Patent Application: US-201515523451-A

Abstract:
blockade of immune checkpoints such as cytotoxic t - lymphocyte antigen - 4 and programmed death - 1 shows promise in patients with cancer . inhibitory antibodies directed at these receptors have been shown to break immune tolerance and promote anti - tumor immunity . these agents work particularly well in patients with a certain category of tumor . such tumors may be particularly susceptible to treatment because of the multitude of neoantigens which they produce .

Description:
the inventors have found that immune checkpoint inhibitors work best in tumors with high mutation burdens . furthermore , tumors deficient in mismatch repair are particularly susceptible to a particular form of immunotherapy because this phenotype results in ongoing accumulation of mutations at a high frequency . the inventors have developed a treatment for cancer patients that display the microsatellite instability phenotype or other high mutational burden . the treatment involves an inhibitory antibody for an immune checkpoint . such checkpoints include pd - 1 , ido , ctla - 4 , pd - l1 , and lag - 3 . other immune checkpoints can be used as well . antibodies can be administered by any means that is convenient , including but not limited to intravenous infusion , oral administration , subcutaneous administration , sublingual administration , ocular administration , nasal administration , etc . microsatellite instability ( msi ) tumors are deficient in dna mismatch repair which leads to a high rate of spontaneous mutations and the potential for the expression of neo - antigens . furthermore , similar to melanoma , in msi positive colon cancers , there is often prominent lymphocyte infiltration . any tumors that are msi or otherwise high mutational burden may be treated according to the invention . they may be tested for the attribute of msi according to any method known in the art , including but not limited that described in example 1 below . any of one or more msi markers can be tested to determine an msi phenotype . samples may be tested for high mutational burden by identifying tumors with 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 tumor genome . high mutational burden means a large number of somatic mutations in the tumor relative to normal tissues of the individual . an average number of somatic mutations in a non - msi tumor is about 70 somatic mutations . any type of tumor that displays the msi phenotype or a high mutational burden may be tested and / or treated according to the invention . these include without limitation cancers of the colon , gastric , endometrial , cholangiocarcinoma , pancreatic , and prostate cancer . tumors of the ampulla , biliary , brain , including glioma , breast , lung , skin , esophagus , liver , kidney , ovaries , sarcoma , uterus , cervix , bladder , testes , oral cavity , tongue , and small and large bowel may also be tested and / or treated . testing of msi can be accomplished by any means known in the art . one or more of the following markers may be tested : five nearly monomorphic mononucleotide repeat markers ( bat - 25 , bat - 26 , mon0 - 27 , nr - 21 and nr - 24 ) and two highly polymorphic pentanucleotide repeat markers ( penta c and penta d ). in one commercial system which can be used , fluorescently labeled primers ( marker panel ) are used for co - amplification of all seven of the above named markers . fragments are detected after amplification for assignment of genotype / phenotype . samples that can be tested for msi include tumor tissue as well as body fluids that contain nucleic acids shed from tumors . testing for tumor dna in such tissues and body fluids is well known . types of antibodies which can be used include any that are developed for the immune checkpoint inhibitors . these can be monoclonal or polyclonal . they may be single chain fragments or other fragments of full antibodies , including those made by enzymatic cleavage or recombinant dna techniques . they may be of any isotype , including but not limited to igg , igm , ige . the antibodies may be of any species source , including human , goat , rabbit , mouse , cow , chimpanzee . the antibodies may be humanized or chimeric . the antibodies may be conjugated or engineered to be attached to another moiety , whether a therapeutic molecule or a tracer molecule . the therapeutic molecule may be a toxin , for example . the data from the small phase 2 trial of pembrolizumab to treat tumors with and without deficiency of mmr supports the hypothesis that mmr - deficient tumors are more responsive to pd - 1 blockade than are mmr - proficient tumors . mmr - deficiency occurs in many cancers , including those of the colorectum , uterus , stomach , biliary tract , pancreas , ovary , prostate and small intestine 18 , 34 - 42 . patients with mmr - deficient tumors of these types also benefit from anti - pd - 1 therapy , as may patients whose tumors contain other dna repair deficiencies , such as those with mutations in pold , pole , or myh . 18 , 43 , 44 the hypothesis that mmr - deficient tumors stimulate the immune system is not a new idea 45 , and has been supported by the dense immune infiltration and th1 - associated cytokine - rich environment observed in mmr - deficient tumors . 19 - 22 , 46 a recent study refined these classic observations by showing that the mmr - deficient tumor microenvironment strongly expressed several immune checkpoint ligands including pd - 1 , pd - l1 , ctla - 4 , lag - 3 and ido , indicating that their active immune microenvironment is counterbalanced by immune inhibitory signals that resists tumor elimination 47 . that the immune infiltrate associated with mmr - deficient carcinomas was directed at neoantigens was the most likely explanation for both the old and new findings . the correlation of higher mutational load and higher response rate to anti - ctla - 4 in melanoma 41 and anti - pd - 1 in lung cancer 48 provide further support for the idea that mana recognition is an important component of the endogenous anti - tumor immune response . based on the results of the current and previous studies , we suggest that the greatly (& gt ; 20 - fold ) increased number of mutation - associated neoantigens resulting from mmr deficiency ( fig1 ( table s4 ); also available on line at new england journal of medicine ; incorporated by reference herein ) is the basis for the enhanced anti - pd - 1 responsiveness of this genetically defined subset of cancers . though our estimates for the number of mutation - associated neoantigens in tumors is based only on in silico predictions of binding - affinity , this suggestion is consistent with the observation that mmr - proficient tumors have far less infiltration of lymphocytes than mmr - deficient tumors ( fig7 ( s 6 ), fig8 ( s 7 ) and fig1 ( table s5 ); available on line at new england journal of medicine ; incorporated by reference herein ). recent studies 49 , 50 show that only a tiny proportion of predicted neo - epitopes are actually presented on the cell surface with mhc and are targets of endogenous t cell responses . it seems likely , though that the number of predicted mutation - associated neoantigens is proportionate to the number of actual mutation - associated neoantigens , and tumors with a high number of actual mutation - associated neoantigens are more likely to stimulate the immune system to react against the tumor . alternative mechanisms underlying the difference in anti - pd - 1 responsiveness between mmr - deficient and mmr - proficient tumors should also be considered . for example , different signaling pathways activated in mmr - deficient and mmr - proficient tumors may result in differences in secretion of soluble factors that could result in differential activation of the pd - 1 pathway within the tumor microenvironment 26 - 28 . genetic differences could effect epigenetic differences that alter the expression of tumor - associated self - antigens that in turn could alter the antigenicity of the tumor . experimental analyses of antigen - specific immune responses as well as changes in immune microenvironments should help to define the relative contribution of these factors to the striking 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 , like cea , corresponded with clinical benefit after a single dose of therapy . declines in cea levels preceded objective radiographic evidence by several months ; perhaps other biomarkers such as circulating tumor dna ( ctdna ) may also be beneficial as surrogate markers of early response . 51 , 52 second , our results suggest that the evaluation of tumor genomes can help guide immunotherapy . they support the view that the number and type of alterations may prove useful for judging the potential utility of immune checkpoint inhibitors , even in mmr - proficient cancers 41 , 48 , 53 most importantly , our results demonstrate a new approach for the treatment of a specific class of tumors based solely on genetic status : i . e ., without regard to underlying tumor type . the above disclosure generally describes the present 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 invention . msi testing is already standardized and performed in clia - certified laboratories without need for assay development . archived tumor samples or newly obtained biopsies will be used for determining msi . msi status will be performed locally by clia certified immunohistochemistry ( ihc ) or pcr based tests for eligibility . evaluable patients will be confirmed using the msi analysis system from promega at johns hopkins . this test will determine msi status through the insertion or deletion of repeating units in the five nearly monomorphic mononucleotide repeat markers ( bat - 25 , bat - 26 , mon0 - 27 , nr - 21 and nr - 24 ). at least 2 msi loci are required to be evaluable in cohorts a and c . patients may be assigned to a new cohort and / or replaced based on the promega test results . treatment - refractory progressive metastatic cancer patients for this phase 2 study were recruited from three participating centers ( table 1 ). three cohorts were evaluated : cohort a was composed of patients with mmr - deficient colorectal adenocarcinomas ; cohort b was composed of patients with mmr - proficient colorectal adenocarcinomas ; and cohort c was composed of patients with mmr - deficient cancers of types other than colorectal . the protocol , which can be found at nejm . org , was approved by each site &# 39 ; s institutional review boards , and the study was conducted in accordance with the declaration of helsinki and the international conference on harmonization guidelines for good clinical practice . all the patients provided written informed consent before study entry . the principal investigator ( d . l .) and study sponsor ( l . a . d .) were responsible for oversight of the study . merck donated the study drug , reviewed the final drafts of the protocol and of this manuscript . the clinical study was primarily funded through philanthropic support . this phase 2 trial was conducted using a green - dahlberg two - stage design and consisted of the three parallel cohorts described above . the study agent , pembrolizumab ( merck ), was administered at 10 mg / kg intravenously every 14 days . pembrolizumab is a humanized monoclonal anti - pd - 1 antibody of the igg4 / kappa isotype that blocks the interaction between pd - 1 and its ligands , pd - l1 and pd - l2 . safety assessments were performed before each treatment . assessments of total tumor burden via measurements of serum biomarkers were performed at the start of each cycle . radiologic assessments were made at 12 weeks and every 8 weeks thereafter . further details concerning the clinical protocol are provided in the example 3 . tumors with genetic defects in mmr pathways are known to harbor thousands of somatic mutations , especially in regions of repetitive dna known as microsatellites . the accumulation of mutations in these regions of the genome is termed microsatellite instability ( msi ) 26 - 28 . mmr - status was assessed using the msi analysis system from promega in tumors , through the evaluation of selected microsatellite sequences particularly prone to copying errors when mmr is compromised 26 - 28 . see supplementary appendix for additional details . primary tumor samples and matched normal peripheral - blood specimens were obtained from a subset of subjects with mmr - deficient and others with mmr - proficient carcinomas where sufficient tumor tissue was available for exome sequencing 30 and hla haplotyping . to assess the potential for mutant peptide binding , somatic exome data combined with the individual patient &# 39 ; s mhc class i hla haplotype was applied to the an epitope prediction algorithm 31 , 32 . this algorithm provided an estimate of the total number of mutation - associated neoantigens in each tumor . additional details are provided in the supplementary appendix ( available on line at new england journal of medicine ; incorporated by reference herein ). the primary endpoints for cohorts a and b were immune - related objective response rate ( irorr ) and immune - related progression - free survival ( irpfs ) rate at 20 weeks assessed using immune - related response criteria ( irrc ) 33 . the primary endpoint for cohort c was irpfs rate at 20 weeks . immune - related criteria ( i . e , criteria used to evaluate immune - based therapies ) are based on radiographic responses , and unlike recist criteria , capture extent of disease after disease progression ; these criteria are defined and compared to recist v1 . 1 in fig1 ( table s1 ). response rate and pfs rate at 20 weeks were evaluated and reported in this study using recist v1 . 1 and irrc ( fig1 ( table s1 )). pfs and overall survival was summarized by kaplan - meier method . details of the hypothesis , the decision rules to reject the null hypotheses and early - stopping rules for efficacy and futility , and statistical methods are provided in the supplementary appendix . to be eligible for participation in this study , patients had to be at least 18 years of age , have histologically confirmed evidence of previously - treated , progressive carcinoma . all patients underwent mmr status testing prior to enrollment . all patients had at least one measurable lesion as defined by the response evaluation criteria in solid tumors ( recist ), version 1 . 1 , an eastern cooperative oncology group ( ecog ) performance - status score of 0 or 1 , and adequate hematologic , hepatic , and renal function . eligible patients with crc must have received at least 2 prior cancer therapies and patients with other cancer types must have received at least 1 prior cancer therapy . patients with untreated brain metastases , history of hiv , hepatitis b , hepatitis c , clinically significant ascites / effusions , or autoimmune disease were excluded . initial drafts of the manuscript were prepared by a subset of the authors and all authors contributed to the final manuscript . all the authors made the decision to submit the manuscript for publication . the principal investigator and study sponsor vouch for the accuracy and completeness of the data reported as well as adherence to the protocol . hla - a , hla - b and hla - c sequence based typing can be divided into three distinct steps , as described below . a generic , a * 02 specific , b generic , b group specific , c generic and c * 07 specific pcr and sequencing mixes were made in the jhu core facility . celera &# 39 ; s alleleseqr hla - b sequence based typing kit was used for b generic sbt . the hla - a typing scheme is composed of two pcr reactions , a generic and a * 02 specific . a generic amplicon encompasses partial exon 1 — partial exon 5 . a * 02 amplicon encompasses partial intron 1 — partial exon 5 . hla - b typing scheme is composed of two pcr reactions , b generic and b group specific . the b generic pcr is a multiplexed reaction containing two pcr amplicons encompassing exon 2 — exon 3 and exon 4 — exon 7 . b group specific amplicon encompasses partial intron 1 — partial exon 5 . hla - c typing scheme is composed of two pcr reactions , c generic and c * 07 specific . c generic and c * 07 specific amplicons encompasses exons 1 - 7 . the specificity of the hla - a and b pcr employed amplitaq gold dna polymerase . the geneamp high fidelity enzyme is used for the hla - c and c * 07 pcr mixes . this enzyme is a mix of two polymerases : amplitaq dna polymerase ( non - proofreading polymerase ) and a proofreading polymerase . this enzyme mix is necessary to produce efficient and robust amplification of the larger full length hla - c amplicon . pcr product purification was performed using exonuclease i and shrimp alkaline phosphatase the a generic and b generic amplicons were bi - directionally sequenced for exons 2 , 3 , 4 . the c generic amplicon was bi - directionally sequenced for exons 2 , 3 and sequenced in a single direction for exons 1 , 4 , 5 , 6 , 7 . a * 02 specific , b group specific and c * 07 specific amplicons were sequenced in a single direction for exons 2 , 3 . all sequencing reactions were performed with big dye terminator v1 . 1 from applied biosystems and sequenced with an abi prism 3500xl genetic analyzer . conexio genomic &# 39 ; s “ assign sbt ” allele assignment software was used to process the data files . six slides of tumor and normal ( uninvolved lymph node or margin of resection ) were cut ( 5 microns each ), deparaffinized ( xylene ), and one stained with hematoxylin and eosin ( h + e ). a tumor area containing at least 20 % neoplastic cells , designated by a board - certified anatomic pathologist was macrodissected using the pinpoint dna isolation system ( zymo research , irvine , calif . ), digested in proteinase k for 8 hours and dna was isolated using a qiaamp dna mini kit ( qiagen , valencia , calif .). msi was assessed using the msi analysis system ( promega , madison , wis . ), composed of 5 pseudomonomorphic mononucleotide repeats ( bat - 25 , bat - 26 , nr - 21 , nr - 24 and mono - 27 ) to detect msi and 2 - pentanucleotide repeat loci ( pentac and pentad ) to confirm identity between normal and tumor samples , per manufacturer &# 39 ; s instructions . following amplification of 50 - 100 ng dna , the fluorescent pcr products were sized on an applied biosystems 3130xl capillary electrophoresis instrument ( invitrogen , calsbad , calif .). pentanucleotide loci confirmed identity in all cases . controls included water as a negative control and a mixture of 80 % germline dna with 20 % msi cancer dna as a positive control . the size in bases was determined for each microsatellite locus and tumors were designated as msi if two or more mononucleotide loci varied in length compared to the germline dna . samples provided as ffpe blocks or frozen tissue underwent pathological review to determine tumor cellularity . tumors were macrodissected to remove contaminating normal tissue , resulting in samples containing & gt ; 20 % neoplastic cells . matched normal samples were provided as blood , saliva or normal tissue obtained from surgery . sample preparation , library construction , exome capture , next generation sequencing , and bioinformatics analyses of tumor and normal samples were performed at personal genome diagnostics , inc . ( baltimore , md .). in brief , dna was extracted from frozen or formalin - fixed paraffin embedded ( ffpe ) tissue , along with matched blood or saliva samples using the qiagen dna ffpe tissue kit or qiagen dna blood mini kit ( qiagen , ca ). genomic dna from tumor and normal samples were fragmented and used for rumina truseq library construction ( illumina , san diego , calif .) according to the manufacturer &# 39 ; s instructions or as previously described4 . briefly , 50 nanograms ( ng )— 3 micrograms ( μg ) of genomic dna in 100 microliters ( μl ) of te was fragmented in a covaris sonicator ( covaris , woburn , mass .) to a size of 150 - 450 bp . to remove fragments smaller than 150 bp , dna was purified using agencourt ampure xp beads ( beckman coulter , in ) in a ratio of 1 . 0 to 0 . 9 of pcr product to beads twice and washed using 70 % ethanol per the manufacturer &# 39 ; s instructions . purified , fragmented dna was mixed with 36 μl of h2o , 10 μl of end repair reaction buffer , 5 μl of end repair enzyme mix ( cat # e6050 , neb , ipswich , mass .). the 100 μl end - repair mixture was incubated at 20 ° c . for 30 min , and purified using agencourt ampure xp beads ( beckman coulter , in ) in a ratio of 1 . 0 to 1 . 25 of pcr product to beads and washed using 70 % ethanol per the manufacturer &# 39 ; s instructions . to a - tail , 42 μl of end - repaired dna was mixed with 5 μl of 10 × da tailing reaction buffer and 3 μl of klenow ( exo -)( cat # e6053 , neb , ipswich , mass .). the 50 μl mixture was incubated at 37 ° c . for 30 min and purified using agencourt ampure xp beads ( beckman coulter , in ) in a ratio of 1 . 0 to 1 . 0 of pcr product to beads and washed using 70 % ethanol per the manufacturer &# 39 ; s instructions . for adaptor ligation , 25 μl of a - tailed dna was mixed with 6 . 7 μl of h2o , 3 . 3 μl of pe - adaptor ( ilumina ), 10 μl of 5 × ligation buffer and 5 μl of quick t4 dna ligase ( cat # e6056 , neb , ipswich , mass .). the ligation mixture was incubated at 20 ° c . for 15 min and purified using agencourt ampure xp beads ( beckman coulter , in ) in a ratio of 1 . 0 to 0 . 95 and 1 . 0 of pcr product to beads twice and washed using 70 % ethanol per the manufacturer &# 39 ; s instructions . to obtain an amplified library , twelve pcrs of 25 μl each were set up , each including 15 . 5 μl of h2o , 5 μl of 5 × phusion hf buffer , 0 . 5 μl of a dntp mix containing 10 mm of each dntp , 1 . 25 μl of dmso , 0 . 25 μl of illumina pe primer # 1 , 0 . 25 μl of illumina pe primer # 2 , 0 . 25 μl of hotstart phusion polymerase , and 2 μl of the dna . the pcr program used was : 98 ° c . for 2 minutes ; 12 cycles of 98 ° c . for 15 seconds , 65 ° c . for 30 seconds , 72 ° c . for 30 seconds ; and 72 ° c . for 5 min . dna was purified using agencourt ampure xp beads ( beckman coulter , in ) in a ratio of 1 . 0 to 1 . 0 of pcr product to beads and washed using 70 % ethanol per the manufacturer &# 39 ; s instructions . exonic or targeted regions were captured in solution using the agilent sureselect v . 4 kit according to the manufacturer &# 39 ; s instructions ( agilent , santa clara , calif .). the captured library was then purified with a qiagen minelute column purification kit and eluted in 17 μl of 70 ° c . eb to obtain 15 μl of captured dna library . ( 5 ) the captured dna library was amplified in the following way : eight 30 ul pcr reactions each containing 19 μl of h2o , 6 μl of 5 × phusion hf buffer , 0 . 6 μl of 10 mm dntp , 1 . 5 μl of dmso , 0 . 30 μl of illumina pe primer # 1 , 0 . 30 μl of illumina pe primer # 2 , 0 . 30 μl of hotstart phusion polymerase , and 2 μl of captured exome library were set up . the pcr program used was : 98 ° c . for 30 seconds ; 14 cycles ( exome ) or 16 cycles ( targeted ) of 98 ° c . for 10 seconds , 65 ° c . for 30 seconds , 72 ° c . for 30 seconds ; and 72 ° c . for 5 min . to purify pcr products , a nucleospin extract ii purification kit ( macherey - nagel , pa ) was used following the manufacturer &# 39 ; s instructions . paired - end sequencing , resulting in 100 bases from each end of the fragments for exome libraries and 150 bases from each end of the fragment for targeted libraries , was performed using illumina hiseq 2000 / 2500 and iilumina miseq instrumentation ( ilumina , san diego , calif .). primary processing of next - generation sequencing data and identification of putative somatic mutations3 somatic mutations were identified using variantdx custom software ( personal genome diagnostics , baltimore , md .) for identifying mutations in matched tumor and normal samples . prior to mutation calling , primary processing of sequence data for both tumor and normal samples were performed using illumina casava software ( v1 . 8 ), including masking of adapter sequences . sequence reads were aligned against the human reference genome ( version hg18 ) using eland with additional realignment of select regions using the needleman - wunsch method 5 . candidate somatic mutations , consisting of point mutations , insertions , and deletions were then identified using variantdx across the either the whole exome or regions of interest . variantdx examines sequence alignments of tumor samples against a matched normal while applying filters to exclude alignment and sequencing artifacts . in brief , an alignment filter was applied to exclude quality failed reads , unpaired reads , and poorly mapped reads in the tumor . a base quality filter was applied to limit inclusion of bases with reported phred quality score & gt ; 30 for the tumor and & gt ; 20 for the normal . a mutation in the tumor was identified as a candidate somatic mutation only when ( i ) distinct paired reads contained the mutation in the tumor ; ( ii ) the number of distinct paired reads containing a particular mutation in the tumor was at least 10 % of read pairs ; ( iii ) the mismatched base was not present in & gt ; 1 % of the reads in the matched normal sample as well as not present in a custom database of common germline variants derived from dbsnp ; and ( iv ) the position was covered in both the tumor and normal at & gt ; 150 ×. mutations arising from misplaced genome alignments , including paralogous sequences , were identified and excluded by searching the reference genome . candidate somatic mutations were further filtered based on gene annotation to identify those occurring in protein - coding regions . functional consequences were predicted using snpeff and a custom database of ccds , refseq and ensembl annotations using the latest transcript versions available on hg18 from ucsc ( https :// genome . ucsc . edu /). predictions were ordered to prefer transcripts with canonical start and stop codons and ccds or refseq transcripts over ensembl when available . finally mutations were filtered to exclude intronic and silent changes , while retaining mutations resulting in missense mutations , nonsense mutations , frameshifts , or splice site alterations . a manual visual inspection step was used to further remove artifactual changes . somatic frameshift , insertions , deletions , and missense mutations predicted to result in an amino acid change were analyzed for potential mhc class i binding based on the individual patient &# 39 ; s hla haplotype . our initial analysis focused on hla - a and hla - b . amino acid mutations were linked to their corresponding ccds accession number and in instances where this was unavailable , either a refseq or ensemble transcript was used to extract the protein sequence . to identify 8mer , 9mer , and 10mer epitopes , 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 a predicted affinity of & lt ; 50 nm were considered to be strong potential binders and epitopes with a predicted affinity of & lt ; 500 nm were considered to be weak potential binders as suggested by the netmhc group6 . to further refine the total neoantigen burden , we repeated that same process for the complementary wild - type peptide for each mutant peptide . we then filtered for mutant peptides that were strong potential binders when the complementary wild - type peptide was predicted a weak potential binder . these mutant peptides are referred to as mutation - associated neoantigens ( mana ). in the event that a patient had a ( e . g ., cases 1 , 17 and 21 ) single mhc haplotype not supported by netmhc 3 . 4 , the individual haplotype was not included in our analysis . this trial was conducted using a parallel two - stage design to simultaneously evaluate the efficacy of mk - 3475 and msi as a treatment selection marker for anti - pd - 1 therapy . it consisted of two - stage phase 2 studies in parallel in the three cohorts of patients described in the text . the study agent , mk - 3475 , was administered at 10 mg / kg intravenously every 14 days . for each of cohort a and b , the co - primary endpoints were progression - free - survival ( irpfs ) at 20 weeks and objective response ( iror ) assessed using immune related criteria . a step - down gatekeeping procedure was used to preserve the overall type i error . a two - stage green - dahlberg design was used to evaluate irpfs , with interim and final analysis after 15 and 25 patients , respectively . at stage 1 , ≧ 1 of 15 free - of - progression at 20 weeks were required to proceed to the second stage , and ≧ 4 of 25 free - of - progression at 20 weeks were then required to proceed to test for iror , with ≧ 4 of 25 responders ( ircr or irpr ) indicating promising efficacy in that cohort . each cohort could be terminated for efficacy as soon as ≧ 4 free - of - progression at 20 weeks and ≧ 4 responses were confirmed , or be terminated for futility as soon as 0 of 15 in stage 1 were free - of - progression at 20 weeks or ≧ 22 subjects had disease progression by 20 weeks . this design achieves 90 % power to detect a 20 - week irpfs rate of 25 % and 80 % power to detect an iror rate ( irorr ) of 21 %, with an overall type i error of 0 . 05 at the null hypothesis of 20 - week irpfs rate of 5 % and irorr of 5 %. for cohort c , the primary endpoint was irpfs at 20 weeks . a two - stage green - dahlberg two - stage design was used , with an interim and final analysis after 14 and 21 patients ; at stage 1 , ≧ 1 of 14 free - of - progression at 20 weeks were required to proceed to the second stage , with ≧ 4 of 21 free - of - progression at 20 weeks at the end indicating adequate efficacy in cohort c . the cohort could be terminated as soon as ≧ 4 free - of - progression at 20 weeks were confirmed . the design has 81 % power to detect a 20 - week irpfs rate of 25 % with a 5 % type i error at the null hypothesis of 20 - week irpfs rate of 5 %. response and progression were evaluated using recist v1 . 1 and the immune - related response criteria ( irrc ) adopted from wolchok et al . 8 , which uses the sum of the products of bidimensional tumor measurements and incorporates new lesions into the sum . progression - free survival ( pfs ) rates and irpfs rate at 20 - weeks was estimated as the proportion of patients who were free - of - disease progression and alive at 20 weeks after the initiation of pembrolizumab . patients who had disease progression prior to 20 weeks or were enrolled for & gt ; 20 weeks at the time the study data were collated were included in the analysis for estimating 20 - week pfs ( irpfs ) rate . patients who dropped out early due to toxicities or worsening disease and therefore did not have 20 - week tumor assessment were considered as having progressive disease . orr ( irorr ) was the proportion of patients who achieved best overall response of cr or pr ( ircr or irpr ). patients who were in the study long enough to have tumor response evaluations were included in the analysis for estimating response rates . among those who responded ( cr or pr ), duration of response was the time of first recist response to the time of disease progression , and was censored at the last evaluable tumor assessment for responders who had not progressed . pfs and irpfs were defined as the time from the date of initial dose to the date of disease progression or the date of death due to any cause , whichever occurred first . pfs and irpfs were censored on the date of the last evaluable tumor assessment documenting absence of progressive disease for patients who were alive and progression - free . overall survival ( os ) was defined as the time from the date of initial dose to death due to any cause . for patients who were still alive at the time of analysis , the os time was censored on the last date the patients were known to be alive . survival times were summarized by the kaplan - meier method . as a post hoc analysis , log - rank tests were used to compare cohort a and b and hazard ratios were estimated based on cox models . the association of percent cea decline after 1 cycle with pfs or os was assessed using landmark analysis based on cox regression models . for correlative studies , non - parametric wilcoxon test was used to compare mutational load between mmr - deficient and mmr - proficient patients . the effects of baseline mutational burden and immune markers on response and survival times were examined using logistic regression and cox regression , respectively . the fraction of malignant cells exhibiting a membranous pattern of b7 - h1 expression and the percentage at the invasive front were quantified by three pathologists ( r . a . a ., f . b ., and j . m . t .) as previously reported9 , 10 . image analysis was used to determine the number of cd8 diaminobenzidine ( dab )- stained cells . using the h & amp ; e - stained slide for each case , we identified the following regions : i ) tumor , ii ) invasive front ( the boundary between malignant and non - malignant tissue ), and iii ) normal tissue . the cd8 - stained slides were scanned at 20 × equivalent magnification ( 0 . 49 micrometers per pixel ) on an aperio scanscope at . regions corresponding to tumor , invasive front and normal tissue ( above , from the h & amp ; e ) were annotated on separate layers using aperio imagescope v12 . 1 . 0 . 5029 . cd8 - positive lymphocyte density was calculated in each of the above regions using a custom algorithm implemented in pip11 . results were converted to deepzoom images using the vips libraryl2 and visualized using the openseadragon viewer ( http :// openseadragon . github . io ). 1 . bacher j w , flanagan l a , smalley r l , et al . development of a fluorescent multiplex assay for detection of msi - high tumors . disease markers 2004 ; 20 : 237 - 50 . 2 . murphy k m , zhang s , geiger t , et al . comparison of the microsatellite instability analysis system and the bethesda panel for the determination of microsatellite instability in colorectal cancers . the journal of molecular diagnostics : jmd 2006 ; 8 : 305 - 11 . 3 . jones s , anagnostou v , lytle k , et al . personalized genomic analyses for cancer mutation discovery and interpretation . science translational medicine 2015 ; 7 : 283ra53 . 4 . sausen m , leary r j , jones s , et al . integrated genomic analyses identify arid1a and arid1b alterations in the childhood cancer neuroblastoma . nature genetics 2013 ; 45 : 12 - 7 . 5 . needleman s b , wunsch c d . a general method applicable to the search for similarities in the amino acid sequence of two proteins . journal of molecular biology 1970 ; 48 : 443 - 53 . 6 . lundegaard c , lamberth k , harndahl m , buus s , lund o , nielsen m . netmhc - 3 . 0 : accurate web accessible predictions of human , mouse and monkey mhc class i affinities for peptides of length 8 - 11 . nucleic acids research 2008 ; 36 : w509 - 12 . 7 . buyse m , michiels s , sargent d j , grothey a , matheson a , de gramont a . integrating biomarkers in clinical trials . expert review of molecular diagnostics 2011 ; 11 : 171 - 82 . 8 . wolchok j d , hoos a , o &# 39 ; day s , et al . guidelines for the evaluation of immune therapy activity in solid tumors : immune - related response criteria . clinical cancer research : an official journal of the american association for cancer research 2009 ; 15 : 7412 - 20 . 9 . llosa n j , cruise m , tam a , et al . the vigorous immune microenvironment of microsatellite instable colon cancer is balanced by multiple counter - inhibitory checkpoints . cancer discov 2015 : 43 - 51 . 10 . taube j m , anders r a , young g d , et al . colocalization of inflammatory response with b7 - h1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape . science translational medicine 2012 ; 4 : 127ra37 . 11 . cuka n , hempel h , sfanos k , de marzo a , cornish t . pip : an open source framework for multithreaded image analysis of whole slide images . laboratory investigation 2014 ; 94 : 398a - a . 12 . cupitt j , martinez k . vips : an image processing system for large images . electronic imaging : science & amp ; technology ; 1996 : international society for optics and photonics . p . 19 - 28 . 41 consecutive patients were enrolled and treated between september 2013 and january 2015 . ( table 1 ). recruitment included patients in pursuit of a clinical trial option who were known to have tumors with mismatch repair , or who had tumors of unknown status who were then tested . one patient in the mmr - deficient crc cohort was enrolled under an irb eligibility waiver allowing a grade 3 bilirubin level . a total of 32 crc patients were enrolled into cohorts a and b . all crc patients received ≧ 2 prior chemotherapy regimens ( median = 4 ) except for one mmr - proficient patient who had received one chemotherapeutic and one ( non - pd1 - based ) immunotherapeutic regimen . nine subjects diagnosed with mmr - deficient solid tumors other than crc were enrolled onto cohort c . all cohort c patients received ≧ 1 prior cancer treatments ( median = 2 ). the irorr and irpfs at 20 weeks ( fig1 ( table s2 )) for cohort a were 40 % ( 4 of 10 patients ; 95 % ci , 12 to 74 %) and 78 % ( 7 of 9 patients ; 95 % ci , 40 to 97 %) and for cohort c were 71 % ( 5 of 7 patients ; 95 % ci , 29 to 96 %) and 67 % ( 4 of 6 patients ; 95 % ci , 22 to 96 %). in cohort b , comprised of patients with mmr - proficient crcs , irorr and 20 - week irpfs were 0 % ( 95 % ci , 0 to 20 %) and 11 % ( 2 of 18 patients ; 95 % ci , 1 to 35 %). both the mmr - deficient cohorts a and c reached their predefined early stopping rule for efficacy when four subjects were free - of - disease progression at 20 weeks and four objective responses were observed based on immune - related response criteria ( fig1 ( table s2 ); available on line at new england journal of medicine ; incorporated by reference herein ; and supplementary methods , above ). the median time of follow - up for patients was 32 weeks ( range , 5 - 51 weeks ) for patients with mmr - deficient crc ( cohort a ), 12 weeks ( range , 2 - 56 weeks ) for patients with mmr - proficient crc ( cohort b ) and 12 weeks ( range , 4 - 42 weeks ) for patients with mmr - deficient non - crc tumors ( cohort c ). all patients evaluable for 20 - week irpfs were followed for at least 20 weeks . of the ten evaluable mmr - deficient crc patients in cohort a , four ( 40 %; 95 % ci , 12 - 74 %) achieved objective responses by recist criteria ( table 2 , fig1 and fig3 ( s 2 )). patients were considered not evaluable unless they underwent a 12 - week scan . the disease control rate was defined as the fraction of patients who achieved an objective response or whose disease was stable , and was 90 % in cohort a ( 9 of 10 patients ; 95 % ci , 55 - 100 %). of the seven evaluable patients with mmr - deficient cancer types other than crc enrolled in cohort c , five ( 71 %; 95 % ci , 29 - 96 %) achieved objective responses ( table 2 , fig3 ( s 2 ) and fig1 ) using recist criteria and the disease control rate was 71 % ( 5 of 7 patients ; 95 % ci , 29 - 96 %). patients in cohort c responded faster than patients in cohort a ( median time to response by recist of 12 vs . 28 weeks , p = 0 . 03 ). furthermore , all six mmr - deficient tumors that were not associated with lynch syndrome ( 100 %) achieved an objective response , whereas only three of eleven tumors ( 27 %) associated with lynch syndrome responded ( table s3 ; p = 0 . 009 ; available on - line at new england journal of medicine ; and incorporated by reference herein ). no other baseline characteristics showed statistically significant association with objective responses . of the 18 patients with mmr - proficient crcs in cohort b , no objective responses were observed ( table 2 , fig3 ( s 2 ) and fig1 ) using recist criteria and the disease control rate was 11 % ( 2 of 18 patients ; 95 % ci , 1 to 35 %). all patients who achieved a response by recist criteria ( fig1 ( table 2 )) also achieved a response by immune - related response criteria ( fig1 ( table s2 )). in cohort a , the patients with mmr - deficient crc , median progression - free survival ( pfs ) and median overall survival ( os ) were not reached ( fig2 ). in contrast , the patients with mmr - proficient cancers in cohort b achieved a pfs of only 2 . 2 months ( 95 % ci , 1 . 4 - 2 . 8 ) and a median os of 5 . 0 months ( 95 % ci , 3 . 0 to not estimable ). in cohort c ( mmr - deficient non - crc ), the median pfs was 5 . 4 months ( 95 % ci , 3 to not estimable ) and the median os was not reached . a post hoc ( fig2 ) comparison of the mmr - deficient and proficient crc cohorts showed hazard ratios ( hr ) for disease progression ( hr = 0 . 10 ; 95 % ci , 0 . 03 - 0 . 37 ; p & lt ; 0 . 001 ) and overall survival ( hr = 0 . 22 ; 95 % ci , 0 . 05 - 1 . 00 ; p = 0 . 05 ), favoring patients with mmr - deficient crc . to evaluate whether the difference in survival might be due to prognostic differences , we measured the duration of time patients had been diagnosed with metastatic disease and the clinical performance of patients on their previous regimen prior to enrollment . we found that there was no significant difference between mmr - deficient vs . mmr - proficient crc patients with respect to their duration of metastatic disease ( p = 0 . 77 ; log - rank test ) or median pfs ( p = 0 . 60 , log - rank test ) on their prior regimens ( fig4 ( s 3 )). we also performed an additional multivariate analysis of pfs and os to examine the difference in outcomes between mmr - deficient crc and mmr - proficient tumors adjusting for elapsed time since initial diagnosis . the magnitude of the hazard ratios for pfs ( hr 0 . 04 , 95 % ci 0 . 01 - 0 . 21 , p & lt ; 0 . 001 ) and os ( hr 0 . 18 , 95 % ci 0 . 03 - 1 . 01 , p = 0 . 05 ), representing the different effect of pembrolizumab between mmr - deficient and mmr - proficient tumors , was maintained after adjusting for this potential difference . adverse events occurring in & gt ; 5 % of patients are listed in table 3 . select adverse events included rash / pruritus ( 24 %), thyroiditis / hypothyroidism / hypophysitis ( 10 %), and asymptomatic pancreatitis ( 15 %). while the numbers were small , thyroid function abnormalities were limited to the mmr - deficient cohorts ( table 3 ). in the two crc cohorts , baseline cea levels were evaluable and above the upper limit of normal ( 3 mg / dl ), in 29 of 32 patients prior to enrollment . major cea declines occurred in seven of the ten patients with mmr - deficient crc and in none of the 19 patients with mmr - proficient crcin which cea was evaluable ( fig1 and fig5 ( s 4 )). in non - crc mmr - deficient patients , tumor marker levels ( cea , ca19 - 9 or ca - 125 ) were elevated above the upper limit of normal in four patients . ca19 - 9 or ca - 125 declines of & gt ; 70 % occurred in three of these four patients . tumor marker kinetics of all 3 cohorts are shown in fig1 . the level of cea decline after 1 dose ( between days 14 and 28 ) of pembrolizumab was predictive of both progression - free ( p = 0 . 01 ) and overall survival outcomes ( p = 0 . 02 ). the cea response occurred well in advance of radiographic confirmation of disease control ( range , 10 to 35 weeks ). in contrast , patients who progressed showed rapid biomarker elevation within 30 days of initiating therapy . thus , changes in cea levels significantly preceded and correlated with ultimate radiographic changes . analysis of whole - exome sequences showed an average of 1 , 782 somatic mutations per tumor in mmr - deficient patients ( n = 9 ) compared with 73 mutations per tumor in mmr - proficient patients ( n = 6 ) ( non - parametric wilcoxon test , p = 0 . 007 ) ( fig6 a - 6b ( s 5 ); see also table s3 which is available on - line at new england journal of medicine ; incorporated by reference herein ). most ( 63 %) of these mutations are predicted to alter amino acids . these mutations were then assessed for their immunogenic potential in the context of each patient &# 39 ; s individual mhc haplotype . we thereby identified an average of 578 and 21 potential mutation - associated neoantigens from the tumors of mmr - deficient and mmr - proficient patients , respectively ( table s3 ; which is available on - line at new england journal of medicine ; incorporated by reference herein ). the fraction of potential mutation - associated neoantigens among all somatic mutations was similar in both cohorts ( averaging 32 % and 29 % in mmr - deficient and - proficient patients , respectively ). high numbers of somatic mutations and potential mutation - associated neoantigens were associated with improved progression - free survival and with a trend in favor of objective response ( fig1 ( s 5 ) and fig1 ( table s4 ); also available on line at new england journal of medicine ; incorporated by reference herein ). expression of cd8 and pd - l1 were evaluated by immunohistochemistry within the tumor and at the invasive fronts of the tumor in the 30 cases in which tumor tissue was available ( fig7 ( s 6 ); 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