1 monoclonal antibody MDPL3280A and PD-L1 Other human monoclonal antibodies to IgG1 are described in U.S. Pat. No. 7,943,743 and U.S. Pat. It is disclosed in Publication No. 20120039906.

[0191] additional therapy The treatments described herein may be combined with different therapies, e.g., non-anti-PD-1 and / or non- In patients who have had an unsatisfactory response to anti-PD-L1 therapeutic agents or treatments In one embodiment, the subject may be receiving a different therapy, e.g., a non-anti-PD-1 and / or or are currently being treated with or have been treated with a non-anti-PD-L1 therapeutic substance or method The different therapeutic approaches include small molecules, proteins, polypeptides, peptides, and antibodies. molecules, nucleic acids (e.g., siRNA, antisense, or microRNA) or cells. Possible.

[0192] In one embodiment, the different therapies are chemotherapy, radiation therapy, immunotherapy, radiotherapy, or a therapeutic agent selected from radioimmunotherapy, oncolytic virus therapy, surgical procedure, or any combination thereof; In one embodiment, the different therapy is dacarbazine. zine), temozolomide, interleukin-2 (IL- 2), interferon, CTLA-4 inhibitors (e.g., anti-CTLA-4 antibodies, For example, ipilimumab, BRAF inhibitors, MEK inhibitors Bitter, Talimogene Laherparepbec epvec), adoptive cell transfer, or any combination thereof. In this condition, the different therapies include dacarbazine, temozolomide, temozolomide, interleukin 2 (IL-2) (e.g., recombinant interferon alfa 2b or peginterferon alfa 2b), interferon Ferron, ipilimumab, BRAF inhibitors [e.g., Murafenib (vemurafenib) or dabrafenib (dabrafenib) )], MEK inhibitors [e.g., cobimetinib or trametinib), talimogene laherparepvec mogene laherparepvec), adoptive cell transfer (e.g., modified T cells or modified dendritic cells), or any combination thereof.

[0193] The agents, e.g., therapeutic agents, described herein may be administered alone or in combination, e.g., In some cases, therapies may be used in combination with other chemotherapeutic agents or procedures to reduce or inhibit tumor cell growth in a subject. The compound may be administered in an amount sufficient to inhibit the growth of the tumor and / or to treat or prevent cancer.

[0194] Nucleic Acid Inhibitors In another embodiment, the agent is an antisense molecule, a ribozyme, a double-stranded R a NA molecule, a triple helix molecule (which hybridizes to the nucleic acid encoding the mutation), or Nucleic acid inhibitors selected from transcriptional regulatory regions that block or reduce mRNA expression of mutants - is.

[0195] In one embodiment, the nucleic acid antagonist inhibits the mRNA encoding the mutation. Other types of antagonist nucleic acids, such as dsRNA, ribonucleotides (siRNA), Binding enzymes, triple helix formers or antisense nucleic acids may also be used. Nucleic acid inhibitors, e.g., antisense, RNAi, against nucleic acid molecules encoding the mutations The present invention provides an isolated nucleic acid molecule which is

[0196] An "antisense" nucleic acid is complementary to a "sense" nucleic acid encoding a protein (e.g., For example, it may be complementary to the coding strand of a double-stranded cDNA molecule or may be complementary to an mRNA sequence. The antisense nucleic acid may comprise a nucleotide sequence encoding one or more mutations. It may be complementary to the entire nucleotide sequence or only a portion of it. In the present invention, the antisense nucleic acid molecule is an antisense nucleic acid molecule of a nucleotide sequence encoding one or more mutations. Antisense to "non-coding regions" of the coding strand (e.g., 5' and 3' untranslated regions) The antisense substance is, for example, about 8 to about 80 nucleic acid bases (i.e., about 8 to about 80 nucleotides), for example, about 8 to about 50 nucleic acid bases, or about 12 to about 30 nucleic acid bases Antisense compounds may include ribozymes, external guide sequence (EGS) oligonucleotides, and the like. oligonucleotides (oligozymes), and oligonucleotides that hybridize to target nucleic acids and regulate their expression Antisense compounds include other short catalytic RNAs or catalytic oligonucleotides. , may comprise a stretch of at least 8 contiguous nucleobases complementary to a sequence in the target gene. To be specifically hybridizable, an oligonucleotide must have a specific sequence identity to its target nucleic acid sequence. It is not necessary for the oligonucleotide to be 100% complementary to the target sequence. It is said to be a target molecule because binding of the oligonucleotide to the target molecule does not interfere with the normal function of the target molecule. This leads to loss of usefulness and is not possible under conditions where specific binding is desired, i.e., in vivo assays. under physiological conditions in the case of se or therapeutic treatments, or in the case of in vitro assays Avoid non-specific binding of the oligonucleotide to non-target sequences under the conditions under which the assay is performed. When there is a sufficient degree of complementarity between the two,

[0197] Hybridization of antisense oligonucleotides with mRNA results in the transcription of mRNA. It may interfere with one or more of the normal functions of mRNA. The functions that may be interfered with include all central functions, e.g. For example, RNA transfer to the protein translation site, translation of RNA into protein, and the This includes splicing of RNA to give mRNA species and catalytic activities that RNA may be involved in. The binding of specific proteins to RNA is also known as antisense oligonucleotides to RNA. Hybridization can be prevented.

[0198] Typical antisense compounds include those that bind to a target nucleic acid (e.g., a nucleic acid sequence described herein). It contains a DNA or RNA sequence that specifically hybridizes to the mRNA encoding the mutation. The complementary region can extend over about 8 to about 80 nucleotides. Modified nucleobases are known in the art. See, for example, U.S. Patent Nos. 4,987,071 and 5,116,742. Nos. and 5,093,246; Woolf et al. (1992) Proc Natl Ac ad Sci USA;Antisense RNA and DNA, DAMel ton, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1988);89:7305-9;Hasel hoff and Gerlach (1988) Nature 334:585-59; lene, C. (1991) Anticancer Drug Des.6:569-8 4;Helene(1992)Ann.NYAcad.Sci.660:27-36 ; and see Maher (1992) Bioassays 14:807-15. sea ​​bream.

[0199] Antisense nucleic acid molecules are typically administered (e.g., by direct injection at a tissue site) It is administered to a subject or produced in situ, which suddenly hybridize to cellular mRNA and / or genomic DNA encoding the mutation, This allows the expression of proteins to be inhibited, for example, by inhibiting transcription and / or translation. Alternatively, antisense nucleic acid molecules can be modified to target selected cells. For systemic administration, the antibody can be administered via, for example, a cell surface receptor. by linking antisense nucleic acid molecules to peptides or antibodies that bind to receptors or antigens. This allows the antisense molecule to bind specifically to a receptor or antigen expressed on the surface of a selected cell. Antisense molecules can be modified to specifically bind to the target nucleic acid. They can also be delivered to cells using the vectors described herein. To obtain sufficient intracellular concentrations of the sense molecule, a strong pol II or pol III A vector construct in which the antisense nucleic acid molecule is placed under the control of a promoter is preferred.

[0200] In yet another embodiment, the antisense nucleic acid molecule is an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA; In this case, contrary to the usual β-units, the chains run parallel to each other (Gaultier et al. (1999) 87) Nucleic Acids. Res. 15:6625-6641). Antisense Nucleic acid molecules are 2'-o-methylribonucleotides (Inoue et al. (1987) Nucleic Acid ic Acids Res.15:6131-6148) or chimeric RNA-DNAs including analogs (Inoue et al. (1987) FEBS Lett. 215:327-330). I can see it.

[0201] siRNAs are small double-stranded RNAs (dsRNAs) that may optionally contain overhangs For example, the double-stranded region of the siRNA is about 18 to 25 nucleotides long, e.g., about 19, 20 , 21, 22, 23 or 24 nucleotides in length. Typically, the siRNA sequence is In particular, dsRNA and siRNA are specifically designed to target the target mRNA in mammalian cells. It can be used to silence gene expression in cells (e.g., human cells). The siRNA consists of a short hairpin with a 29 base pair stem and a 2-nucleotide 3' overhang. Also included are shRNAs. See, for example, Clemens et al. (2000) Proc. atl.Acad.Sci.USA 97:6499-6503;Billy et al. (200 1)Proc.Natl.Sci.USA 98:14428-14433;Elbas hir et al. (2001) Nature. 411:494-8; Yang et al. (2002) Pr oc.Natl.Acad.Sci.USA 99:9942-9947;Siolas (2005), Nat. Biotechnol. 23(2):227-31; U.S. Patent Publication No. 20040086884, No. 20030166282, No. 200301432 See No. 04, No. 20040038278 and No. 20030224432 .

[0202] In yet another embodiment, the antisense nucleic acids featured in the present invention are Ribozymes with specificity for nucleic acids encoding mutations are known in the art. one or more sequences complementary to the nucleotide sequences of the mutant cDNAs disclosed in the specification, and and sequences with known catalytic sequences that result in mRNA cleavage (U.S. Patent No. 5,099,499). 3,246 or Haselhoff and Gerlach (1988) Nature 334:585-591). For example, the nucleotide sequence of the active site is A nucleotide sequence complementary to the nucleotide sequence to be cleaved in the mutant encoding mRNA. Derivatives of Trahymena L-19 IVS RNA can be constructed. See, e.g., Cech et al., See U.S. Patent No. 4,987,071 and Cech et al., U.S. Patent No. 5,116,742. Alternatively, catalytic RNAs with specific ribonuclease activity can be introduced into RNA molecules. Mutant mRNAs can be used to select from the pool. See, e.g., Bartel, D. and Szostak, J.W. (1993) Science 261:1411- See 1418.

[0203] Mutant gene suppression is achieved by targeting nucleotide sequences complementary to the regulatory region of the mutation. by forming a triple helix structure that prevents the transcription of the mutant gene in the target cell. Generally, Helene, C. (1991) Anticancer Drug Des.6:569-84;Helene,Ci(1992)Ann.N. Y. Acad. Sci. 660:27-36; and Maher, L. J. (1992). Bioassays 14:807-15. The potential sequences that can be ligated are the so-called "switchback" nucleic acid molecules. The switchback molecule can be increased by creating an alternating ing) 5'-3', 3'-5' form, which is first synthesized by combining one strand of the double strand with the salt base pairing with the other strand to form a large stretch of purines or pyrimidines eliminates the need for The present invention also provides detectably labeled oligonucleotide primers and probes. Typically, such labels are chemiluminescent, fluorescent, radioactive or It is colorimetric.

[0204] Mutant nucleic acid molecules may be used to modify, for example, the stability, hybridization, or solubility of the molecule. The base moiety, sugar moiety, or phosphate backbone may be modified to improve the activity of the polypeptide. For non-limiting examples of synthetic oligonucleotides, see Toulme (2001) Nature. re Biotech.19:17 and Faria et al. (2001) Nature Bi See, for example, Olefin Tech. 19:40-44. Reotide may be an effective antisense agent.

[0205] For example, the deoxyribose phosphate backbone of a nucleic acid molecule can be modified to give peptide nucleic acids. It is possible (Hyrup B. et al. (1996) Bioorganic & Medici As used herein, "peptide nucleic acid" or The term "PNA" refers to a peptide in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone. and refers to nucleic acid mimics, e.g., DNA mimics, in which only the four natural nucleobases are retained. The neutral backbone of PNA allows specific binding to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be carried out by the method of Hyrup B. (1996) supra and Perry-O'Keefe et al., Proc. Natl. Aca d. Standard solid-phase peptide synthesis method described in Sci. 93:14670-675 This can be done using

[0206] PNAs of mutant nucleic acid molecules can be used for therapeutic and diagnostic applications. For example, PNAs can be For example, by inducing transcription or translation arrest or by inhibiting replication may be used as antisense or antigene agents for sequence-specific regulation of gene expression. PNAs of mutant nucleic acid molecules can also be used to analyze single base pair mutations in genes (e.g. , by PNA-directed PCR clamping) or by other enzymes (e.g., S When used in combination with nuclease (Hyrup B. et al. (1996) supra), as "restriction enzymes" or as protease enzymes for DNA sequencing or hybridization probe or primer (Hyrup B. et al. (1996) supra; Perry-O'Ke efe, supra).

[0207] In other embodiments, the oligonucleotide may contain other additional groups, such as peptides (e.g., for example, when targeting host cell receptors in vivo), or to the cell membrane (e.g., Letsi nger et al. (1989) Proc. Natl. Acad. Sci. USA 86:655 3-6556;Lemaitre et al. (1987) Proc.Natl.Acad.Sci USA 84:648-652; see WO88 / 09810) or blood Substances that enhance transport across the brain barrier (see, e.g., WO 89 / 10134) The oligonucleotide may also contain a hybridization-triggered cleavage agent (e.g., See Krol et al. (1988) Bio-Techniques 6:958-976. (See, e.g., Zon (1988) Pharm. Res. 5:539-549). is linked to another molecule (e.g., a peptide, hybridization-inducing cross-linking agent, transport agent, or hybridization agent). The hybridization-triggered cleavage agent may be conjugated to a cleavage site.

[0208] In some embodiments, the nucleic acid inhibitors described herein are inhibitors. The present invention provides for the inhibition of expression of a nucleic acid containing a mutation in a nucleotide sequence.

[0209] cancer The invention described herein can be used to treat cancer or to evaluate a subject with cancer. It can be used to evaluate

[0210] Typical cancers include, but are not limited to: melanoma B-cell cancers, such as multiple myeloma, breast cancer, lung cancer (e.g., non-small cell lung cancer or NSCLC), C), bronchial cancer, colorectal cancer, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer Nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral or pharyngeal cancer , liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendiceal cancer, salivary gland cancer, thyroid cancer, adrenal gland cancer, bone Sarcoma, chondrosarcoma, cancer of the blood tissue, adenocarcinoma, inflammatory myofibroblastoma, gastrointestinal stromal tumor (GIS) T), colon cancer, multiple myeloma (MM), myelodysplastic syndrome (MDS), myeloproliferative disorders ( MPD), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia Leukemia (CML), chronic lymphocytic leukemia (CLL), polycythemia vera, Hodgkin's lymphoma sarcoma, non-Hodgkin's lymphoma (NHL), soft tissue sarcoma, fibrosarcoma, myxosarcoma, liposarcoma, osteosarcoma sarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial cell sarcoma, synovioma, mesothelioma tumor, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma , sebaceous carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, liver cancer, cholangiocarcinoma, Hair carcinoma, seminoma, embryonal carcinoma, Wilms' tumor, bladder cancer, epithelial carcinoma, glioma, astrocytoma , medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma , neuroblastoma, retinoblastoma, follicular lymphoma, diffuse large B-cell lymphoma, hepatocellular Cancer, thyroid cancer, gastric cancer, head and neck cancer, small cell cancer, essential thrombocythemia, idiopathic myeloid metaplasia, eosinophilia Hypereosinophilic syndrome, systemic mastocytosis, familial hypereosinophilia, chronic eosinophilic leukemia, neuroendocrine Secretory carcinoma, carcinoid tumors and their metastatic lesions.

[0211] melanoma The present invention provides, at least in part, a method for treating cancer, e.g., melanoma, in a subject. In certain embodiments, the method provides a method for detecting a mutation described herein (e.g., Mutations in genes disclosed in Table 1, such as the NF1 gene described herein and the treatment of cancers containing mutations in the LRP1B gene or the LRP1B gene, such as melanoma. The method includes administering a therapeutic agent, e.g., an immunotherapeutic agent, e.g., an immunoreactive protein (PD-1) or a PD-L1 inhibitor. The method includes administering an inhibitor to a subject.

[0212] In some embodiments, the cancer is melanoma. In some embodiments, the cancer is advanced In some embodiments, the cancer is metastatic melanoma. In some embodiments, the cancer, e.g., melanoma, is at risk of recurrence or relapse. In some embodiments, the cancer, e.g., melanoma, is at risk of metastasis. For purposes of this description, melanoma may be any type of melanoma defined according to any suitable melanoma classification system known to those of skill in the art. melanoma of any stage or risk group.

[0213] In certain embodiments, the melanoma is stage 0, stage IA, stage IB, stage IV ... Stage IIA, stage IIB, stage IIC, stage III, or stage IV melanoma In other embodiments, the melanoma is one of the cancers identified by the American Joint Committee on Cancer (AJC). American Joint Committee on Cancer (AJCC) Stage 0, stage IA, stage IB, stage IIA, and stage II as defined by the Breast Cancer Staging System B, stage IIC, stage III, or stage IV melanoma. In some embodiments, the melanoma is classified according to any suitable melanoma classification system known to those of skill in the art. In another embodiment, the melanoma is any stage defined according to are listed in Tables 2A-2B as Stage 0, Stage IA, Stage IB, Stage IIA, and Stage II. B, stage IIC, stage III, or stage IV melanoma.

[0214] In certain embodiments, the melanoma is stage III or stage IV melanoma. In some embodiments, the melanoma is stage III melanoma. In the present study, melanoma is classified as stage I according to any suitable melanoma classification system known to those of skill in the art. In certain embodiments, the cancer is a Class II melanoma. In one embodiment, the melanoma is stage III melanoma. In certain embodiments, the melanoma is any suitable tumor known to those of skill in the art. In one embodiment, the cancer is stage IV melanoma according to the melanoma classification system. Stage IV melanoma as described in Tables 2A-2B.

[0215] In some embodiments, the melanoma is an aggressive melanoma. In this context, advanced melanoma is classified as stage III or stage IV melanoma, e.g., as described in Tables 2A-2B. In one embodiment, the melanoma is stage IV melanoma. In another embodiment, the melanoma is metastatic melanoma. In some embodiments, the metastatic melanoma is a melanoma, e.g., a melanoma described in Tables 2A-2B. stage III or stage IV melanoma, as described in the National Cancer Institute. [Table 1] [Table 2]

[0216] In some embodiments, the melanoma is cutaneous melanoma. In this case, the melanoma is a non-cutaneous melanoma.

[0217] In certain embodiments, cancer, e.g., melanoma, e.g., advanced or metastatic melanoma, Mutations, e.g., mutations in the genes disclosed herein, e.g., The mutation is identified or determined to contain, or to have, a mutation that is known to be present in the human genome. In certain embodiments, cancer, e.g., melanoma, e.g., advanced or metastatic melanoma, is a cancer characterized by a phenotype similar to that described in Table 1. Containing or confirmed or determined to have a mutation in the gene described. In other embodiments, cancer, e.g., melanoma, e.g., advanced or metastatic Melanomas contain or contain multiple mutations in the genes listed in Table 1. In other embodiments, the cancer is identified or determined to be a cancer, e.g., melanoma. For example, advanced or metastatic melanoma may be associated with multiple mutations in multiple genes listed in Table 1. It contains, or has been identified or determined to have, multiple mutations.

[0218] In certain embodiments, cancer, e.g., melanoma, e.g., advanced or metastatic melanoma, The NF1 gene may be a mutation in the NF1 gene, such as those described herein. The present invention relates to a method for detecting a mutation in a human ovarian tumor.

[0219] In one embodiment, the mutation in the NF1 gene results in a decrease in the activity of NF1 compared to wild-type activity. This results in a decrease in the activity of the NF1 gene product (e.g., NF1 protein) when For example, the mutation may affect the GTPase activator activity of NF1, phosphatidylcholine binding, a change (e.g., a decrease) in the activity and / or phosphatidylethanolamine binding activity of In certain embodiments, the mutation results in increased activation of RAS.

[0220] In one embodiment, the mutation in the NF1 gene is a mutation (e.g., a somatic mutations), such as substitutions (e.g., base substitutions), insertions or deletions Typical NF1 mutations associated with melanoma are described, for example, in Wiesner et al., Am J Surg Pathol.2015;39(10):1357-62;Krautham mer et al., Nat Genet. 2015;47(9):996-1002 Additional mutations in the NF1 gene are listed, for example, in the COSMIC database (ca ncer.sanger.ac.uk / cosmic; Forbes et al., Nucl.Ac ids Res.2015;43(D1):D805-D811).

[0221] In certain embodiments, cancer, e.g., melanoma, e.g., advanced or metastatic melanoma, The mutated LRP1B gene may be a mutation in the LRP1B gene, such as those described herein. Containing or confirmed or determined to have a mutation in a gene.

[0222] In certain embodiments, the mutation in the LRP1B gene prevents the wild-type activity of LRP1B. a reduced activity of the LRP1B gene product (e.g., LRP1B protein) compared to For example, the mutation may result in a decrease in calcium ion binding and / or a decrease in the activity of LRP1B. This may result in an alteration (eg, a decrease) in high density lipoprotein receptor activity.

[0223] In one embodiment, the alteration in the LRP1B gene is a mutation (e.g., a somatic a cellular mutation), such as a substitution (e.g., base substitution), insertion or deletion, or Typical LRP1B mutations associated with melanoma include, for example, those described by Nikolaev et al. Nat Genet. 2011;44(2):133-9. LRP1B Additional mutations in genes can be found, for example, in the COSMIC database (cancer.sa nger.ac.uk / cosmic;Forbes et al., Nucl.Acids Res .2015;43(D1):D805-D811).

[0224] In certain embodiments, cancer, e.g., melanoma, e.g., advanced or metastatic melanoma, The mutations include those in the BRAF gene, such as those described herein. The present invention relates to a method for detecting a mutation in a human ovarian tumor.

[0225] In one embodiment, the mutation in the BRAF gene is a mutation that results in a BRAF gene that is mutated relative to wild-type activity of BRAF. resulting in increased activity of the BRAF gene product (e.g., BRAF protein) compared to For example, the mutation may result in an alteration in one or more of the following activities of BRAF: TP-binding calcium, ion binding, same protein binding, MAP kinase kinase kinase enzyme activity, protein kinase activity, or protein serine / threonine kinase activity. In certain embodiments, the mutation also results in an alteration (e.g., an increase) in the kinase activity of BRAF. Drop.

[0226] In one embodiment, the BRAF mutation is a mutation (e.g., a somatic mutation), such as For example, the mutation may be or include a substitution (e.g., base substitution), insertion, or deletion. In some embodiments, the mutation is a base substitution.

[0227] In one embodiment, the mutation in BRAF is located at codon V600. In some embodiments, the mutation in BRAF is a V600E mutation. In one embodiment, the mutation in BRAF is a V600K mutation. In one embodiment, the mutation in BRAF is a V600R mutation. The difference is the V600D mutation.

[0228] In one embodiment, the mutation in BRAF is a mutation other than a V600 mutation. In some embodiments, the mutation in BRAF is a K601 mutation. In one embodiment, the mutation in BRAF is a K601E mutation. The mutation in BRAF is a G469 mutation. In one embodiment, the mutation in BRAF is a G469E mutation. In one embodiment, the mutation in BRAF is a D594G mutation. In one embodiment, the mutation in BRAF is the L597 mutation. In one embodiment, the mutation in BRAF is a L597S mutation. The mutation in RAF is an S467 mutation. The mutation is S467L.

[0229] In other embodiments, the present invention relates to a cancer, e.g., a melanoma, e.g., an advanced or metastatic melanoma. The mutations include those in the NRAS gene, such as those described herein. The present invention relates to a method for detecting a mutation in a human ovarian tumor.

[0230] In one embodiment, the mutation in the NRAS gene results in a decrease in the activity of NRAS compared to wild-type activity. Increased activity of the NRAS gene product (e.g., NRAS protein) compared to For example, the mutation results in an alteration (e.g., a decrease) in the GTPase activity of NRAS. In one embodiment, the NRAS protein reduces intrinsic GTP hydrolysis. These include mutations that result in constitutively active NRAS, for example.

[0231] In one embodiment, the NRAS mutation is a mutation (e.g., a somatic mutation), such as For example, the mutation may be or include a substitution (e.g., base substitution), insertion, or deletion. In some embodiments, the mutation is a base substitution.

[0232] In one embodiment, the mutation in NRAS is located at codon Q61. In some embodiments, the mutation in NRAS is a Q61H mutation. In one embodiment, the mutation in NRAS is a Q61K mutation. In one embodiment, the mutation in NRAS is a Q61L mutation. In one embodiment, the mutation in NRAS is a Q61Q mutation. In one embodiment, the mutation in NRAS is a Q61R mutation.

[0233] In other embodiments, the mutation in NRAS is a G12 mutation. In one embodiment, the mutation in NRAS is a G12A mutation. In one embodiment, the mutation in NRAS is a G12C mutation. In one embodiment, the mutation in NRAS is a G12D mutation. In one embodiment, the mutation in NRAS is a G12S mutation. In one embodiment, the mutation in NRAS is a G12V mutation. In one embodiment, the mutation in NRAS is a G13 mutation. In one embodiment, the mutation in NRAS is a G13C mutation. In one embodiment, the mutation in NRAS is a G13R mutation. In one embodiment, the mutation in NRAS is a G13V mutation. In one embodiment, the mutation in NRAS is an S17 mutation. The mutation in NRAS is the S17N mutation.

[0234] Mutation Load As used herein, the term "mutation load" or "mutation burden" refers to the amount of mutations in a given gene. per preselected unit in a set (e.g., coding region of a given set of genes) (e.g., per megabase) mutations (e.g., one or more mutations, e.g., one or more somatic The mutation load refers to the level, e.g., number, of mutations in a genome, e.g., the whole genome or exon. The measurement can be based on the genome or on a subset of the genome or exome. In certain embodiments, the prognosis measured based on a subset of the genome or exome is The natural mutation load can be extrapolated to determine the mutation load of the whole genome or exome is.

[0235] In certain embodiments, the mutation load is determined based on the number of mutations in a subject, e.g., a subject described herein. Samples from elephants, e.g., tumor samples (e.g., melanoma samples, or melanoma In some embodiments, the measurement is performed in samples obtained from melanoma or derived from melanoma. In morphology, the mutation load is: (i) the level of somatic mutations in a given set of genes listed in Table 1; (ii) the presence of a somatic mutation in the NF1 gene; (iii) the number of somatic mutations in the LRP1B gene, or (iv) Number of C to T changes The present invention is measured by determining one, two, three or all of the following:

[0236] In one embodiment, somatic mutations in a predetermined set of genes listed in Table 1 The level of mutations correlates with the mutation weight (e.g., whole genome or exome mutation load). In one embodiment, the presence of a somatic mutation in the NF1 gene is determined by mutation weighting ( For example, based on the level of somatic mutations in a given set of genes listed in Table 1 correlates with the mutation load measured in the genome or whole genome or exome In one embodiment, the number of somatic mutations in the LRP1B gene is mutation-weighted. (e.g., based on the level of somatic mutations in a given set of genes listed in Table 1) and mutation load measured in the whole genome or exome) In one embodiment, the number of C to T changes is weighted by a mutation weight (e.g., as shown in Table 1). The prognosis is measured based on the level of somatic mutations in a given set of described genes. It correlates with the mutation load (or whole genome or exome mutation load).

[0237] The terms "mutation load" and "mutation burden" are used interchangeably herein. In the context of tumors, mutation load is referred to herein as "tumor mutation load"; Also referred to as "tumor mutation burden" or "TMB."

[0238] Typical genes associated with cancer The invention described herein involves the selection of a predetermined set of genes associated with cancer (e.g., the level of somatic mutations (e.g., one or more somatic mutations) in the This involves measuring the mutation load by determining the mutation rate.

[0239] In one embodiment, the genes or gene products listed in Table 1 are In one embodiment, the level of cell mutation is determined. Determine the level of somatic mutations in the genomic region. [Table 3] TIFF0007759177000004.tif118149

[0240] Neurofibromin 1 (NF1) The invention described herein relates to, for example, mutations in the NF1 gene (e.g., determining the presence of a mutation in the NF1 gene (e.g., a somatic mutation) or This involves measuring the mutation load by determining the presence of cellular mutations.

[0241] Neurofibromin 1 (NF1), also known as WSS, NFNS, or VRNF ) gene encodes a protein that functions as a negative regulator of the Ras signaling pathway Mutations in this gene are known to cause, for example, neurofibromatosis type 1 and juvenile myelomonocytic leukemia. It has been linked to myeloid leukemia and Watson syndrome. The mRNA for this gene is expressed too early. RNA editing (CGA>UGA->Arg1306Term) leads to translation termination. Alternatively, alternatively spliced ​​transcripts encoding different isoforms may be expressed. Mutations have also been described for this gene.

[0242] The nucleotide and amino acid sequences of human NF1 are set forth, for example, in: Wallace et al., Science. 1990;249:181-186, Erratu m: Wallace et al., Science. 1990;250:1749-1749 (I isoform 1); Marchuk et al., Genomics. 1991;11:931-94 0 (isoform 1); Cawthon et al., Cell. 1990;62:193-20 1 (isoform 1); Bernards et al., DNA Cell Biol. 1992 ;11:727-734 (isoforms 1 and 2); Li et al., Genomics.1 995;25:9-18 (isoforms 1 and 2); Nishi et al., Oncogen e.1991;6:1555-1559(isoforms 1 and 2);Anderse n et al., Mol. Cell. Biol. 1993;13:487-495 (isoforms 2);Suzuki et al.,Biochem.Biophys.Res.Commun.19 91;181:955-961 (isoform 2); Suzuki et al., Biochem Biophys.Res.Commun.1992;187:984-990(Iso Form 3); Martin et al., Cell. 1990; 63:843-849 (Isoph Form 4); Suzuki et al., Tohoku J. Exp. Med. 1995; 175: 225-233 (isoform 5); and Xu et al., Cell. 1990;62:59 9-608 (isoforms 1 and 6).

[0243] Additional variants, mutations, and polymorphisms of human NF1 are also described, for example, in :Upadhyaya et al., Hum.Mutat.1994;4:83-101;Sh en et al., J.Med.Genet.1996;33:2-17;Li et al., Cell.19 92;69:275-281(mutant Glu-1444);Upadhyaya et al.,Hu m. Mol. Genet. 1992;1:735-740 (mutant NF1 Met-21 64 and Asn-2192); Tassabehji et al., Am. J. Hum. Gene t.1993;53:90-95 (variant Gly-His-Glu-Gln-Gln-L ys-Leu-Pro-Ala-Ala-Thr-Leu-Ala-Leu-1733 ins);Shen et al.,Hum.Mol.Genet.1993;2:1861-186 4 (mutant Met-991 del); Purandare et al., Hum. Mol. Gen et.1994;3:1109-1115 (mutant NF1 Asp-1166 and Ar g-1440);Abernathy et al., Hum. Mutat.1994;3:347- 352 (mutant NF1 2387-Asn-Phe-2388 del);Upadhy Aya et al., J. Med. Genet. 1995;32:706-710 (mutant NF1 Ala-2631); Gasparini et al., Hum. Genet. 1996;97:4 92-495 (mutant NF1 Arg-629); Wu et al., Hum. Mutat. 199 6;8:51-56(Mutant NF1 Arg-1035);Upadhyaya et al.,Hu m. Genet. 1997;99:88-92 (mutant NF1 Ser-1412; Gl n-1440; Glu-1444 and Gly-1489); Maynard et al., Hum Genet. 1997;99:674-676 (mutant NF1 Arg-844 and Pro-898);Hudson et al., Hum. Mutat.1997;9:366-36 7 (mutant NF1 Arg-1952); Upadhyaya et al., Hum. Mutat. 1997;10:248-250 (mutant NF1 GLY-338 and TRP-161 1); Klose et al., Hum. Mol. Genet. 1998;7:1261-1268 (Mutant NF1 Pro-1276); Krkljus et al., Hum. Mutat. 199 8;11:411-411 (mutant NF1 Gly-1204, mutant His-765) Messiaen et al., Genet. Med. 1999;1:248-253 (variant N F1 Pro-508); Peters et al., Hum. Mutat. 1999;13:33 7-337 (mutant NF1 Pro-1446); Fahsold et al., Am. J. Hum Genet.2000;66:790-818(mutant NF1 Pro-216;Pr o-357;Cys-491;Pro-549;Thr-581;Arg-583;Ph e-665;Pro-695;Pro-763;Ser-777;Lys-780;Pr o-781;Pro-847;Ser-1156;Pro-1250;Gln-1276 ;Pro-1276;Pro-1446;Val-1605 and Ile-2507, Variant Glu-176); Ars et al., Hum. Mol. Genet. 2000;9:237 -247 (mutant NF1 Ser-117;Trp-1204;Pro-1446 and 2387-Asn-Phe-2388 del), Erratum Ars et al., Hum. Mol. Genet. 2000;9:659-659; Boulandet et al., Hum. Mutat.2000;16:274-275(Mutant NF1 Phe-844);Ka ufmann et al., Am.J.Hum.Genet.2001;69:1395-1400 (Mutant spinal FSNF Pro-2088); Han et al., Hum. Genet. .2001;109:487-497 (Mutant NF1 Lys-780;Cys-784 ;Pro-1147;Cys-1193;Arg-1444;Ser-1785;Asn -2012 and Lys-2357);Kluwe et al., Hum.Mutat.2002; 19:309-309 (mutant NF1 Phe-82; Arg-784 and Glu-1 444);Baralle et al., Am.J.Med.Genet.A 2003;119: 1-8 (mutant NFNS Glu-1459 del); Wang et al., Hum. Gene t.2003;112:117-123(mutant NF1 Tyr-93;Val-604 ;Arg-844 and Pro-898, variants Asp-74;Glu-176;Arg -712 and Gln-1276); De Luca et al., Hum. Mutat. 2003 ;21:171-172(Mutant NF1 Lys-780;Pro-847;Glu-8 48 and Arg-968; Asn-1444; Leu-1953 del and Arg -2001); Kluwe et al., J. Med. Genet. 2003;40:368-37 1 (mutant NF1 Arg-578; Pro-920 and Ala-2221); Zat Kova et al., Hum. Mutat. 24:491-501 (mutant NF1 Val-18 6, Characterization of the mutant NF1 Val-186); De Luca et al., Hum. Muta t.2004;23:629-629(Mutant NF1 Asn-157;Arg-629 ;Ser-777;Lys-780;Arg-784;Pro-847;Glu-848 ;Arg-968;Asn-1444;Leu-1953 del and Arg-200 1, mutant Glu-176); Mattocks et al., J. Med. Genet. 2004 ;41:E48-E48 (mutant NF1 Arg-31;Pro-145;Arg-32 4;Val-337;Cys-489;Pro-532;Arg-574;Arg-62 9;Phe-665;Phe-844;Pro-844;Met-991 del;Va l-1073;Arg-1196;Gly-1276;Gln-1276;Glu-14 30; Glu-1459 del and Gly-1489, mutant Glu-176 and Cys-873);Ferner et al., J.Med.Genet.2004;41:837 -841 (mutant NF1 Pro-1243); Bertola et al., Am. J. Med. Genet.A 2005;136:242-245(Mutant NF1 Arg-844) ;De Luca et al.,Am.J.Hum.Genet.2005;77:1092-11 01 (mutant NFNS Arg-194;Glu-1444;Thr-1451;Leu -1453 and Glu-1459 del); Sjoeblom et al., Science. 2006;314:268-274 (mutants Ile-1187; Leu-1951 and Arg-2745);Upadhyaya et al., Am.J.Hum.Genet.2007 ;80:140-151(mutant NF1 Met-991 del);Nystrom et al. , Clin. Genet. 76:524-534 (mutant NFNS Phe-1411) ;Ponti et al.,Hered.Cancer Clin.Pract.2011;9:6 -6 (mutant NF1 Thr-160); Thomas et al., Eur. J. Hum. Gen et.2012;20:411-419 (mutant Glu-176; Thr-330; As p-393; Leu-393; Pro-519; Thr-776 and Phe-1484 );Nemethova et al., Ann.Hum.Genet.2013;77:364-3 79 (mutant Nf1 Trp-93;Arg-1048;Arg-1189;Arg-1 661 (isoform 1) and Thr-1918 (isoform 1); and B en-Salem et al.,Childs Nerv.Syst.2014;30:1183- 1189 (mutant NF1 Pro-2125).

[0244] Low-density lipoprotein receptor-related protein 1B (LRP1B) The invention described herein relates to, for example, mutations in the LRP1B gene (e.g., , somatic mutations), or to determine the number of mutations (e.g., This involves measuring the mutation load by determining the presence of mutations (e.g., somatic mutations).

[0245] Low-density lipoprotein (LRP-DIT), also known as LRP-1B or LRPDIT The protein receptor-related protein 1B (LRP1B) gene encodes the low-density lipoprotein (LDL) These receptors belong to the α- and β-catenin receptor gene family. These receptors interact with multiple ligands, In addition, they play a wide variety of roles in normal cell function and development.

[0246] The nucleotide and amino acid sequences of human LRP1B are described, for example, in Liu et al., Cancer r Res.2000;60:1961-1967; Lie et al., Genomics 20 00;69:271-274.

[0247] Subject evaluation A subject, e.g., a patient, can receive a therapy described herein, e.g., a cancer immunotherapy, e.g., PD Patients may be evaluated for response to therapy, including inhibitors of PD-L1 or PD-L1. For example, the assessment can include determining the mutational burden and and / or mutations, such as those described herein or The patient may be, for example, a patient. This can be assessed by obtaining knowledge of the mutation load in tumor samples. Additionally, patients may be screened for genotype, e.g., by sequencing the patient's genome, e.g., by NGS methods. Alternatively or additionally, evaluation of the patient can include, for example, screening for mutant nucleic acids (e.g., DNA or RNA) for example by Southern blot, Northern blot or RT-PCR, Assays for detecting, for example, by qRT-PCR, can be used to determine the presence of mutations in patients. Alternatively or additionally, the patient may be directly assayed for the presence of For example, immunohistochemistry, Western blot, immunoprecipitation, or immunomagnetic bead assays can be used to identify the target. The patient can be evaluated for the presence of protein mutations.

[0248] In one embodiment, the results of clinical trials, e.g., successful or unsuccessful clinical trials, The results of this study could be used to identify substances to treat cancer, for example, in a subset of patient populations. One typical method is to review candidate substances used in clinical trials. The test is evaluated to determine whether the substance in the test has a particular level of mutation load or is a specific mutation. It is possible to determine whether a tumor containing the mutation is effective in treating the tumor. For example, subjects participating in clinical trials of substances such as immune checkpoint modulators Improvement in symptoms, such as cancer (e.g., melanoma) symptoms, e.g., tumor size Patients who showed a decrease in mutation burden and / or tumor growth rate Patients who do not show improvement in their cancer symptoms may also be evaluated for mutation burden and / or In one embodiment, the tumor sample may be evaluated for the presence of a mutation. A patient with a predetermined level of mutation load in were found to be more likely to respond to the test substance than patients without is an appropriate treatment option for patients with a given level of mutational burden. In another embodiment, a patient containing a mutation is determined to be such were more likely to respond to the test substance than patients who did not harbor the mutation If known, the substance may be an appropriate treatment option for patients containing the mutation. It is determined that:

[0249] A "reassessment" of a patient may involve, for example, the administration of a tumor sample, e.g., a melanoma sample, from the subject. Alternatively or additionally, re-evaluation of the patient may include obtaining knowledge of the mutation load in the patient. by determining the genome sequence of a patient or a subset of clinical trial patients, for example, by NGS. Alternatively or additionally, re-evaluation of the patient may include, for example, determining whether the patient has a mutated nucleic acid (e.g., R by an assay for detecting NA, e.g., by RT-PCR, e.g., qRT-PCR. This may involve directly assaying for the presence of the mutation in the patient. Alternatively or additionally, the patient may be subjected to immunohistochemistry, Western blot, immunoprecipitation or immunoassay. The proteins can be reassessed for the presence of mutations by immunomagnetic bead assay.

[0250] Nucleic acid and polypeptide detection methods Methods for assessing mutant genes, mutations and / or gene products are known to those of skill in the art. In one embodiment, the variation (e.g., mutation) is ion assay, selective suppression of PCR (SSP), HPLC or mass spectrometry genotyping The nucleic acid molecule is detected by one or more of the following methods:

[0251] Additional exemplary methods that may be used include traditional "direct probe" methods, such as Southern blots, and "comparative probe" methods, such as comparative genomic hybridization (CG H), such as cDNA-based or oligonucleotide-based CGH. The methods include substrate (e.g., membrane or glass) binding methods or array-based approaches (these The invention can be used in a wide variety of forms, including but not limited to:

[0252] In certain embodiments, the evaluation method includes a protocol for the mutations described herein. Includes probe / primer.

[0253] In one embodiment, the probe / primer detects a mutation or its reciprocal These probes can be designed to detect reciprocals. / Primers are suitable for example for PCR amplification. DNA salts present in various parts of the chromosome A probe is used which contains a DNA segment that is substantially complementary to the base sequence. Probes useful in the identification and characterization of probes and hybridization of probes to samples Examples of such probes are described in two U.S. patents to Vysis, Inc. and U.S. patents to Bittner et al. Described in Patent Nos. 5,491,224 and 6,277,569.

[0254] Chromosomal probes are typically about 50 to about 10 5 nucleotides in length. The lobes comprise smaller fragments, typically about 100 to about 500 nucleotides in length. Probes that hybridize to centromeric DNA and locus-specific DNA can be used, e.g., Vysis, Inc. (Downers Grove, Ill.), Molecule ar Probes, Inc. (Eugene, Oreg.) or Cytocell ( The probes are commercially available from Sigma-Aldrich (Oxfordshire, UK). They can be produced non-commercially from chromosomal or genomic DNA by conventional techniques. For example, Sources of DNA that can be used include genomic DNA, cloned DNA sequences, and DNA from a single chromosome ( an organism that contains a portion of one chromosome (e.g., a human chromosome) along with the host's normal chromosome set Cell hybrids and chromosomes purified by flow cytometry or microdissection The region of interest can be isolated by cloning or by polymerase chain reaction (PCR). They can be isolated by site-specific amplification via PCR (see, e.g., Nath and Johns on,Biotechnic Histochem.,1998,73(1):6-22 , Wheeless et al., Cytometry 1994, 17:319-326 and See US Patent No. 5,491,224.

[0255] The probes used hybridize to specific regions of the chromosome to identify cytogenetic abnormalities. Determine whether a cytogenetic abnormality exists in this region. One type of cytogenetic abnormality is a deletion. Deletions usually involve only one or more chromosomes, although deletions can involve one or more entire chromosomes. This includes loss of a portion of the chromosome. The entire region of the chromosome contained within the probe is deleted from the cell. Hybridization of the probe to DNA from the cell does not normally occur, No signal is present on the chromosome. The region of the chromosome that is partially contained within the probe is If the probe is deleted from the cell, hybridization of the probe to DNA from the cell may still occur, but the signal may be less. Probe hybridization to DNA from control cells that do not contain the genetic abnormality being detected In some embodiments, at least one , 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 12 0, 130, 140, 150, 160, 170, 180, 190, 200 or more The upper cells are counted for the presence of cytogenetic abnormalities.

[0256] Cytogenetic abnormalities detected include nonreciprocal translocations, balanced translocations, intrachromosomal inversions, and point mutations. These include deletions, gene copy number changes, changes in gene expression levels, and germline mutations. In particular, one type of cytogenetic abnormality is A duplication can be of an entire chromosome or of a region smaller than an entire chromosome. If the region of the chromosome contained within the probe is duplicated in the cell, D Hybridization of the probe to the NA usually involves the addition of a dye contained within the probe. At least one additional signal is detected compared to the number of signals present in control cells that do not contain abnormalities in the somatic region. Generates an additive signal.

[0257] Chromosomal probes are labeled so that the chromosomal regions to which they hybridize can be detected. The probe typically absorbs lower wavelength / higher energy light and then They are directly labeled with fluorophores, which are organic molecules that fluoresce when detected by a secondary detection molecule. This allows the probe to be visualized in the absence of nucleotides. After ligation, the nucleotides are subjected to nick translation, random priming, and can be incorporated directly into the probe using standard techniques such as PCR labeling. Alternatively, deoxycytidine nucleotides in the probe are transaminated with a linker. A fluorophore is then covalently attached to the transaminated deoxycytidine nucleotide. See U.S. Patent No. 5,491,224.

[0258] U.S. Patent No. 5,491,224 describes a method for producing a cytosine derivative comprising the steps of: The number of fluorescently labeled cytosine bases is determined by the number of detectable cytosine bases. While sufficient to generate a sufficient fluorescent signal, the individual DNA segments so labeled The fragments are specifically complementary to the chromosome or chromosomal region to be detected (hybridization). The unlabeled DNA probe segments are taken and the segments are then subjected to a quantification process. Some deoxycytidine nucleotides in the cleavage region are transaminated with a linking group, or by covalently attaching a fluorescent label to at least a portion of the transaminase base. Such probes are produced.

[0259] Probes can be labeled by nick translation, random primer labeling, or PCR labeling. Labeling can also be achieved by fluorescent (direct) or hapten (indirect) labeled nucleosides. Representative, non-limiting examples of labels include AMCA-6- dUTP, Cascade Blue-4-dUTP, Fluorescein-12-dUTP, Loader Min-6-dUTP, Texas Red-6-dUTP, Cy3-6-dUTP, Cy5- dUTP, biotin (BIO)-11-dUTP, digoxigenin (DIG)-11-d UTP or dinitrophenyl (DNP)-11-dUTP.

[0260] Probes can also be indirectly labeled with biotin or digoxigenin, or radiolabeled. Radioactive isotopes, e.g. 32 P and 3 It is possible to label the probe with H, but the probe is visually A secondary detection molecule or further processing is required to detect the marker. For example, biotin-labeled markers are used. The probe can be detected by avidin conjugated to a detectable marker. Vidin can also be used to bind enzyme markers, such as alkaline phosphatase or horseradish peroxidase. The enzyme marker can be bound to an enzyme using a substrate and / or catalyst for the enzyme. , which can be detected in a standard colorimetric reaction. Catalysts for alkaline phosphatase include: 5-Bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium Diaminobenzoates act as catalysts for horseradish peroxidase. It can be used.

[0261] Before or during hybridization, fluorescent or other labels may be attached to the DNA. After hybridization, the probe hybridized to the chromosome is detected. Probes can also be prepared to include fluorescent or other labels for detection. For example, a probe having an antigen molecule incorporated into the DNA can be used. After immunization, these antigen molecules are detected using specific antibodies that react with the antigen molecules. Such antibodies may themselves contain fluorescent dyes or may be conjugated to It can be detected using a second antibody bearing a fluorescent dye.

[0262] No matter how it is treated or modified, the probe DNA is Remove any unreacted residual products (e.g., fluorescent dyes not incorporated into DNA) before use in the system. It is generally purified to remove small molecules.

[0263] Prior to hybridization, the chromosomal probes are prepared according to methods well known in the art. The probe hybridizes or denatures to chromosomal DNA under hybridizing conditions. "Hybridizing conditions" refer to the conditions under which a probe and a target chromosomal DNA can be annealed. The annealing of various probes depends on the length of the probe. The probe concentration, hybridization temperature, salt concentration, etc. Annealing can be promoted by varying the concentration and other factors well known in the art. It will be promoted.

[0264] Hybridization conditions include probe concentration, base composition, complexity, and length, and This is facilitated by varying the salt concentration, temperature and length of incubation. For example, in situ hybridization is typically performed in 1-2x SSC, 5x PBS, and 5x PBS. 0–65% formamide and blocking DNA (to prevent nonspecific hybridization) The hybridization is carried out in a hybridization buffer containing a nucleotide sequence (for example, a nucleotide sequence for silencing). The hybridization conditions are a temperature of about 25°C to about 55°C and a time of about 0.5 hours. ~ Including approximately 96 hours of incubation time.

[0265] Nonspecific binding of chromosomal probes to DNA outside the target region can be removed by a series of washes. The temperature and salt concentration in each wash were varied to control the stringency of the wash. For example, under high stringency conditions, the temperature is about 65°C to about 80°C, and the temperature is 0. 2× to about 2×SSC, and about 0.1% to about 1% non-ionic surfactant, e.g., Non Cleaning can be performed using Idet P-40 (NP40). The temperature of the cleaning solution can be reduced or the salt concentration in the cleaning solution can be increased. In some applications, hybridization of repetitive sequences can be reduced. Therefore, in some embodiments, it is necessary to block the ability of non-specific To prevent hybridization, tRNA, human genomic DNA, or Cot- After washing, the slides were drained and air dried, then mounted in mounting medium. Apply a counterstain, e.g., DAPI, and a coverslip to the slide. can be observed immediately or stored at -20°C before examination.

[0266] In CGH, a first collection of nucleic acids (e.g., a sample, e.g., a potential tumor a second population of nucleic acids (e.g., from a control, e.g., healthy cell line) is labeled with a first label; The hybridization ratio of the nucleic acids in the array is then determined by the ratio of the number of hybridizations of the nucleic acids in the array. The ratio of the two (first and second) labels bound to each strand is used to determine chromosome deletion. or if multiplexing is present, the difference in the ratio of signals from those two labels is detected, The ratio is a measure of copy number. Array-based CGH can also be performed with single-color labeling (although Control and potential tumor samples were labeled with two different dyes and subjected to hybridization. Mixing them before application allows for competitive hybridization of the probes on the array. In single-color CGH, the controls are labeled and hybridized to one array. The absolute signal is read, potential tumor samples are labeled, and a second array ( The two arrays are hybridized to the same array (identical contents) and the absolute signal is read. Copy number differences are calculated based on these absolute signals.

[0267] Hybridization methods suitable for use in the methods of interest herein include, for example, , Albertson (1984)EMBO J.3:1227-1234;Pinke l(1988)Proc.Natl.Acad.Sci.USA 85:9138-91 42;EPO Publication No. 430,402;Methods in Molecular B iology,Vol.33:In situ Hybridization Prot ocols, Choo, Humana Press, Totowa, NJ (199 4) and the like. In one embodiment, Pinkel et al. (1998) N ature Genetics 20:207-211, or Kallioniemi (1992) Proc. Natl Acad Sci USA 89:5321-532 5 (1992) hybridization method is used. Array-based CGH is performed in the United States. Patent No. 6,455,258, the contents of which are incorporated herein by reference. is described in.

[0268] In yet another embodiment, an amplification-based assay is used to determine presence / absence and In such amplification-based assays, the nucleic acid sequence can be , which serves as a template in an amplification reaction (e.g., polymerase chain reaction (PCR)). In reactive amplification, the amount of amplification product is proportional to the amount of template in the original sample. Comparison with a control, for example healthy tissue, provides a measure of copy number.

[0269] Methods of "quantitative" amplification are well known to those skilled in the art. For example, quantitative PCR is a method for This involves simultaneously co-amplifying a known amount of a control sequence using a primer. This provides an internal standard that can be used to calibrate the reaction. The protocol is based on Innis et al. (1990) PCR Protocols, A Guide to Methods and Applications,Academic Pre ss, Inc. NY. Microsatellite analysis using quantitative PCR analysis Measurement of DNA copy number at the α locus was performed by Ginzonger et al. (2000) Cancer er Research 60:5405-5409. This allows those skilled in the art to routinely select primers for amplifying the portion. For this purpose, the known nucleic acid sequence of the gene is sufficient. Fluorogenic quantitative PCR may also be used. In bioquantitative PCR, quantitation is achieved by fluorescent signals, e.g., TaqMan and Sybr Based on the amount of vinegar.

[0270] Other suitable amplification methods include ligase chain reaction (LCR) (Wu and Wallace ( 1989) Genomics 4:560, Landegren et al. (1988) Scie nce 241:1077, and Barringer et al. (1990) Gene 89 :117), transcriptional amplification (Kwoh et al. (1989) Proc. Natl. A cad.Sci.USA 86:1173), self-sustained sequence replication (Guatelli et al. (1990) Proc. Nat. Acad. Sci. USA 87:1874), dot These include, but are not limited to, PCR and linker-adapter PCR. do not have.

[0271] Nucleic acid samples A variety of tissue samples can be the source of the nucleic acid sample used in the present method. Genomic or subgenomic DNA fragments are obtained from a subject's sample (e.g., tumor sample, normal adjacent tissue, In some embodiments, the antibody may be isolated from a normal tissue (NAT), a blood sample, or any normal control. In this study, tissue samples were collected as frozen samples or in formaldehyde or paraformaldehyde-based media. Preserved as a morphaldehyde-fixed, paraffin-embedded (FFPE) tissue preparation. For example, The sample may be embedded in a matrix (e.g., an FFPE block or frozen sample). The isolation process can involve flow sorting of individual chromosomes and / or the target sample ( For example, microdissection of tumor samples, NAT, blood samples) may be included. In the present study, the sample contains circulating tumor DNA (ctDNA).

[0272] Protocols for the isolation, fragmentation and processing of DNA from tissue samples are available, e.g. , WO 2012 / 092426 (title of invention "Optimization of Mu ltigene Analysis of Tumor Samples) (the entire (which is incorporated herein by reference) Formaldehyde or paraformaldehyde-fixed paraffin-embedded (FFP) E) Additional methods for isolating nucleic acids (e.g., DNA) from tissue include, for example, Cronin. M. et al. (2004) Am J Pathol.164(1):35-42;Masuda N. et al. (1999) Nucleic Acids Res.27(22):4436- 4443;Specht K. et al. (2001) Am J Pathol.158(2): 419-429;Ambion RecoverAll(TM) Total Nucle ic Acid Isolation Protocol (Ambion Catalog No. A M1975, September 2008); and QIAamp® DNA FFPE tissue Tissue Handbook (Qiagen Catalog No. 3762 5, October 2007). The Total Nucleic Acid Isolation Kit is a Hot xylene to solubilize fin-embedded samples and gas to capture nucleic acids Uses glass fiber filters. QIAamp® DNA FFPE Tissue Kit (Tissue Kit) is a Q-series kit for the purification of genomic and mitochondrial DNA. IAamp® DNA Microtechnology y) is used.

[0273] Bait design The bait is capable of hybridizing to (e.g., complementary to) the target nucleic acid. a nucleic acid molecule, e.g., a DNA or RNA molecule, that allows capture of a target nucleic acid by In one embodiment, the bait is an RNA molecule. In another embodiment, In this case, the bait is a hybrid formed by the bait and the nucleic acid hybridized to the bait. A conjugate, e.g., an af, that allows capture and separation of the lid, e.g., by binding to the conjugate. In one embodiment, the bait comprises an affinity tag. Suitable for

[0274] Bait is US 2010 / 0029498, Gnirke, A. et al. (2009) Na t Biotechnol.27(2):182-189 and WO 2012 / 092 426 (Name of invention: "Optimization of Multigene Analyses" "Decomposition of Tumor Samples" (each of which is incorporated herein by reference) The methods and hybridization conditions described in It can be manufactured and used by

[0275] Sequencing The present invention also includes methods for sequencing nucleic acids. In one embodiment, methods known in the art include Directly sequencing at least a portion of the mutation using any of a variety of sequencing reactions. In one embodiment, the mutant sequence can be compared with a corresponding reference (control) sequence. Compare with a column.

[0276] In one embodiment, the sequence of the nucleic acid molecule comprising a mutation described herein is: determined by methods including one or more of the following: oligonucleotides, e.g., those described herein; An allele-specific oligonucleotide for one of the mutations listed in the table is inserted into the nucleic acid molecule. a primer that amplifies the region of the allele containing the mutation; or hybridizing a primer set (e.g., a primer pair); amplifying, e.g., specifically amplifying, a region of the allele containing the mutation; attaching an adaptor oligonucleotide to one end of the nucleic acid containing the mutation; generating an optical signal, e.g., a colorimetric signal, specific to the presence of the mutation; The nucleic acid containing the mutation is hybridized to a second nucleic acid, e.g., a second nucleic acid bound to a substrate. generating a signal, e.g., an electrical or fluorescent signal, specific to the presence of said mutation; and an oligonucleotide that hybridizes to the nucleic acid containing the mutation. Incorporation of nucleotides into

[0277] In another embodiment, the sequence is determined by a method comprising one or more of the following: Determining the nucleotide sequence from individual nucleic acid molecules (e.g., multiple clonal expansions) For example, the signal corresponding to the sequence is determined by a single molecule, rather than by the sum of signals from the molecules. the nucleotide sequence of the clonally expanded proxy for each nucleic acid molecule; Determining; Massively Parallel Short-Read Sequencing; Template-Based Sequencing; Pyrosequencing Imaging the sequential incorporation of dye-labeled nucleotides during DNA synthesis Real-time sequencing including: nanopore sequencing; and sequencing by hybridization. determination; nanotransistor array based sequencing; polony sequencing; Sequencing based on scanning tunneling microscopes (STM); or nanowire-based molecular sensors Sequencing based on the sequence.

[0278] Any sequencing method known in the art may be used. A typical sequencing reaction includes: Maxam and Gilbert (Proc. Natl Acad Sci USA (1 977) 74:560) or Sanger (Sanger et al. (1977) Proc. This includes technology developed by the University of California, San Diego, CA (Acad. Sci. 74:5463). When performing the assay (Biotechniques (1995) 19:448), A variety of automated sequencing methods can be used, including sequencing by mass spectrometry (see, e.g., U.S. Pat. No. 6,413,199). Patent No. 5,547,835 and International Patent Application Publication No. WO 94 / 16101(HK Title of invention: DNA Sequencing by Mass Spectroscopy ectrometry); U.S. Patent No. 5,547,835 and International Patent Application Publication No. WO 94 / 21822 (H. Koster, title: DNA Sequence cing by Mass Spectrometry Via Exonucleas and U.S. Patent Application No. 5,605,798 and International Patent Application No. PCT / US96 / 03651 (by H. Koster, entitled: DNA Diagnostics Based on Mass Spectromet ry); Cohen et al. (1996) Adv Chromatogr 36:127-16 2; and Griffin et al. (1993) Appl Biochem Biotech Nol 38:147-159).

[0279] The sequencing of nucleic acid molecules is performed using next-generation sequencing (NGS). Next generation sequencing can also be performed by sequencing individual nucleic acid molecules (e.g., single molecules). Nucleotides in the clonal expansion of individual nucleic acid molecules (as in child sequencing) Arrays can be run in a highly parallel manner (e.g., 10 5 more than 100 molecules are sequenced simultaneously) In one embodiment, the nucleic acid species in the library are The relative abundances are those of their cognates in the data obtained by the sequencing experiment. Next generation sequencing methods can be used to estimate the relative number of sequences present. These methods are well known in the art and are described, for example, in Metzker, M. (2010) Nature Bio Technology Reviews 11:31-46 (which is incorporated herein by reference) (which is incorporated herein by reference).

[0280] In one embodiment, next generation sequencing involves determining the nucleotide sequence of individual nucleic acid molecules. Allows for sequence determination (e.g., HeliS from Helicos BioSciences) cope Gene Sequencing System and Pacific BioSc In another embodiment, the sequencing method is The method determines the nucleotide sequence of a clonally propagated proxy for each nucleic acid molecule (e.g., For example, Solexa sequencer, Illumina Inc., San Diego, CA alif;454 Life Sciences (Branford, Conn.) and and Ion Torrent). For example, massively parallel short-read sequencing (e.g., S OLEXA sequencer, Illumina Inc., San Diego, California f.) produces fewer and longer reads than other sequencing methods, per sequencing unit. 454 Life Sciences (Branford, Conn.), Appl. ied Biosystems(Foster City,Calif.;SOLiD System -Sequencer) and Helicos BioSciences Corporation (Cambridge, Mass.), which However, the present invention is not limited to the above.

[0281] Platforms for next-generation sequencing include Roche / 454's Geno me Sequencer(GS)FLX System, Illumina / Sole xa Genome Analyzer (GA), Life / APG Support Oligonucleotide Ligation Detection (SOLi D) System, Polonator G.007 System, Helicos BioSc iences' HeliScope Gene Sequencing System, and This includes Pacific Biosciences' PacBio RS system, NGS techniques include, but are not limited to, those described in WO 2012 / 092426 (published The name of the study is "Optimization of Multigene Analysis" of Tumor Samples,” which is incorporated herein by reference. (see below) any of the steps described in (see below), such as template preparation, sequencing and imaging, and data analysis.

[0282] Various types of mutations, such as somatic mutations and germline mutations, are described herein. In certain embodiments, the nucleic acid sequence can be detected by conventional methods (e.g., sequencing methods). Germline mutations are further identified by methods using the SGZ algorithm. The algorithm is described in International Application Publication No. WO2014 / 183078 and U.S. Patent Application Publication No. No. 2014 / 0336996 (the entire contents of which are incorporated herein by reference). (hereinafter referred to as "the Company")

[0283] Data analysis After NGS reads are generated, they are aligned against a known reference sequence. These can be assembled either de novo or as a pre-assembled product.

[0284] For example, aligning NGS reads to a reference sequence (e.g., wild-type sequence) can Genetic variations such as single nucleotide polymorphisms and structural variants in samples (e.g., tumor samples) Identification of mutations can be achieved. Methods of sequence alignment for NGS are described, for example, in Trap nell C. and Salzberg SL, Nature Biotech., 2009, 27:455-457. Examples of de novo assembly are described in Warren R. et al., Bioinformatics, 2007, 23:500- 501;Butler J. et al., Genome Res., 2008, 18:810-8 20; and Zerbino D.R. and Birney E., Genome R. es., 2008, 18:821-829. Assembly uses read data from one or more NGS platforms, e.g. , combining Roche / 454 and Illumina / Solexa read data, Algorithms and methods for data analysis can be found in WO 2012 / 09242 6 (Name of invention: “Optimization of Multigene Analysis” "Synthesis of Tumor Samples" (which is incorporated herein by reference). (hereinafter referred to as "the Company")

[0285] report The methods described herein may be administered to a patient or another person or entity, e.g., a caregiver. , e.g., a physician, e.g., an oncologist, a hospital, a clinic, a third-party payer, an insurance company, or a government agency. The report may include a report to the Output from the method, e.g., identification of values ​​for response status to therapy, values ​​for mutation load Identification, identification of the number of mutations in the genes described herein, and / or The present invention may include an indication of the presence or absence of a mutation described in the document, or of the wild-type sequence. In one embodiment, a value for response status to therapy, a value for mutation load, The number of mutations in the gene described herein or the presence of the mutations described herein The presence or absence of a mutation is identified, and, if desired, the value, number, or mutation-containing sequence is obtained. A report is generated, e.g., in paper or electronic form, including the patient identifier.

[0286] The report also reports the use of the mutations described herein in cancers, such as melanoma, Such information may include information regarding the role of the wild-type sequence. and / or recommended treatment options, e.g., agents described herein, e.g., cancer treatments. (e.g., cancer immunotherapy, e.g., PD-1 or PD-L1 inhibitors) The report may include information about the possible effectiveness of a treatment option, the tolerability of a treatment option, or the patient (e.g., For example, a predetermined value for response status to therapy, a predetermined level of mutation load, the suitability or otherwise of a treatment option for a patient with a given number of mutations in a given gene were identified in the study, and in some embodiments, in the report. For example, the report may include information about the sequence, variation, or mutation present in the patient. administration of a drug to treat a disease, e.g., a preselected dosage or a preselected treatment regimen (e.g., The information or recommendations may include information or recommendations regarding administration in combination with other drugs. In one embodiment, all mutations identified in the method are identified in the report. For example, the report does not provide any information on the occurrence, prognosis, stage, or treatment sensitivity of cancer. have a preselected level of correlation to, for example, a preselected treatment option The report may be limited to mutations in the gene responsible for the mutation. and within 7, 14, or 21 days of receipt of the sample, The document may be delivered to the relevant department.

[0287] In another aspect, the present invention provides a method for detecting a tumor in a sample, such as a tumor sample (e.g., a melanoma). obtaining a response status value for the therapy; determining the mutation load in a gene, determining the number of mutations in the gene, or Detecting the mutations described herein in a sample and providing a therapeutic response the response state value, the value for the mutation load, the number of mutations in the gene or the detected mutations Method for generating reports, e.g., personalized cancer treatment reports, by selecting treatment based on mutations - Patent Application 20070122999 In one embodiment, the value of the response status to therapy, the mutation load, Based on the value of the gene, the number of mutations in the gene, or the mutations detected, the treatment selected or lists two or more treatment options, e.g., in order of preference. In another embodiment, the subject, e.g., patient, is further provided with a report of the selected The treatment method will be implemented.

[0288] kit In one aspect, the present invention provides a method for producing a mutant nucleotide sequence similar to that described herein, e.g., A chimeric antigen receptor containing an oligonucleotide having a mutation in a gene described herein. Optionally, the kit also includes a wild-type counterpart of the mutant oligonucleotide. It may also contain oligonucleotides.

[0289] In some embodiments, the kit comprises: (a)(i) somatic mutations in a given set of genes listed in Table 1; (ii) somatic mutations in the NF1 gene; (iii) a somatic mutation in the LRP1B gene, or (iv) C to T change one or more detection reagents capable of detecting one or more of the following: (b) the mutation load and / or the presence of one or more of said mutations in the melanoma sample; Includes instructions for use in determining presence or absence.

[0290] In some embodiments, the kit further comprises an inhibitor of PD-1 or PD-L1. In some embodiments, the kit further comprises a method for detecting a tumor in a subject, the method comprising administering to the subject a tumor suppressor or a composition thereof. This document includes instructions for use in the treatment of melanoma.

[0291] The kit may comprise a carrier, e.g., a compartmentalized means for close containment of one or more container means. In one embodiment, the container contains oligonucleotides, such as The components of the kit may include, for example, a primer or probe for detecting a tumor in a patient. The probes or Primers may be used in any sequencing or nucleotide detection assay known in the art, e.g. For example, sequencing assays, such as NGS, RT-PCR or in situ hybridization. It can be used in redization.

[0292] In some embodiments, the components of the kit are useful for, e.g., diagnosing or identifying mutations. and / or assessment of mutational load in patient tumor samples and accordingly It is also useful in identifying suitable therapeutic agents for cancer treatment.

[0293] Kits of interest in the present invention may include, for example, assay positive and negative controls, nucleoside peptides, enzymes (e.g., RNA or DNA polymerases or ligases), solvents or buffers buffers, stabilizers, preservatives, secondary antibodies, e.g., anti-HRP antibodies (IgG), and detection reagents may include:

[0294] The oligonucleotides may be in any form, e.g., liquid, dry, semi-dry or lyophilized form, Alternatively, it may be provided in a form suitable for storage under frozen conditions.

[0295] Typically, the oligonucleotides and other components in the kit are provided in sterile form. Oligonucleotides, e.g., oligonucleotides containing the mutations described herein, may be used. The nucleotides, or oligonucleotides complementary to the mutations described herein, are The liquid solution is generally an aqueous solution, for example, a sterile aqueous solution. When the oxides are provided in a dried form, reconstitution (reduction) is generally achieved by the addition of a suitable solvent. The solvent, e.g., a sterile buffer, can optionally be provided in the kit.

[0296] The kit includes a composition containing oligonucleotides at concentrations suitable for use in the assay. It may contain one or more containers or may be used for dilution in an assay. In some embodiments, the kit includes an oligonucleotide and an The assay system may contain separate containers, dividers or compartments for the assay components, as well as informational material. The oligonucleotides may be contained in a bottle or vial and may include informational material. can be contained within a plastic sleeve or packet. In the kit, the separate elements are contained within a single, undivided container. The oligonucleotide composition is contained in a bottle or vial accompanied by informational material in the form of a label. In some embodiments, the kit comprises a plurality of individual containers, each of which Each may be one or more unit forms of oligonucleotides (e.g., for use in one assay). For example, the kit may contain multiple ampoules, foil packets or contains blister packs, each of which contains the sequence of mutations in a tumor sample. The kit contains a single unit of oligonucleotide to be used in the determination or detection. The container may be airtight and / or waterproof. The container may be accompanied by a label for use.

[0297] In the case of an antibody-based kit, the kit comprises: (1) a first antibody that binds to the mutant polypeptide; (2) an antibody (e.g., bound to a solid support), and optionally, a detectable Conjugated to a substance, either the polypeptide or the first antibody The antibody may comprise a second, different antibody that binds to

[0298] In one embodiment, the kit includes information for performing and interpreting sequencing or diagnostics. In another embodiment, the kit may include a reporting facility for the assay results (e.g., The kit may include instructions for administering the drug to a patient (e.g., a treatment facility or healthcare provider). the form of reporting the results of sequencing or diagnostic assays that are being used, as well as the or to send other relevant information to address and contact information, or to provide results online. UR for reporting in a database or online application (e.g., app) Uniform Resource Locator In another embodiment, the information material may include an access provides guidance on whether patients should be treated with specific chemotherapy drugs depending on the results of the It may include.

[0299] The informational material in the kit is not limited in its form. In many cases, the informational material, e.g., instructions The indications include printed matter, such as type, drawings and / or photographs, e.g. labels or printed sheets. However, informational material may also be provided in other formats, such as computer-readable material, video It may also be provided as a video or audio recording. The information material must include contact information, such as a physical address, email address, website or telephone number. These are protocol numbers in which users of the kits may use the kits for sequencing or diagnostic assays and and / or obtain substantial information regarding its use in the methods described herein. Also, the informational material may be provided in any combination of formats.

[0300] In some embodiments, the biological sample is delivered to an assay provider, e.g., a service provider. provided to a donor (e.g., a third-party facility) or healthcare provider, who may use the assay For example, in one embodiment, the assay The donor may provide a biological sample from the subject, e.g., a blood or tissue sample, e.g., a biopsy sample. The samples are received and subjected to an assay described herein (e.g., a sequencing assay or The sample is evaluated using in situ hybridization (i.e., in situ hybridization) to determine whether the sample is The assay provider, e.g., a service provider or a medical a medical provider determines that the subject is a candidate for a particular drug or a particular cancer treatment regimen, or It can be concluded that it is not a candidate.

[0301] Detection Reagents In another aspect, the present invention relates to detection reagents, e.g., purified or isolated preparations thereof. The detection reagent may be, for example, a mutation described herein (e.g., a mutation described in Table 1). somatic mutations in a defined set of genes involved in the treatment of NF1, LRP 1B gene), or a C to T change. Nucleic acid or protein sequences having the mutations described in may be identified.

[0302] The detection reagent, e.g., a nucleic acid-based detection reagent, is used to detect a target nucleic acid, e.g., a sample (e.g., Nuclei obtained or derived from melanoma, e.g., advanced or metastatic melanoma DNA, e.g., genomic DNA or cDNA or RNA in the acid sample Detection reagents, for example, antibody-based detection reagents, can be used to identify mutations in the For example, a sample (e.g., melanoma cells, e.g., advanced or metastatic melanoma cells) a sample of proteins obtained from, derived from, or produced by the cells The method can be used to identify mutations in a target protein in a mammalian cell line.

[0303] In one embodiment, the detection reagent comprises a sequence complementary to a nucleic acid sequence on the target nucleic acid. Nucleic acid molecules, including DNA, RNA, or mixed DNA / RNA molecules (binding to detection reagents) The sequence on the target nucleic acid that binds to the detection reagent is referred to herein as the "detection reagent binding site." The portion of the detection reagent that corresponds to the binding site is referred to as the "target binding site." In this case, the detection reagent binding site is located relative to the interrogation site. thereby preventing binding of the detection reagent to the detection reagent binding site (or in some embodiments The lack of binding in some cases is due to the mutants described herein (as described herein). Mutations, e.g., somatic mutations in a given set of genes listed in Table 1, NF1 gene Somatic mutations in the gene, somatic mutations in the LRP1B gene and / or C to T The detection of a nucleic acid molecule containing a mutation in a reference sequence allows for the identification of mutant and reference sequences. The reagents can be modified, for example, with a label or other moiety, eg, a moiety that allows for capture.

[0304] In one embodiment, the detection reagent is a nucleic acid molecule, e.g., DNA, RNA, or a mixture thereof. and a synthetic DNA / RNA molecule, which, for example, in its target binding site, and comparing a mutation, e.g., a mutation described herein, with a reference sequence (e.g., a target nucleic acid sequence). By affinity of the detection reagent to the acid or by reaction, e.g., by linkage with the detection reagent. In some embodiments, the interrogation position can be identified by: The terminal nucleotide of the detection reagent or target binding site, e.g., the 3' or 5' terminal nucleotide nucleotide, the nucleotide immediately adjacent to the 3' or 5' terminal nucleotide, or another internal It may correspond to a nucleotide.

[0305] In some embodiments, a target nucleic acid containing a mutation described herein and a reference The difference in the affinity of the detection reagent for the target nucleic acid containing the sequence is due to mutations (or allows the determination of the presence or absence of a (reference) sequence. Typically, the presence or absence of that sequence under assay conditions is determined by the Such detection reagents are substantially specific to the mutant or to the reference sequence. , showing a higher level of binding, e.g., to the mutation only or to the reference sequence Only with α-glucan, do they show significant levels of binding.

[0306] In some embodiments, binding allows for subsequent reactions, such as the addition of a detection reagent or target nucleic acid. For example, binding can be used to inhibit (or inhibit) subsequent reactions, such as those involving nucleic acid polymerases. The enzyme allows for the attachment or addition of one or more nucleotides to a nucleic acid, e.g., a detection reagent. This can be used to distinguish mutants from baselines. In the present invention, the interrogation site is at or sufficiently at the terminus of the detection reagent or its target binding site. to be located in close proximity to the target site, resulting in hybridization or chemical reactions, e.g., detection reagents. The addition of one or more nucleotides to the query (e.g., by a DNA polymerase) Search at position or at nucleotides within 1, 2, or 3 nucleotides of the query position This occurs only if there is a perfect match between the target nucleic acid and the target reagent, or occurs at a substantially higher rate in such cases.

[0307] In one embodiment, the detection reagent is a nucleic acid, such as DNA, RNA, or mixed DNA. A / RNA molecule, where the molecule or its target binding site is adjacent to the interrogation position ( or flanked by the target nucleic acid, which is Mutations can be distinguished from the reference sequence.

[0308] In some embodiments, the detection reagent binding site is adjacent to the interrogation site, e.g., , the 5' or 3' nucleotide of the detection reagent or its target binding site may be, for example, at the interrogation position. Position from 0 (directly adjacent) to 1,000, 500, 400, 200, 100, 50, 10, 5 In some embodiments, the reaction The results of the analysis show that the nucleotide sequence at the query position allows for the discrimination between the mutant and the reference sequence. Varies depending on the type of nucleotide. For example, the presence of the first nucleotide at the query position In the following, the first reaction is prioritized over the second reaction. For example, in a ligation or primer extension reaction, In this case, the product may be selected based on, for example, charge, sequence, size, or other reactions (e.g., restriction cleavage). In some embodiments, the detection reagents differ in their sensitivity to the interrogation site. paired molecules (e.g., In such an embodiment, The presence of a mutation is determined by comparing the sequence containing the query position and the sequence containing the reference nucleotide at the query position. The properties of the amplification products resulting from the corresponding sequences, such as size, sequence, charge, or reaction In some embodiments, the difference in sensitivity can be determined by the difference in the amplification product. The presence or absence of a nucleotide indicates the type of nucleotide at the query site, and thus Allows for the detection of mutations.

[0309] In some embodiments, the detection reagent or its target binding portion is directly adjacent to the interrogation position. For example, the 5' or 3' terminal nucleotide of the detection reagent is immediately adjacent to the interrogation position. In some embodiments, the nucleotide type at the query position is The nature of the reaction, eg, a reaction that includes a detection reagent, eg, modification of one end of the detection reagent, is determined. For example, in the presence of a first nucleotide at the query position, the first reaction is more likely to be positive than the second reaction. Priority is given to the first nucleotide at the query position, e.g., the first nucleotide associated with the mutation. The presence of a complementary nucleotide triggers a first reaction, e.g., the addition of a complementary nucleotide to the detection reagent. For example, the presence of A at the interrogation position may facilitate, for example, the identification of a T having a first colorimetric label. whereas the presence of a G at the interrogation position causes the incorporation of a second colorimetric label, e.g. In one embodiment, the first nucleic acid at the position The presence of the nucleotide results in the linkage of the detection reagent to the second nucleic acid. If the third nucleic acid has an exact match at the interrogation site, the third nucleic acid is a target nucleic acid sufficiently close to the interrogation site. The ligation product may be hybridized to a nucleic acid, which may be ligated to a detection reagent. detection of its absence indicates the type of nucleotide at the query site, and thus Allows for the detection of mutations.

[0310] A variety of readouts can be used, for example, binding of a detection reagent to the mutant or reference sequence. This is followed by binding of a moiety associated with the detection reagent, such as a label, e.g., a radioactive or enzyme label. In some embodiments, the label comprises a quencher and a signaling agent, Hybridization results in a change in the distance between those two elements, e.g. In some embodiments, the distance increases, dequenching the signaling agent. The detection reagent may contain a moiety that allows for separation from other components of the reaction mixture. In embodiments, binding is achieved by, for example, an enzyme, such as a DNA polymerase, nuclease. The cleavage allows for cleavage of the bound detection reagent by enzyme activity or by a restriction enzyme. The appearance or disappearance of nucleic acids or the interaction of signaling and quenching agents associated with the detection reagents In some embodiments, binding can be detected by further chemical reactions, e.g., For example, to label or protect the target against degradation (e.g., by a restriction enzyme) or to In some embodiments, binding to a detection reagent sensitizes the target. allowing capture, separation or physical manipulation of target nucleic acids, thereby enabling their identification In some embodiments, binding results in detectable localization of the detection reagent or target. For example, binding can result in capture of the target nucleic acid or displacement of a third nucleic acid. Binding may result in an expansion or other size change in the component, e.g., the detection reagent. This allows for differentiation of mutants from the reference sequence. It may be possible to distinguish from the sequence, for example by generating an amplification product by PCR.

[0311] In one embodiment, the detection reagent or target binding site is 5 to 500, 5 to 300 , 5~250, 5~200, 5~150, 5~100, 5~50, 5~25, 5~20, In one embodiment, the detection reagent is 5 to 15, or 5 to 10 nucleotides in length. Or the target binding sites are 10-500, 10-300, 10-250, 10-200, 1 0-150, 10-100, 10-50, 10-25, 10-20, or 10-15 In one embodiment, the detection reagent or target binding site is 20 nucleotides in length. ~500, 20~300, 20~250, 20~200, 20~150, 20~100, In one embodiment, the detection the reagent or target binding site is long enough to distinguish between the mutant and the reference sequence; It is less than 100, 200, 300, 400 or 500 nucleotides in length.

[0312] The muteins described herein can be used to bind to reagents, e.g., substrates, e.g., catalytic activity. Substrates for specific or functional activity, or antibodies (mutants and differentially expressed from the reference protein) (which reacts with a reference, e.g., a non-mutant or wild-type protein) In one aspect, the present invention provides a method for generating a mutant having a predetermined level of mutation load. and / or by subjecting a sample containing a mutant protein described herein to such a reagent. and determining whether the mutant protein is present in the sample. .

[0313] In one embodiment of the reaction mixture or method for producing a reaction mixture, the detection reagent is Probes or primers specific for nucleic acids containing the mutations described herein, or or an antibody specific for a mutein described herein. In one embodiment of the method for preparing the reaction mixture, the mutation is a gene that is present in, for example, a gene listed in Table 1, e.g., the NF1 gene or LRP1B It is a somatic mutation in a gene.

[0314] Nucleic acid preparations and uses thereof In another aspect, the present invention provides a method for determining the mutation load of a sample or the disclosed Query positions described herein useful for determining the presence or absence of a mutation Neoplastic or tumor cell nucleic acid, e.g., DNA, e.g., genomic DNA or cDNA, containing The present invention relates to a purified or isolated preparation of nucleic acid or RNA, the nucleic acid comprising an interrogation site, and Typically, the nucleic acid contains additional sequences on either or both sides of the query position. Typically, a heterologous sequence, such as an adapter or priming sequence, is attached to one or both sides of the nucleic acid. The nucleic acid may also contain a label or other moiety, e.g., a sequence for isolation or localization. Includes the enabling part.

[0315] In certain embodiments, nucleic acids, such as nucleic acids from melanoma samples, are purified or The isolated preparation is (i) somatic mutations in a given set of genes listed in Table 1; (ii) somatic mutations in the NF1 gene; (iii) somatic mutations in the LRP1B gene; (iv) C to T change Contains one or more of the following.

[0316] In some embodiments, the preparation comprises a cytoplasmic sample, such as a melanoma sample. In some embodiments, the preparation is used to determine the mutation load. Located within a sequence determination device or within a sample holder used in such a device .

[0317] In some embodiments, the nucleic acid is 20 to 1,000, 30 to 900, 40 to 80 0, 50-700, 60-600, 70-500, 80-400, 90-300, or 100-200 nucleotides in length (with or without heterologous sequences). In the above, the nucleic acid is 40 to 1,000, 50 to 900, 60 to 800, or 70 to 700 , 80-600, 90-500, 100-400, 110-300, or 120-20 0 nucleotides in length (with or without heterologous sequences). The nucleic acid is 50 to 1,000, 50 to 900, 50 to 800, 50 to 700, 50 to 600, 50-500, 50-400, 50-300, or 50-200 nucleotides In some embodiments, the nucleic acid is a sequence Sequence determination (e.g., by chemical sequencing or T between mutant and reference preparations) m), but long enough to allow for less than 100, 200, 300, 400, or 500 nucleotides in length (without heterologous sequences) Such preparations may be prepared from samples, e.g., neoplasm or tumor samples. In one embodiment, the purified nucleic acid can be used to sequence the nucleic acid from the purified The preparation is provided by in situ amplification of nucleic acids provided on a substrate. In embodiments, the purified preparation is spatially separated from other nucleic acids, e.g., other amplified nucleic acids, on the substrate. It is separate.

[0318] In one embodiment, the purified or isolated preparation of nucleic acid is a melanoma, e.g., Derived from melanoma. Such preparations are useful for determining the mutation load in melanoma, and and / or the presence or absence of a mutant sequence in the melanoma, such as a mutation described herein. It can be used to determine the presence or absence of a

[0319] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (a) a nucleic acid, e.g., DNA, containing a query position described herein, e.g., a gel preparing purified or isolated preparations of genomic DNA or cDNA, or RNA; (b) a detection reagent or a target sequence, e.g., by chemical bonding, e.g., covalent or non-covalent bonding; The nucleic acid is sequenced by a method that breaks or forms chemical bonds to identify the nucleic acid at the interrogation position. The present invention includes determining the type of nucleotide (i.e., what the nucleotide is). The present invention also relates to a method for determining the sequence of a query position for a mutation described in the specification. , allowing one to determine whether the mutations described herein are present.

[0320] In one embodiment, sequencing is performed on nucleic acids containing a mutation described herein. , including contacting with a detection reagent described herein.

[0321] In one embodiment, sequencing is performed to determine the physical identity of a mutant that can be distinguished from a reference sequence. properties, e.g., the stability of the double-stranded form of a nucleic acid containing a mutation described herein, e.g., T m This includes determining:

[0322] Reaction mixture and apparatus In one aspect, the present invention provides a method for detecting the mutation load of a sample or the mutations disclosed. , including the query positions described herein, useful for determining the presence or absence of and detecting a nucleic acid, such as DNA, e.g., genomic DNA or cDNA, or RNA, In one embodiment, the nucleic acid is a melanoma nucleic acid. , for example obtained or derived from an advanced melanoma.

[0323] In another aspect, the present invention provides a method for determining the mutation load of a sample or the disclosed Query positions described herein useful for determining the presence or absence of a mutation and nucleic acids, such as DNA, e.g., genomic DNA or cDNA, or RNA, containing a reaction mixture containing a detection reagent, which detects the physical or chemical properties of the duplex, e.g., Qualitative, e.g., T m for use in or on an apparatus for determining In one embodiment, the device is In one embodiment, the nucleic acid containing the mutations described herein is a obtained or derived from a melanoma, such as an advanced melanoma.

[0324] In certain embodiments, the reaction mixture described herein comprises: (a)(i) somatic mutations in a given set of genes listed in Table 1; (ii) somatic mutations in the NF1 gene; (iii) somatic mutations in the LRP1B gene; (iv) C to T change one or more detection reagents capable of detecting one or more of the following: (b)(i) somatic mutations in a given set of genes listed in Table 1; (ii) somatic mutations in the NF1 gene; (iii) somatic mutations in the LRP1B gene; (iv) C to T changes (e.g., one or more C to T changes) The present invention also includes nucleic acid derived from a melanoma sample comprising one or more of:

[0325] The detection reagents described herein can be used to detect the mutation load of a sample or the mutations in a sample. can be used to determine the presence or absence of the disclosed mutations. In an embodiment, the sample contains nucleic acid derived from melanoma, e.g., advanced melanoma. The cells may be derived from a neoplasm or tumor sample, e.g., a sample obtained from a neoplasm or tumor. biopsy; circulating tumor cells, e.g., from peripheral blood; or from blood or plasma samples In one embodiment, the nucleic acid is a nucleic acid encoding a gene encoding a melanoma, e.g., an aggressive It comes from melanoma.

[0326] Thus, in one aspect, the present invention provides a method for detecting the mutations described herein. The detection reagents that can be used include sequences with query positions for the mutations described herein. The present invention relates to a method for preparing a reaction mixture, which comprises combining nucleic acids from a sample containing the nucleic acid. In some embodiments, the method comprises: (i) detecting a gene in a predetermined set of genes listed in Table 1; (ii) somatic mutations in the NF1 gene; (iii) somatic mutations in the LRP1B gene (iv) one or more C to T changes in the offspring. The detection reagents were selected from the group consisting of (i) somatic mutations in a given set of genes listed in Table 1; i) somatic mutations in the NF1 gene, (iii) somatic mutations in the LRP1B gene or (iv) tumor cells or samples containing one or more C to T alterations, e.g., melanoma ... This involves combining the nucleic acid from the normal cell or sample.

[0327] In one embodiment of the reaction mixture or method of making the reaction mixture, the detection reagent is , a nucleic acid, e.g., DNA, RNA, which is complementary to a nucleic acid sequence (detection reagent binding site) on the target nucleic acid A or a mixed DNA / RNA molecule, wherein the detection reagent binding site is associated with the interrogation position. The detection reagent binding site is positioned adjacent to the detection reagent binding site, such that the binding of the detection reagent to the detection reagent binding site is Allows identification of mutant and reference sequences for a described mutation sequence or event To do so.

[0328] In one embodiment of the reaction mixture or method for producing the reaction mixture, a target nucleic acid sequence is The columns are derived from melanoma, e.g., advanced melanoma (as described herein). In one embodiment of the reaction mixture or method for producing the reaction mixture, is a nucleotide sequence as described herein, including substitutions, e.g., base substitutions as described herein. It is a mutation.

[0329] The variations described herein react with enzymes that react differentially with the mutation and the reference. can be distinguished from a reference, e.g., a non-mutant or wild-type sequence, by They were identified by cleavage with restriction enzymes that have different activities on the mutant and reference sequences. For example, the present invention provides nucleic acids containing the mutations described herein as such. The mutant is contacted with an enzyme, and the presence of a cleavage product that distinguishes the mutant from the reference sequence is determined. This includes a method for determining

[0330] In one embodiment, the present invention provides a restriction enzyme cleavage sequence that can distinguish between a mutant and a reference sequence. and providing a purified preparation of the product, wherein one end of the cleavage product is a mutant and a reference sequence. In one embodiment, the cleavage products are determined by enzymes that differentially cleave contains the query location.

[0331] Detection of mutant polypeptides Mutant polypeptides (e.g., polypeptides encoded by the genes listed in Table 1) Activity of the peptide (e.g., mutant NF1 polypeptide or LRP1B polypeptide) or levels may also be detected and / or quantified by detecting or quantifying expressed polypeptides. The mutant polypeptide can be detected by any of a number of means known to those of skill in the art. These include analytical biochemical methods such as electrophoresis, capillary Electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC) ), superdiffusion chromatography, etc., or various immunological methods, e.g., fluid or gel chromatography. immunoprecipitation reaction, immunodiffusion (one-dimensional or two-dimensional), immunoelectrophoresis, radioimmunoassay ( RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, These methods may include immunoblotting, immunohistochemistry (IHC), etc. Those skilled in the art will recognize that known protein Protein / antibody detection methods can be applied.

[0332] Another agent for detecting mutant polypeptides is the polypeptide corresponding to the polypeptide. and an antibody molecule capable of binding to a polypeptide (e.g., an antibody bearing a detectable label). Techniques for producing "labels" for probes or antibodies are described herein. The term "coupling" refers to coupling a detectable substance to a probe or antibody (i.e. Direct labeling of probes or antibodies by direct labeling (e.g., physically linking) and direct labeling It is intended to include indirect labeling of a probe or antibody by reactivity with another reagent that is Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody, and fluorescently labeled Includes end-labeling of DNA probes with biotin, which can be detected with streptavidin .

[0333] In another embodiment, the antibody is labeled, e.g., radiolabeled, chromophore labeled, fluorophore labeled. In another embodiment, the mutant protein is labeled with a label or an enzyme label. Antibody derivatives that specifically bind to proteins (e.g., antibodies bound to a substrate, or protein- Protein or ligand of a ligand pair (e.g., biotin-streptavidin) antibody fragments (e.g., single chain antibodies, isolated antibody hypervariable regions, etc.) etc.) are used.

[0334] The mutant polypeptide from the cells can be isolated using techniques known to those skilled in the art. Possible protein isolation methods include those described, for example, by Harlow and Lane (Harlow and Bi Lane, 1988, Antibodies: A Laboratory Manua l,Cold Spring Harbor Laboratory Press,Co (Illegible Spring Harbor, New York) Possible.

[0335] Means for detecting proteins using electrophoretic techniques are well known to those skilled in the art (see, generally See R. Scopes (1982) Protein Purification, Sp Ringer-Verlag, NY;Deutscher(1990)Method s in Enzymology Vol.182:Guide to Protein Purification, Academic Press, Inc., NY I want to be illuminated.

[0336] In another embodiment, Western blot (immunoblot) analysis is used to , to detect and quantify the presence of the polypeptide in the sample.

[0337] In another embodiment, an immunoassay is used to detect the polypeptide. As used herein, an immunoassay refers to an assay that uses an antibody to specifically bind to an analyte. Thus, an immunoassay is a method for detecting the specific binding of a polypeptide to an antibody. and other physical or chemical methods for isolating, targeting, and quantifying analytes. This is in contrast to the use of chemical properties.

[0338] The mutant polypeptides can be detected and purified using any of a number of immunological binding assays. and / or quantified (e.g., U.S. Pat. Nos. 4,366,241, 4,376,110 (See US Pat. Nos. 4,517,288 and 4,837,168). For a review of immunoassays, see Asai (1993) Methods in Cell Biology Volume 37: Antibodies in Cell Bi ology, Academic Press, Inc. New York; and Sti tes & Terr(1991)Basic and Clinical Immun. See also: ology 7th Edition.

[0339] Screening Method In another aspect, the present invention provides a method for detecting and / or treating a strain of a mammalian cell with a predetermined level of mutation load. or substances that can be used to treat tumors harboring the mutations described herein. The present invention relates to a method or assay for screening for a compound.

[0340] The method includes: A cell or tissue, such as a tumor cell or tissue, having a mutation is contacted with a candidate substance, and the cell or tissue is then subjected to changes in parameters associated with cells or tissue, e.g., tumor cells or tissue, e.g., tumors The method includes detecting changes in tumor growth, angiogenesis, apoptosis, or metastasis. optionally comparing the value of the parameter to a reference value, e.g., determining the presence or absence of the candidate substance. The parameter values ​​obtained from the tumor samples in the absence of the candidate substance were compared with those obtained from the tumor samples in the absence of the candidate substance. In one embodiment, the tumor may be compared to a parameter value obtained from Tumor-related parameters, such as tumor growth, angiogenesis, apoptosis, or metastasis If an alteration is detected, the candidate substance is identified.

[0341] Typical parameters that are assessed include (i) changes in the activity of cells, e.g., tumor cells; (ii) changes in cell proliferation, morphology, or tumorigenicity; (iii) changes in the tumor, e.g., tumor size, appearance, growth; or (iv) changes in the cells, e.g., For example, the level of a nucleic acid or polypeptide or protein associated with tumor cell activity, e.g. These include one or more of the following: a change in expression level;

[0342] In one embodiment, the contacting step is performed by contacting the cells in the culture with a predetermined level of mutation, e.g., in cells with a different load and / or with a mutation described herein In another embodiment, the contacting step is performed in vivo in a cell (such as a tumor present in a subject). cells), e.g., in an animal subject (e.g., an in vivo animal model).

[0343] In other embodiments, the change in cellular activity is determined by measuring the activity of cells in culture, e.g., at a predetermined level. Cells with a low mutational load and / or harboring the mutations described herein (e.g., mammalian cells, tumor cells or cell lines, recombinant cells). In one embodiment, the cells are isolated from a tumor having a predetermined level of mutational load. In one embodiment, the cell comprises a mutation described herein. Recombinant cells modified to express nucleic acids, e.g., those containing the mutations described herein, A transfected cell is a recombinant cell that has been transfected with a nucleic acid containing the Changes in response to the mutations introduced, such as increased proliferation, changes in morphology, increased tumorigenicity, and and / or the acquisition of a transformed phenotype. A change in any of the cellular activities can be detected, for example, an increase in the activity of the cells in the presence of a candidate substance. A reduction in one or more of proliferation, tumorigenicity, and transformation phenotypes of a given level of mutation load is and / or the utility of said agents in treating tumors harboring the mutations described herein. It may be effective.

[0344] In yet other embodiments, tumors present in an animal subject (e.g., an in vivo animal model) are In one embodiment, the animal model is a tumor-bearing animal, Alternatively, the mutations may be present in a population having a predetermined level of mutation load and / or a mutation described herein. Cells with mutations (e.g., having a predetermined level of mutational load and / or The candidate substance is administered to the animal pair. In one embodiment, the method can be administered to an elephant to detect changes in the tumor. The changes in the tumor include one or more of tumor growth, tumor size, tumor burden, and survival. A reduction in one or more of tumor growth, tumor size, tumor burden, or an increase in survival rate is evaluated. having a predetermined level of mutation load and / or having the mutations described herein The candidate substance is shown to be effective in treating tumors.

[0345] In certain embodiments, the screening methods described herein are repeated, and / or combinations thereof. In one embodiment, the Candidate agents evaluated in cell-based assays can be further tested in animal subjects.

[0346] In one embodiment, the candidate substance is a modulator of an immune checkpoint molecule. , for example, an inhibitor, for example, an inhibitor of PD-1 or PD-L1. In embodiments, candidate substances include small molecule compounds, nucleic acids (e.g., antisense, siRNA, RNA, aptamer, ribozyme, microRNA), or antibody molecule (e.g., mutation Intact antibodies or antigen-binding fragments thereof that bind to proteins encoded by the DNA The candidate substance is a compound selected from the group consisting of a chimeric, humanized, or human antibody molecule. available (e.g., from a commercial library of inhibitors) or rationally designed It can be done.

[0347] Candidate substances can be identified using combinatorial library methods known in the art, including: These can be obtained using any of a number of approaches: biological libraries; pepto- Ide library (novel non-peptide peptides that are resistant to enzymatic degradation but still bioactive) Libraries of molecules with peptide functionality, including those with a peptide backbone (e.g., Zucker See mann, RN et al. (1994) J. Med. Chem. 37:2678-85 spatially addressable parallel solid-phase or liquid-phase libraries; deconvolution synthetic library methods requiring fusion; "one bead one compound" library methods; and Synthetic library method using affinity chromatography selection. Biological libraries. and peptoid library approaches are limited to peptide libraries, while the remaining 4 One approach is to develop compound libraries of peptides, non-peptide oligomers, or small molecules. Applicable (Lam (1997) Anticancer Drug Des. 12: 145).

[0348] Phage display and combinatorial methods for generating antibodies are known in the art. It is known (e.g., U.S. Pat. No. 5,223,409, International Application Publication No. WO 92 / 092666). 18619, WO 91 / 17271, WO 92 / 20791, WO 92 / 1567 9, WO 93 / 01288, WO 92 / 01047, WO 92 / 09690, WO 90 / 02809; Huse et al. (1989) Science 246:1275-12 81;Fuchs et al. (1991) Bio / Technology 9:1370-137 2; Clackson et al. (1991) Nature 352:624-628; Garr ad et al. (1991)Bio / Technology 9:1373-1377;Hoog Enboom et al. (1991) Nuc Acid Res 19:4133-4137;B Arbas et al. (1991) PNAS 88:7978-7982; Hawkins et al. (1 992) J Mol Biol 226:889-896; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85;Gram et al. (1992) P NAS 89:3576-3580; and Griffths et al. (1993) EMBO J 12:725-734).

[0349] Chimeric antibodies can be produced by recombinant DNA techniques known in the art (International Publication No. Nos. WO 86 / 01533, WO 87 / 02671, Cabilly et al., U.S. Pat. No. 4,816,567; Better et al. (1988 Science 240:1041 -1043);Liu et al. (1987) PNAS 84:3439-3443;Liu et al. 1987, J. Immunol. 139:3521-3526; Sun et al. (1987) P NAS 84:214-218; Nishimura et al., 1987, Canc. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-44 9; and Shaw et al., 1988, J. Natl Cancer Inst. 80:15 53-1559).

[0350] Humanized or CDR-grafted antibodies are CDR-grafted or CDR-substituted (in this case, immunoglobulin The CDRs of the amino acid sequence of the ribonucleotide chain may be replaced (one, two or all of the CDRs of the amino acid sequence of the ribonucleotide chain may be replaced). For example, U.S. Patent No. 5,225,539; Jones et al., 1986 Nature 321:5 52-525;Verhoeyan et al., 1988 Science 239:1534; See Beidler et al., 1988 J. Immunol. 141:4053-4060. Humanized antibodies in which specific amino acids are substituted, deleted, or added are also included in the present invention. The criteria for selecting amino acids from the donor are described in US Pat. 9. Other techniques for humanizing antibodies are described in U.S. Patent No. 5,693,761. No. 5,693,762, European Patent No. EP519596, Morrison, S. L., 1985, Science 229:1202-1207, and Oi et al., 198 6, BioTechniques 4:214.

[0351] Human monoclonal antibodies are antibodies derived from mice that contain human immunoglobulin genes rather than murine systems. These can be produced using transgenic mice immunized with an antigen of interest. Using splenocytes from transgenic mice, epitopes derived from human proteins were identified. to produce hybridomas secreting human mAbs with specific affinity for (See, for example, International Application Publication Nos. WO 91 / 00906, WO 91 / 10741, WO 92 / 03918, WO 92 / 03917; Bruggeman et al., 1991 Eur J Immunol 21:1323-1326;Bruggeman et al., 1993 Year Immunol 7:33-40;Tuaillon et al., 1993 PNAS 90:3720-3724; Lonberg, N. et al., 1994 Nature 36 8:856-859; Green, LL et al., 1994 Nature Genet. 7:13-21; and Morrison, SL et al., 1994 Proc. Natl Acad. Sci. USA 81:6851-6855).

[0352] Examples of methods for the synthesis of molecular libraries are available in the art, for example, in DeWi tt et al. (1993) Proc. Natl. Acad. Sci. USA 90:690 9; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11 422;Zuckermann et al. (1994). J. Med. Chem. 37:2678 ;Cho et al. (1993) Science 261:1303;Carrell et al. (199 4)Angew.Chem.Int.Ed.Engl.33:2059;Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; and (1994) J. Med. Chem. 37:1233.

[0353] Libraries of compounds can be prepared in solution (e.g., Houghten (1992) Biotech hniques 13:412-421) or on beads (Lam (1991) Nature re 354:82-84), on chip (Fodor (1993) Nature 364 :555-556), on bacteria (Ladner, U.S. Pat. No. 5,223,409), on spores on (Ladner, U.S. Pat. No. 5,223,409), on plasmids (Cull et al. (1991) 992)Proc Natl Acad Sci USA 89:1865-1869) or on phages (Scott and Smith (1990) Science 249: 386-390;Devlin(1990)Science 249:404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378- 6382; and Felici (1991) J. Mol. Biol. 222:301- 310; Ladner, supra).

[0354] Other embodiments of the present invention include the following.

[0355] The present invention also provides isolated nucleic acid molecules or single strands of nucleic acid molecules containing the mutations described herein. Such nucleic acid molecules or preparations thereof are described herein. can contain mutations or can be used to detect sequence mutations, for example .

[0356] The present invention also relates to a method for the treatment of rhesus mast cell carcinoma (HMS) comprising or adjacent to the mutations described herein, and hybridizes to the mutation, or is useful for identifying the mutation, or is based on the mutation Nucleic acid molecules suitable as probes, primers, baits or library members based on In certain embodiments, the probe, primer or vector The nucleotide molecule may contain a mutation described herein, e.g., a gene listed in Table 1 (e.g., capture of nucleic acid molecules containing mutations in the NF1 gene or the LRP1B gene; It is an oligonucleotide that allows for detection or isolation.

[0357] The oligonucleotides may be used to identify nucleic acid molecules or nucleic acid fragments containing the mutations described herein. The nucleic acid molecule, e.g., the oligonucleotide, may comprise a nucleotide sequence substantially complementary to a fragment of the nucleic acid molecule. Sequence identity between the nucleotides and the target sequence determines whether those sequences are suitable for capturing, detecting, or otherwise targeting the target sequence. The sequence need not be precise, as long as it is sufficiently complementary to allow isolation. In one embodiment, the nucleic acid fragments are about 5 to 25, for example 10 to 20, or 10 to 1 The probe or primer comprises an oligonucleotide of 5 nucleotides in length. In embodiments, the nucleic acid fragment is about 100 to 300 nucleotides, 130 to 230 nucleotides, or The bait contains a nucleotide or an oligonucleotide of 150-200 nucleotides in length. .

[0358] In one embodiment, the nucleic acid fragment contains, for example, a mutation described herein, e.g., For example, a gene listed in Table 1 (e.g., the NF1 gene or the LRP1B gene) to identify or capture, e.g., by hybridization, nucleic acid molecules containing mutations in For example, nucleic acid fragments can be used to express the mutations described herein, e.g., in hybridization. The probe, primer, or It could be bait.

[0359] The probes or primers described herein can be used, for example, in PCR amplification. In one exemplary embodiment where detection is PCR-based, the mutation Amplification can be performed using a platform, for example, to amplify sequences flanking the mutations described herein. This can be done using a dimer or a primer pair.

[0360] In other embodiments, the nucleic acid fragment comprises a mutation described herein. A nucleic acid molecule that hybridizes to the nucleic acid molecule, thereby enabling capture or isolation of the nucleic acid molecule. In one embodiment, the bait comprises a nucleotide sequence. In another embodiment, the bait is suitable for hybridization. The capture and separation of hybrids formed by the nucleic acid and the bait can be performed by, for example, binding to a binder. Binding-enabling conjugates include, for example, affinity tags.

[0361] In other embodiments, the nucleic acid fragments are libraries comprising the nucleic acid molecules described herein. In one embodiment, the library members include mutagenized Mutations include, for example, base substitutions that result in the mutations described herein.

[0362] The nucleic acid fragments may be labeled, for example, by radioactive labels, fluorescent labels, bioluminescent labels, chemiluminescent labels, enzyme labels, or the like. It can be detectably labeled with a label, a binding pair label, or an affinity tag. , tags, or identifiers (e.g., adapters, barcodes, or other sequence identifiers). do.

[0363] In another aspect, the invention provides a polypeptide comprising a mutation described herein. polypeptides (e.g., purified polypeptides containing the mutations described herein), their biological activity, and antibody or antigenic fragments, as well as reagents (e.g., polynucleotides containing the mutations described herein). the activity of polypeptides containing the mutations described herein, Methods for modulating sexuality and the use of polynucleotides containing the mutations described herein. Regarding the detection of peptides.

[0364] In another embodiment, the polypeptide or fragment is a polypeptide as described herein. Such peptides or proteins contain mutations that are present in the target gene. Such immunogenic peptides or proteins include polypeptides containing the mutations described herein. The peptide or protein may be used to generate antibodies specific for the peptide or protein. In the present invention, such immunogenic peptides or proteins are used in the production of vaccines. The vaccine formulation may contain other components, such as an adjuvant.

[0365] In another aspect, the invention provides a polypeptide comprising a mutation described herein. The present invention relates to an antibody molecule or fragment thereof that binds to a tide. In this embodiment, the antibody comprises a mutant polypeptide, such as one described herein. The wild-type polypeptide can be distinguished from polypeptides containing mutations that Techniques for this are known in the art and are described, for example, in WO 2012 / 092426 (title of the invention). Title: “Optimization of Multigene Analysis of "Tumor Samples" (which is incorporated herein by reference) ) is described in

[0366] The invention may be defined, for example, in any of the following numbered paragraphs:

[0367] 1. (a) for a subject to a therapy comprising an inhibitor of PD-1 or PD-L1 a response status value is obtained, wherein the response status value is obtained by measuring the melanoma sample or melanoma samples from the subject. Include a measure of tumor mutation burden (TMB) in tumor-derived samples, (b) an increase in the value of the response state, e.g., in response to an increase in the value of the response state compared to a baseline value of the response state; and administering a therapy to the subject, thereby treating the subject, if necessary. Targeted treatment methods.

[0368] 2. Administering a therapy containing an inhibitor of PD-1 or PD-L1 to the subject, thereby 1. A method of treating a subject with melanoma, comprising treating the subject with: The subject exhibits an increase in the value of the response state, e.g., as compared to the baseline value of the response state; and has been confirmed to exhibit this increase, The response status value is determined by the following: The method includes a measure of tumor mutation burden.

[0369] 3. Obtaining a response status value to a therapy for a subject, thereby selecting a therapy. and therapies comprising inhibitors of PD-1 or PD-L1 for subjects with melanoma, including A choice of law method comprising: The response status value is determined by the following: Includes a measure of tumor mutation burden, An increase in the value of the response state, e.g., when compared to a baseline value of the response state, indicates that the subject , is or is likely to be a responder to the therapy, or the subject responds to the therapy or A method that shows that this is highly likely.

[0370] 4. (a) for a subject to a therapy comprising an inhibitor of PD-1 or PD-L1 a response status value is obtained, wherein the response status value is obtained by measuring the melanoma sample or melanoma samples from the subject. a measure of tumor mutation burden in tumor-derived samples, (b) determining whether a subject is a responder (e.g., a complete or partial responder) or non-responder to a therapy; 1. A method for assessing a subject having melanoma, comprising identifying a subject as a responder, the method comprising: A response status value equal to or greater than the reference response status value indicates that the subject is responding to the therapy. or that the subject is or is likely to be an answer to the therapy. Show that, or A response status value less than the reference response status value indicates that the subject is a non-responder to the therapy or indicates that the subject is likely to be unresponsive to the therapy, or that the subject is likely to be unresponsive to the therapy, method.

[0371] 5. The tumor mutation burden measure is one of the following in a sample from the subject: (i) somatic mutations in a given set of genes listed in Table 1 (e.g., one or more levels of somatic mutations in (ii) the presence of a somatic mutation (e.g., one or more somatic mutations) in the NF1 gene; (iii) the number of somatic mutations (e.g., one or more somatic mutations) in the LRP1B gene; ,or (iv) C to T changes in a given set of genes listed in Table 1 (e.g., , the number of C to T changes of 1 or more The method according to any one of claims 1 to 4, comprising one, two, three or all of the determination results. Law.

[0372] 6. The following in a sample from the subject: (i) somatic mutations in a given set of genes listed in Table 1 (e.g., one or more The pre-defined set of genes listed in Table 1 compared to the baseline level of somatic mutations in the an increased level of somatic mutations (e.g., one or more somatic mutations) in the patient; (ii) the presence of a somatic mutation (e.g., one or more somatic mutations) in the NF1 gene; (iii) a source of somatic mutations (e.g., one or more somatic mutations) in the LRP1B gene; somatic mutations in the LRP1B gene (e.g., one or more somatic mutations) compared with the normal population an increase in the number of mutations, or (iv) the method of any one of claims 1-5, wherein therapy is administered to or selected for the subject in response to one, two, three, or all of an increase in the number of C to T transitions (e.g., one or more C to T transitions) in a given set of genes listed in Table 1 compared to a reference number of C to T transitions (e.g., one or more C to T transitions) in a given set of genes listed in Table 1.

[0373] 7. (a) In a melanoma sample or melanoma-derived sample from a subject, The following, namely: (i) somatic mutations in a given set of genes listed in Table 1 (e.g., 1 or more somatic mutations), (ii) the presence of a somatic mutation (e.g., one or more somatic mutations) in the NF1 gene Presence or non-existence, (iii) a somatic mutation in the LRP1B gene (e.g., one or more somatic mutations) ) or (iv) C to T changes in a given set of genes listed in Table 1 ( For example, the number of C to T changes of 1 or more Determine one or more of (b) The following: (i) somatic mutations in a given set of genes listed in Table 1 (e.g., increased levels of one or more somatic mutations in a given set of genes listed in Table 1, the level of somatic mutations (e.g., one or more somatic mutations) in a patient compared to a baseline level Increase in (ii) the presence of a somatic mutation (e.g., one or more somatic mutations) in the NF1 gene Currently, (iii) a somatic mutation in the LRP1B gene (e.g., one or more somatic mutations) ), e.g., an increase in the number of somatic mutations in the LRP1B gene (e.g., one or more somatic an increase in the number of mutations compared to the baseline number, or (iv) C to T changes in a given set of genes listed in Table 1 ( an increase in the number of mutations (e.g., one or more C to T changes), e.g., genes listed in Table 1 The reference number of C to T changes (e.g., 1 or more C to T changes) in a given set of Its increase compared to and administering to the subject a therapy comprising an inhibitor of PD-1 or PD-L1 in response to one or more of the following: and thereby treating the subject.

[0374] 8. Claim 1, wherein the reference value of response status is a value of response status for a non-responder to the therapy. The method described in any one of items 1 to 7.

[0375] 9. The tumor mutation burden in a sample from a subject compared to a baseline level In response to an increasing level of tumor mutation burden, a therapy is administered to the subject or selected for the subject. The method according to any one of claims 1 to 8, wherein the method comprises selecting

[0376] 10. Melanoma samples from non-responders to therapy with baseline levels of tumor mutation burden 10. The method of claim 9, wherein the level of tumor mutation burden in a sample derived from a human or melanoma is How to post.

[0377] 11. The melanoma sample from the subject is: (i) Relative to baseline levels of somatic mutations in a given set of genes listed in Table 1 The level of somatic mutations in a given set of genes listed in Table 1 compared increase, (ii) the presence of a somatic mutation in the NF1 gene; (iii) LR compared with baseline levels of somatic mutations in the LRP1B gene an increased number of somatic mutations in the P1B gene, or (iv) the baseline level of C to T transitions in a given set of genes listed in Table 1 C to T in a given set of genes listed in Table 1 compared to the Increasing number of changes A subject is identified as a responder to therapy if they exhibit one, two, three, or all of the following: The method according to any one of claims 1 to 10.

[0378] 12. The sample from the subject is: (i) Relative to baseline levels of somatic mutations in a given set of genes listed in Table 1 somatic mutations in a given set of genes listed in Table 1 that are reduced or unchanged compared to Different levels, (ii) the absence of somatic mutations in the NF1 gene; (iii) Similar, consistent, and comparable levels of somatic mutations in the LRP1B gene compared to baseline levels. the same or reduced number of somatic mutations in the LRP1B gene, or (iv) the baseline level of C to T transitions in a given set of genes listed in Table 1 A given set of genes listed in Table 1 that are similar, the same, or decreased compared to the baseline Number of C to T changes in A subject is identified as a non-responder to therapy if they exhibit one, two, three, or all of the following: The method according to any one of claims 1 to 11,

[0379] 13. The predetermined set of genes is at least about 50 or more of the genes listed in Table 1. , about 100 or more, about 150 or more, about 200 or more, about 250 or more, about 300 or more 13. The method according to any one of claims 1 to 12, comprising:

[0380] 14. Baseline levels of somatic mutations in a given set of genes listed in Table 1 are: in melanoma samples or melanoma-derived samples from non-responders to therapy , the level of somatic mutations in a predetermined set of genes listed in Table 1. 14. The method according to any one of items 9 to 13.

[0381] 15. Baseline levels of somatic mutations in the LRP1B gene indicate non-responders to therapy. In these melanoma samples or melanoma-derived samples, The method according to any one of claims 6 to 14, wherein the level of somatic mutation in the gene is measured.

[0382] 16. Baseline levels of C to T transitions in a given set of genes listed in Table 1 The assay was performed on melanoma samples or melanoma-derived samples from non-responders to therapy. The method according to any one of claims 6 to 15, wherein the level of C to T change is

[0383] 17. The level of somatic mutations in a given set of genes listed in Table 1 Sequencing a predetermined set of genes listed in Table 1, e.g., a method for determining the coding region of a predetermined set of genes that are The method according to any one of claims 5 to 16.

[0384] 18. The presence or absence of somatic mutations in the NF1 gene can be determined by sequencing the NF1 gene. For example, by sequencing the coding region of the NF1 gene. The method according to any one of claims 5 to 17, wherein the method is determined as follows:

[0385] 19. The number of somatic mutations in the LRP1B gene was determined by sequencing the LRP1B gene. for example, by a method comprising sequencing the coding region of the LRP1B gene. The method according to any one of claims 5 to 18, wherein

[0386] 20. The number of C to T transitions in a given set of genes listed in Table 1 is 1. The method of claim 1, wherein the sequence of the predetermined set of genes is determined by a method comprising: The method according to any one of claims 5 to 19.

[0387] 21. According to the tumor mutation load measure, the following: (a) administering a modified dose of a therapy to a subject; (b) altering the schedule or time course of therapy for a subject; (c) administering an additional agent in combination with the therapy, e.g., to non-responders or partial responders; And, or (d) prognosticating the time course of melanoma progression in a subject 21. The method according to claim 1, further comprising performing one, two, three or all of the following: How to post.

[0388] 22. Determining the level of somatic mutations in a given set of genes listed in Table 1 , about 25 or more, for example, about 50 or more, about 100 or more of the genes listed in Table 1; Approximately 150 or more, approximately 200 or more, approximately 250 or more, approximately 300 or more, or all of the above 22. The method of any one of claims 5 to 21, comprising determining the level of somatic mutation.

[0389] 23. Determining the level of somatic mutations in a given set of genes listed in Table 1 , per preselected unit (e.g., in the coding region of a given set of genes, e.g., (e.g., per megabase in the coding region of a given set of sequenced genes) 23. The method of claim 5, further comprising determining the number of mutations.

[0390] 24. Relative to baseline levels of somatic mutations in a given set of genes listed in Table 1 The level of somatic mutations in a given set of genes listed in Table 1 compared An increase, e.g., at least about 2-fold, at least about 3-fold, at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about In response to a 40-fold or at least about 50-fold increase, administering therapy to the subject or administering therapy to the subject The method according to any one of claims 1 to 23, wherein the method is selected for the purpose of

[0391] 25. The number of somatic mutations in a given set of genes listed in Table 1 is More than about 3.3 somatic mutations per megabase in a given set of coding regions, e.g., about 5 more than 10 pieces, about 15 pieces or more, about 20 pieces or more, about 25 pieces or more, about 30 pieces or more, about Depending on the determination that the number is 35 or more, approximately 40 or more, approximately 45 or more, or approximately 50 or more 25. The method of claim 1, wherein the therapy is administered to or selected for the subject. The method described.

[0392] 26. The number of somatic mutations in a given set of genes listed in Table 1 is More than about 23.1 somatic mutations per megabase in a given set of coding regions, e.g., about 25 or more, approximately 30 or more, approximately 35 or more, approximately 40 or more, approximately 45 or more, or approximately 50 responsive to the determination that the subject is at least partially or completely responsive to the determination, administering or selecting a therapy for the subject; The method according to any one of claims 1 to 25.

[0393] 27. The number of somatic mutations in a given set of genes listed in Table 1 is Approximately 3.3 to 23.1 somatic mutations per megabase in a given set of coding regions, e.g. For example, about 5 to about 20, about 10 to about 20, or about 15 to about 20. 27. The method of claim 1, wherein the therapy is administered to or selected for the subject according to the method. 1. The method described in paragraph 1.

[0394] 28. Relative to baseline levels of somatic mutations in a given set of genes listed in Table 1 The level of somatic mutations in a given set of genes listed in Table 1 compared An increase, e.g., at least about 2-fold, at least about 3-fold, at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about A 40-fold or at least about a 50-fold increase indicates that the subject is a responder to the therapy. 28. The method according to any one of claims 1 to 27.

[0395] 29. Relative to baseline levels of somatic mutations in a given set of genes listed in Table 1 Similar, the same, or decreased compared to a given set of genes listed in Table 1 Claims 1-28, wherein the level of somatic mutations indicates that the subject is a non-responder to the therapy. The method according to any one of the preceding claims.

[0396] 30. The number of somatic mutations in a given set of genes listed in Table 1 is More than about 3.3 somatic mutations per megabase in a given set of coding regions, e.g., about 5 more than 10 pieces, about 15 pieces or more, about 20 pieces or more, about 25 pieces or more, about 30 pieces or more, about A determination that the number of items is 35 or more, approximately 40 or more, approximately 45 or more, or approximately 50 or more is indicates a responder, e.g., a complete or partial responder, to the therapy. The method described in any one of items 1 to 29.

[0397] 31. The number of somatic mutations in a given set of genes listed in Table 1 is There are less than approximately 3.3 somatic mutations per megabase in a given set of coding regions. 31. Any one of claims 1 to 30, wherein the determination indicates that the subject is a non-responder to the therapy. The method described.

[0398] 32. The number of somatic mutations in a given set of genes listed in Table 1 is More than about 23.1 somatic mutations per megabase in a given set of coding regions, e.g., about 25 or more, approximately 30 or more, approximately 35 or more, approximately 40 or more, approximately 45 or more, or approximately 50 A determination that the subject is a responder, e.g., a complete responder, to the therapy. The method according to any one of claims 1 to 31.

[0399] 33. The number of somatic mutations in a given set of genes listed in Table 1 is Approximately 2 somatic mutations per megabase in the coding region of a given set of listed genes 3. A determination of less than one indicates that the subject is a partial responder or non-responder to therapy. The method according to any one of claims 1 to 32, wherein

[0400] 34. The number of somatic mutations in a given set of genes listed in Table 1 is Approximately 3 somatic mutations per megabase in the coding region of a given set of listed genes 0.2 (e.g., about 3.3) to about 20, e.g., about 5 to about 20, about 10 to about 20, or A determination that the number of serotonins is about 15 to about 20 indicates that the subject is a partial responder to the therapy. The method according to any one of claims 1 to 33.

[0401] 35. In response to a determination that a somatic mutation is present in the coding region of the NF1 gene, therapy should be The method according to any one of claims 1 to 34, which is administered to or selected for a subject. Law.

[0402] 36. The presence of a somatic mutation in the coding region of the NF1 gene may contribute to a subject's response to therapy. that a subject is or is likely to be an answer to a therapy, or that a subject is or is likely to respond to a therapy The method according to any one of claims 1 to 35, wherein the method indicates a high

[0403] 37.(a) A somatic mutation is present in the coding region of the NF1 gene, and (b) The level of somatic mutations in a given set of genes listed in Table 1 is Somatic mutations per megabase in the coding region of a given set of genes listed in Approximately 35 or more, approximately 38.5 or more, approximately 40 or more, approximately 45 or more, or approximately 50 or more R In response to the determination, a therapy is administered to or selected for the subject. 36. The method of any one of claims 36 to 38.

[0404] 38.(a) The presence of a somatic mutation in the coding region of the NF1 gene; and (b) Somatic mutations in the BRAF gene and NRAS gene , or mela that is triple WT (wild type) for BRAF, NRAS, and NF1 genes Location of genes listed in Table 1 in melanoma samples or melanoma-derived samples The levels of somatic mutations in the genes listed in Table 1 compared with the levels in a fixed set An increase in the level of somatic mutations in a given set, e.g., at least about 2-fold, at least about a 3-fold, at least about 5-fold, or at least about 10-fold increase and administering or selecting a therapy for the subject in response to the determination of claim 1-37. The method according to any one of the preceding claims.

[0405] 39. Approximately 1 or more, approximately 2 or more, approximately 3 or more, or approximately 4 or more in the LRP1B gene In response to determining the presence of at least about five somatic mutations, administering a therapy to the subject or administering a therapy to the subject The method of any one of claims 1 to 38, wherein the method is selected for:

[0406] 40. Approximately 1 or more, approximately 2 or more, approximately 3 or more, or approximately 4 or more in the LRP1B gene The presence of about 5 or more somatic mutations indicates that the subject is or is likely to be a responder to therapy. or indicates that the subject is likely to respond to the therapy or is likely to respond to the therapy. 9. The method of any one of claims 9 to 10.

[0407] 41. The number of somatic mutations in LRP1B compared to the reference number in the LRP1B gene an increase in the number of somatic mutations in the at least about 2.8 times, at least about 3 times, at least about 3.5 times, at least about 4 times, or In response to a determination of at least about a five-fold increase, a therapy is administered to the subject or selected for the subject. The method according to any one of claims 1 to 40, wherein

[0408] 42. The reference number of somatic mutations in the LRP1B gene is 100%. The LRP1B gene expression profile in melanoma and melanoma-derived samples 42. The method of claim 41, wherein the number of mutations is the number of mutations.

[0409] 43. The baseline number of somatic mutations in the LRP1B gene is 0 or 1 somatic mutation. 43. The method of claim 41 or 42,

[0410] 44. About 20 or more, about 25 or more of a given set of genes listed in Table 1 , upon determining the presence of about 30 or more, about 40 or more, or about 50 or more C to T changes. 44. The method of claim 1, wherein the therapy is administered to or selected for the subject based on the results of the method. 1. The method described in paragraph 1.

[0411] 45. About 20 or more, about 25 or more of a given set of genes listed in Table 1 , determining the presence of about 30 or more, about 40 or more, or about 50 or more C to T changes; The subject is a responder to a therapy comprising an inhibitor of PD-1 or PD-L1. The method according to any one of claims 1 to 44, wherein

[0412] 46. ​​The standard number of C to T transitions in a given set of genes listed in Table 1 and of C to T transitions in a given set of genes listed in Table 1 when compared an increase in number, e.g., at least about 8-fold, at least about 10-fold, at least about 12-fold, In response to determining an increase of at least about 15-fold or at least about 20-fold, administering therapy to the subject. 46. ​​The method of any one of claims 1 to 45, wherein the method is selected for or on behalf of a subject.

[0413] 47. The reference number of C to T changes was determined for melanoma samples from non-responders to therapy or or the number of C to T transitions in a melanoma-derived sample. Law.

[0414] 48. Claim 46 or claim 47, wherein the reference number of C to T transitions is about 2 C to T transitions. is the method described in 47.

[0415] 49. The subject is currently receiving a therapeutic agent or treatment other than an inhibitor of PD-1 or PD-L1. Any of claims 1 to 48, wherein the patient is undergoing or has undergone a different therapy, including a method or the method described in paragraph 1.

[0416] 50. The following, namely: (i) Relative to baseline levels of somatic mutations in a given set of genes listed in Table 1 The level of somatic mutations in a given set of genes listed in Table 1 compared increase, (ii) the presence of a somatic mutation in the NF1 gene; (iii) LR compared with baseline levels of somatic mutations in the LRP1B gene an increased number of somatic mutations in the P1B gene, or (iv) the baseline level of C to T transitions in a given set of genes listed in Table 1 C to T in a given set of genes listed in Table 1 compared to the Increasing number of changes and discontinuing the different therapies in response to one, two, three, or all of the determinations of claim 4. 9. The method described in 9.

[0417] 51. The method of claim 49 or 50, wherein the therapy is administered after discontinuation of the different therapy. Law.

[0418] 52. The method of claim 49 or 50, wherein the therapy is administered in combination with a different therapy. Law.

[0419] 53. The different therapies include chemotherapy, radiotherapy, immunotherapy, radioimmunotherapy, oncolysis, 49 to 50. The method according to claim 49, wherein the therapeutic agent is selected from a therapeutic virus therapy, a surgical procedure, or any combination thereof. 52. The method of any one of claims 52.

[0420] 54. The different therapies are dacarbazine, temozolomide, (temozolomide), interleukin 2 (IL-2), interferon, Ipilimumab, BRAF inhibitor, MEK inhibitor, Talimogene laherparepvec c), adoptive cell transfer, or any combination thereof. 1. The method according to any one of the preceding items.

[0421] 55. If interferon is recombinant interferon alfa-2b or peginterferon 55. The method of claim 54, wherein the antibody is steroid alpha 2b.

[0422] 56. BRAF inhibitors are vemurafenib or dabu 55. The method of claim 54, wherein the compound is dabrafenib.

[0423] 57. MEK inhibitors are cobimetinib or trametinib 55. The method of claim 54, wherein the compound is trametinib.

[0424] 58. The method of claim 54, wherein the adoptive cell transfer comprises modified T cells or modified dendritic cells.

[0425] 59. Any one of claims 1 to 58, wherein the PD-1 inhibitor is an anti-PD-1 antibody. The method described in section.

[0426] 60. PD-1 inhibitors include nivolumab (ONO-45 38, BMS-936558 or MDX1106), pembrolizumab (pembro lizumab) (MK-3475 or lambrolizumab b), pidilizumab (CT-011), MEDI0680 (AMP-514), PDR001, REGN2810, BGB-108, BGB-A3 17, SHR-1210 (HR-301210, SHR1210 or SHR-1210 PF-06801591 or AMP-224. Law.

[0427] 61. Any of claims 1 to 58, wherein the PD-L1 inhibitor is an anti-PD-L1 antibody. or the method described in paragraph 1.

[0428] 62. PD-L1 inhibitors include atezolizumab ( MPDL3280A, RG7446 or RO5541267), YW243.55.S 70, MDX-1105, durvalumab (MEDI4736 ) or avelumab (MSB0010718C), 62. The method of claim 61.

[0429] 63. Somatic mutations, including silent mutations (e.g., synonymous mutations), are associated with cancer phenotypes. somatic mutations not identified, passenger mutations (e.g., clonal adaptation) mutations that have no detectable effect on disease severity), variants of unknown significance (VUS) (e.g., mutations that mutations for which the pathogenicity cannot be confirmed or excluded), point mutations, coding shorts (c short) variants (e.g., base substitutions or indels), nonsynonymous single base variations 63. The method according to any one of claims 1 to 62, comprising one or more of: single nucleotide variants (SNVs); and splice variants. How to do it.

[0430] 64. Mutations (e.g., somatic mutations) can be followed by rearrangements (e.g., , translocation, functional mutation, or germline mutation. The method of any one of claims 1 to 63, wherein

[0431] 65. Any of claims 1 to 64, wherein the somatic mutation is a silent mutation, e.g., a synonymous mutation. 1. The method according to any one of the preceding items.

[0432] 66. Claims 1-64, in which somatic mutations have not been identified as being associated with a cancer phenotype. The method according to any one of the preceding claims.

[0433] 67. Somatic mutations can be detected as passenger mutations, e.g., to improve clonal fitness. The method according to any one of claims 1 to 64, wherein the mutation has no adverse effect.

[0434] 68. Somatic mutations are variants of unknown significance (VUS), e.g., where no pathogenicity can be identified. 65. The method of any one of claims 1 to 64, wherein the mutation is not excludable.

[0435] 69. The method of any one of claims 1 to 64, wherein the somatic mutation is a point mutation.

[0436] 70. Any of claims 1 to 64, wherein the somatic mutation is other than a rearrangement, e.g., other than a translocation. The method described in any one of claims 1 to 10.

[0437] 71. If the somatic mutation is a coding short variant, e.g., a base substitution or indel, The method according to any one of claims 1 to 64.

[0438] 72. Any of claims 1 to 64, wherein the somatic mutation is a nonsynonymous single nucleotide variant (SNV). 1. The method described in paragraph 1.

[0439] 73. The method of any one of claims 1 to 64, wherein the somatic mutation is a splice variant. Law.

[0440] 74. The method of any one of claims 1 to 64, wherein the somatic mutation is not a functional mutation.

[0441] 75. The method of any one of claims 1 to 64, wherein the mutation is not a germline mutation.

[0442] 76. Melanoma, including at least one processor operatively connected to a memory 1. A system for evaluating a subject having: Then, (a) Response to a therapy comprising an inhibitor of PD-1 or PD-L1 for a subject a state value is obtained, wherein the response state value is obtained by measuring the amount of melanoma sample or melanoma from the subject. a measure of tumor mutation burden in the tumor-derived sample, (b) determining whether a subject is a responder (e.g., a complete or partial responder) or non-responder to a therapy; It is designed to identify the answer, A response status value equal to or greater than the reference response status value indicates that the subject is responding to the therapy. or that the subject is or is likely to be an answer to the therapy. Show that, or A response status value less than the reference response status value indicates that the subject is a non-responder to the therapy or indicates that the subject is likely to be unresponsive to the therapy, or that the subject is likely to be unresponsive to the therapy, A system that evaluates the subject.

[0443] 77. The measure of tumor mutation burden is: , (i) somatic mutations in a given set of genes listed in Table 1 (e.g., one or more levels of somatic mutations in (ii) the presence of a somatic mutation (e.g., one or more somatic mutations) in the NF1 gene; (iii) the number of somatic mutations (e.g., one or more somatic mutations) in the LRP1B gene; ,or (iv) the number of C to T changes in a given set of genes listed in Table 1 77. The system of claim 76, comprising one, two, three or all of the determination results of:

[0444] 78. The following in a sample from the subject: (i) Relative to baseline levels of somatic mutations in a given set of genes listed in Table 1 The level of somatic mutations in a given set of genes listed in Table 1 compared increase, (ii) the presence of a somatic mutation in the NF1 gene; (iii) LR compared with baseline levels of somatic mutations in the LRP1B gene an increased number of somatic mutations in the P1B gene, or (iv) the baseline level of C to T transitions in a given set of genes listed in Table 1 C to T in a given set of genes listed in Table 1 compared to the Increasing number of changes Therapy is administered to or selected for the subject depending on one, two, three, or all of the following: 78. The system of claim 76 or 77, wherein

[0445] 79.(a)(i) Somatic mutations in a given set of genes listed in Table 1 (e.g., (e.g., one or more somatic mutations), (ii) a somatic mutation in the NF1 gene (e.g., one or more somatic mutations); (iii) a somatic mutation in the LRP1B gene (e.g., one or more somatic mutations); , (iv) C to T changes in a given set of genes listed in Table 1 (e.g., (e.g., one or more C to T changes) one or more detection reagents capable of detecting one or more of the following: (b) Tumor mutation burden in melanoma samples or melanoma-derived samples instructions for use in determining and / or treating melanoma in a subject, and (c) optionally, an inhibitor of PD-1 or PD-L1 or composition Kit including:

[0446] 80.(i) Somatic mutations in a given set of genes listed in Table 1 (e.g., one or more somatic mutations), (ii) a somatic mutation in the NF1 gene (e.g., one or more somatic mutations); (iii) a somatic mutation in the LRP1B gene (e.g., one or more somatic mutations); (iv) C to T changes in a given set of genes listed in Table 1 (e.g., , 1 or more C to T changes) A nucleic acid extract from a melanoma sample or a melanoma-derived sample, comprising one or more of: a prepared or isolated preparation comprising: The preparation may be used in a sequencing instrument or in a sample holder for use in such an instrument. A preparation used to determine the tumor mutation load of the sample placed in the preparation.

[0447] 81.(a)(i) Somatic mutations in a given set of genes listed in Table 1 (e.g., (e.g., one or more somatic mutations), (ii) a somatic mutation in the NF1 gene (e.g., one or more somatic mutations); (iii) a somatic mutation in the LRP1B gene (e.g., one or more somatic mutations); ,or (iv) C to T changes in a given set of genes listed in Table 1 (e.g., (e.g., one or more C to T changes) one or more detection reagents capable of detecting one or more of the following: (b)(i) somatic mutations in a given set of genes listed in Table 1 (e.g., one or more somatic mutations), (ii) a somatic mutation in the NF1 gene (e.g., one or more somatic mutations); (iii) a somatic mutation in the LRP1B gene (e.g., one or more somatic mutations); ,or (iv) C to T changes in a given set of genes listed in Table 1 (e.g., (e.g., one or more C to T changes) and nucleic acid derived from a melanoma sample or melanoma-derived sample containing one or more of: A reaction mixture comprising: The reaction mixture may be placed in a sequencing instrument or in a sample holder used in such an instrument. A reaction mixture containing the sample is used to determine the tumor mutation load of the sample placed in the reaction mixture. mixture.

[0448] 82.(i) Somatic mutations in a given set of genes listed in Table 1 (e.g., one or more somatic mutations), (ii) a somatic mutation in the NF1 gene (e.g., one or more somatic mutations); (iii) a somatic mutation in the LRP1B gene (e.g., one or more somatic mutations), or or (iv) C to T changes in a given set of genes listed in Table 1 (e.g., , 1 or more C to T changes) one or more detection reagents capable of detecting one or more of the following: (i) somatic mutations in a given set of genes listed in Table 1 (e.g., one or more somatic mutations), (ii) a somatic mutation in the NF1 gene (e.g., one or more somatic mutations); (iii) a somatic mutation in the LRP1B gene (e.g., one or more somatic mutations), or or (iv) C to T changes in a given set of genes listed in Table 1 (e.g., , 1 or more C to T changes) together with nucleic acids from a melanoma sample or melanoma-derived sample containing one or more of: and thereby obtaining a reaction mixture. [Brief explanation of the drawings]

[0449] [Figure 1A] FIG. 1A shows the mutation burden in responders compared with non-responders in the initial cohort. [Figure 1B] Figure 1B shows the mutation load in responders compared with non-responders in the validation cohort. [Figure 1C] Figure 1C shows progression-free survival in patients with high, intermediate, and low mutation burden. [Figure 1D] Figure 1D shows overall survival in patients with high, intermediate, and low mutation burden. [Figure 2A] Figure 2A shows the mutation load profile across a range of potential thresholds. The vertical lines indicate thresholds selected based on local best performance and clinical relevance. [Figure 2B] Figure 2B shows the receiver operating characteristic curves (ROC) for mutation load cutoffs of 3.3 mutations / MB (low mutation load group) and 23.1 mutations / MB (high mutation load group). [Figure 3A] Figure 3A shows the mutation burden in skin / unknown primary site tumors in responders compared to non-responders. [Figure 3B] Figure 3B shows the mutation burden in tumors of non-cutaneous primary sites (acral, mucosal, uveal) in responders compared with non-responders. [Figure 3C] FIG. 3C shows gene amplification and deletion in responders compared to non-responders. [Figure 4A] Figure 4A shows the total number of mutations observed in responders compared to non-responders. [Figure 4B] FIG. 4B shows the total number of C to T transitions observed in responders compared to non-responders. [Figure 4C] FIG. 4C shows the types of nucleotide variations observed in responders compared to non-responders. [Figure 4D] Figure 4D shows the mutation burden of patients with BRAF mutation, NRAS mutation, NF1 mutation / loss, and "triple WT" (defined as wild type for BRAF, NRAS, and NF1). BRAF non-V600 mutations were included in the BRAF cohort, except for one patient with a coexisting NF1 mutation. One patient with an NRASQ61 R mutation and a coexisting NF1 mutation was included in the NRAS cohort. [Figure 5A]Figure 5A shows that mutation load in TCGA cutaneous melanoma (SKCM) samples using the 315 genes included in the hybrid capture NGS panel is highly correlated with mutations assessed by whole-exome sequencing. [Figure 5B] Figure 5B shows mutation burden groups and survival rates in TCGA using whole exome sequencing (WES). [Figure 5C] Figure 5C shows the mutation load groups and survival rates in TCGA using the 315 (FM) genes tested. [Figure 6] Figure 6 shows the calculated mutational load per sample (top), a color-coded matrix of individual mutations, copy number variations and clinical characteristics (middle), and the mutational spectrum of individual samples (bottom). [Figure 7A] FIG. 7A shows LRP1B mutations / variants of unknown significance in responders compared to non-responders. [Figure 7B] Figure 7B shows the total number of mutations across melanomas in the presence and absence of LRP1B mutations. [Figure 7C] Figure 7C shows the association between the number of LRP1B mutations and total mutations in the melanoma TCGA. [Figure 8A] FIG. 8A shows T cell receptor (TCR) clonality in responders compared to non-responders. [Figure 8B] FIG. 8B shows the T cell fractions in responders compared to non-responders. [Figure 8C] Figure 8C shows TCR clonality in responders compared to non-responders in "ideal" samples, defined as those obtained within 4 months of anti-PD-1 / anti-PD-L1 treatment with no other prior therapy. [Figure 8D] Figure 8D shows the T cell fraction in these "ideal" samples, defined as those obtained within 4 months of anti-PD-1 / anti-PD-L1 treatment with no other prior therapy. [Figure 9A] Figure 9A shows the correlation between mutation load and T cell receptor (TCR) clonality. [Figure 9B] FIG. 9B shows the correlation between mutation load and T cell fraction.

[0450] Example Example 1: Targeted Next-Generation Sequencing Identifies Markers of Response to PD-1 Blockade R overview Therapeutic antibodies that block programmed death-1 and its ligands (PD-1 / PD-L1) induces durable responses in a significant proportion of melanoma patients. Mutations identified using a lid capture-based next-generation sequencing (NGS) panel Does the number and / or type of heterozygotes correlate with response to anti-PD-1 in melanoma? I sought to determine whether this was the case.

[0451] Using archived melanoma samples from anti-PD-1 / PD-L1 treated patients Hybrid capture-based NGS was performed on 236–315 genes, and two T cell receptor (TCR) sequences were compared for the initial and validation cohorts from multiple centers. The row decision was made.

[0452] Patients who responded to anti-PD-1 or anti-PD-L1 compared with non-responders in the initial cohort (median 45.6 vs. 3.9 mutations / MB; P = 0.003) and the validation cohort (3 higher mutation burden in the BRCA1 / BRCA2 subtype (7.1 vs. 12.8 mutations / MB; P = 0.002). The response rate, progression-free survival (PFS) and overall survival (OS) were high. The mutation burden group was superior compared to the intermediate and low mutation burden groups. Melanomas with mutations had a high mutation burden (median 62.7 mutations / MB) and Response rate (74%) was lower in BRAF / NRAS / NF1 wild-type melanoma In these archived samples, TCR clonality predicted response. The mutation counts of 315 genes in the NGS platform were Detected by whole-exome sequencing in Cancer Genome Atlas samples was strongly associated with the number of patients with HIV infection but was not associated with survival.

[0453] Therefore, the abruption determined by the hybrid capture-based NGS platform Mutational burden effectively stratified patients by likelihood of response. This approach was and / or may provide a clinically feasible predictor of response to anti-PD-L1 therapy. do.

[0454] method patient Protected health information under HIPAA (Health Insurance Portability and Accountability Act) The information was reviewed in accordance with the guidelines of the IQI and Accountability Act. Patients and samples were retrospectively selected based on an RB-approved protocol. Patients with metastatic melanoma who are receiving anti-PD-1 therapy (nivolumab) as part of a clinical trial or as standard of care or pembrolizumab) or anti-PD-L1 (atezolizumab) treatment. Patients will have a measurable response as determined by radiography or will undergo further imaging showed a rapid clinical course that hindered follow-up. The patients were randomly assigned to receive the CT scan at 8-12 week intervals. A review of medical records and tumor images determined baseline characteristics, treatment Treatment response, progression-free survival (PFS) and overall survival (OS) were obtained. T 1.1(Eisenhauer EA et al. Eur J Cancer 2009;4 5:228-47; n=30) or at least a classical partial or complete response. If both groups showed an atypical immune-related response (n=2) that persisted for 12 months, patients were classified as responders. If the patient did not respond, the patient was classified as a non-responder. .

[0455] Most formalin-fixed, paraffin-embedded (FFPE) specimens are for research purposes only. These samples were analyzed to determine whether they demonstrated an evaluable response to treatment. All patients with usable residual FFPE were included. Anti-PD-1 / PD-L1 antibodies tested in 100 patients (n=25) to identify possible mutations Most samples were from patients treated with 12 months or more before treatment initiation. The remaining samples were obtained 12 months or more before treatment initiation (n=15) or later. were obtained immediately after treatment initiation (n=7). All pretreatment samples with available tissue were subjected to ImmunoSeq strictly for research purposes.

[0456] Next-generation sequencing (NGS), such as targeted NGS, and The Cancer Genome Atlas (T CGA) analysis Extensively validated Clinical Laboratory Improvement Methods Hybrid capture-based N GS Platform (FoundationOne (registered trademark), Foundation n Medicine, Cambridge MA) (Frampton GM et al., Na t Biotechnol 31:1023-31,2013) to determine the DNA sequence. The initial cohort (n=32) was analyzed using exons and sequences from 236 cancer-related genes. and 19 genes were sequenced by a method that evaluated the introns. The samples (n=33) were analyzed using exons from 315 genes and introns from 28 genes. The cohort was sequenced by a subsequent study to evaluate the phenotype (hereafter referred to as the "validation cohort"). Methods for DNA extraction and sequencing have already been extensively validated and published (Fram pton et al., Nat Biotechnol 31:1023-31, 2013).

[0457] To calculate the total mutation load, the mutation load detected in the test including the genes listed in Table 1 Quantify the number of somatic mutations detected and add the value to the exon using the following algorithm: All short mutations and base substitutions detected in this study were extrapolated to the entire genome. Mutations and indels were also counted, but non-coding mutations were not. Present as a known somatic mutation in the IC database; cancer.sang er.ac.uk / cosmic) and potential (transgenic in tumor suppressor genes Mutations with a functional state of (inactivated) were not counted. This correction contributes to an upward shift in mutation load. This was done to avoid upward skewing because preferentially profile genes known to be recurrently mutated in cancer. The dbSNP database (www.ncbi.nlm.nih.g ov / SNP), ExAC database (those with a count of 2 or more) (exac. broadinstitute.org) and SGZ (somatic germline ne zygosity) algorithm (see, for example, International Application Publication No. WO2014 / 18 3078 and U.S. Patent Application Publication No. 2014 / 0336996 (the entire contents of which are incorporated herein by reference). (which is incorporated herein by reference) Germline variants were excluded and filtered. The SGZ algorithm was used to filter out germline variants. To further reduce the likelihood of calling a cohort of over 60,000 clinical specimens, The mutation load per megabase (MB) was calculated using The total number of mutations counted was divided into 236 and 315 gene forms, respectively. The coding region of the test covers 0.91 and 1.25 megabases. I did the multiplication.

[0458] 345 cutaneous melanoma tumor samples from TCGA (including 263 with clinical data) Matched somatic mutation and clinical data from the sample were analyzed using the cancer genome database. Cancer Genome Data Server-R (CGDS- Using the R API, CbioPortal (www.cbioportal.org / public-portal) (This is the function for extracting data from CGDS. The sequences sequenced in this study were searched using TCGA. The number of nonsynonymous mutations in 315 genes was determined by whole exome sequencing (WES). The mutations were compared to all mutations identified by the analysis of all coding genes (n=20,022). Data was also evaluated for these samples.

[0459] T cell receptor sequencing TCR sequencing and clonality quantification, as well as measurement of T cell fractions, were performed as previously described. As reported in Adaptive Biotechnologies (Tumeh et al., ature 515:568-71,2014;Gerlinger et al., J Patho l, 2013), using ImmunoSeq™ to measure pre-treatment FFPE tumors. T cell clonality was calculated as follows: Entropy was calculated for the clonal abundance of all productive TCR sequences in the dataset. Shannon entropy was calculated by dividing the logarithm of the number of unique productive TCR sequences. This normalized entropy value was then inverted to obtain (1-normalized entropy) (P) clonality metrics were obtained.

[0460] statistical analysis Compare the mutation load between responders and non-responders using the Mann-Whitney U test. The mutation load characteristics over a range of values ​​were calculated and ROC curves were used to evaluate clinical Across the range of significant values, thresholds for low, medium, and high groups were selected from the local maxima. To identify optimal mutation cutoffs from the initial cohort for evaluation in the study ROC was used to calculate the response rate among patients with specific genomic alterations. 2 Compare using a test T cell clonality and T cell fractions were determined using the Mann method and were not corrected for multiple comparisons. Comparisons were made between responders and non-responders using the Whitney U test. In this study, the number of mutations identified in the 315 tested genes was calculated using the Spearman test (C ancer Genome Atlas Network.Cell 2015;161 :1681-96) was used to correlate with all coding genes (n=20,022). Survival rates in TCGA also correlated with the mutation load calculated by WES and the tested genes. PFS and OS were compared between mutation burden groups using Cox proportional hazards. Patients were assessed by the Kaplan-Meier method and, if progression-free and / or alive, were included in the final PFS and PFS for high, intermediate, and low mutations were excluded (censored) at follow-up. OS was compared between mutation groups using the log-rank test. To assess the impact of adjusted mutation burden for prior ipilimumab, Cox proportional ANOVA was performed. Hazard analyses were performed. All analyses were performed using GraphPad Prism version 6.0. 5 and the R statistical computing package version 3.2.1.

[0461] result Mutation burden assessed by hybrid capture-based NGS was either anti-PD-1 or anti-PD-1. PD-L1 correlates with response to therapy Hybrid capture-based NGS was performed on patients treated with anti-PD-1 or anti-PD-L1 therapy In the initial cohort, anti-PD-1 or The mutation burden in anti-PD-L1 responders was significantly greater than in non-responders (median 45.6 vs. 3.9 mutations / MB; P = 0.003, Figure 1A). Similar differences were observed (median 37.1 vs. 12.8 mutations / MB, P = 0.002; Figure 1B). Results showed that in the "optimal" sample obtained within 12 months of treatment initiation, The results were similar compared to the samples (Table 4). [Table 4] TIFF0007759177000006.tif66154 [Table 5]

[0462] Specific cutoffs are used to classify patients according to their likelihood of response to treatment. When using the optimized ROC in the initial cohort, 3 Stratification of patients into two groups had superior predictive ability compared with binary cutoff classification. The patients were classified as high (>23.1 mutations / MB), medium ( 3.3–23.1 mutations / MB) and low (<3.3 mutations / MB) mutation burden groups Using these thresholds, excellent objective response rates (ORR) were observed in patients with high mutation rates. was observed in the heavy load group, followed by the medium and light load groups (82% vs. 36% vs. 10% response rate;χ 2 P = 0.003, Table 5). These cutoffs were applied to the validation cohort. A significantly superior ORR was found in the high mutation burden group (88% vs. 29% vs. 25%, χ 2 P=0.001). The ORR was 0.001 when the initial and validation cohorts were combined. The highest mutation burden was in the high mutation burden group (85%), followed by the medium (29%) and low (14%) mutation burden groups. followed by the mutation burden group (P<0.001). [Table 6] TIFF0007759177000009.tif41150

[0463] Other clinical outcomes were then evaluated. Across both cohorts, progression-free survival (PFS) was ) correlated with mutation load. High mutation load groups showed superiority compared with medium and low mutation load groups. Improved PFS was observed (median not reached vs. 89 days vs. 86 days, P < 0.001; Figure 1C Overall survival (OS) showed a similar pattern (median not reached vs. 300 days vs. 37 days). 5 days, P<0.001; Figure 1D). Notably, ORR was significantly higher in the intermediate mutation burden group than in the low mutation burden group. Although PFS and OS appeared to be higher than in the unweighted group, they were similar between these groups. Cox proportional hazards analysis adjusted for age, sex, stage of disease, and prior ipilimumab High mutation load was also associated with superior OS and PFS when used with EGFR. For OS, high vs. low HR was 0.14, P < 0.001; for OS, HR was 0.09, P<0.001; Tables 6-7). [Table 7] [Table 8]

[0464] Non-cutaneous melanomas (including acrolear subtypes) are far more common than those of cutaneous origin. have fewer mutations and possibly a lower frequency of response to immunotherapy , the mutational profile of melanoma of unknown primary origin closely resembles that of cutaneous melanoma (Hod is E, Watson IR, Kryukov GV et al., Cell 150:251- 63,2012;Krauthammer et al., Nat Genet 44:1006-1 4, 2012; Postow et al., Oncologist 18:726-32, 2013 Luke et al., Cancer, 2013; Johnson et al., Oncologist 2 0:648-52, 2015). To eliminate confounding from non-cutaneous melanoma, Mutation burden in melanoma of unknown etiology was assessed separately, with higher mutation burden associated with response. observed in the body (median 39.0 vs. 14.4 mutations, P < 0.001; Fig. 3A ). 14 patients with non-cutaneous melanoma (only 2 were responders) No difference in mutation load was observed (median 4.5 vs. 2.2 mutations / MB, P=0.714; Figure 3B).

[0465] We next assessed whether specific types of genomic alterations correlated with response. Mutations detected (including those with known or putative functional significance) correlate with response to treatment. was strongly associated (median 46.5 vs. 6.0 mutations, p<0.001; Figure 4A). Also, the C to T transition (strongly associated with UV damage) was more prevalent in responders. (median 33.5 vs. 3.0 change, p<0.001; Figure 4B). Most other nucleotide variants were also more prevalent in responders, albeit at lower frequencies. In contrast, gene amplifications and deletions were similar between groups (Fig. 4C). (Figure 3C).

[0466] Then, the mutation weight is determined by a particular "driver mutation" We investigated whether the differences between BRAF, NRAS, NF1, and "Triple WT" (wild type ) significant differences were observed between melanomas (median 12.0 vs. 17.6 vs. 6, respectively) 2.7 vs. 2.2 mutations / MB, p<0.001) (Figure 4D). Melanoma has already been associated with chronic UV damage and a high mutation load (Can cer Genome Atlas Network.Cell 161:1681-9 6, 2015; Krauthammer et al., Nat Genet 44:1006-14 , 2012). In contrast, the “triple WT” group has an extremely low mutation load. Ta.

[0467] 236-315 sequenced in this hybrid capture-based NGS panel The mutation load of each gene acts as a robust correlate of the whole-genome mutation load by WES. To determine whether or not the TCGA samples could be used, 345 archived samples were evaluated (Can cer Genome Atlas Network.Electronic addr ess imo,Cancer Genome Atlas N:Genomic Cl assification of Cutaneous Melanoma.Cell 161:1681-96, 2015). The total number of mutations identified in these genes The number of mutations correlated strongly with the total exome mutation count in these samples (R = 0.995 , p<0.001; Figure 5A). This correlation was observed in samples with high mutation load. The mutation burden appeared to be particularly robust in unselected patients (anti-PD-1 / To assess whether PD-L1 is associated with improved outcomes in patients receiving PD-L1 Survival was assessed in TCGA samples with evaluable survival data (n=263). When using the tested genes (P = 0.14) or WES (P = 0.06), For the low, medium, and high mutation load groups, WE S (median OS 43.4 vs. 103.0 vs. 68.0 months, P = 0.001) and When using the test gene (median 47.3 vs. 112.5 vs. 61.5 months, P = 0.008), Differences in OS between patients with intermediate mutation burden when using the three mutation burden groups. Patients with intermediate vs. low blood pressure showed the longest survival (HR = 2.1, P = 0.008; Figure 5B- 5C).

[0468] Specific mutations and response to anti-PD-1 or anti-PD-L1 therapy Next, mutations in specific cancer-related genes will be examined to determine their role in response to anti-PD-1. This analysis was performed to assess their effects on the previously described The focus is on mutations and excludes variants of unknown significance (VUS) unless otherwise indicated. Several genes were identified that were more frequently mutated in responders or non-responders. Several genomic variations correlated with the mutation load. For example, NF 1 mutation was more common in responders (50% vs. 21%, p = 0.015; O RR 74%), and "triple WT" patients were more common in the non-responder group (13% vs. 35%). , p=0.045). In smaller numbers, the response was associated with an immunotherapy response (NRAS; Johns Hopkins University Hospital). nson et al., Cancer Immunol Res 3:288-95, 2015), T cell exclusion (CTNNB1; Spranger et al., Nature, 523:231-5, 2015) or PD-L1 regulation (MYC; Casey et al., Science 352:2 PTEN loss correlated with a genetic mutation already associated with the disease (2016). Loss of PD-L1 expression (in glioblastoma) and immunosuppressive sites (in melanoma) associated with kine profiles (Peng et al., Cancer Discov, 2015). Patients with PTEN loss did not respond, but responders and non-responders were There was a significant difference in the frequency of PTEN-inactivating mutations when compared (13% vs. 15%, p=0.76). MYC amplification appeared to be more common in non-responders, but The results were limited by the sample size. BRCA2 mutations (including VUS) were associated with a higher risk of responding. Responders appear to be more common in patients (5 of 32) than in non-responders (2 of 33). Melanomas with BRCA2 mutations were more likely to be BRCA2-positive than those lacking these mutations. had a higher mutation burden (median 68.2 vs. 15.9 mutations / MB; P = 0.01). 028; Hugo et al., Cell 165:35-44, 2016). [Table 9]

[0469] LRP1B mutations and total mutation burden Frequent mutations in LRP1B, a putative tumor suppressor, were observed in general fragility. Common fragile sites (regions of significant genomic instability; Sm A large 1 called (Ith et al., Cancer Lett 232:48-57, 2006) The hypothesis is that the mutations detected here are the total mutation load and as a single-gene surrogate for response (a "biometer" of whole-exon mutation load) Among the responders, 11 patients had LRP1B mutations. , whereas there was one non-responder (34% vs. 3%, P = 0.008). In this case, responders had an average of 2.8 mutations / VUS, while non-responders had 0 0.9 (P = 0.016; Figure 7A). 75% of responders had one or more mutations / V had US, whereas 38% of non-responders had it (p=0.002). To investigate the relevant populations, TCGA was queried (Cancer Genome Atlas). las Network. Cell 161:1681-96, 2015). LRP1B Melanomas with mutations had significantly more total blasts than those lacking LRP1B mutations. had a higher mutation burden in the tumor (median 542 vs. 219, p<0.001; Figure 7B). The number of LRP1B mutations per 1000 individuals correlated with the number of LRP1B mutations per 1000 individuals (Spearman's R = 0.54, p < 0.00 1; Figure 7C). These data suggest that sequencing even a single frequently mutated gene can be useful. To provide insight into genome-wide mutation burden and correlate it with anti-PD-1 response It remains unclear whether LRP1B mutations have an intrinsic immune effect. It is clear.

[0470] T cell receptor (TCR) clonality does not correlate with clinical benefit or mutation burden T cell infiltration and clonality were then correlated with mutation load, demonstrating the predictive value of this approach. To determine whether TCR NG could enhance tumor cell proliferation, we performed TCR NG in available tumor samples (n=42). The clonality (and diversity) of the TCR β-chain repertoire was investigated by performing This may indicate a pre-existing infiltration of tumor-specific antigen populations, and may be due to anti-PD-1 (Tumeh et al., Nature 515:568-71 , 2014). It was observed that TCR clonality did not correlate with response to anti-PD-1. (median 0.11 vs. 0.11, P = 0.54; Fig. 8A). Furthermore, the T cell fraction correlated with the response. There was no significant difference in time or intervention (median 0.13 vs. 0.09, P = 0.11; Figure 8B). This method can regulate TCR clonality / T cell infiltration, allowing for anti-P therapy without interval therapy. A subset of samples obtained within 4 months of initiating D-1 therapy was evaluated. This group (n = 1 4) showed a non-statistically significant trend toward increasing clonality and T cell fraction among responders. The orientation was observed (Figures 8C-8D; Tumeh et al., Nature 515:568-71, 2014). Furthermore, T cell clonality and T cell fraction did not correlate with mutation burden. (Figures 9A-9B).

[0471] In this study, we used an NGS platform based on hybrid capture of hundreds of genes. The number of mutations detected by the study strongly correlated with benefit from anti-PD-1 / anti-PD-L1. A link between mutational load and anti-PD-1 response in melanoma has been demonstrated. In particular, patients were divided into groups (e.g., "high," "intermediate," and "low" mutation burden groups). stratification into groups (groups) may provide insight into anti-cancer outcomes in advanced melanoma and possibly other cancers. This study provided a clinically feasible marker of response to PD-1 / anti-PD-L1. In the study, no difference in survival was observed between the "medium" and "low" mutation load groups. , which may suggest the existence of a "threshold effect" and a significant impact on response and survival. This would suggest that the effect may be most pronounced in the "high" mutation load group.

[0472] Mutations in several genes correlated with benefit from anti-PD-1. Although this finding may have a direct effect on the immune microenvironment, it is unclear whether other mutations simply affect the mutation load ( may correlate with or contribute to increased expression of genes (e.g., NF1, LRP1B, and BRCA2) This suggests that:

[0473] In summary, progress determined by hybrid capture-based NGS platforms Mutation burden in primary melanoma was assessed by anti-PD-1 or anti-PD-1 therapy in two independent cohorts. was strongly correlated with response to anti-PD-L1 therapy, which was associated with a "high" mutation burden group (which This was particularly evident in the 1990s and 2000s (who constituted over 40% of the study sample). rigorously validated approaches to testing mutation burden to improve treatment decision-making, This could enable more rational use of expensive drugs and enhance this new era of precision immunotherapy. This suggests that:

[0474] References All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety as if fully set forth ... , patents or patent applications are specifically and individually indicated to be incorporated by reference herein. In case of conflict, The present application, including any definitions herein, takes precedence.

[0475] Public databases, e.g., on the World Wide Web at tigr.org Maintained by the Institute for Genomic Research (TIGR) and / or the World Wide Web at ncbi.nlm.nih.gov. National Center for Biotechnology on the web and correlations in the National Center for Biological Information (NCBI) maintained by the National Center for Biological Information (NCBI). Any polynucleotide and polypeptide sequences that reference an accession number that No. 6,299,333, filed Dec. 1, 2003, all of which are incorporated herein by reference in their entirety.

[0476] equivalent Those skilled in the art will be able to derive, using no more than routine experimentation, the specific embodiments described herein. Many equivalents to the above-described embodiments may be recognized or identified. Such equivalents are disclosed in ...

Claims

1. A method for assessing the response status of a subject having melanoma to a therapy comprising an inhibitor of PD-1 or PD-L1, the method comprising: (a) obtaining a value of response status to said therapy for the subject, wherein the response status value comprises a measure of tumor mutation burden (TMB) in a melanoma sample or melanoma-derived sample from the subject, wherein the measure of TMB comprises a determination of the number of somatic mutations in at least 236 genes selected from the genes set forth in Table 1, and wherein the somatic mutations do not comprise one or more of rearrangements, translocations, functional mutations, and germline mutations, wherein the somatic mutations are detected in the sample by hybrid capture followed by sequencing; (b) identifying the subject as a responder (e.g., a complete or partial responder) or non-responder to the therapy; wherein a value of the response status equal to or greater than the reference value of the response status indicates that the subject is or is likely to be a responder to the therapy, or that the subject will or is likely to respond to the therapy; or A method wherein a value of the response status less than the reference value of the response status indicates that the subject is or is likely to be a non-responder to the therapy, or that the subject will not respond to the therapy or is likely to be non-responsive.

2. 1. A method for selecting a therapy comprising an inhibitor of PD-1 or PD-L1 for a subject having melanoma, comprising obtaining a value of response status to the therapy for the subject and selecting the therapy accordingly; the response status value comprises a measure of tumor mutation burden (TMB) in a melanoma sample or melanoma-derived sample from the subject, wherein the measure of TMB comprises a determination of the number of somatic mutations in at least 236 genes selected from the genes set forth in Table 1, and wherein the somatic mutations do not include one or more of rearrangements, translocations, functional mutations, and germline mutations, wherein the somatic mutations are detected in the sample by hybrid capture followed by sequencing; A method wherein an increase in the value of the response status compared to the baseline value of the response status indicates that the subject is or is likely to be a responder to the therapy, or that the subject will or is likely to respond to the therapy.

3. The measures of TMB were the following in a sample from subjects: (i) the presence of one or more somatic mutations in the NF1 gene; (ii) the number of one or more somatic mutations in the LRP1B gene; or (iii) the number of one or more C to T transitions in a given set of genes listed in Table 1 The method of claim 1 or 2, further comprising determining one, two or three of the following:

4. Samples from subjects include: (i) an increase in the number of multiple somatic mutations in a given set of genes listed in Table 1 compared to a baseline level of one or more somatic mutations in the given set of genes listed in Table 1; (ii) the presence of one or more somatic mutations in the NF1 gene; (iii) an increase in the number of one or more somatic mutations in the LRP1B gene compared to a baseline level of one or more somatic mutations in the LRP1B gene; or (iv) an increase in the number of one or more C to T transitions in a given set of genes listed in Table 1 compared to a baseline level of one or more C to T transitions in a given set of genes listed in Table 1.

4. The method of claim 1, wherein the subject is identified as a responder to the therapy if the subject exhibits one, two, three or all of the following:

5. Samples from subjects include: (i) a number of somatic mutations in a given set of genes listed in Table 1 that is reduced or unchanged compared to a baseline level of one or more somatic mutations in the given set of genes listed in Table 1; (ii) the absence of one or more somatic mutations in the NF1 gene; (iii) a similar, the same, or decreased number of one or more somatic mutations in the LRP1B gene compared to a baseline level of one or more somatic mutations in the LRP1B gene; or (iv) a similar, the same, or decreased number of one or more C to T transitions in a given set of genes listed in Table 1 compared to a baseline level of one or more C to T transitions in a given set of genes listed in Table 1 The method of any one of claims 1 to 4, wherein the subject is identified as a non-responder to the therapy if the subject exhibits one, two, three or all of the following:

6. A method described in any one of claims 3 to 5, wherein the at least 236 genes include approximately 250 or more, approximately 300 or more, or all of the genes listed in Table 1.

7. 7. The method of any one of claims 1 to 6, wherein the therapy is selected for the subject depending on: (i), (ii) or (iii) (i) an increase in the number of multiple somatic mutations in a given set of genes listed in Table 1 compared to a baseline level of one or more somatic mutations in the given set of genes listed in Table 1; (ii) determining that the number of somatic mutations in a given set of genes listed in Table 1 is about 3.3 or more, e.g., about 5 or more, about 10 or more, about 15 or more, about 20 or more, about 25 or more, about 30 or more, about 35 or more, about 40 or more, about 45 or more, or about 50 or more somatic mutations per megabase in the coding region of the given set of genes; and (iii) determining that the number of somatic mutations in a given set of genes listed in Table 1 is about 23.1 or more, e.g., about 25 or more, about 30 or more, about 35 or more, about 40 or more, about 45 or more, or about 50 or more somatic mutations per megabase in the coding region of the given set of genes.

8. The method of any one of claims 1 to 7, wherein a therapy is selected for the subject depending on a determination that one or more somatic mutations are present in the coding region of the NF1 gene.

9. (i) determining the presence of about one or more, about two or more, about three or more, about four or more, or about five or more somatic mutations in the LRP1B gene; or (ii) Determining an increase of at least about 2-fold, at least about 2.5-fold, at least about 2.8-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, or at least about 5-fold in the number of one or more somatic mutations in the LRP1B gene compared to a reference number of one or more somatic mutations in the LRP1B gene. The method of any one of claims 1 to 8, wherein a therapy is selected for a subject depending on the

10. (i) determining the presence of about 20 or more, about 25 or more, about 30 or more, about 40 or more, or about 50 or more C to T changes in a given set of genes listed in Table 1; or (ii) determining an at least about 8-fold, at least about 10-fold, at least about 12-fold, at least about 15-fold, or at least about 20-fold increase in one or more C to T transitions in a given set of genes listed in Table 1 compared to a reference number of one or more C to T transitions in the given set of genes listed in Table 1. The method of any one of claims 1 to 9, wherein a therapy is selected for a subject depending on the

11. 11. The method of any one of claims 1 to 10, wherein the subject is receiving or has received a different therapy including a therapeutic substance or method other than an inhibitor of PD-1 or PD-L1: (i) the therapy may be administered after discontinuation of said different therapy or in combination with said different therapy; (ii) the different therapy may be selected from chemotherapy, radiation therapy, immunotherapy, radioimmunotherapy, oncolytic virus therapy, surgical procedure, or any combination thereof; or (iii) the above method, wherein the different therapy may comprise one or more of dacarbazine, temozolomide, interleukin 2 (IL-2), interferon, ipilimumab, a BRAF inhibitor, a MEK inhibitor, talimogene laherparepvec, adoptive cell transfer, or any combination thereof.

12. (i) whether the inhibitor of PD-1 is an anti-PD-1 antibody; (ii) the PD-1 inhibitor is selected from nivolumab (ONO-4538, BMS-936558, or MDX1106), pembrolizumab (MK-3475 or lambrolizumab), pidilizumab (CT-011), MEDI0680 (AMP-514), PDR001, REGN2810, BGB-108, BGB-A317, SHR-1210 (HR-301210, SHR1210, or SHR-1210), PF-06801591, or AMP-224; (iii) the inhibitor of PD-L1 is an anti-PD-L1 antibody; or (iv) the PD-L1 inhibitor is selected from atezolizumab (MPDL3280A, RG7446 or RO5541267), YW243.55.S70, MDX-1105, durvalumab (MEDI4736) or avelumab (MSB0010718C); The method according to any one of claims 1 to 11.

13. 1. A system for assessing a subject for having melanoma, comprising at least one processor operably connected to a memory, said at least one processor, when executed, performing: (a) obtaining a value of response status to a therapy comprising an inhibitor of PD-1 or PD-L1 for the subject, wherein the response status value comprises a measure of tumor mutation burden (TMB) in a melanoma sample or melanoma-derived sample from the subject, wherein the measure of TMB comprises a determination of the number of somatic mutations in at least 236 genes selected from the genes set forth in Table 1, and wherein the somatic mutations do not comprise one or more of rearrangements, translocations, functional mutations, and germline mutations, wherein the somatic mutations are detected in the sample by hybrid capture followed by sequencing; (b) is designed to identify subjects as responders, complete responders, partial responders, or non-responders to a therapy; a response status value equal to or greater than the reference response status value indicates that the subject is or is likely to be a responder to the therapy, or that the subject will or is likely to respond to the therapy; or a response status value less than the reference response status value indicates that the subject is or is likely to be a non-responder to the therapy, or that the subject will not or is likely to not respond to the therapy; A system that evaluates the subject.

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