Checkpoint blockade and microsatellite instability

文档序号：1880584 发布日期：2021-11-26 浏览：12次中文

阅读说明：本技术 检查点阻断和微卫星不稳定性 (Checkpoint blockade and microsatellite instability ) 是由 L·迪亚斯 B·沃格尔斯坦 K·W·凯泽 N·帕帕佐普洛斯 D·勒于 2015-11-12 设计创作，主要内容包括：本申请涉及检查点阻断和微卫星不稳定性。如细胞毒素T-淋巴细胞抗原-4(CTLA-4)和程序性死亡-1(PD-1)等免疫检查点的阻断示出患有癌症的患者的前景。已证实针对这些受体的抑制抗体可破坏免疫耐受并且促进抗肿瘤免疫力。这些试剂在患有某一类别的肿瘤的患者中特别好地起作用。这类肿瘤由于它们产生的众多的新抗原可尤其对治疗敏感。(The present application relates to checkpoint blockages and microsatellite instability. Blockade of immune checkpoints such as cytotoxic T-lymphocyte antigen-4 (CTLA-4) and programmed death-1 (PD-1) show promise in patients with cancer. Inhibitory antibodies directed against these receptors have been shown to break immune tolerance and promote anti-tumor immunity. These agents work particularly well in patients with a certain class of tumors. Such tumors can be particularly susceptible to treatment due to the numerous neoantigens they produce.)

1. Use of an immune checkpoint inhibitory antibody in the manufacture of a medicament for treating a cancer patient, wherein the cancer patient has microsatellite unstable cancer (MSI).

2. The use of claim 1, wherein the cancer is selected from the group consisting of: colon, gastric, endometrial, biliary, pancreatic and prostate cancer.

3. The use of claim 1, wherein the antibody is an anti-PD-1 antibody.

4. The use of claim 1, wherein the antibody is an anti-IDO antibody.

5. The use of claim 1, wherein the antibody is an anti-CTLA-4 antibody.

6. The use of claim 1, wherein the antibody is an anti-PD-L1 antibody.

7. The use of claim 1, wherein the antibody is an anti-LAG-3 antibody.

8. The use of claim 1, wherein a sample from the MSI cancer patient is tested to assess the stability of one or more microsatellite markers prior to the administering step.

9. The use of claim 1, wherein the antibody is a humanized monoclonal antibody.

10. The use of claim 1, wherein the antibody is MK-3475.

11. The use of claim 1, wherein the antibody is an IgG4 antibody.

12. The use of claim 8, wherein a plurality of microsatellite markers are tested.

13. The use of claim 8, wherein the one or more microsatellite markers are selected from the group consisting of: BAT-25, BAT-26, MON0-27, NR-21, NR-24, Penta C and Penta D.

14. The use of claim 12, wherein the plurality of microsatellite markers comprises BAT-25, BAT-26, MON0-27, NR-21, NR-24, Penta C and Penta D.

15. Use of an anti-PD-1antibody for the preparation of a medicament for the treatment of a patient suffering from cancer,

wherein a sample from a cancer patient has been tested and determined to have instability of one or more microsatellite markers selected from the group consisting of: BAT-25, BAT-26, MON0-27, NR-21, NR-24, Penta C and Penta D;

wherein the cancer is selected from the group consisting of: colon, gastric, endometrial, biliary, pancreatic and prostate cancer.

16. Use of an immune checkpoint inhibitory antibody in the manufacture of a medicament for treating a cancer patient, wherein the cancer patient has a mutation burden of at least 100 individual cell mutations per tumor.

17. The use of claim 16, wherein the antibody is an anti-PD-1 antibody.

18. The use of claim 16, wherein the antibody is an anti-IDO antibody.

19. The use of claim 16, wherein the antibody is an anti-CTLA-4 antibody.

20. The use of claim 16, wherein the antibody is an anti-PD-L1 antibody.

21. The use of claim 16, wherein the antibody is an anti-LAG-3 antibody.

22. The use of claim 16, further comprising determining a high mutation load in the tumor prior to the administering step.

Technical Field

The present invention relates to the field of cancer. In particular, it relates to cancer therapy.

Background

Microsatellite instability (MSI) is the accumulation of sequencing errors in microsatellites. This occurs in tumors that have defects in DNA mismatch repair. MSI is present in Lynch syndrome, an inherited cancer syndrome that predisposes patients to colon, endometrial, gastric, ovarian, small bowel, liver and gall bladder, upper urinary tract, brain and prostate cancer. MSI is also present in 10% to 20% of sporadic colorectal, gastric, prostate, lung, ampulla and endometrial cancers. Pancreatic cancers between 0.3% and 13% are also reported as MSI.

The importance of complete immune monitoring in controlling the outgrowth of neoplastic transformations is known for decades. Accumulating evidence shows a correlation between tumor-infiltrating lymphocytes (TILs) in cancer tissues and a better prognosis for various malignant diseases. In particular, the presence of CD8+ T cells and the ratio of CD8+ effector T cells/FoxP 3+ regulatory T cells appear to correlate with improved prognosis and long-term survival of solid malignancies (e.g., ovarian, colorectal and pancreatic cancers, hepatocellular carcinoma, malignant MEL and RCC). TILs can be expanded and reinjected ex vivo to induce a long lasting targeted tumor response in cancers such as melanoma.

PD-1 receptor-ligand interactions are the major pathway hijacked by tumors to suppress immune control. The normal function of PD-1 expressed on the cell surface of activated T cells under healthy conditions down regulates unwanted or excessive immune responses, including autoimmune reactions. The ligands for PD-1 (PD-L1 and PD-L2) are constitutively expressed or can be induced in various tumors. Binding of PD-1ligand to PD-1 inhibits T cell activation triggered by T cell receptors. PD-L1 is expressed at low levels on various non-hematopoietic tissues, most notably on vascular endothelium, whereas PD-L2 protein is only detectably expressed on antigen presenting cells found in lymphoid tissues or in a chronic inflammatory setting. PD-L2 is thought to control immune T cell activation in lymphoid organs, while PD-L1 serves to attenuate unwanted T cell function in peripheral tissues. While healthy organs express little, if any, PD-L1, many cancers are demonstrated to express sufficient levels of this T cell inhibitor. High expression of PD-L1 (and to a lesser extent PD-L2) on tumor cells has been found to be associated with poorer prognosis and survival of various cancer types, including Renal Cell Carcinoma (RCC), pancreatic cancer, hepatocellular carcinoma, ovarian cancer, and non-small cell lung cancer (NSCLC). In addition, PD-1 has been proposed to modulate tumor-specific T cell expansion in patients with malignant MEL. The correlation of the observed clinical prognosis with PD-L1 expression in a variety of cancers suggests that the PD-1/PD-L1 pathway plays an important role in tumor immune evasion and should be considered as an attractive target for therapeutic intervention.

Blockade of immune checkpoints such as the cytotoxic T-lymphocyte antigen-4 (CTLA-4) and programmed death-1 (PD-1) show promise in patients with cancer. CTLA-4 and PD-1 are upregulated on activated T cells and provide inhibitory signals to the activating T cells. Inhibitory antibodies introduced at these receptors have been shown to break immune tolerance and promote anti-tumor immunity. MK-3475 is a humanized monoclonal IgG4 antibody to PD-1 and shows activity in a variety of tumor types including melanoma and non-small cell lung cancer (NSCLC). Previously, the activity of different PD-1 blocking antibodies, BMS-936558, fully humanized monoclonal IgG4 antibody also showed activity in melanoma, NSCLC and a complete response in individual patients with colorectal cancer.

MK-3475 (previously referred to as SCH 900475) is a potent and highly selective humanized mAb of the IgG 4/kappa isotype designed to block directly the interaction between PD-1 and its ligand, PD-L1 and PD-L2. MK-3475 contains the S228P stabilizing mutation and has no antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) activity. MK-3475 potently enhances the T lymphocyte immune response in cultured blood cells from healthy human donors, cancer patients and primates. EC50 ranged from 0.1nM to 0.3nM in a T cell activation assay using human donor blood cells. MK-3475 also modulates the levels of interleukin-2 (IL-2), tumor necrosis factor alpha (TNF α), interferon gamma (IFN γ), and other interleukins. Antibodies enhance the existing immune response only in the presence of antigen and do not non-specifically activate T cells.

The programmed death 1(PD-1) pathway is a negative feedback system that suppresses the Th1 cytotoxic immune response, which, if unregulated, can damage the host^1-3. It is upregulated in many tumors and in their surrounding microenvironment. Blocking this pathway by antibodies to PD-1 or its ligands has resulted in significant clinical responses in some patients with many different cancer types, including melanoma, non-small cell lung cancer, renal cell carcinoma, bladder cancer, and hodgkin's lymphoma^4-10. Expression of ligands of PD-1 (PD-L1 or PD-L2) on the surface of tumor cells or immune cells is an important but not decisive predictive biomarker for the response to PD-1blockade^4,6-8,11。

We were inspired that in the report of the effect of PD-1blockade in human tumors, only one of 33 colorectal cancer (CRC) patients responded to this treatment compared to a large proportion of patients with melanoma, renal cell carcinoma and lung tumors^10，12. What is this single patient difference? We assume that this patient has MMR deficiency, because MMR deficiency occurs in a small fraction of late CRC,^13，14somatic mutations found in tumors are recognized by the patient's own immune system,¹⁵and MMR-deficient cancers have 10-fold to 100-fold more cell mutations than MMR-active CRC.^16-18In addition, MMR-deficient cancers contain significant lymphocyte infiltration, consistent with an immune response^19-22. And in Topalian et al¹⁰Two of the most responding tumor types blocked by PD-1 in the study had high numbers of somatic mutations due to exposure to cigarette smoke (lung cancer) or UV radiation (melanoma))^23，24. Our assumptions are just: tumors of individual CRC patients in response to PD-1blockade were MMR deficient.²⁵We therefore hypothesized that MMR-deficient tumors respond more to PD-1blockade than MMR-active tumors.

To test this hypothesis, we initiated a phase 2 clinical trial to assess immune checkpoint blockade in patients whose tumors either had or did not have MMR defects. Because MMR deficiency in tumors occurs via two routes^26-28We therefore recruited patients with hereditary non-polyposis colorectal cancer (HNPCC, also known as Lynch syndrome) resulting from an inherited germline defect in one of the four MMR genes followed by a second inactive somatic alteration in the remaining wild-type allele. We also recruited patients with sporadic MMR-deficient tumors in which both alleles of the MMR gene were inactivated by somatic mutation or by epigenetic silencing²⁹. In either case, the resulting tumors have hundreds or thousands of mutations^16，18。

There is a continuing need in the art to improve cancer therapy so that the patient's life is not diminished and thus the quality of life is not diminished.

Disclosure of Invention

According to one embodiment of the present invention, a method of treating a cancer patient is provided. Cancer patients have a high mutational burden as found in microsatellite unstable cancer (MSI). Administering an immune checkpoint inhibitory antibody to a cancer patient.

According to another embodiment of the present invention, a method of treating a cancer patient is provided. Testing a sample from a cancer patient for one or more microsatellite markers selected from the group consisting of: BAT-25, BAT-26, MON0-27, NR-21, NR-24, Penta C and Penta D, and was determined to have microsatellite instability. The cancer is selected from the group consisting of: colon, gastric, endometrial, biliary, pancreatic and prostate cancer. Administering an anti-PD-1antibody to a cancer patient.

According to another embodiment of the present invention, a method for classifying a tumor in a human is provided. Samples from humans were tested to assess the stability of one or more microsatellite markers. Microsatellite instability was determined in the sample. Tumors were identified as good candidates for treatment with immune checkpoint inhibitory antibodies.

According to another embodiment of the present invention, a method for classifying a tumor in a human is provided. Samples from humans were tested to assess the stability of one or more microsatellite markers. The microsatellite stability in the sample is determined. Tumors were identified as poor candidates for treatment with immune checkpoint inhibitory antibodies.

These and other embodiments, which will be apparent to those skilled in the art upon reading the present specification, provide methods for treating microsatellite unstable cancers in the field.

Drawings

Fig. 1A-1 b clinical responses to pelvizumab. (FIG. 1A) biochemical response. Serum protein biomarker levels were measured, where each cycle sum value represents the percentage change from baseline. If the baseline tumor marker value is greater than the upper normal limit, then the patient is included. CA-125 is used in patients with endometrial cancer; CA19-9 for a cholangiocarcinoma and an ampulla carcinoma; and CEA for all other patients. Green, red and black lines indicate patients with MMR-deficient CRC, MMR-active CRC and MMR-deficient non-CRC, respectively. (FIG. 1B) radiation response. Tumor response was measured at intervals and the values show the best fraction change from baseline measurement for the sum of the longest diameters (SLD) of each measurable tumor.

Fig. 2A-2 d. clinical benefit of parilizumab according to MMR status. Kaplan-Meier curves (Kaplan-Meier curves) show (fig. 2A) progression free survival of colorectal cancer population, (fig. 2B) overall survival of colorectal cancer population, (fig. 2C) progression free survival of patients with MMR-deficient cancer other than colorectal (median PFS ═ 5.4 months; 95% CI, 3% to no estimate), and (fig. 2D) overall survival of patients with MMR-deficient cancer other than colorectal cancer. In both populations with MMR-deficient tumors (CRC and non-CRC), median overall survival was not reached. Patients in the population with MMR-active cancer had a median PFS of 2.2 months (95% CI 1.4% to 2.8%) and a median OS of 5.0 months (95% CI 3.0 to unestimable).

Fig. 3 (fig. S2.) spider web plot of radiation response. Tumor responses were measured at regular intervals and values show the percentage change from baseline measurement of the sum of the longest diameters (SLD) of each measurable tumor. Patients were included only if a baseline and treatment scan in the study was available. Green and red indicate patients with MMR deficient CRC and MMR active CRC, respectively. Blue indicates patients with MMR deficient cancer other than CRC.

Fig. 4A-4B (fig. S3.) MMR active CRC and MMR deficient CRC had comparable treatment and duration of metastatic disease prior to addition to the study. Kaplan-meier estimates for the treatment time immediately before addition to the study (HR 0.81, 95% CI 0.38 to 1.752, p ═ 60) and (fig. 4B) the duration of metastatic disease before addition (HR 1.13, 95% CI 0.49 to 2.62, p ═ 78) for this parislizumab study were comparable between the MMR deficient CRC population and the MMR active CRC population. A short duration of prior treatment is desired in the treatment-refractory CRC population.

Fig. 5 (fig. S4.) waterfall graph of biochemical responses. Serum protein biomarker levels were measured, with each cycle sum value representing the best percentage of change from baseline. If the baseline tumor marker value is greater than the upper limit of normal, then the patient is included. CA-125 is used in patients with endometrial cancer; CA19-9 was used for 1 cholangiocarcinoma and 1 ampulla carcinoma; and CEA for all other patients. Green and red indicate patients with MMR deficient CRC and MMR active CRC, respectively. Blue indicates patients with MMR deficient cancer other than CRC.

Fig. 6A to 6B (fig. S5.) somatic mutations in MMR deficient tumors and MMR active tumors. Gross cellular mutations per tumor were identified by exome sequencing of tumors and matched normal DNA (fig. 6A) and correlation with objective responses (fig. 6B) (nonparametric Wilcoxon test, p 0.007 and jonckherere-Terpstra test for trending, p 0.02).

FIG. 7 (FIG. S6). immunohistochemistry for CD8 and PD-L1 expression. Aggressive leading edges (yellow dashed line) from MMR deficient CRC (subject #16, top) and MMR active CRC (subject #3, bottom). The yellow dashed line separates tumor (T) and normal (N) tissues. There was significant expression of PD-L1 (blue arrow) and CD8 (brown dot) in MMR-deficient tumor (upper panel) patients, while there was minimal expression of either marker in MMR-active tumors (lower panel). Representative images of Tumor Infiltrating Lymphocytes (TILs) in another MMR deficient CRC (subject #19, top) and MMR active CRC (subject #3, bottom) that was immunolabeled with antibodies to CD8 (brown dots). Note the infiltration of CD8 cells in MMR-deficient tumors. Initial magnification of aggressive front 10 x and TIL20 x.

Figure 8 (figure S7.) CD8 and PD-L1 expression in MMR deficient and MMR active tumor microenvironments. T cell Density units cell/mm²The tumor of (2). The invasive frontier refers to immune cells (TILs and macrophages) at the junction of tumor and normal tissues. P values were obtained using unpaired t-test.

Figure 9 (figure S8.) CD8 expression and clinical benefit of parilizumab. Intratumoral CD8⁺T cell Density (cells/mm)²) And objective responses (Jonckheere-Terpstra test for trends, p ═ 0.02).

Figure 10 (table S1.) comparison of immune correlation and RECIST response criteria (adapted from Wolchok et al, clinical cancer research (clin.

FIG. 11 (Table S2.) immune-related responses to treatment

FIG. 12 (Table S4.) correlation of Total cellular mutations and mutation-associated neo-antigen (MANA) with clinical outcomes FIG. 13 (Table S5.) correlation of immune markers with clinical outcomes

Detailed Description

The inventors have found that immune checkpoint inhibitors work best in tumors with a high mutation load. Furthermore, tumor defects of mismatch repair are particularly sensitive to specific forms of immunotherapy, as this phenotype produces a persistent accumulation of mutations at high frequency. The present inventors have developed treatments for cancer patients exhibiting microsatellite instability phenotypes or other high mutational loads. Treatments include inhibitory antibodies for immune checkpoints. Such checkpoints include PD-1, IDO, CTLA-4, PD-L1, and LAG-3. Other immune checkpoints may also be used. The antibody may be administered by any convenient means including, but not limited to, intravenous infusion, oral administration, subcutaneous administration, sublingual administration, ocular administration, nasal administration, and the like.

Microsatellite instability (MSI) tumors are defective in DNA mismatch repair, leading to high spontaneous mutation rates and the potential for expression of neoantigens. Furthermore, similar to melanoma, in MSI-positive colon cancers, there is often significant lymphocyte infiltration. Any tumor that is MSI or otherwise highly mutationally loaded may be treated according to the present invention. They may test the attributes of MSI according to any method known in the art including, but not limited to, those described in example 1 below. Any of the one or more MSI markers may be tested to determine the MSI phenotype. The sample can be tested for high mutation load by identifying tumors that have at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, at least 1500, or at least 1600 mutations per genome. High mutational load means a large number of somatic mutations in the tumor relative to normal tissue of the individual. The average number of somatic mutations in non-MSI tumors was about 70 individual cell mutations.

Any type of tumor that shows MSI phenotype or high mutational load can be tested and/or treated according to the present invention. These include, but are not limited to, colon, gastric, endometrial, biliary, pancreatic, and prostate cancers. Tumors of the ampulla, gall bladder, brain, including gliomas, breast, lung, skin, esophagus, liver, kidney, ovary, sarcoma, uterus, cervix, bladder, testes, mouth, tongue, and small and large intestine may also be tested and/or treated.

Testing of MSI may be accomplished by any means known in the art. One or more of the following markers may be tested: five nearly monomorphic single nucleotide repeat markers (BAT-25, BAT-26, MON0-27, NR-21, and NR-24) and two highly polymorphic five nucleotide repeat markers (Penta C and Penta D). In one commercially available system that can be used, fluorescently labeled primers (labeling plates) are used for the co-amplification of all seven or more of the enumerated labels. After amplification the fragments were tested for genotyping/assigning phenotypes.

Samples that can be tested for MSI include tumor tissue as well as body fluids containing nucleic acids shed from tumors. It is well known to test tumor DNA in such tissues and body fluids.

The types of antibodies that can be used include any of the types developed for use as immune checkpoint inhibitors. These may be monoclonal or polyclonal. They may be single chain fragments or other fragments of whole antibodies, including those prepared by enzymatic cleavage or recombinant DNA techniques. They may be of any isotype, including but not limited to IgG, IgM, IgE. The antibody may be of any species origin, including human, goat, rabbit, mouse, cow, chimpanzee. The antibody may be humanized or chimeric. The antibody may be conjugated or engineered to attach to another moiety, whether a therapeutic molecule or a tracer molecule. For example, the therapeutic molecule can be a toxin.

Data from a small number of stage 2 trials of pelvizumab treating tumors with and without defects in MMR support the hypothesis that MMR-deficient tumors respond more to PD-1blockade than MMR-active tumors. MMR deficiency occurs in many cancers, including those of the colorectal, uterine, gastric, biliary, pancreatic, ovarian, prostate and small intestine^18,34-42. Patients with these types of MMR-deficient tumors also benefit from anti-PD-1therapy, as can patients whose tumors contain other DNA repair defects, such as those with POLD, POLE or MYH mutations.^18，43，44

The assumption that MMR-deficient tumors stimulate the immune system is not a new idea⁴⁵And has been supported by the dense immunoinfiltration and Th 1-associated cytotoxin-rich environment observed in MMR-deficient tumors.^19-22,46Recent studies have shown that the MMR-deficient tumor microenvironment strongly expresses several immune checkpoint ligands (including PD-1, PD-L1, CTLA-4, LAG-3 and IDO), suggesting that its active immune microenvironment passes throughImmunosuppressive signal balance against tumor removal⁴⁷Improving these traditional observations. The introduction of immunoinfiltration associated with MMR-deficient carcinoma at neoantigens is the most likely explanation for both old and new findings. For melanoma⁴¹anti-CTLA-4 and in lung cancer⁴⁸The correlation of higher mutation burden and higher response rate against PD-1 in MANA provides additional support for the idea that MANA recognizes as an important component of the endogenous anti-tumor immune response.

Based on the results of current and previous studies, we propose to do so by (1)>20-fold) increased number of mutations generated by MMR deficiency associated with neoantigens (fig. 12 (table S4); also available on-line in New England Journal of Medicine (New England of Medicine); incorporated herein by reference) is the basis for the improved anti-PD-1 responsiveness of a defined subset of this gene for cancer. While our estimates of the number of mutation-associated neo-antigens in tumors were based only on computer predictions of binding-affinity, this suggestion was consistent with the observation that MMR-active tumors had much less infiltration of lymphocytes than MMR-deficient tumors (fig. 7(S6), fig. 8(S7), and fig. 13 (table S5); obtained online in New England Journal of Medicine (New England of Medicine); incorporated herein by reference). Recent research^49，50It is shown that only a minor proportion of the predicted neoepitope is actually presented on the MHC-bearing cell surface and is the target of the endogenous T cell response. It seems likely that, although the number of predicted mutation-associated neoantigens is proportional to the number of actual mutation-associated neoantigens, tumors with a high number of actual mutation-associated neoantigens are more likely to stimulate the immune system to respond to the tumor. Alternative mechanisms that elicit differences in anti-PD-1 responsiveness between MMR deficient and MMR active tumors should also be considered. For example, different signaling pathways activated in MMR deficient and MMR active tumors may produce differences in secretion of soluble factors, which may produce differential activation of PD-1 pathways within the tumor microenvironment^26-28. Genetic differences can affect epigenetic differences that alter the expression of tumor-associated self-antigens, which in turn can alter the antigenicity of the tumor. AntigensExperimental analysis of specific immune responses and changes in immune microenvironment should help define the relative contribution of these factors to the significant responsiveness of MMR-deficient tumors to PD-1 antibodies.

Several notable observations were made during the course of this study. First, changes in serum protein biomarkers (such as CEA) following a single dose of treatment are consistent with clinical benefit. Reduction of CEA levels several months prior to the target radiologic evidence; it is possible that other biomarkers (such as circulating tumor dna (ctdna)) may also be beneficial as surrogate markers for early response.^51，52Second, our results suggest that the assessment of the tumor genome can help guide immunotherapy. They support the notion that the number and type of variation may prove useful in determining the possible utility of immune checkpoint inhibitors even in MMR-active cancers^41，48，53Most importantly, our results show a new approach to treating a specific class of tumors based on gene status alone: i.e. irrespective of the underlying tumor type.

The foregoing disclosure generally describes the invention. All references disclosed herein are expressly incorporated by reference. A more complete understanding can be obtained by reference to the following specific examples, which are provided herein for purposes of illustration only and are not intended to limit the scope of the present disclosure.

Example 1

MSI test

The MSI test has been standardized and performed in CLIA certified laboratories without assay development. Tumor samples or newly obtained biopsies will be archived for determination of MSI. MSI status will be performed locally by Immunohistochemical (IHC) or PCR-type tests for qualified CLIA certification. Patients can be assessed to be confirmed using the MSI analysis system from Promega (Promega) in john Hopkins (Johns Hopkins). This test will determine MSI status by the insertion or deletion of repeat units in five nearly singlet mononucleotide repeat markers (BAT-25, BAT-26, MON0-27, NR-21, and NR-24). At least 2 MSI loci are required in middle population a and population C to be evaluable. Patients may be assigned to new populations and/or replaced based on the results of the promegate test.

Example 2

Method

Patient's health

Treatment-refractory progressive metastatic cancer patients for this phase 2 study were recruited from three participating centers (table 1). Three populations were evaluated: population a consists of patients with MMR deficient colorectal adenocarcinoma; population B consisted of patients with MMR active colorectal adenocarcinoma; and population C consists of patients with MMR-deficient cancers of a type other than colorectal.

Study supervision

The protocol found at nejm.org was approved by the ethical review board at each site and the study was conducted according to the Declaration of Helsinki (Declaration of help) and the International coordination of Good Clinical Practice norms (the International Conference on harmony Guidelines for Good Clinical Practice). All patients provided written informed consent prior to study entry. The first consultant investigator (D.L.) and the study sponsor (l.a.d.) are responsible for the supervision of the study. Merck corporation (Merck) donates research medications, review protocols, and final draft for this manuscript. Clinical studies are funded primarily by charitable support.

Design of research

This phase 2 experiment was performed using a Green-Dahlberg two-step design and consisted of the three parallel populations described above. The study agent, parilizumab (merck corporation), was administered every 14 days at 10mg/kg intravenously. Pelizumab is a humanized monoclonal anti-PD-1antibody of the IgG 4/kappa isotype that blocks the interaction between PD-1 and its ligands (PD-L1 and PD-L2).

Safety assessments were performed prior to each treatment. Assessment of total tumor burden via measurement of serum biomarkers was performed at the beginning of each cycle. Radioactivity assessments were performed at 12 weeks and every 8 weeks thereafter. Additional details regarding the clinical protocol are provided in example 3.

Analysis of mismatch repair status

Tumors with genetic defects in the MMR pathway are known to have thousands of somatic mutations, particularly in the quiltA region of repetitive DNA called a microsatellite. The accumulation of mutations in these regions of the genome is called microsatellite instability (MSI)^26-28. Evaluation of MMR status in tumors using MSI analysis system from Promega, sequence being particularly prone to replication errors when MMR is impaired by evaluation of selected microsatellites^26-28. See supplementary appendix for additional details.

Genomic and bioinformatic analysis

Primary tumor samples and matched normal peripheral blood specimens were obtained from a subset of subjects with MMR deficiency and other subjects with MMR-active carcinomas in which sufficient tumor tissue is available for exome sequencing³⁰And HLA haplotype analysis. To assess possible mutant peptide binding, somatic exome data in combination with individual patient MHC class I HLA haplotypes were applied to an epitope prediction algorithm^31，32. This algorithm provides an estimate of the total number of mutation-associated neoantigens in each tumor. Additional details are provided in the supplemental appendix (obtained online in New England Journal of Medicine; incorporated herein by reference).

Statistical analysis

The primary endpoint for both population A and population B was the use of immune-related response criteria (irRC)³³Assessed immune-related objective response rate (irORR) and immune-related progression-free survival (irPFS) rate at 20 weeks. The main endpoint for population C was irPFS rate at 20 weeks. Immune-related criteria (i.e., criteria for evaluating immune-based therapies) are based on radiation responses and, unlike RECIST criteria, the degree of capture of disease after disease progression; these criteria are defined and compared to RECIST v1.1 in fig. 10 (table S1). Response rates and PFS rates at 20 weeks were evaluated using RECIST v1.1 and irRC (fig. 10 (table S1)) and reported in this study. PFS and overall survival were summarized by the kaplan-meier method. Assumptions, decision rules to reject null assumptions, and early stop rules for valid and invalid, and details of the statistical method are provided in the supplemental appendix.

Example 3

Replenishment method

Patient's health

In agreement with the conditions in this study, patients must be at least 18 years of age, with histologically confirmed evidence of previously treated advanced carcinoma. All patients were tested for MMR status prior to enrollment. All patients had at least one measurable lesion and sufficient blood function, liver function and kidney function as defined by response assessment criteria for solid tumors (RECIST), version 1.1, eastern cooperative group of tumors (ECOG) performance status score of 0 or 1. Eligible patients with CRC must have received at least 2 prior cancer treatments and patients with other cancer types must have received at least 1 prior cancer treatment. Patients with untreated brain cancer metastasis, history of HIV, hepatitis B, hepatitis C, clinically significant ascites/fluid accumulation, or autoimmune disease were excluded.

Study supervision

The initial draft of the manuscript is prepared by a subset of authors and all authors provide the final manuscript. All authors make decisions to submit manuscripts for publication. The chief advisor investigators and study promoters ensure the accuracy and integrity of the reported data and compliance with the protocol.

HLA typing

Sequence-based typing of HLA-A, HLA-B and HLA-C can be divided into three distinct steps, as described below. A general, a × 02 specific, B general, B group specific, C general and C × 07 specific PCR and sequencing mixtures were made in JHU core mechanism. AlleleSEQR HLA-B of Celera is used for the B general SBT based on the parting kit of the sequence. HLA-a typing protocol consists of two PCR reactions, universal a and specific a x 02. The universal amplicon A contains partial exon 1-partial exon 5. The a × 02 amplicon encompasses part of intron 1-part of exon 5. HLA-B typing procedure consists of two PCR reactions, B general and B group specificity. B universal PCR is a multiplexed reaction containing two PCR amplicons encompassing exon 2-exon 3 and exon 4-exon 7. The B group specific amplicon encompasses part intron 1-part exon 5. HLA-C typing protocol consisted of two PCR reactions, C universal and C × 07 specific. C universal and C × 07 specific amplicons encompass exons 1-7.

Specificity of HLA-A and B PCR AmpliTaq Gold DNA polymerase was used. GeneAmp high fidelity enzyme was used for HLA-C and C × 07PCR mixtures. This enzyme is a mixture of two polymerases: AmpliTaq DNA polymerase (uncorrected polymerase) and corrected polymerase. This enzyme cocktail is necessary for efficient and robust amplification to produce large full-length HLA-C amplicons.

PCR product purification A Universal and B Universal amplicons were performed using exonuclease I and shrimp alkaline phosphatase to sequence exons 2, 3, 4 in both directions. C universal amplicons were sequenced in both directions for exons 2, 3 and in single directions for exons 1, 4, 5, 6, 7. A x 02 specific, B group specific and C x 07 specific amplicons were sequenced in a single direction against exons 2, 3. All sequencing reactions were performed with Big Dye Terminator V1.1 from Applied Biosystems and sequenced with an ABI Prism 3500XL Gene Analyzer. Conexio Genomic "assign SBT (assign SBT)", allele assignment software for processing data files.

Mismatch repair status test^1，2

Six slides of tumor and normal (excluding lymph node or resection boundaries) were cut (5 microns each), deparaffinized (xylene), and one stained with hematoxylin and eosin (H + E). Tumor regions containing at least 20% neoplastic cells, designated by committee-certified anatomical pathologists, were excised entirely using the Pinpoint DNA isolation system (Zymo Research, Irvine, CA) and digested in proteinase K for 8 hours and DNA isolated using QIAamp DNA mini kit (Qiagen, Valencia, CA) in barnacia, california. MSI was assessed using the MSI analysis system (Promega, Madison, Wis.) and consisted of 5 mimonoid single nucleotide repeats (BAT-25, BAT-26, NR-21, NR-24, and MONO-27) that detected MSI and 2-pentanucleotide repeat loci (PentaC and PentaD) that confirmed the identity between normal and tumor samples, according to the manufacturer's instructions. After amplification of 50ng to 100DNA, fluorescent PCR product sizes were determined on an applied biosystems 3130xl capillary electrophoresis instrument (Invitrogen, Calsbad, Calif.). The five nucleotide locus confirmed the identity in all cases. Controls included water as a negative control and a mixture of 80% germline DNA and 20% MSI cancer DNA as a positive control. The size of the bases is determined for each microsatellite locus and if two or more single nucleotide loci vary in length compared to germline DNA, the tumor is called MSI.

Sequencing analysis

Sample (I)

Samples are provided as FFPE patches or frozen tissue for pathological examination to determine tumor cellularity. Tumors were excised entirely to remove contaminated normal tissue, yielding samples containing > 20% neoplastic cells. Providing a matched normal sample, such as blood, saliva, or normal tissue obtained from surgery.

Sample preparation and next generation sequencing³

Sample preparation, library construction, exome capture, next generation sequencing and bioinformatic analysis of tumor and normal samples were performed at Personal Genome Diagnostics, Inc. Briefly, DNA was extracted from frozen or Formalin Fixed Paraffin Embedded (FFPE) tissue along with a matching blood or saliva sample using a Qiagen DNA FFPE tissue kit or a Qiagen DNA blood mini kit (Qiagen, ca). Genomic DNA from tumor and normal samples was fragmented and used for Illumina TruSeq library construction (ilomina, san diego, california) according to the manufacturer's instructions or as described previously 4. Briefly, 50 nanograms (ng) to 3 micrograms (μ g) of genomic DNA in 100 microliters (μ l) of TE was fragmented in a Covaris ultrasonicator (Covaris of Woburn, MA) to a size of 150bp to 450 bp. To remove fragments less than 150bp, DNA was purified twice using Agencourt AMPure XP beads (Beckman Coulter, IN, indiana) at a PCR product to bead ratio of 1.0 to 0.9 and washed with 70% ethanol according to the manufacturer's instructions. The purified, fragmented DNA was mixed with 36. mu.l H2O, 10. mu.l of end-repair reaction buffer, and 5. mu.l of end-repair enzyme mixture (Cat. E6050, NEB, Ipswich, MA) by Epowermay, Mass. According to the manufacturer's instructions, 100 μ l of the end repair mixture was incubated at 20 ℃ for 30min and purified using Agencour AMPure XP beads (Beckmann Coulter, Ind.) at a ratio of PCR product to beads of 1.0 to 1.25 and washed with 70% ethanol. For the A-tail, 42. mu.l of the end-repaired DNA was mixed with 5. mu.l of 10 XDA tailing reaction buffer and 3. mu.l of Klenow (extracellular-) (Cat. E6053, NEB by Epowerman, Mass.). According to the manufacturer's instructions, 50 μ l of the mixture was incubated at 37 ℃ for 30min and purified using Agencour AMPure XP beads (Beckmann Coulter, Ind.) at a ratio of 1.0 to 1.0 PCR product to beads and washed with 70% ethanol. For linker ligation, 25. mu.l of A-tailed DNA was mixed with 6.7. mu.l of H2O, 3.3. mu.l of PE-linker (Illomana), 10. mu.l of 5 Xligation buffer, and 5. mu.l of Quick T4 DNA ligase (Cat. No. E6056, NEB of Italy, Mass.). The ligation reaction mixture was incubated at 20 ℃ for 15min according to the manufacturer's instructions and purified using Agencour AMPure XP beads (Beckmann Coulter, Ind.) at 1.0 to 0.95 and 1.0 PCR product to bead ratios and washed with 70% ethanol. To obtain the amplified library, twelve PCRs of 25. mu.l each were set, each including 15.5. mu.l of H2O, 5. mu.l of 5 XPisun HF buffer, 0.5. mu.l of a dNTP mix containing 10mM each dNTP, 1.25. mu.l of DMSO, 0.25. mu.l of Illumina PE primer #1, 0.25. mu.l of Illumina PE primer #2, 0.25. mu.l of Hotstart Phusion polymerase, and 2. mu.l of DNA. The PCR procedure used was: maintaining at 98 deg.c for 2 min; maintaining at 98 ℃ for 15 seconds, 65 ℃ for 30 seconds and 72 ℃ for 30 seconds for 12 cycles; and maintained at 72 ℃ for 5 min. DNA was purified using Agencourt AMPure XP beads (beckmann coulter, indiana) at a 1.0 to 1.0 ratio of PCR product to beads and washed with 70% ethanol according to the manufacturer's instructions. Exons or target regions were captured in solution using the Agilent SureSelect v.4 kit according to the manufacturer's instructions (Agilent, Santa Clara, CA). The captured library was then purified using Qiagen MinElute column purification kit and eluted in 17. mu.l of 70 ℃ EB to obtain 15. mu.l of the captured DNA library. (5) The captured DNA library was amplified in the following manner: eight 30uL PCR reactions were set up, each containing 19. mu.l of H2O, 6. mu.l of 5 XPisuon HF buffer, 0.6. mu.l of 10mM dNTP, 1.5. mu.l of DMSO, 0.30. mu.l of Illumina PE primer #1, 0.30. mu.l of Illumina PE primer #2, 0.30. mu.l of Hotstart Phusion polymerase, and 2. mu.l of the captured exome library. The PCR procedure used was: maintaining at 98 deg.c for 30 sec; 98 ℃ for 10 seconds, 65 ℃ for 30 seconds, 72 ℃ for 30 seconds for 14 cycles (exome) or 16 cycles (target); and 72 ℃ for 5 min. To purify the PCR product, NucleoSpin Extract II purification kit (Macherey-Nagel, Pa.) was used according to the manufacturer's instructions. Paired-end sequencing was performed using Illumina HiSeq 2000/2500 and Illumina MiSeq instrument (ilomina, san diego, california) to generate 100 bases from each end of the fragment for exome libraries and 150 bases from each end of the fragment for the target library.

Primary processing of Next Generation sequencing data and identification of putative somatic mutations 3

Somatic mutations were identified using VariantDx custom software (personal genome diagnosis of morgans, barren, ma) for identifying mutations in matched tumor and normal samples. Prior to what was termed mutation, primary processing of sequence data, including masking of linker sequences, was performed on both tumor and normal samples using Illumina CASAVA software (v 1.8). Sequence reads were aligned using Needleman-Wunsch method 5 using ELAND control human reference genome (version hg18) with additional realignment of selected regions. Candidate somatic mutations consisting of point mutations, insertions and deletions were then identified using VariantDx at either end of the entire exome or the region of interest. VariantDx checks the tumor samples against a matching normal sequence alignment while applying a filter to exclude alignment and sequencing artifacts. Briefly, a contrast filter is applied to exclude quality-failing reads, unpaired reads, and poorly mapped reads in the tumor. An alkaline mass filter was applied to limit the inclusion of bases with reported phred quality scores >30 (for tumors) and >20 (for normal). (ii) only if (i) the different paired reads contain a mutation in the tumor; (ii) the number of different paired reads containing a particular mutation in the tumor is at least 10% of the read pairs; (iii) mismatched bases were not present in > 1% of reads in matched normal samples and in custom databases derived from common germline variants of dbSNP; and (iv) mutations in tumors were identified as candidate somatic mutations when positions were overlaid by >150 x in both tumor and normal. Mutations (including parallel homologous sequences) resulting from a misplaced genomic alignment are identified and excluded by searching the reference genome.

Candidate somatic mutations were further filtered based on gene annotation to identify those that appeared in the protein coding region. Functional outcomes were predicted using snpEff and using a custom database annotated with CCDS, Refseq and Ensembl for the latest transcript version available on hg18 from UCSC (https:// genome. When obtained, predictions are ordered to favor transcripts with typical start and stop codons and CCDS or Refseq transcripts on Ensembl. Finally, the mutations are filtered out to exclude introns and silent changes while retaining mutations that produce missense, nonsense, frameshift or splice site variations. The manual visual inspection step is used to further remove the human changes.

Mutant peptide MHC binding prediction

Analysis of potential MHC class I binding based on individual patient HLA haplotype pairs to predict somatic frameshifts, insertions, deletions and missense mutations that produce amino acid changes. Our initial analysis focused on HLA-A and HLA-B. Amino acid mutations are related to their corresponding CCDS accession numbers, and in cases where this is not available, Refseq or collective transcription is used to extract protein sequences. To identify the 8mer, 9mer and 10mer epitopes, the amino acid fragments surrounding each mutation were identified. These 15, 17 and 19 mutant amino acid fragments were analyzed by the epitope prediction program NetMHC 3.4. 6 epitopes with predicted affinities <50nm were considered to be strong potential binders and epitopes with predicted affinities <500nm were considered to be weak potential binders as indicated by the NetMHC group 6.

To further improve the total neoantigen load, we repeated the same process for complementing the wild-type peptide for each mutant peptide. When the complementary wild-type peptide is predicted to be a weakly potential binder, then we filter the mutant peptide that is a strongly potential binder. These mutant peptides are referred to as mutation-associated neoantigens (MANA). In cases where patients with (e.g., cases 1, 17 and 21) a single MHC haplotype are not supported by NetMHC 3.4, individual haplotypes are not included in our analysis.

Statistical method

Design of the experiment⁷

This assay was performed using a parallel two-step design to simultaneously assess the efficacy of MK-3475 and MSI as therapeutic selection markers for anti-PD-1 therapy. It consists of a two-step phase 2 study described herein in parallel in three patient populations. Study agent (MK-3475) was administered intravenously every 14 days at 10 mg/kg.

For each of population a and population B, the co-primary endpoint was progression-free survival (irPFS) at 20 weeks, and objective responses (irOR) were assessed using immune-related criteria. A step-down gate keeper is used to preserve the overall type I error. The two-step Green-Dahlberg design was used to evaluate irPFS with transition and final analysis after 15 and 25 patients, respectively. In phase 1, 15 out of 15 >1 no progression at 20 weeks was required to continue the second phase, and then 25 out of 4 no progression at 20 weeks was required to continue the test for irOR, with > 4 out of 25 responders (irCR or irPR) indicating promising efficacy in this population. Each population can be terminated for efficacy once ≧ 4 no progression and ≧ 4 responses are confirmed at 20 weeks, or for ineffectiveness once 0 out of 15 at 20 weeks in phase 1 is no progression or ≧ 22 subjects have disease progression by 20 weeks. This design achieved a 90% ability to detect a 20-week irPFS rate of 25% and an 80% ability to detect an irOR rate (irORR) of 21%, with an overall type I error of 0.05 at a false set of a 20-week irPFS rate of 5% and an irORR of 5%.

For population C, the primary endpoint was irPFS at 20 weeks. Using a two-step Green-Dahlberg two-step design, with transitions and final analysis after 14 and 21 patients; in phase 1, 14 out of 14 >1 no progression at 20 weeks was required to continue the second phase, with > 4 out of 21 at the end of 20 weeks indicating adequate efficacy in population C. Once > 4 no progression at 20 weeks was confirmed, the population could be terminated. The design has 81% capability to detect a 20-cycle irPFS rate of 25%, with a type I error of 5% at a false setting of a 20-cycle irPFS rate of 5%.

Statistical analysis

Responses and progression were assessed using RECIST v1.1 and an immune-related response criterion (irRC) extracted from Wolchok et al 8, which uses the sum of the results of two-dimensional tumor measurements and incorporates novel lesions into the sum. The Progression Free Survival (PFS) rate and irPFS rate at 20-weeks was estimated as the proportion of patients who were disease free to progress and survive at 20 weeks after the start of palivizumab. Patients with disease progression prior to 20 weeks or participated in >20 weeks when checking study data were included in the analysis used to estimate the 20-week pfs (irpfs) rate. Patients who withdraw prematurely due to toxic or malignant disease and therefore do not have a 20-week tumor rating are considered to have progressive disease. Orr (irorr) is the proportion of patients who achieve the best overall response for CR or PR (irCR or irPR). Patients long enough to have an assessment of tumor response in the study were included in the analysis used to estimate response rates. In those that respond (CR or PR), the duration of the response is the time of the first RECIST response to the time of disease progression and is examined at the last evaluable tumor assessment for non-progressing responders.

PFS and irPFS are defined as the time from the date of initial dose to the date of disease progression or death for any reason, whichever occurs first. PFS and irPFS were examined on the date of recording the last evaluable tumor assessment for the absence of progressive disease in surviving and progression free patients. Overall Survival (OS) is defined as the time from the initial dose date to death for any reason. For patients who remained alive at the time of analysis, the OS time was checked on the last date that the patient was known to be alive. Survival time was summarized by the kaplan-meier method. As a post hoc analysis, a log rank test was used to compare population a and population B, and the hazard ratio was estimated based on the Cox model.

Correlation of% CEA reduction to PFS or OS after 1 cycle was assessed based on Cox regression model using signature analysis. For the relevant studies, a non-parametric wilcoxon test was used to compare mutation load between MMR deficient and MMR active patients. The effects of baseline mutational burden and immune markers on response and survival time were examined using logistic regression and Cox regression, respectively.

Immunohistochemistry and image analysis

The fraction of malignant cells presenting the membranous pattern of B7-H1 expression and the percentage at the invasive leading edge were quantified by three pathologists (r.a.a., f.b., and j.m.t.) as previously reported 9, 10. Image analysis was used to determine the number of CD8 Diaminobenzidine (DAB) stained cells. For slides stained with H & E in each case, we identified the following regions: i) tumors ii) invasive frontiers (boundaries between malignant and non-malignant tissues), and iii) normal tissues. CD8 stained slides were scanned AT 20 x equivalent magnification (0.49 microns per pixel) on an Aperio ScanScope AT. Regions corresponding to tumor, invasive leading edge and normal tissue (superior, from H & E) were infused on separate layers using Aperio ImageScope v12.1.0.5029.

CD8 positive lymphocyte density in each of the above regions was calculated using a custom algorithm implemented in PIP 11. The results were converted to Deepzoom images using the VIPS library 12 and observed using an OpenSeadragon viewer (http:// OpenSeadragon.

Reference only to example 3

Bacher JW, Flanagan LA, Smalley RL, et al. Development of a fluorescent multiplex assay for detecting MSI-High tumors (Development of a fluorescent multiplex assay for detection of MSI-High tumors). Disease markers 2004; 20: 237-50.

Murphy KM, Zhang S, Geiger T, et al. Comparison of the microsatellite instability analysis system and the Bethesda plate for the determination of microsatellite instability in colorectal cancer (compare of the microsatellite instability analysis system and the Bethesda plate for the determination of microsatellite instability in microbial cameras). Journal of molecular diagnostics (The Journal of molecular diagnostics): JMD 2006; 8: 305-11.

Jones S, Anagnostou V, Lytle K, et al. Personalized genomic analysis for cancer mutation discovery and interpretation (Personalized genetic analysis and interpretion). 2015 in scientific translational medicine (Science translational medicine); 7: 283ra 53.

Sausen M, Leary RJ, Jones S, et al. Integrated genomic analysis identified ARID1A and ARID1B variations (Integrated genetic analytes ARID1A and ARID1B alterations in the childhood cancer neuroblastoma). 2013 in Nature genetics (Nature genetics); 45: 12-7.

Needleman SB, Wunsch CD. A general method (A general method of applying reagents to the search for peptides in the amino acid sequence of two proteins) is suitable for exploring the similarity of the amino acid sequences of two proteins. 1970 journal of molecular biology (j.moi.biol.); 48: 443-53.

Lundegaard C, Lamberth K, Harndahl M, Buus S, Lund O, Nielsen M. NetMHC-3.0: accessible predicted accurate net of MHC class I affinities for humans, mice and monkeys for peptides of length 8 to 11 (NetMHC-3.0: secure web accessible ligands of human, mouse and monkey MHC class I affinities for peptides of length 8-11). Nucleic acids research (Nucleic acids research) 2008; 36: w509-12.

Buyse M, Michiels S, Sargent DJ, Grothey A, Matheson A, de Gramont A. Biomarkers (Integrating biomarkers in clinical trials) were integrated in clinical trials. Expert review of molecular diagnostics 2011; 11: 171-82.

Wolchok JD, hoss A, O' Day S, et al. Guidelines for the assessment of immunotherapeutic activity in solid tumors: criteria for immune-related responses (Guidelines for the evaluation of immune therapy activity in soluble tumors: immune-related response criteria). Clinical cancer study: an American Cancer Research institute's journal of officials (Clinical Cancer Research: an of the American Association for Cancer Research) 2009; 15: 7412-20.

Llosa NJ, Cruise M, Tam A, et al. The strong immune microenvironment of microsatellite-contained colon cancer by multiple countermeasures against suppressor checkpoints balances The microsatellite-unstable colon cancer. Cancer findings (Cancer Discov) 2015: 43-51.

Taube JM, Anders RA, Young GD, et al. Co-localization with the Inflammatory Response expressed by B7-H1 in Human Melanocytic Lesions Supports an Adaptive Resistance Mechanism for Immune evasion (coordination of Inflammatory responses with B7-H1 Expression in Human melanotic facilities Supports an Adaptive Resistance Mechanism for Immune Escape). 2012 scientific translation Medicine; 4: 127ra 37.

Cuka N, Hempel H, Sfanos K, De Marzo A, Cornish T. PIP: open Source Framework for multi-threaded Image Analysis of Whole Slide Images (PIP: An Open Source frame for Multi-threaded Image Analysis of white Slide Images). 2014, LABORATORY INVESTIGATIONs (LABORATORY information); 94: 398A-A.

Cupitt J, Martinez K. VIPS: image Processing Systems (VIPS) for large images. Electronic imaging: science and Technology (Electronic Imaging: Science & Technology); 1996: the International Society of Optics and Photonics (International Society for Optics and Photonics). Pages 19-28.

Example 4

Patient's health

41 consecutive patients were enrolled and treated between 2013, month 9 and 2015, month 1. (Table 1). Patients known to have tumors with mismatch repair or to have tumors of unknown status that were subsequently tested were enrolled, including those that pursued clinical trial selection. One patient in the MMR-deficient CRC population participated in IRB eligibility to allow a level 3 bilirubin level. A total of 32 CRC patients were enrolled in both population a and population B. All CRC patients received >2 previous courses of chemotherapy (median 4) except one MMR active patient who had received one course of chemotherapy and one (non-PD 1-class) course of immunotherapy.

Nine subjects diagnosed with MMR-deficient solid tumors other than CRC participated in population C. All population C patients received >1 prior cancer treatments (median 2).

Example 5

Primary endpoint assessment

irORR and irPFS at 20 weeks (fig. 11 (table S2)) were 40% (4 of 10 patients; 95% CI, 12% to 74%) and 78% (7 of 9 patients; 95% CI, 40% to 97%) for population a, and 71% (5 of 7 patients; 95% CI, 29% to 96%) and 67% (4 of 6 patients; 95% CI, 22% to 96%) for population C. In population B, which consisted of patients with MMR-active CRC, irorR and 20-week irPFS were 0% (95% CI, 0% to 20%) and 11% (2 out of 18 patients; 95% CI, 1% to 35%). Both MMR-deficient population a and population C reached their predetermined early-arrest rules for efficacy when four subjects were disease-free at 20 weeks and four objective responses were observed based on immune-related response criteria (fig. 11 (table S2); obtained online in New England Journal of Medicine (New England of Medicine); incorporated by reference herein; and the supplementary methods described above).

Median time to follow for patients was 32 weeks (range, 5 to 51 weeks) for patients with MMR-deficient CRC (population a), 12 weeks (range, 2 to 56 weeks) for patients with MMR-active CRC (population B), and 12 weeks (range, 4 to 42 weeks) for patients with MMR-deficient non-CRC tumors (population C). All patients with 20 weeks irPFS can be evaluated for at least 20 weeks of follow-up.

Example 6

Radiation evaluation

Objective responses were achieved by four RECIST criteria (40%; 95% CI, 12% to 74%) among ten evaluable MMR-deficient CRC patients in population a (table 2, fig. 1 and fig. 3 (S2)). Unless the patient is scanned for 12-weeks, the patient is considered to be unevaluable. Disease control rate is defined as the fraction of patients achieving objective response or their disease stabilization and is 90% in population a (9 out of 10 patients; 95% CI, 55% to 100%).

Of seven evaluable patients with MMR-deficient cancer types other than CRC, five (71%; 95% CI, 29% to 96%) involved in population C achieved objective responses using the RECIST criterion (table 2, fig. 3(S2) and fig. 1) and disease control rate was 71% (5 out of 7 patients; 95% CI, 29% to 96%).

Patients in population C responded faster than patients in population a (median time to response by RECIST 12 weeks versus 28 weeks, p ═ 0.03). Furthermore, objective responses were achieved for all six MMR-deficient tumors not associated with Lynch syndrome (100%), while only three of the eleven tumors associated with Lynch syndrome (27%) (Table S3; p ═ 0.009; available online in the New England Journal of Medicine; and incorporated herein by reference). No other baseline features showed statistically significant correlation with objective responses.

In 18 patients with MMR active CRC in population B, no objective response was observed using RECIST criteria (table 2, fig. 3(S2) and fig. 1), and the disease control rate was 11% (2 out of 18 patients; 95% CI, 1% to 35%).

All patients who achieved a response by RECIST criteria (fig. 11 (table 2)) also achieved a response by immune-related response criteria (fig. 11 (table S2)).

Example 7

Survival period

In population a, patients with MMR-deficient CRC did not reach median progression-free survival (PFS) and median Overall Survival (OS) (fig. 2). In contrast, patients with MMR-active cancer in population B achieved PFS for only 2.2 months (95% CI, 1.4 to 2.8) and a median OS of 5.0 months (95% CI, 3.0 to no estimate). In population C (MMR-deficient non-CRC), the median PFS was 5.4 months (95% CI, 3 to no-estimable) and did not reach the median OS.

A post hoc (figure 2) comparison of MMR-deficient and active CRC populations shows Hazard Ratio (HR) (HR ═ 0.10; 95% CI, 0.03 to 0.37; p <0.001) and overall survival (HR ═ 0.22; 95% CI, 0.05 to 1.00; p ═ 0.05) for disease progression, favoring patients with MMR-deficient CRC.

To assess whether differences in survival could be due to predicted differences, we measured the duration of patients who had been diagnosed with metastatic disease and the clinical performance of patients on their previous course of treatment prior to enrollment. We found that there was no significant difference in the duration of metastatic disease (p 0.77; log rank test) or median PFS (p 0.60, log rank test) for their previous regimen between MMR deficient versus MMR active CRC patients (fig. 4 (S3)). We also performed additional multivariate analyses of PFS and OS to examine the differences in outcome between MMR deficient CRC and MMR active tumors that modulate the time elapsed since the initial diagnosis. After adjusting for this possible difference, the magnitude of the hazard ratio was maintained for PFS (HR 0.04, 95% CI 0.01 to 0.21, P <0.001) and OS (HR 0.18, 95% CI 0.03 to 1.01, P ═ 0.05), which represent the different effects of parizumab between MMR deficient and active tumors.

Example 8

Safety assessment

Adverse events occurring in > 5% of patients are listed in table 3. Adverse events were selected to include rash/pruritus (24%), thyroiditis/hypothyroidism/hypophysitis (10%) and asymptomatic pancreatitis (15%). Although in small numbers, thyroid dysfunction is limited to MMR deficient populations (table 3).

Example 9

Tumor markers

In both CRC populations, the baseline CEA was evaluable and above the upper limit of normal (3mg/d1) in 29 of the 32 patients prior to enrollment. Major CEA reduction occurred in seven of ten patients with MMR-deficient CRC, but not in 19 patients with MMR-active CRC, with CEA being evaluable (fig. 1 and fig. 5 (S4)). In non-CRC MMR deficient patients, the tumor marker levels (CEA, CA19-9, or CA-125) were higher than the upper limit of normal in four patients. A > 70% reduction in CA19-9 or CA-125 occurred in three of these four patients. Tumor marker kinetics for all 3 populations are shown in figure 1. The level of CEA reduction after 1 dose (between 14 and 28 days) of pellizumab predicts both progression-free (p-0.01) and overall survival outcome p-0.02). CEA responses appear well before radiologic confirmation of disease control (range, 10 to 35 weeks). In contrast, patients who progressed showed a rapid biomarker rise within 30 days of starting treatment. Thus, the change in CEA levels was significantly prior and correlated with the final radiation change.

Example 10

Genomic analysis

Analysis of all exome sequences showed an average of 1,782 individual cell mutations per tumor (non-parametric wilcoxon test p 0.007) in MMR deficient patients (n 9) compared to 73 mutations per tumor in MMR active patients (n 6) (fig. 6A-6B (S5); see also table S3, obtained online in New England Journal of Medicine (New England Journal of Medicine); incorporated herein by reference). Most of these mutations (63%) were predicted to change amino acids.

The immunogenicity potential of these mutations was then assessed in the context of individual MHC haplotypes for each patient. We thus identified mean values of 578 and 21 potential mutations associated with New antigens from tumors of MMR-deficient and MMR-active patients, respectively (table S3; obtained online in New England Journal of Medicine (New England Journal of Medicine; incorporated herein by reference). The fraction of potential mutation-associated neoantigens among all somatic mutations was similar in both populations (mean values of 32% and 29% among MMR-deficient and active patients, respectively). High numbers of somatic and potential mutation-associated neoantigens were associated with improved progression-free survival and with trends favoring objective responses (FIG. 13(S5) and FIG. 12 (Table S4); also obtained online in New England Journal of Medicine (New England of Medicine); incorporated by reference herein).

Example 11

Immunohistochemistry

Expression of CD8 and PD-L1 was assessed intratumorally and at the invasive front of the tumor by immunohistochemistry in 30 cases where tumor tissue was available (fig. 7 (S6); also obtained online in New England Journal of Medicine (New England Journal of Medicine); incorporated herein by reference). Tumors from patients in populations a and C contained CD8 positive lymphocytes that were indeed more dense than tumors from patients in population B (fig. 8 (S7); p ═ 0.10), and CD8 markers were associated with trends that contributed to objective responses and stabilized disease (fig. 9(S8) and fig. 13 (table S5); also obtained online in New England Journal of Medicine; incorporated herein by reference). This CD 8-positive lymphatic infiltration was particularly pronounced at the aggressive front of the tumor (fig. 8 (S7); p ═ 0.04). Significant membranous PD-L1 expression appeared only in MMR deficient patients and was significant on Tumor Infiltrating Lymphocytes (TIL) and tumor associated macrophages located at the invasive leading edge of the tumor (fig. 8 (S7); p ═ 0.04). Expression of CD8 and PD-L1 was not statistically correlated with PFS or OS (fig. 13 (table S5)).

TABLE 1 demographic and baseline characteristics of patients

MMR, mismatch repair; CRC, colorectal cancer

¹MMR deficient CRC vs MMR active CRC

²ECOG, eastern tumor cooperative group

TABLE 2 Targeted RECIST response

¹Original PR at week 12 converted to CR at week 20

²One PR at 12 weeks

³If the patient does not take a week 12 scan due to clinical progression, the patient is considered to be unevaluable.

⁴Disease control rate is defined as the percentage of patients who have a complete response, partial response, or stable disease for 12 weeks or more.

⁵Non-responsive record for MMR active CRC patients

TABLE 3 drug related adverse events

¹Adverse events occurred in greater than 5% of patients

²All cases of pancreatitis are asymptomatic

³One pneumonia incidence (2%)

Reference to the literature

The disclosure of each of the cited documents is expressly incorporated herein.

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