Microsatellite Instability-high Colorectal Carcinomas Research Articles

Intratumoral spatial heterogeneity of tumor-infiltrating lymphocytes is a significant factor for precisely stratifying prognostic immune subgroups of microsatellite instability-high colorectal carcinomas

Although the density of tumor-infiltrating lymphocytes (TILs) is known to be linked to prognosis in various cancers, the prognostic impact and immunologic significance of the spatial heterogeneity of TILs have been rarely investigated. In this study, CD3+ and CD8+ TILs were quantified in independent cohorts (discovery, n = 73; and external validation, n = 93) of colorectal carcinomas (CRCs) with microsatellite instability-high (MSI-H) utilizing whole-slide image analysis of CD3/CD8 immunohistochemistry. The Shannon and Simpson indices, which measure intratumoral patch-to-patch evenness of TIL densities, were used to quantitatively assess the spatial heterogeneity of TILs in each case. To uncover immune-related gene expression signatures of spatial heterogeneity-based TIL subgroups of MSI-H CRCs, representative cases were subjected to GeoMx digital spatial profiler (DSP) analysis. As expected, a low density of TILs was significantly associated with poor disease-free survival (DFS) in MSI-H CRCs. The TIL-low tumors were further classified into two subgroups based on the spatial heterogeneity of TILs: TIL-low/heterogeneity-high and TIL-low/heterogeneity-low subgroups. In both discovery and validation cohorts, the TIL-low/heterogeneity-high, TIL-low/heterogeneity-low, and TIL-high subgroups were significantly associated with poor, intermediate, and good DFS, respectively. In the DSP analysis, the TIL-low/heterogeneity-high subgroup showed higher spatial diversity in the expression of immune-related genes than that of the TIL-low/heterogeneity-low subgroup and exhibited upregulation of genes related to immune checkpoints, chemokine/cytokine receptors, and myeloid cells. TIL-low/heterogeneity-high tumors were also enriched with gene sets related to good response to immune checkpoint inhibitor therapy. In conclusion, TIL-low MSI-H CRCs are prognostically heterogeneous and can be divided into prognostically and immunologically distinct subgroups by considering the spatial heterogeneity of TILs. Our data suggest that intratumoral spatial heterogeneity of TILs can be used as a key element for clinically relevant immunologic subtyping of tumors.

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.

B2M and JAK1/2-mutated MSI-H Colorectal Carcinomas Can Benefit From Anti-PD-1 Therapy.

β2-microglobulin (B2M) and Janus kinases 1 and 2 (JAK1/2) mutations have been suggested as genetic mechanisms of immune evasion for anti–programmed cell death protein 1 (PD-1) therapy. Whether B2M and JAK1/2 lose-of-function mutation can cause primary resistance to anti-PD-1 therapy in colorectal carcinoma (CRC) patients remains controversial. Here, we sought to compare the efficacy of anti-PD-1 therapy in DNA mismatch repair deficient/microsatellite instability–high CRC patients with or without B2M or JAK1/2 mutations. Thirty-Five CRC patients who received anti-PD-1 therapy were enrolled in this study. All tumor samples underwent next-generation sequencing. The clinical and molecular data from 110 CRC patients sequenced with the Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) assay and accessed through cBioportal were also analyzed in this study. Of the 35 CRC patients from our center, 10 (28.6%) had a B2M loss-of-function mutation, and 8 (22.9%) had a JAK1/2 loss-of-function mutation. Compared with B2M wild-type CRCs, B2M-mutated CRCs did not show a higher frequency of resistance to anti-PD-1 therapy (P=0.71). There was even better response to anti-PD-1 therapy in patients with JAK1/2 mutation than in those without (P=0.015). Of the 110 CRC patients in the MSK-IMPACT datasets, 13 (11.8%) had a B2M mutation, and 15 (13.6%) had a JAK1/2 mutation. After analyzing the response to anti-PD-1 therapy in these 110 patients, we found similar results (P=0.438 and 0.071, respectively). Moreover, patients with B2M or JAK1/2 mutation had a lower tumor mutational burden score compared with those without. B2M and JAK1/2 loss-of-function mutations occur frequently in microsatellite instability–high CRC. Our study demonstrated that patients with CRC harboring B2M or JAK1/2 mutations should not be excluded from anti-PD-1 therapy.

DNA mismatch repair and microsatellite instability in colorectal tumors: An observational study in the Veterans Affairs Health Care System.

e15562 Background: Microsatellite instability-high (MSI-H) and deficient mismatch repair (dMMR) cancers exhibit high mutational load and are observed in colorectal carcinomas (CRC). The objectives of this study were to determine the proportion of MSI-H/dMMR in CRC and to describe clinical and demographic characteristics among MSI-H/dMMR and non-MSI-H/dMMR CRC patients receiving standard of care in an equal access health care system. Methods: Using the Veteran Affairs (VA) Healthcare system data, we conducted a retrospective study of patients, aged ≥18 years old, diagnosed with CRC who underwent MSI/MMR testing from January 1, 2010 to December 31, 2018. MSI/MMR testing was defined as having received an MSI status determined by polymerase chain reaction and/or next-generation sequencing test or an MMR status determined by immunohistochemistry test. Median and interquartile range (IQR) were calculated for continuous variables while frequencies and percentages were calculated for categorical variables. Results: A total of 291 patients diagnosed with CRC who underwent MSI/MMR testing between 2010-2018 were identified from VA centers. The majority of patients were white (69.8%) and male (95.5%), with median (IQR) age of 65 (56-70) years at diagnosis. Sixty-four (22.0%) patients had stage I CRC at diagnosis, 84 (28.9%) stage II, 87 (29.9%) stage III, and 45 (15.5%) stage IV; 11 (3.7%) had no stage information. Fifty-four (18.6%) patients were reported to have MSI-H and/or dMMR CRC, with rates similar for MSI-H [17.4% (21/121)] vs. dMMR [19.7% (44/223)] alone. The proportion of MSI-H/dMMR varied by CRC stage, and was more common in stage II (26%) and stage III (23%) than in stage IV (6%). Patients were similar between MSI-H/dMMR and non-MSI-H/dMMR with regards to age at diagnosis, gender, race, and ethnicity. Of 54 patients with MSI-H/dMMR CRC, three had metastatic CRC (mCRC) at diagnosis and seven progressed to mCRC. Among 51 MSI-H/dMMR patients without metastatic disease at diagnosis, 18 (35%) underwent chemotherapy and 44 (86%) underwent curative surgery. A similar pattern was also observed among non-MSI-H/dMMR CRC patients. Conclusions: In this real-world observational study in a VA population, we found MSI-H/dMMR was present in 18.6% of CRC patients who received MSI testing, highest in stage II (26%) and stage III (23%) and lowest in stage IV (6%) disease. Results are consistent with published estimates seen across other populations and geographies. Additional research is needed in larger cohorts of patients to further assess utilization patterns and real-world clinical outcomes of newer treatments (immunotherapies) approved for MSI-H/dMMR CRC.

Comparison of Clinical and Histopathological Parameters amongst Microsatellite Unstable and Microsatellite Stable Cases of Colorectal Carcinomas in an Indian Setting

Introduction:Inthiseraofprognosisbasedmedicine,itisimportant to identify microsatellite unstable Colorectal Cancers (CRCs) as they offer good prospects to the patient and they respond poorly to 5-fluorouracil and platinum based chemotherapeutic regime. Aim: To find out the prevalence of Microsatellite Instability-High (MSI-H) in CRC, to identify clinicopathological features associated with Microsatellite Instability (MSI) and assess the value of surgical pathology in predicting MSI-H. Materials and Methods: The present study was a case-control study conducted in a tertiary care centre of Pune in Western India from January 2013 to December 2020. Thirty-five CRCs deficient in Mismatch Repair (MMR) proteins contrasted with 206 Microsatellite Stable (MSS) CRCs were studied and analysed for a given set of clinical and histopathological parameters to find out any correlation between the occurrence of microsatellite unstable tumours and these variables were presented as percentages. Results: In the present study, the prevalence rate of MSI-H was found to be 14.5% and the statistical analysis was carried out using the software Statistical Package for the Social Sciences (SPSS) version 27.0. Univariate analysis revealed that right-sided/proximal location of tumours, age at diagnosis less than 50 years, no lymph node deposits (N0 disease), presence of Tumour Infiltrating Lymphocytes (TILs), peri-tumoural reaction, mucinous component, increased stromal plasma cells, histological heterogeneity, signet ring/medullary component and Crohn-like reaction were all statistically significant predictors of microsatellite instability (p-value <0.05). Multivariate analysis of these significant parameters revealed right-sided location of tumours, age at diagnosis less than 50 years, N0 disease, and presence of TILs, increased stromal plasma cells, histological heterogeneity and Crohn-like reaction to be independent predictors. Conclusion: Clinical parameters and histological evaluation is handy in screening for the MSI-H colorectal carcinomas. This would go a long way in selecting the patients who will require confirmatory molecular testing and thus precluding the need of Immunohistochemistry (IHC), which will be helpful in day-to-day practice as it is uncomplicated, cost-effective and easy to replicate.

Immune subtype of mismatch repair deficient colorectal carcinoma.

e15194 Background: Half of microsatellite instability-high (MSI-H) colorectal carcinoma (CRCs) patients do not response to checkpoint blockade (ICB) immunotherapy. We aimed to unleash the underlying mechanism of distinct efficacy to ICB by sophisticated depicting the immune microenvironment of MSI-H CRC. Methods: Public omics and clinical data of patients with MSI labels were collected from TCGA data portal (364 samples) and Gene Expression Omnibus (GSE39582, GSE13294, GSE13067, GSE35896, GSE24514, GSE25071, GSE79959, GSE103340, GSE29638, GSE33113 cohorts, 1119 samples). GEO datasets were used as discovering sets, and TCGA dataset was used as validating sets. The immune cell infiltrations and signaling pathways in the tumor microenvironment were quantified by single sample Gene Set Enrichment Analysis (ssGSEA), and the bio-similarities were estimated by t-Distributed Stochastic Neighbor Embedding (tSNE). Clustering was identified by k-mean unsupervised strategy with optimal k. Statistical analysis and data visualization were all carried out using the R. Results: Based on the whole transcriptome and immune cell infiltration profiling, both GEO and TCGA cohorts showed that MSI-H patients harbored unique pattern which significantly differed from the MSS counterpart. Unsupervised clustering further classified the MSI-H patients into “Hot” and “Cold” subgroups with remarkable different IFNG-related signatures, inflammatory response score, interferon-γ signaling score, and survival outcome. Screening assay identified 23 genes ( ADAMTSL3, DLGAP4, DNAH11, FFAR3, GABRA4, KCNA6, KCTD9, KIAA1033, KLHL6, LARGE, MORC1, NDST3, OR5H14, PAX5, PHF3, PIK3R5, RNF25, SPATA20, STAT4, TLR9, TMC3, TTC12, ZNF790) enriched and 5 genes ( CELSR3, DGKD, HECTD4, RECQL4, SLC9A4) spared in immune “hot” tumors. Moreover, “hot” tumor showed less WNT signaling pathway score, which was negatively correlated with the IFNG signaling and inflammatory response within the MSI-H CRC tumors. Conclusions: We firstly identified immune hot and cold groups from MSI-H CRCs, which may be related to different immunotherapy response. This result may help further understanding the anti-immunotherapy of MSI-H CRCs.

Minimal microsatellite shift in microsatellite instability high endometrial cancer: a significant pitfall in diagnostic interpretation

Mismatch-repair deficiency testing plays a critical role in the identification of proband in Lynch Syndrome families and triaging patients with high stage or recurrent solid malignancies for check point inhibitor (Pembrolizumab) immunotherapy. We compared microsatellite shift patterns of microsatellite instability PCR analysis at 5 NCI recommended loci between microsatellite instability high endometrial carcinoma (n = 50) and microsatellite instability high colorectal cancer (n = 19). The endometrial cancer cohort included 45 endometrioid, 1 serous, and 4 clear cell carcinomas. Overall, 52% (26/50) of microsatellite instability high endometrial cancers showed minimal microsatellite shift (defined as a one to three nucleotide repeat shift at an involved locus) observed at least at one locus. Among microsatellite instability high endometrial cancers with minimal microsatellite shift, the frequencies at each involved locus were D2S123 (21/21, 100%), D17S250 (10/11, 89%), D5S346 (11/12, 92%), BAT25 (9/12, 80%), and BAT26 (8/21, 45%). Noticeably, 11 of the 26 cases (42%) showed only minimal shift. Among microsatellite instability high endometrial cancers with minimal microsatellite shift, 65% (17/26) had combined MLH1 and PMS2 loss, 8% (2/26) had combined MSH2 and MSH6 loss, 13% (3/26) had MSH6 loss and 15% (4/26) had loss of PMS2 by immunohistochemistry. In contrast, only 16% (3/19) had minimal microsatellite shift seen in colorectal cancer cohort with corresponding loss of MLH1/PMS2, MSH2/MSH6, or MSH6. Overall, 15% (7/50) of microsatellite instability high endometrial carcinomas showed isolated loss of MSH6 in contrast to 7% (1/15) seen in microsatellite instability high colorectal carcinomas. In conclusion, microsatellite instability high endometrial carcinomas have a significantly higher frequency of minimal microsatellite shift that coincides with a high percentage of combined loss of MLH1/PMS2. Microsatellite instability high endometrial cancers also have more frequent loss of MSH-6. Diagnostically, recognition of minimal microsatellite shift is crucial for accurate interpretation of microsatellite instability PCR data of endometrial carcinoma.

Colorectal Carcinomas Containing Hypermethylated MLH1 Promoter and Wild-Type BRAF/KRAS Are Enriched for Targetable Kinase Fusions.

Kinase fusions are rare and poorly characterized in colorectal carcinoma, yet they present unique opportunities for targeted therapy. In this study, we characterized kinase fusions from patients with advanced colorectal carcinoma who had MSK-IMPACT testing of their tumors between January 2014 and June 2018. Patients were analyzed for the presence of fusions, microsatellite instability (MSI), and RAS/BRAF mutations. Mismatch repair (MMR), IHC, and promoter hypermethylation status of MLH1 (MLH1ph) in microsatellite instability-high (MSI-H) colorectal carcinoma with fusions were investigated. Fusion transcripts were confirmed using a targeted RNA-seq panel assay. Of 2,314 colorectal carcinomas with MSK-IMPACT testing, 21 harbored kinase fusions. Overall 57% (12/21) of colorectal carcinoma fusions were MSI-H/MMR-D. Loss of MLH1 and MLH1ph was confirmed in all 12 and all 10 cases with available material, respectively. Fusions were present in 5% of MSI-H/MMR-D colorectal carcinoma compared with 0.4% of MSS/MMR-P colorectal carcinoma (P < 0.001) and 15% of MSI-H/MMR-D colorectal carcinoma with wild-type RAS/BRAF. Of 24 total MLH1-deficient colorectal carcinomas with MLH1ph and wild-type RAS/BRAF, 10 (42%) harbored kinase fusions. Kinase fusions in MSI-H colorectal carcinoma were associated with sporadic MLH1ph rather than with Lynch syndrome, and these patients may be eligible for kinase inhibitors, particularly following resistance or toxicity in response to immunotherapy. These findings identify a molecular subset of colorectal carcinoma with kinase fusions that may be responsive to kinase inhibitors.Significance: A high frequency of targetable kinase fusions in BRAF/RAS wild-type, MSI-H colorectal carcinoma offers a rationale for routine screening to identify patients with colorectal carcinoma with kinase fusions that may be responsive to kinase inhibitors.See related commentary by Valeri, p. 1041.

Majority of B2M-Mutant and -Deficient Colorectal Carcinomas Achieve Clinical Benefit From Immune Checkpoint Inhibitor Therapy and Are Microsatellite Instability-High.

Microsatellite instability-high (MSI-H) colorectal carcinomas (CRCs) show high rates of response to immune checkpoint inhibitors (IOs). B2M mutations and protein loss have been proposed as causes of resistance to IOs, yet they are enriched in MSI-H CRC. We aimed to characterize B2M-mutant, IO-naive CRC. All CRCs with results for Memorial Sloan Kettering Integrated Mutation Profiling of Actionable Cancer Targets, a next-generation sequencing assay that interrogates > 400 genes for mutations as well as MSI status, were surveyed for B2M mutations. All B2M-mutant CRCs were assessed for expression of B2M, major histocompatibility complex class I, and programmed death-1 ligand (PD-L1) via immunohistochemistry and average CD3+ and CD8+ tumor-infiltrating lymphocyte counts against a control group of MSI-H B2M wild-type CRCs. Fifty-nine (3.4%) of 1,751 patients with CRC harbored B2M mutations, with 84% (77 of 92) of the mutations predicted to be truncating. B2M mutations were significantly enriched in MSI-H CRCs, with 44 (24%) of 182 MSI-H CRCs harboring B2M mutations (P < .001). Thirty-two of 44 B2M-mutant CRCs with available material (73%) had complete loss of B2M expression, whereas all 26 CRCs with wild-type B2M retained expression (P < .001). B2M mutation status was not associated with major histocompatibility complex class I expression, KRAS or BRAF mutation, tumor-infiltrating lymphocyte level, or PD-L1 expression after adjustment for MSI status. Of 13 patients with B2M-mutant CRC who received programmed death-1 or PD-L1 IOs, 11 (85%) achieved clinical benefit, defined as stable disease or partial response using Response Evaluation Criteria in Solid Tumors criteria. B2M mutations occur in approximately 24% of MSI-H CRCs and are usually associated with loss of B2M expression. Most patients with B2M-mutant MSI-H CRC with loss of protein expression obtain clinical benefit from IOs.

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.

Dominant high expression of wild-type HSP110 defines a poor prognostic subgroup of colorectal carcinomas with microsatellite instability: a whole-section immunohistochemical analysis.

The aim of this study is to establish heat shock protein 110 (HSP110) as a prognostic biomarker of colorectal carcinomas (CRCs) with microsatellite instability-high (MSI-H) by considering the intratumoral heterogeneity of HSP110 expression. We performed whole-section immunohistochemistry (IHC) for wild-type HSP110 (HSP110wt) in 164 MSI-H CRCs. The intensity of the HSP110wt expression in tumor cells was semiquantitatively scored (0/1/2/3), and the HSP110wt expression status of each tumor was classified as low or high using the following four scoring criteria: H-score, dominant intensity score, lowest intensity score, and highest intensity score. Among the four criteria, only the dominant intensity score-based dichotomous classification of HSP110wt expression was significantly associated with a difference in disease-free survival (log-rank p=0.035) in 164 MSI-H CRCs. The HSP110wt-low MSI-H CRCs were significantly correlated with larger deletions in the HSP110 T17 mononucleotide repeat (≥4bp; p<0.001). In conclusion, the dominant intensity score-based assessment of HSP110wt IHC can be a simple and useful method for the prognostic stratification of MSI-H CRCs. It is expected that HSP110wt IHC may be used to identify a subgroup of MSI-H CRCs with poor prognosis and/or candidates for further treatment, such as immunotherapy using immune checkpoint inhibitors in MSI-H CRCs.

Abstract 4578: Does poor prognosis in African Americans with MSI-H colorectal cancer associate with altered immune markers

Abstract BACKGROUND: Microsatellite instability high (MSI-H) of sporadic colorectal carcinomas (CRC) is usually associated with improved prognosis and a high density of tumor-infiltrating lymphocytes. However, African Americans with MSI-H have poor prognosis. AIM: To evaluate whether or not expression of different immune and tumor markers individually or in combination in African American MSI-H CRC associate with the prognosis status. METHODS: Tissues Microarray (TMA) were prepared by microdissection from FFPE (Formalin Fixed Paraffin Embedded) blocks of 15 MSI-H patients. Immune markers (CD8+, Granzymes, Perforins, CD4+, STAT1, IRF1 and IRF5) and PD-L1 status were analyzed by immunohistochemistry (IHC). Demography and clinical data including TNM, tumor grading (WHO standard), histological type of the tumor, date of diagnosis, date of the last follow-up examination, treatment, comorbidities, metastasis, recurrence, 5-year disease-free survival and death were collected. RESULTS: There were 4 patients with improved prognosis (27%) with relatively high CD4 density (2 with &amp;gt;50% and 2 with 11-50%) regardless of PD-L1 status (3+/1-), stage (II,III), and other immune markers (CD8, CD4, Granzymes, Perforins, IRF1, IRF5 and STAT1). One of the improved prognosis patients with KRAS mutation has elevated expression of all considered immune markers besides CD4. There were 9 patients (6 dead, 3 alive) with poor prognosis, with different immune and tumor markers level (3 were PD-L1+ and 5 have relatively high CD4 count; but with low level of STAT1. There were also 2 patients with expected improved prognosis based on their immune markers level (stage II), however, they died within the 5 year period post-diagnosis, due to their age (98 and 87 years old). Conclusion: MSI-H Colorectal cancer from African American patients has poor prognosis which may correlate with the nature of tumor-associated immune response. Other factors such as MSH3 defects might cancel the positive prognosis of MSI-H status. Nonetheless, low STAT1 and low CD4 may be indicators of poorer prognosis. Citation Format: Umamaheshwari Golconda, Lena Sokol, Babak Shokrani, Edward Lee, Donna Hansel, Oluwole Fadare, Sandip Patel, Mehdi Nouraie, Hooman Soleimani, Zaki Sharif, Ali Afsari, Fareed Daremipouran, Hassan Brim, Hassan Ashktorab. Does poor prognosis in African Americans with MSI-H colorectal cancer associate with altered immune markers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4578. doi:10.1158/1538-7445.AM2017-4578

Intratumoral Fusobacterium nucleatum abundance correlates with macrophage infiltration and CDKN2A methylation in microsatellite-unstable colorectal carcinoma.

Fusobacterium nucleatum (Fn), a specific species of gut microbiota, has been suggested to be enriched in the microsatellite instability-high (MSI-H) molecular subtype of colorectal carcinomas (CRCs). However, the clinicopathologic and molecular factors that interact with Fn in MSI-H CRCs are poorly understood. In this study, 16S ribosomal RNA gene DNA sequence of Fn was quantitatively measured by real-time polymerase chain reaction in tumor DNA samples from a total of 160 surgically resected MSI-H CRC tissues. Each case was classified into one of the three categories based on the Fn DNA amount: Fn-high, Fn-low, and Fn-negative. The clinicopathologic and molecular associations of Fn in MSI-H CRCs were statistically analyzed. Among the 160 MSI-H CRC samples, 15 (9%), 92 (58%), and 53 (33%) cases were Fn-high, Fn-low, and Fn-negative, respectively. Compared with Fn-low/negative tumors, Fn-high MSI-H CRCs were significantly associated with a high density of CD68+ tumor-infiltrating macrophages (P=0.019) and promoter CpG island hypermethylation of the CDKN2A (p16) gene (P=0.008). There were also tendencies toward associations of Fn-high with the BRAF V600E mutation (P=0.047) and active Crohn-like lymphoid reactions (P=0.052) in MSI-H CRCs. However, Fn-high was not significantly associated with CD3+ T cell density, CD163+ M2 macrophage density or PD-L1 expression status. In conclusion, high amounts of intratumoral Fn are correlated with increased macrophage infiltration and CDKN2A promoter methylation in MSI-H CRCs.

Comparative validation of assessment criteria for Crohn-like lymphoid reaction in colorectal carcinoma

AimsCrohn-like lymphoid reaction (CLR) in colorectal carcinoma (CRC) is associated with a favourable prognosis and microsatellite instability-high (MSI-H) status. However, there is a lack of consensus on optimal criteria for...

Annexin A10 expression correlates with serrated pathway features in colorectal carcinoma with microsatellite instability.

Annexin A10 (ANXA10) has recently been identified as a marker of sessile serrated adenomas/polyps of the colorectum. Although the serrated neoplasia pathway is thought to be involved in the majority of microsatellite instability-high (MSI-H) sporadic colorectal carcinomas (CRCs), the clinicopathological implications of ANXA10 expression in CRC are unknown. Here, we evaluated ANXA10 expression status in 168 MSI-H CRCs by immunohistochemistry. Among 168 MSI-H CRCs, nuclear staining for ANXA10 in tumor cells revealed 28 cases (17%) with ANXA10-positive (ANXA10+) tumors. Most of the ANXA10+ tumors were located in the proximal colon (96%, p < 0.001). The ANXA10+ phenotype in MSI-H CRC was significantly associated with female gender (68%, p = 0.016), CpG island methylator phenotype-high (CIMP-H) (68%, p < 0.001), MLH1 promoter hypermethylation (61%, p < 0.001), loss of MLH1 expression (82%, p = 0.019), and wild-type KRAS status (96%, p = 0.023). Survival analysis revealed no prognostic significance of ANXA10 expression in MSI-H CRC. In conclusion, ANXA10+ MSI-H colon carcinomas are characterized by serrated pathway features, including proximal location, female predominance, and high frequencies of CIMP-H status and MLH1 methylation.

Differential Features of Microsatellite-Unstable Colorectal Carcinomas Depending on EPCAM Expression Status

BackgroundRecent studies have revealed that a small subset of Lynch syndrome-associated colorectal carcinomas (CRCs) is caused by a germline EPCAM deletion-induced MSH2 epimutation. Based on the finding of this genetic alteration, we investigated the implications of EPCAM expression changes in microsatellite instability-high (MSI-H) CRCs.MethodsExpression of EPCAM and DNA mismatch repair proteins was assessed by immunohistochemistry in 168 MSI-H CRCs. Using DNA samples of these tumors, MLH1 promoter methylation status was also determined by methylation-specific real-time polymerase chain reaction method (MethyLight).ResultsAmong 168 MSI-H CRCs, complete loss (CL) and focal loss (FL) of EPCAM expression was observed in two (1.2%) and 22 (13.1%) cases, respectively. Both of the EPCAM-CL cases were found in MSH2-negative tumors without MLH1 promoter methylation. However, only nine of the 22 EPCAM-FL tumors had MSH2 deficiency. Of the 22 EPCAM-FL tumors, 13 showed MLH1 loss, and among them, nine cases were determined to have MLH1 methylation. EPCAM-FL was significantly associated with advanced stage (p=.043), distant metastasis (p=.003), poor differentiation (p=.001), and signet ring cell component (p=.004).ConclusionsLoss of EPCAM expression is differentially associated with clinicopathological and molecular features, depending on the completeness of the loss, in MSI-H CRCs.

Different gene expression profiles between microsatellite instability-high and microsatellite stable colorectal carcinomas.

Recent molecular genetic studies have revealed that two major types of genomic instabilities, chromosomal instability (CIN) and microsatellite instability (MSI), exist in colorectal carcinomas. In order to clarify the molecular signature related to the CIN and MSI in colorectal carcinomas, we performed transcriptomic expression analysis on eight microsatellite instability-high (MSI-H) colorectal carcinomas and compared the results obtained with that of nine microsatellite stable (MSS) colorectal carcinomas using oligonucleotide microarrays containing 17 334 known genes and 1331 unknown genes or expression sequence tags (ESTs). Unsupervised two-way hierarchical clustering with 5724 genes successfully classified tumors from normal mucosa, and displayed a distinctive MSI-H carcinomas subgroup. Based on intensive filtering, 57 known genes and eight ESTs were found to be highly relevant to the differentiation of MSI-H and MSS colorectal carcinomas. These genes successfully distinguish the new test set of six MSI-H and five MSS colorectal carcinomas. Many up- and downregulated genes in MSI-H colorectal carcinomas were related to the previously reported phenotypic characteristics; increased mucin production and intense peritumoral immune response in MSI-H carcinomas. Some of these differences were confirmed by semiquantitative reverse transcription-PCR and immunohistochemical analysis. Our findings indicate that there are many different genetic and transcriptomic characteristics between MSI-H and MSS colorectal carcinomas, and some of these differently expressed genes can be used as diagnostic or prognostic markers.