Microsatellite Stable Colorectal Carcinomas Research Articles

CEA-CD3 bispecific antibody cibisatamab with or without atezolizumab in patients with CEA-positive solid tumours: results of two multi-institutional Phase 1 trials

Cibisatamab is a bispecific antibody-based construct targeting carcinoembryonic antigen (CEA) on tumour cells and CD3 epsilon chain as a T-cell engager. Here we evaluated cibisatamab for advanced CEA-positive solid tumours in two open-label Phase 1 dose-escalation and -expansion studies: as a single agent with or without obinutuzumab in S1 (NCT02324257) and with atezolizumab in S2 (NCT02650713). Primary endpoints were safety, dose finding, and pharmacokinetics in S1; safety and dose finding in S2. Secondary endpoints were anti-tumour activity (including overall response rate, ORR) and pharmacodynamics in S1; anti-tumour activity, pharmacodynamics and pharmacokinetics in S2. S1 and S2 enrolled a total of 149 and 228 patients, respectively. Grade ≥3 cibisatamab-related adverse events occurred in 36% of S1 and 49% of S2 patients. The ORR was 4% in S1 and 7% in S2. In S2, patients with microsatellite stable colorectal carcinoma (MSS-CRC) given flat doses of cibisatamab and atezolizumab demonstrated an ORR of 14%. In S1 and S2, 40% and 52% of patients, respectively, developed persistent anti-drug antibodies (ADAs). ADA appearance could be mitigated by obinutuzumab-pretreatment, with 8% of patients having persistent ADAs. Overall, cibisatamab warrants further exploration in immunotherapy combination strategies for MSS-CRC.

Association of Microsatellite Instability and Gene Expression Profile in Colorectal Carcinoma and Potential Implications for Therapy.

Background and Objective: In sporadic colorectal carcinomas (CRC), microsatellite instability (MSI) pathways play important roles. Previously, we showed differences in DNA methylation patterns in microsatellite stable (MSS) colorectal carcinomas and MSI-CRC. In the current study, we explore the similarities and differences in gene expression profiles in MSS and MSI at the gene level and at the pathway level to better understand CRC pathogenesis and/or the potential for therapeutic opportunities. Material and Methods: Seventy-one CRC patients (MSI = 18, MSS = 53) were studied. Paired tumor and adjacent normal tissues were used for genome-wide gene expression assays. Result: At the gene level, we compared the list of differentially expressed genes (fold change (FC) ≥ 3 and FDR < 0.05) in tumor tissues compared to corresponding normal tissue in CRC patients with MSI tumors (190 genes) and MSS tumors (129 genes). Of these, 107 genes overlapped. The list of genes that were differentially expressed in MSI tumors only showed enrichment predominantly in two broad categories of pathways-(a) Inflammation-related pathways including the interleukin-17 (IL-17) signaling pathway, tumor necrosis factor (TNF) signaling pathway, chemokine signaling, nuclear factor kappa B (NFκB) signaling, and cytokine-cytokine interactions, and (b) metabolism-related pathways, including retinol metabolism, steroid hormone biosynthesis, drug metabolism, pentose and glucoronate interconversions, and ascorbate and aldarate metabolism. The genes in inflammation-related pathways were up-regulated whereas genes in metabolism-related pathways were down-regulated in MSI tumor tissue. Pathway-level analysis also revealed similar results confirming the gene enrichment findings. For example, the 150 genes involved in the IL-17 signaling pathway were on average up-regulated by 1.19 fold (CI 1.16-1.21) in MSI compared to 1.14 fold (CI 1.13-1.16) in MSS patients (interaction p = 0.0009). Conclusions: We document an association between MSI status and differential gene expression that broadens our understanding of CRC pathogenesis. Furthermore, targeting one or more of these dysregulated pathways could provide the basis for improved therapies for MSI and MSS CRC.

CXD101 and nivolumab in patients with metastatic microsatellite-stable colorectal cancer (CAROSELL): a multicentre, open-label, single-arm, phase II trial

Patients with microsatellite stable (MSS) colorectal carcinoma (CRC) do not respond to immune checkpoint inhibitors. Preclinical models suggested synergistic anti-tumour activity combining CXD101 and anti-programmed cell death protein 1 treatment; therefore, we assessed the clinical combination of CXD101 and nivolumab in heavily pre-treated patients with MSS metastatic CRC (mCRC). This single-arm, open-label study enrolled patients aged 18 years or older with biopsy-confirmed MSS CRC; at least two lines of systemic anticancer therapies (including oxaliplatin and irinotecan); at least one measurable lesion; Eastern Cooperative Oncology Group performance status of 0, 1 or 2; predicted life expectancy above 3 months; and adequate organ and bone marrow function. Nine patients were enrolled in a safety run-in study to define a tolerable combination schedule of CXD101 and nivolumab, followed by 46 patients in the efficacy assessment phase. Patients in the efficacy assessment cohort were treated orally with 20 mg CXD101 twice daily for 5 consecutive days every 3 weeks, and intravenously with 240 mg nivolumab every 2 weeks. The primary endpoint was immune disease control rate (iDCR). Between 2018 and 2020, 55 patients were treated with CXD101 and nivolumab. The combination therapy was well tolerated with the most frequent grade 3 or 4 adverse events being neutropenia (18%) and anaemia (7%). Immune-related adverse reactions commonly ascribed to checkpoint inhibitors were surprisingly rare although we did see single cases of pneumonitis, hypothyroidism and hypopituitarism. There were no treatment-related deaths. Of 46 patients assessable for efficacy, 4 (9%) achieved partial response and 18 (39%) achieved stable disease, translating to an immune disease control rate of 48%. The median overall survival (OS) was 7.0 months (95% confidence interval 5.13-10.22 months). The primary endpoint was met in this phase II study, which showed that the combination of CXD101 and nivolumab, at full individual doses in the treatment of advanced or metastatic MSS CRC, was both well tolerated and efficacious.

Somatic mutations in FAT cadherin family members constitute an underrecognized subtype of colorectal adenocarcinoma with unique clinicopathologic features.

The FAT cadherin family members (FAT1, FAT2, FAT3 and FAT4) are conserved tumor suppressors that are recurrently mutated in several types of human cancers, including colorectal carcinoma (CRC). To characterize the clinicopathologic features of CRC patients with somatic mutations in FAT cadherin family members. We analyzed 526 CRC cases from The Cancer Genome Atlas PanCancer Atlas dataset. CRC samples were subclassified into 2 groups based on the presence or absence of somatic mutations in FAT1, FAT2, FAT3 and FAT4. Individual clinicopathological data were collected after digital slide review. Statistical analysis was performed using t tests and chi-square tests. This CRC study cohort had frequent mutations in the FAT1 (10.5%), FAT2 (11.2%), FAT3 (15.4%) and FAT4 (23.4%) genes. Two hundred CRC patients (38.0%) harbored somatic mutations in one or more of the FAT family genes and were grouped into the FAT mutated CRC subtype. The FAT-mutated CRC subtype was more commonly located on the right side of the colon (51.0%) than in the rest of the cohort (30.1%, P < 0.001). It showed favorable clinicopathologic features, including a lower rate of positive lymph nodes (pN1-2: 33.5% vs 46.4%, P = 0.005), a lower rate of metastasis to another site or organ (pM1: 7.5% vs 16.3%, P = 0.006), and a trend toward an early tumor stage (pT1-2: 25.0% vs 18.7%, P = 0.093). FAT somatic mutations were significantly enriched in microsatellite instability CRC (28.0% vs 2.1%, P < 0.001). However, FAT somatic mutations in microsatellite stable CRC demonstrated similar clinicopathologic behaviors, as well as a trend of a better disease-free survival rate (hazard ratio = 0.539; 95% confidence interval: 0.301-0.967; log-rank P = 0.073). FAT cadherin family genes are frequently mutated in CRC, and their mutation profile defines a subtype of CRC with favorable clinicopathologic characteristics.

Comprehensive assessment of actionable genomic alterations in primary colorectal carcinoma using targeted next-generation sequencing

BackgroundThe clinical utility of comprehensive genomic profiling (CGP) for guiding treatment has gradually become the standard-of-care procedure for colorectal carcinoma (CRC). Here, we comprehensively assess emerging targeted therapy biomarkers using CGP in primary CRC.MethodsA total of 575 primary CRCs were sequenced by ACTOnco® assay for genomic alterations, tumour mutational burden (TMB), and microsatellite instability (MSI).ResultsEighteen percent of patients were detected as MSI-High (MSI-H), and the remaining cases were classified as microsatellite stable (MSS). Driver mutation prevalence in MSS CRCs were APC (74%), TP53 (67%), KRAS (47%), PIK3CA (21%) and BRAF (13%). The median TMBs for MSI-H and MSS patients were 37.8 mutations per mega base (mut/Mb) and 3.9 mut/Mb, respectively. Forty-seven percent of MSI-H CRC harboured at least one loss-of-function mutations in genes that may hamper immune checkpoint blockade. Among MSS RAS/RAF wild-type CRCs, 59% had at least one actionable mutation that may compromise the efficacy of anti-EGFR therapy. For late-stage CRC, 51% of patients are eligible for standard care actionability and the remaining 49% could be enrolled in clinical trials with investigational drugs.ConclusionsThis study highlights the essential role of CGP for identifying rational targeted therapy options in CRC.

Abstract 1705: Spatial interrogation of tertiary lymphoid structures (TLS) in colorectal carcinoma (CRC) tumor microenvironment (TME) using the MultiOmyx࣪ assay

Abstract Immune checkpoint blockade (ICB) therapy has revolutionized the landscape of cancer therapy in multiple tumor types since Ipilimumab, the first ICB agent, was approved for the treatment of metastatic melanoma is 2011. Current predictive biomarkers for therapy response include PD-L1 expression, tumor mutational burden (TMB) and microsatellite instability (MSI) status. However, responses to ICB vary widely and these predictive markers have demonstrated imperfect predictive power to ICB therapy. For instance, half of microsatellite instability-high (MSI-H) colorectal carcinoma (CRC) patients do not respond to ICB immunotherapy. There is still a crucial need to identify and develop biomarkers predictive of outcome to ICB therapy. Tertiary lymphoid structures (TLS) have been observed in a variety of solid tumors in humans, and their presence is a favorable prognostic indicator for survival in a variety of solid cancers including CRC. Further, it was recently shown that the presence of mature TLS was associated with improved response rate to ICB and overall survival in patients with advanced tumors in a large retrospective study. In this study, we used a 17-plex MultiOmyx panel to detect TLS, explore TLS maturation stage and characterize the tumor microenvironment (TME) in 40 CRC patients with known MSI status. The 17-plex includes CD3, CD4, CD8, CD45RO, FOXP3, CD20, CD56, CD68, CTLA-4, PD1, PD-L1, CXCL13, PNAd, CD21, CD23, DC-LAMP and PanCK. Using this panel, we successfully detected different stages of TLS in these CRC samples. Using proprietary deep-learning-based image analysis algorithm (NeoLYTX), we quantified TLS’s in the CRC samples and classified them by maturation stage based on biomarker expression and spatial organization of immune cells. While there was no difference in the number of early TLS (E-TLS; characterized by CD3, CD20 and pNAd) between MSI-H and microsatellite-stable (MSS) CRC samples, more primary follicle-like TLS (P-TLS; defined by CD3, CD20 and CD21) and secondary follicle-like TLS (S-TLS; defined by CD3, CD20 and CD23) were observed in the MSI-H group than the MSS group. In particular, the abundance of mature S-TLS was significantly higher in MSI-H CRC samples. This 17-plex MultiOmyx assay provides a powerful tool to characterize the cellular composition and spatial organization of the tumor microenvironment. The panel enables quantification of TLS, PD-L1 expression and abundance of tumor infiltrating lymphocytes from one single FFPE slide. The rich datasets generated by the MultiOmyx assay can provide greater understanding of the immune contexture within the TME and deeper insights into the correlations between biomarkers. These findings may have broad application and help identify biomarker signatures with improved predictive performance to immune checkpoint inhibition efficacy in solid tumors. Citation Format: Harry Nunns, Erinn A. Parnell, Michael Lazare, Judy Kuo, Eric Leones, Flora Sahafi, Josette William, Qingyan Au. Spatial interrogation of tertiary lymphoid structures (TLS) in colorectal carcinoma (CRC) tumor microenvironment (TME) using the MultiOmyx࣪ assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1705.

Landscape of DNA damage response gene mutations in microsatellite-stable colorectal carcinoma.

e15558 Background: Colorectal carcinoma (CRC) is one of the most frequent and lethal cancers in the world. Important therapeutic progress of immune checkpoint inhibitors (ICIs) is limited to molecular niche of CRC with microsatellite-stable(MSS). The identification of genomic alterations in the DNA damage response (DDR) pathway have been recognized as pathogenic factors in hereditary cancer predisposition. Few studies showed the effects of ICIs on DDR pathway in patients with MSS CRC. Some reports suggested that DDR could reshape tumor immune microenvironment by regulating tissue repair and local immune response. Here we evaluated the molecular roles of DDR pathway and the immune microenvironment in efficacy of immunotherapy in MSS CRC. Methods: A total of 67 patients with MSS CRC were enrolled into the study. All samples were collected and detected by using NGS with a 1021 gene panel which containing 56 DDR genes (Supplementary table 1). Furthermore, comprehensive bioinformatics analysis were performed by the TCGA and MSK data. We defined pathway alteration as at least one gene in DDR pathway mutated. Results: In our results, three DDR pathway alteration showed poor prognosis (P = 0.0167) compared with DDR wild type in TCGA data (Figure 1A). The MSK data demonstrated that three DDR pathway alteration were associated with a better prognosis in MSS CRC patients through ICIs therapy (P = 0.0416, P = 0.00627, respectively, Figure 1B). The clinical data suggested the patient with DDR pathway alteration had longer OS (354.7 ± 134.0 days, PR) than the others with DDR wild type (247.1± 258.4 days, PD/SD) after PD-1-targeted therapies (P = 1.587e-08). Moreover, the patient with DDR pathway alteration showed significant longer PFS (312.3 ± 179.2 days, PR) compared with DDR wild type (163.3 ± 157.7 days, PD/SD) through immune therapies (P = 4.249e-10). Moreover, significant results of immune infiltrate deconvolution revealed resting memory CD4+ T cells were the most prevalent population between DDR pathway alteration and DDR wild type using CIBERSORT analysis (P = 0.0295, Figure 1C). The activated memory CD4+ T cells in DDR pathway alteration were lower than DDR wild type (P = 0.00858, Figure 1C-D) in patients with MSS CRC. The effects of DDR pathway alteration on immune microenvironment was also necessary for effective PD-1-targeted therapy in future study. Our results suggest that two or more DDR pathways and CD4 + T cells may serve as immunotherapy markers for advanced MSS colorectal cancer. Conclusions: Mutations in DDR pathway may serve as a biomarker for PD-1-targeted therapy through memory CD4+ T cells for MSS CRC patients, which warrants further studies.

Comprehensive clinicopathologic, molecular, and immunologic characterization of colorectal carcinomas with loss of three intestinal markers, CDX2, SATB2, and KRT20.

Caudal-type homeobox 2 (CDX2), special AT-rich sequence-binding protein 2 (SATB2), and keratin 20 (KRT20) are frequently used as intestinal epithelium-specific markers in immunohistochemical studies. However, subsets of colorectal carcinomas (CRCs) show loss of these markers. We analyzed The Cancer Genome Atlas data to explore molecular correlates of CDX2, SATB2, and KRT20 genes in 390 CRCs. The decreased mRNA expression of each of the three genes commonly correlated with microsatellite instability-high (MSI-H), CpG island methylator phenotype-high (CIMP-H), BRAF/RNF43 mutations, consensus molecular subtype 1, and high tumor mutational burden. The downregulation of CDX2 or SATB2 was dependent on both MSI-H and CIMP-H, whereas that of KRT20 was more dependent on MSI-H than on CIMP-H. Next, we evaluated the immunohistochemical expression of CDX2, SATB2, and KRT20 in 436 primary CRCs. In contrast to RNA-level expression, decreased expression of CDX2 and SATB2 was more dependent on CIMP-H than on MSI-H. However, consistent with RNA-level expression, decreased expression of KRT20 was more dependent on MSI-H than on CIMP-H. CIMP-H and lymphatic invasion were consistently associated with both CDX2 loss and SATB2 loss in CRCs, regardless of MSI status. In microsatellite stable CRCs, CDX2 loss correlated with BRAF mutation, whereas SATB2 loss was associated with KRAS mutations and decreased T-cell infiltration. Cases with concurrent loss of all three markers were found exclusively in MLH1-methylated MSI-H/CIMP-H CRCs. In conclusion, MSI-H and/or CIMP-H are major common correlates of decreased CDX2/SATB2/KRT20 expression in CRCs, but the specific features associated with the loss of each marker are different in CRCs.

Aberrant DNA methylation patterns in microsatellite stable human colorectal cancers define a new marker panel for the CpG island methylator phenotype.

A distinct group of colorectal carcinomas (CRCs) referred to as the "CpG island methylator phenotype" (CIMP) shows an extremely high incidence of de novo DNA methylation and may share common pathological, clinical or molecular features. However, there is limited consensus about which CpG islands (CGIs) define a CIMP, particularly in microsatellite stable (MSS) carcinomas. To study this phenotype in a systematic manner, we analyzed genome-wide CGI DNA methylation profiles of 19 MSS CRC using methyl-CpG immunoprecipitation (MCIp) and hybridization on 244K CGI oligonucleotide microarrays, determined KRAS and BRAF mutation status and compared disease-related DNA methylation changes to chromosomal instability as detected by microarray-based comparative genomic hybridization. Results were validated using mass spectrometry analysis of bisulfite-converted DNA at a subset of 76 individual CGIs in 120 CRC and 43 matched normal tissue samples. Both genome-wide profiling and CpG methylation fine mapping segregated a group of CRC showing pronounced and frequent de novo DNA methylation of a distinct group of CGIs that only partially overlapped with previously established classifiers. The CIMP group defined in our study revealed significant association with colon localization, either KRAS or BRAF mutation, and mostly minor chromosomal losses but no association with known histopathological features. Our data provide a basis for defining novel marker panels that may enable a more reliable classification of CIMP in all CRCs, independently of the MS status.

NTRK oncogenic fusions are exclusively associated with the serrated neoplasia pathway in the colorectum and begin to occur in sessile serrated lesions.

Neurotrophic tropomyosin receptor kinase (NTRK) gene fusions are emerging tissue-agnostic drug targets in malignancies including colorectal carcinomas (CRCs), but their detailed landscape in the context of various colorectal carcinogenesis pathways remains to be investigated. In this study, pan-tropomyosin receptor kinase (TRK) protein expression was assessed by immunohistochemistry (IHC) in retrospectively collected colorectal epithelial tumor tissues, including 441 CRCs [133 microsatellite instability-high (MSI-high) and 308 microsatellite stable (MSS)] and 595 premalignant colorectal lesions (330 serrated lesions and 265 conventional adenomas). TRK-positive cases were then subjected to next-generation sequencing and/or fluorescence in situ hybridization to confirm NTRK rearrangements. TRK IHC positivity was not observed in any of the MSS CRCs, conventional adenomas, traditional serrated adenomas, or hyperplastic polyps, whereas TRK positivity was observed in 11 of 58 (19%) MLH1-methylated MSI-high CRCs, 4 of 23 (17%) sessile serrated lesions with dysplasia (SSLDs), and 5 of 132 (4%) sessile serrated lesions (SSLs). The 11 TRK-positive MSI-high CRCs commonly harbored CpG island methylator phenotype-high (CIMP-high), MLH1 methylation, BRAF/KRAS wild-type, and NTRK1 or NTRK3 fusion (TPM3-NTRK1, TPR-NTRK1, LMNA-NTRK1, SFPQ-NTRK1, ETV6-NTRK3, or EML4-NTRK3). Both NTRK1 or NTRK3 rearrangement and BRAF/KRAS wild-type were detected in all nine TRK-positive SSL(D)s, seven of which demonstrated MSS and/or CIMP-low. TRK expression was selectively observed in distorted serrated crypts within SSLs and was occasionally localized at the base of serrated crypts. NTRK fusions were detected only in SSLs of patients aged ≥50 years, whereas BRAF mutation was found in younger age-onset SSLs. In conclusion, NTRK-rearranged colorectal tumors develop exclusively through the serrated neoplasia pathway and can be initiated from non-dysplastic SSLs without BRAF/KRAS mutations prior to full occurrence of MSI-high/CIMP-high. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The landscape of MAP3K1/MAP2K4 alterations in gastrointestinal (GI) malignancies.

4113 Background: Inactivating alterations in MAP3K1/MAP2K4 occur in various solid tumors, sensitize cancer models to MEK inhibitors, and have co-mutation partners which may enable therapeutic targeting. Methods: We retrospectively reviewed 20290 GI malignancy patients (pts), comprised of 9986 colorectal carcinoma (CRC) and 10304 non-CRC, whose tumors were profiled with Caris Life Sciences from 2015-2019. Profiling included immunohistochemistry (IHC) with programmed death ligand-1 (PD-L1), next-generation sequencing (NGS), tumor mutational burden (TMB) and deficient mismatch repair or microsatellite instability-high status (dMMR/MSI-H). Results: MAP3K1/MAP2K4-alteration ( MAP3K1/MAP2K4-MT) was more frequent in CRC than non-CRC pts (2.0% v. 1.2%, p&lt;0.0001), with truncating mutations representing the majority of lesions along both genes. While MAP3K1/MAP2K4-MT CRC pts were similar in age and gender to wild-type (WT), mutated non-CRC pts were older (median age 69 v. 65 years) and more likely female (51% v. 42%) compared to WT (both p&lt;0.05). MAP3K1/MAP2K4-MT CRC (25% v. 7%) and non-CRC (30% v. 3%) were more frequently dMMR/MSI-H than WT pts (both p&lt;0.0001). MAP3K1/MAP2K4-MT CRC cases were affiliated with higher TMB and similar rate of PD-L1 expression compared to WT. A higher rate of MAP3K1/MAP2K4-MT CRC pts were right-sided (36% v. 22%, p&lt;0.0001) and transverse (8% v. 4%, p&lt;0.05) compared to WT, whereas a higher frequency of WT cases were left-sided (20% v. 28%, p&lt;0.05) and rectal (15% v 23%, p&lt;0.05). Of microsatellite stable (MSS) CRC pts, those with MAP3K1/MAP2K4-MT were more likely PIK3CA (26% v. 17%) and APC (85% v. 78%) and less-likely TP53 (64% v. 77%) co-mutated versus WT MSS pts (all p&lt;0.05); no difference was seen in BRAF V600E, ERBB2/ ERBB3 or KRAS co-mutation rate in MSS pts. In both all-comers and MSS CRC, MAP3K1/MAP2K4-MT pts were more frequently co-mutated than WT with ARID1A, POLE, ATM, BRCA2 and PIK3R1 (all ≥7% of MAP3K1/MAP2K4-MT pts, p&lt;0.0001). A higher frequency of all-comer non-CRC GI malignancy pts with MAP3K1/MAP2K4-MT were co-mutated with PIK3CA (13% v. 6%), ERBB2/ERBB3 (8% v. 3%) or APC (13% v. 5%) compared to WT (all p&lt;0.01). For MSS non-CRC GI cases, ARID1A (50% v. 30%) and SMAD4 (21% v. 12%) were more frequently co-mutated in MAP3K1/MAP2K4-MT versus WT pts (all p&lt;0.05). Conclusions: Truncating MAP3K1/MAP2K4 alterations occur in nearly 2% of GI malignancy pts and are more commonly associated with dMMR/MSI-H than WT. Potentially targetable co-mutation partners implicated in MAPK and PI3K pathways as well as POLE, BRCA2 and ATM warrant further evaluation.

Abstract IA01: Turning the heat up on pancreatic cancer: Lessons on overcoming a “cold” immunologic microenvironment

Abstract Immune checkpoint inhibitors (ICIs) are providing durable clinical responses in about 20% of cancer patients, but these agents have minimal effect in cancers without intratumoral T cells. Approaches that turn currently unresponsive cancers into ones that are more “antigenic” are needed to sensitize tumors to ICIs. Tumor genome mutations can express mutant proteins that are tumor-specific and not expressed on normal cells (neoantigens), and cancers with the highest mutational burdens are more likely to respond to single-agent ICIs. However, most cancers have lower mutational loads resulting in lower antigenicity, weaker endogenous T-cell repertoires, and T cells in the tumor. The best example of a high mutation load cancer is mismatch repair deficient (MMRd) cancers; these cancers often have &amp;gt;1,000 mutations/exome and have a &amp;gt;50% response to anti-PD-1 ICIs. However, cancers including pancreatic ductal adenocarcinoma (PDA) and microsatellite stable colorectal carcinoma (MSS CRC) have on average only 50-70 expressed mutations per exome and do not respond to single-agent ICIs. Emerging data suggest that it should be possible to develop precision immunology approaches that combine a neoantigen targeting vaccine to activate and expand the limited repertoire of T cells specific for the expressed neoantigens found in low-mutation cancers, with ICIs to induce clinically relevant antitumor responses. Challenges to successful immunization include knowledge about the repertoire and functional state of pre-existing antitumor T cells, identification of the best adjuvants, and approaches that more precisely predict which expressed neoantigens are the best T-cell targets for immunization. In addition, we now appreciate that there are many different immune-regulatory signals on T cells, monocytes and other tumor microenvironment cell populations that are likely regulated by the genetic alterations specific to a given patient’s tumor. This talk will discuss current knowledge of precision immune oncology and novel clinical trial approaches under development.

Immunohistochemical-based molecular subtyping of colorectal carcinoma using maspin and markers of epithelial-mesenchymal transition.

The aim of the present study was to classify colorectal carcinoma (CRC) into molecular subtypes, based on immunohistochemical (IHC) assessments. A total of 112 CRC samples were molecularly classified based on the expression levels of epithelial-mesenchymal transition (EMT)-associated IHC markers. A total of three molecular subtypes were defined: Epithelial, membrane positivity for E-cadherin and β-catenin, negative for vimentin; mesenchymal, E-cadherin-negative, nuclear β-catenin- and vimentin-positive; and hybrid cases, epithelial tumor core and mesenchymal tumor buds. Most of the cases were diagnosed as moderately differentiated adenocarcinoma (n=89; 79.46%). The majority of cases (n=100; 89.28%) exhibited a mismatch repair proficient status (microsatellite stable CRCs). A predominance of epithelial-type (n=51; 45.54%) and hybrid CRCs (n=47; 41.96%) was observed, whereas a few cases (n=14; 12.50%) were classified as mesenchymal-type CRCs. This molecular classification was associated with pathological stage (P<0.01), pT stage (P=0.04), pN stage (P<0.01), the grade of tumor budding (P=0.04), and maspin expression in both the tumor core (P=0.04) and the invasion front (P<0.01). The mesenchymal-type cases predominantly exhibited lymph node metastases, high-grade budding and a tendency towards maspin nuclear predominance. All epithelial-type cases with maspin-only expression (n=18) were non-metastatic. Patients with CRC of the epithelial subtype and those with a lymph node ratio (LNR) ≤0.15 presented the best overall survival, followed by those with hybrid and mesenchymal subtypes. Nuclear maspin positivity was more frequent in cases with a high-budding degree compared with those with a low-budding degree (P=0.03). The EMT-associated molecular classification of CRCs may be used to identify the most aggressive CRCs, which show a mesenchymal phenotype, high-budding degree, maspin nuclear positivity and lymph node metastases. The pN stage, LNR and budding degree of patients, which can be evaluated with maspin expression, remain the most important prognostic factors.

Determination of the recommended phase II dose of birinapant in combination with pembrolizumab: Results from the dose-escalation phase of BPT-201.

2506 Background: Birinapant is a bivalent SMAC mimetic targeting cIAP1. Synergistic effects of combining birinapant with immune checkpoint inhibitors have been demonstrated in preclinical models. Based on these observations, a clinical trial with birinapant and pembrolizumab was initiated (NCT02587962). Methods: Patients ≥18 years with advanced solid tumors without further suitable standard therapeutic options were eligible for inclusion. Birinapant (5.6-22 mg/m2) was administered IV on day 1 and 8 in addition to pembrolizumab 200 mg on day 1 in a 21-day cycle until disease progression using standard 3+3 dose-escalation. The primary objective was to determine the safety and tolerability of the recommended phase 2 dose (RP2D) of birinapant in combination with pembrolizumab. Secondary and exploratory objectives included antitumor activity assessed by RECIST 1.1 and iRECIST, pharmacokinetics and assessment of biomarkers including serum cytokines, cIAP1, PD-L1 expression and tumor infiltrating lymphocytes. Results: Nineteen patients were enrolled at 4 dose levels of 5.6 (n = 3), 11 (n = 3), 17 (n = 6) and 22 (n = 7) mg/m2. Most common tumors were pancreatic (n = 5), colorectal (n = 4), ovarian (n = 3) and sarcoma (n = 3). Median prior therapies were 4 (0-12). The most common AE related to any of the study drugs was rash occurring in 3 patients. Ten patients had 17 SAE's of which only one (stomatitis) was judged related to birinapant. Increased ALT/AST (G3/G2) leading to missed day 8 dose constituted a DLT at 22 mg/m2. Grade 2 lipase increases were seen in 2 patients. No cases of Bell’s palsy were detected. ORR by RECIST 1.1 was 5.6% (n = 1) in 18 evaluable patients. The responding patient had microsatellite stable colorectal carcinoma (MSS-CRC)) and remains on therapy 13+ months after first dose. By iRECIST, ORR was 11.1%. CBR (PR+SD) by RECIST was 22.2%. The exposure to birinapant generally increased with dose. The RP2D was determined to be 22 mg/m2. Conclusions: Birinapant and pembrolizumab is a safe and tolerable combination that has shown encouraging signals of efficacy. A phase 2 study evaluating efficacy of this combination in MSS-CRC is ongoing. Clinical trial information: NCT02587962.

Microsatellite instability test using peptide nucleic acid probe-mediated melting point analysis: a comparison study

BackgroundAnalysis of high microsatellite instability (MSI-H) phenotype in colorectal carcinoma (CRC) is important for evaluating prognosis and choosing a proper adjuvant therapy. Although the conventional MSI analysis methods such as polymerase chain reaction (PCR) fragment analysis and immunohistochemistry (IHC) show high specificity and sensitivity, there are substantial barriers to their use.MethodsIn this study, we analyzed the MSI detection performance of three molecular tests and IHC. For the molecular tests, we included a recently developed peptide nucleic acid probe (PNA)-mediated real-time PCR-based method using five quasi-monomorphic mononucleotide repeat markers (PNA method) and two conventional PCR fragment analysis methods using NCI markers (NCI method) or five quasi-monomorphic mononucleotide repeat markers (MNR method). IHC analysis was performed with four mismatch repair proteins. The performance of each method was validated in 166 CRC patient samples, which consisted of 76 MSI-H and 90 microsatellite stable (MSS) CRCs previously diagnosed by NCI method.ResultsOf the 166 CRCs, 76 MSI-H and 90 MSS CRCs were determined by PNA method. On the other hand, 75 MSI-H and 91 MSS CRCs were commonly determined by IHC and MNR methods. Based on the originally diagnosed MSI status, PNA showed 100% sensitivity and 100% specificity while IHC and MNR showed 98.68% sensitivity and 100% specificity. When we analyzed the maximum sensitivity of MNR and PNA method, which used the same five markers, PNA method could detect alterations in all five mononucleotide repeat markers in samples containing down to 5% MSI-H DNAs, whereas MNR required at least 20% MSI-H DNAs to achieve the same performance.ConclusionsBased on these findings, we suggest that PNA method can be used as a practical laboratory test for the diagnosis of MSI.

Abstract 1026: A multiplex IHC evaluation of multiple immune checkpoint receptors and mismatch repair proteins in colorectal carcinoma

Abstract Introduction: The recent success of checkpoint inhibitor therapies in clinical oncology practice has generated overwhelming enthusiasm for immunotherapeutics. Recent studies have suggested that patients with mismatch repair deficient (dMMR)/microsatellite instability-high (MSI-H) colorectal carcinoma (CRC) may benefit from anti-PD-1/PD-L1 inhibitors. In this study, we have evaluated a large panel of immune checkpoint receptors (ICRs) on CRC by multiplex immunohistochemistry (IHC). Design: Tissue sections were initially reviewed by H&amp;E to confirm the presence of adequate pathological features including histology, grade and abundance of tumor-infiltrating lymphocytes (TILs). An MMR protein panel (MLH1, MSH2, MSH6, PMS2) was tested by IHC to determine dMMR/MSI-H status through loss of MLH1/PMS2 or MSH2/MSH6. Multiple ICRs, including CD4, CD8, CD137, CTLA-4, FOXP3, GITR, IDO1, LAG-3, PD-1, and PD-L1, were then tested by multiplex IHC on 18 CRC cases consisting of MSI-H CRCs (n=8) and microsatellite stable (MSS) CRCs (n=10) using four chromogenic end-points. Expression of ICRs in TILs was determined at four sites: tumor, invasive margin, peri-tumor, and tertiary lymphoid structures (TLS). ICR expression levels were determined by counting and averaging positive TILs in three high-power fields (400X) and categorized in the following manner: 0 (negative, no expression), 1 (weak, 1-25 cells), 2 (moderate, 26-50 cells), 3 (high, &amp;gt;50 cells). Results: Five cases of MSS (50%) and two cases of MSI-H showed 10-30% PD-L1 positive tumor cell expression. The remaining cases were negative for PD-L1. All cases of MSI-H and MSS demonstrated high expression of CD8 in tumor, invasive margin, peri-tumor and TLS. Five MSI-H cases showed moderate to high PD-1, and three MSI-H cases were weak for PD-1. All MSS cases showed weak to moderate PD-1 expression. CD4, FOXP3, CTLA-4, IDO1, LAG-3, and CD137 were weak to moderate in all MSI-H and MSS cases. Within the TLS, the predominant cellular components were CD8+, CD4+ and PD-1+ T cells in all cases. Conclusion: In the present study, a greater number of MSS CRCs expressed PD-L1 than MSI-H CRCs. PD-1 was positive in all CRC cases with variable expression between MSI-H and MSS CRCs. There appears to be no significant difference in TIL expression of additional immune checkpoint receptors tested in MSI-H colorectal carcinomas and MSS colorectal carcinomas. Multiplex technology appears to allow for superior evaluation of the synergistic relationship between immune checkpoint receptors as well as the interconnection between inhibitory barriers to CD8+ effector T cells and regulatory T cells developed by immune checkpoint receptors within each of the tumoral regions. Further study of expression patterns of immune checkpoint receptors may provide useful information for clinical management of patients with colorectal carcinoma. Citation Format: George Yang, Sara Figueroa, David Tacha, Cristin Douglas. A multiplex IHC evaluation of multiple immune checkpoint receptors and mismatch repair proteins in colorectal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1026.

Abstract B011: Immunoediting in untreated mismatch repair deficient colorectal cancer

Abstract A subset of colorectal carcinomas (CRC) with high microsatellite instability (MSI-high) have been shown to respond to immune checkpoint blockade, while the much more common microsatellite stable (MSS) CRC do not usually respond to these immunotherapies. Most MSI-high tumors grow progressively despite a high mutational load, suggesting that the cancer may have developed means to escape from immune recognition, which is termed immunoediting. This immunoediting process includes three phases: an initial phase of nascent cancer cell elimination due to high immunogenicity, then a period of equilibrium with the immune response, finally leading to escape of the cancer cells that have developed genetic or epigenetic mechanisms allowing them to grow progressively in an immunocompetent host. It is likely that MSI-high cancers would go through similar processes to eventually escape from immune recognition. To better understand the differential immunoediting in MSI-high and MSS CRC, we combined tumor exome data from 592 cases from The Cancer Genome Atlas (TCGA) with previously published tumor exome data from 619 cases from the Nurses’ Health Study (NHS) and the Health Professionals Follow-up Study to yield an unprecedented sample of 1,211 CRC molecularly characterized cases. The large number of samples, including a very large number of MSI-high cases (N = 179), gives us the power to identify the prevalence of the mechanisms of resistance discussed and thereby prioritize downstream efforts. We utilized the MutSigCV algorithm to identify significantly mutated genes in MSS and MSI-high tumors in the TCGA cohort (496 MSS and 75 MSI-high cases), and the NHS/HPFS cohorts (438 MSS and 104 MSI-high cases). We identified a total of 62 significantly mutated genes, of which 9 were identified in MSS tumors only, 40 in MSI-high tumors only, and 13 in both. Twenty-seven of the 53 significantly mutated genes in MSI-high were novel, likely resulting from a large increase in the number of MSI-high tumors relative to previous studies. We identified 13 significantly mutated genes with a documented role in the immune system, specifically in MSI-high tumors, a subtype shown previously to have a high level of T-cell infiltration; ZFP36L2 and B2M were significantly mutated in both MSS and MSI-high. B2M, HLA-A, and HLA-B are significantly mutated antigen presenting genes that were previously reported to be recurrently mutated in MSI-high. Eleven of the significantly mutated immune-related genes had roles in modulating diverse hematopoietic cell types and effects beyond antigen presentation; those genes include XYLT2, a dendritic cell trafficking gene; ZBTB20, a Toll-like receptor mediating immune response; RNF128, a regulator of IL2 and IL4 mediated T-cell maturation; KLF3, a gene involved in B-cell development; ZFP36L2, a gene involved in thymocyte development; CASP8, a gene involved in innate immunity; and CD58, a gene involved in the activation of both T lymphocytes and NK cells. Together, these mutations indicate positive selection for immune escape through other mechanisms beyond mutations in antigen-presenting machinery. Citation Format: Catherine Grasso, Marios Giannakis, Daniel Wells, David Wheeler, Eve Shinbrot, Syed Zaidi, Jeroen Huyghe, Milan Geybels, Stephen Salipante, Gabriel Abril-Rodriguez, Helena Escuin-Ordinas, Cristina Puig-Saus, Daniel Sanghoon Shin, Shuji Ogino, Antoni Ribas, Ulrike Peters. Immunoediting in untreated mismatch repair deficient colorectal cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B011.

Neoadjuvant therapy in microsatellite-stable colorectal carcinoma induces concomitant loss of MSH6 and Ki-67 expression

Universal screening using immunohistochemistry for DNA mismatch-repair proteins (MLH1, MSH2, MSH6, and PMS2) is advocated by major professional medical organizations to identify Lynch syndrome-associated colorectal carcinoma. Loss of MSH6 expression independent of MSH2 expression has been reported in microsatellite-stable (MSS) colorectal carcinoma after neoadjuvant therapy. The mechanism remains unclear. We studied the immunohistochemical expression of MSH2, MSH6, and Ki-67 in MSS colorectal carcinoma with (n=50) or without (n=64) preoperative neoadjuvant therapy and Lynch syndrome-associated colorectal carcinoma with confirmed MSH6 germline mutation (n=3). Twelve of 50 MSS colorectal carcinoma postneoadjuvant resections demonstrated reduced MSH6 expression, with loss of expression ranging from 20% to 100% of tumor cells. Eight of 64 MSS colorectal carcinomas without neoadjuvant therapy also exhibited reduced MSH6 expression but to a lesser degree (10%-50% of tumor cells with loss of expression). In both subgroups, concomitant loss of MSH6 and Ki-67 expressions was demonstrated in the same tumor areas in consecutive tissue sections. However, 3 cases of Lynch syndrome-associated colorectal carcinoma due to germline MSH6 mutation revealed complete loss of MSH6 expression with discordant positive Ki-67 staining in the tumor cells. The MSH2-independent, Ki-67-related expression of MSH6 in colorectal carcinoma helps to explain the heterogeneous MSH6 staining in MSS colorectal carcinoma with or without neoadjuvant therapy.

Medullary carcinoma of the colon: a distinct morphology reveals a distinctive immunoregulatory microenvironment

Medullary carcinoma of the colon is a unique histologic subtype of microsatellite unstable colorectal carcinoma but little is known regarding its tumor-immunoregulatory microenvironment. The aims of this study were to characterize the immune environment of medullary carcinoma and compare it with other microsatellite unstable and microsatellite stable colorectal carcinomas. An initial gene expression microarray analysis of six cases of medullary carcinoma was used to detect potentially differentially expressed genes. We extended this analysis utilizing genomic data from the Cancer Genome Atlas to compare eight cases of medullary carcinoma with other microsatellite unstable and stable carcinomas. Finally, we evaluated expression of key immune pathway proteins and lymphocyte subsets via immunohistochemistry of a large group of medullary carcinomas (n=105) and compared these findings with three other groups: poorly differentiated, microsatellite unstable well-differentiated and microsatellite stable well-differentiated carcinomas. Microarray and the Cancer Genome Atlas data analysis identified significant upregulation of several immunoregulatory genes induced by IFNγ including IDO-1, WARS (tRNA(trp)), GBP1, GBP4, GBP5, PDCD1 (PD-1), and CD274 (PD-L1) in medullary carcinoma compared with other microsatellite unstable and microsatellite stable tumors. By immunohistochemistry, IDO-1 was expressed in 64% of medullary carcinomas compared with 19% (9/47) of poorly differentiated carcinomas, 14% (3/22) of microsatellite unstable, and 7% (2/30) of the microsatellite stable well-differentiated carcinomas (P<0.0001). tRNA(trp) was overexpressed in 81% (84/104) of medullary carcinomas, 19% (9/47) of poorly differentiated, 32% (7/22) of microsatellite unstable, and 3% (1/30) of microsatellite stable well-differentiated carcinomas (P<0.0001). Medullary carcinoma had higher mean CD8+ and PD-L1+ tumor-infiltrating lymphocytes compared with all other groups (P<0.0001). This study demonstrates overexpression of several immunoregulatory genes in microsatellite unstable colorectal carcinomas and that expression of these genes and proteins is more prevalent in the medullary carcinoma subtype, which may be of use both diagnostically and therapeutically.

Serrated tubulovillous adenoma of the large intestine

Most colorectal polyps are classified readily, but a subset of tubulovillous adenomas (TVA) with prominent serrated architecture causes diagnostic confusion. We aimed to (i) identify histological features that separate serrated TVAs from both conventional TVAs and traditional serrated adenomas (TSA) and (ii) perform a clinicopathological and molecular analysis to determine if the serrated TVA has unique features. We collected 48 serrated TVAs, 50 conventional TVAs and 66 BRAF wild-type TSAs for analysis. For each polyp we performed a clinicopathological assessment, BRAF and KRAS mutation profiling, cytosine-phosphate-guanosine (CpG) island methylator phenotype status, MGMT methylation and immunohistochemical assessment of seven markers [MutL homologue 1 (MLH1), p16, p53, β-catenin, Ki67, CK7 and CK20]. We found that serrated TVAs can be diagnosed reliably, and have features distinct from both conventional TVAs and TSAs. Compared to conventional TVAs, serrated TVAs are larger, more often proximal, more histologically advanced, show more CpG island methylation and more frequent KRAS mutation. Compared to TSAs they are more often proximal, show less CpG island methylation, more frequent MGMT methylation and more frequent nuclear staining for β-catenin. The serrated TVA can be diagnosed reliably and has unique features. It represents a precursor of KRAS mutated, microsatellite stable colorectal carcinoma.