Microsatellite Instability Phenotype Research Articles

Abstract 4924: Assessing microsatellite instability dominance in colorectal cancer phenotype: A multi-study initiative using multi-target transformers for genomic biomarker prediction

Abstract We used deep learning (DL) to predict multiple genetic biomarkers of colorectal cancer (CRC) from routine histopathology images, aiming to identify morphological changes associated with the mutational profile of tumors. A new Transformer-based prediction model was developed which can simultaneously predict multiple biomarkers from a single histopathology image. We compared the performance of the multi-target approach to conventional single-target models. We analyzed five patient cohorts from the Genetics and Epidemiology of CRC Consortium (GECCO) with colorectal cancer (N=1,385 patients). Our model was trained on an internal data set (N=739) to predict the presence of >200 genetic alterations, focusing on genes with non-silent mutations and mutational signatures, which were assessed using targeted sequencing. We used a 7-fold cross validation and validated the model on an independent, external data set (N=646). Morphological features of microsatellite instability (MSI) are detectable by DL models in histopathology images, making MSI a potential confounding factor. Thus, we also assessed whether our model can predict genetic alterations regardless of MSI status, or whether the model identifies the MSI phenotype. For the majority of biomarkers, the multi-target transformer reached a higher prediction performance as measured by the mean Area Under the Receiver Operating Characteristic (AUROC) Curve and lower standard deviation than single-target DL models. For the external validation set, the mean AUROC of the multi-target versus the single-target model was 0.78 (+/- 0.01) versus 0.72 (+/- 0.06) for BRAF mutations, 0.88 (+/- 0.01) versus 0.86 (+/- 0.03) for hypermutation status, 0.94 (+/- 0.01) versus 0.91 (+/- 0.02) for MSI, 0.86 (+/- 0.01) versus 0.80 (+/- 0.05) for RNF43 mutations and 0.72 (+/- 0.02) versus 0.69 (+/- 0.05) for TP53 mutations, respectively. However, our analysis of the individual prediction scores suggests that the model does not identify the phenotype of the alterations themselves. Instead, the model seems to determine the status of specific genetic alterations based on their correlation with MSI. Consequently, biomarkers which are associated with microsatellite stability have prediction scores negatively correlated with predicted MSI scores. Conversely, biomarkers which are associated with MSI have prediction scores positively correlated with predicted MSI scores. Our results demonstrate potential benefits of multi-target DL models in improving the predictive power and efficacy of histology-based biomarker extraction compared to single-target DL models. However, our results crucially highlight the importance of considering potential confounders, such as MSI status in CRC, and emphasize the need for extensive analysis beyond AUROC in studies focusing on DL-based biomarker detection. Citation Format: Marco Gustav, Marko van Treeck, Zunamys I. Carrero, Chiara M. Loeffler, Nic G. Reitsam, Bruno Märkl, Asier Rabasco Meneghetti, Lisa A. Boardman, Amy J. French, Ellen L. Goode, Andrea Gsur, Stefanie Brezina, Marc J. Gunter, Neil Murphy, Paul Limburg, Stephen Thibodeau, Sebastian Foersch, Robert Steinfelder, Tabitha Harrison, Ulrike Peters, Amanda Phipps, Jakob N. Kather. Assessing microsatellite instability dominance in colorectal cancer phenotype: A multi-study initiative using multi-target transformers for genomic biomarker prediction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4924.

Large-Scale Cancer Genomic Analysis Reveals Significant Disparities between Microsatellite Instability and Tumor Mutational Burden.

Microsatellite instability (MSI) and tumor mutational burden (TMB) are predictive biomarkers for pan-cancer immunotherapy. The interrelationship between MSI-high (MSI-H) and TMB-high (TMB-H) in human cancers and their predictive value for immunotherapy in lung cancer remain unclear. We analyzed somatic mutation data from the Genomics Evidence Neoplasia Information Exchange (n = 46,320) to determine the relationship between MSI-H and TMB-H in human cancers using adjusted multivariate regression models. Patient survival was examined using the Cox proportional hazards model. The association between MSI and genetic mutations was assessed. Patients (31-89%) with MSI-H had TMB-low phenotypes across 22 cancer types. Colorectal and stomach cancers showed the strongest association between TMB and MSI. TMB-H patients with lung cancer who received immunotherapy exhibited significantly higher overall survival [HR, 0.61; 95% confidence interval (CI), 0.44-0.86] and progression-free survival (HR, 0.65; 95% CI, 0.47-0.91) compared to the TMB-low group; no significant benefit was observed in the MSI-H group. Patients with TMB and MSI phenotypes showed further improvement in overall survival and PFS. We identified several mutated genes associated with MSI-H phenotypes, including known mismatch repair genes and novel mutated genes, such as ARID1A and ARID1B. Our results demonstrate that TMB-H and/or a combination of MSI-H can serve as biomarkers for immunotherapies in lung cancer. These findings suggest that distinct or combined biomarkers should be considered for immunotherapy in human cancers because notable discrepancies exist between MSI-H and TMB-H across different cancer types.

Short tandem repeat mutations regulate gene expression in colorectal cancer

Short tandem repeat (STR) mutations are prevalent in colorectal cancer (CRC), especially in tumours with the microsatellite instability (MSI) phenotype. While STR length variations are known to regulate gene expression under physiological conditions, the functional impact of STR mutations in CRC remains unclear. Here, we integrate STR mutation data with clinical information and gene expression data to study the gene regulatory effects of STR mutations in CRC. We confirm that STR mutability in CRC highly depends on the MSI status, repeat unit size, and repeat length. Furthermore, we present a set of 1244 putative expression STRs (eSTRs) for which the STR length is associated with gene expression levels in CRC tumours. The length of 73 eSTRs is associated with expression levels of cancer-related genes, nine of which are CRC-specific genes. We show that linear models describing eSTR-gene expression relationships allow for predictions of gene expression changes in response to eSTR mutations. Moreover, we found an increased mutability of eSTRs in MSI tumours. Our evidence of gene regulatory roles for eSTRs in CRC highlights a mostly overlooked way through which tumours may modulate their phenotypes. Future extensions of these findings could uncover new STR-based targets in the treatment of cancer.

Characterization of sessile serrated adenomas with dysplasia including intramucosal adenocarcinoma and colorectal carcinoma with a microsatellite instability phenotype

Recent studies have shown that sessile serrated lesions (SSLs) lead to the development of colorectal cancer (CRC) with a microsatellite instability (MSI) phenotype via a dysplasia-carcinoma sequence. However, the pathological and molecular mechanisms of SSL with dysplasia (SSLD) are unclear. Here, we aimed to examine the clinicopathological and molecular alterations in SSLD and to evaluate the significance of such alterations with regard to lesion progression. Fifty-four SSLDs (20 serrated dysplasia cases and 17 intestinal dysplasia cases, including 30 low-grade dysplasia [LGD] cases, 7 high-grade dysplasia [HGD] cases, and 17 intramucosal adenocarcinomas [IMAs]) were evaluated. Molecular alterations, including immunohistochemical expression of various markers, DNA methylation status, and multiple genetic mutations (using next-generation sequencing), were assessed. Additionally, such alterations were also investigated in 41 CRCs with an MSI phenotype (invasion beyond submucosa). The frequency of mismatch repair (MMR) deficiency in SSLD was 12 of 39 cases (32.4 %), whereas the MMR proficient type was observed in 17 of 39 SSLD cases. SSLD with serrated dysplasia showed a significantly higher frequency of loss of MMR protein expression and methylation status. Moreover, loss of MMR protein expression differed significantly between LGD and IMA. Furthermore, the frequency of TP53 mutation was significantly higher in IMA than in LGD. The current findings demonstrated that SSL with serrated dysplasia may be associated with an increased risk of malignant transformation compared with intestinal dysplasia. Loss of MMR proteins and mutation of TP53 may play important roles in tumor progression from dysplasia to carcinomatous lesions.

Deficient mismatch repair/microsatellite unstable colorectal cancer: therapeutic advances and questions.

The microsatellite instability (MSI) phenotype is related to a deficiency of the DNA mismatch repair (dMMR) system and is observed in 5% of metastatic colorectal cancers (mCRCs). MSI/dMMR phenotype testing should be routine for all CRCs regardless of stage. Two complementary techniques with a high concordance (90-97%) allow us to determine the MSI/dMMR status of a tumor: immunohistochemistry and polymerase chain reaction. Since 2020 and the results of the phase III KEYNOTE 177 trial, pembrolizumab [anti-programmed cell death protein 1 (PD1)] is the new standard of care in first-line MSI/dMMR mCRC. To date, no combination of chemtotherapy ± targeted therapy with immune checkpoint inhibitors (ICIs) has been validated in the management of MSI/dMMR mCRC, and it is not known whether this combination would be beneficial. It is also unclear whether dual therapy with two ICIs is more effective than monotherapy. Several phase III trials are ongoing to answer these questions. Despite a high response rate and long-term benefit of a first line by anti-PD1, 30-50% of patients with MSI/dMMR mCRC experience an early or secondary progression. There are currently no validated predictive biomarkers of anti-PD1 ± anti-cytotoxic T lymphocyte antigen-4 resistance in patients with MSI/dMMR mCRC. In case of early progression on ICIs, the first two questions to consider are the possibility of pseudoprogression and the correct diagnosis of MSI/dMMR status. To date, there are no data on the use of adjuvant ICIs for MSI/dMMR resected colon cancers. By contrast, data are accumulating regarding the efficacy of neoadjuvant ICIs, with at least two-thirds of patients in the different trials in pathological complete response, making it possible to envisage 'Watch and wait' strategies in future.

Low incidence of microsatellite instability in gastric cancers and its association with the clinicopathological characteristics: a comparative study

Gastric cancer is a complex heterogeneous disease with different molecular subtypes that have clinical implications. It is characterized by high mortality rates and limited effective therapies. Microsatellite instability (MSI) has been recognized as a subgroup with a good prognosis based on TCGA and ACRG categorizations. Besides its prognostic and predictive value, gastric cancers with high MSI exhibit different clinical behaviors. The prevalence of high MSI has been assessed in gastric cancer worldwide, especially in East Asia, but there is a lack of such information in the Middle East. Therefore, this study aimed to investigate the incidence and status of MSI in Iranian gastric cancer patients using 53 samples collected from 2015 to 2020 at Taleghani Hospital Medical Center. DNA from tumoral and normal tissues were extracted and assessed through multiplex-PCR based on five mononucleotide repeats panel. Clinicopathological variables, including age, sex, Lauren classification, lymph node involvement, TNM stage, differentiation, localization, and tumor size, were also analyzed. With 2 males and 2 females, high microsatellite instability represented a small subgroup of almost 7.5% of the samples with a median age of 60.5 years. High microsatellite instability phenotypes were significantly associated with patients aged 68 years and older (p‑value of 0.0015) and lower lymph node involvement (p‑value of 0.0004). Microsatellite instability was also more frequent in females, with distal gastric location, bigger tumor size, and in the intestinal type of gastric cancer rather than the diffuse type.

Focusing on colorectal cancer in young adults (Review).

Colorectal cancer (CRC) ranks as the third leading cause of cancer-related mortality worldwide. Recent years have witnessed an increase in the incidence of CRC among adults <50 years old on a global scale. The increased incidence is associated with several modifiable risk factors, including obesity, type II diabetes, physical inactivity and frequent antibiotic use. In younger individuals, haematochezia and abdominal pain are the most common symptoms, predominantly affecting the left-side colon. While certain cases of early-onset CRC (eoCRC) are associated with a genetic predisposition, the majority result from sporadic mutations in the genes APC, KRAS, BRAF and TP53, which trigger uncontrolled cell proliferation and tumour formation. Colorectal carcinogenesis involves three major pathways: The chromosomal instability (CIN), microsatellite instability and CpG island methylator phenotype pathways. Dysregulation of the CIN pathway accounts for 85% of sporadic cases of eoCRC. Notably, eoCRC exhibits a distinctive molecular profile, characterized by a decreased prevalence of BRAF mutations, an increased prevalence of KRAS mutations and LINE-1 hypomethylation, and involvement of the Microsatellite and Chromosomal Stable pathway. Prevention strategies for eoCRC primarily centre on lifestyle modifications and the development of screening programs targeting younger populations. Further exploration into the molecular mechanisms involved in the identification of novel risk factors associated with eoCRC is imperative. These efforts, in conjunction with the development of specific screening strategies, hold the potential to reduce morbidity and mortality in the future.

Immunohistochemistry, Molecular Biology, and Clinical Scoring for the Detection of Muir-Torre Syndrome in Cutaneous Sebaceous Tumors: Which Strategy?

Background: Sebaceous neoplasms (SNs) always raise the possibility of an association with Muir-Torre syndrome (MTS) and permit to screen internal malignancies, colorectal and endometrial carcinomas, before they become symptomatic. Immunohistochemistry (IHC), molecular biology, and clinical examination are different approaches for detection of MTS. We conducted a retrospective analysis of non-selected SNs in order to determine the optimal tools to implement for MTS screening. Methods: Deficient MMR phenotype (dMMR) was determined by either IHC using antibodies directed to four mismatch repair (MMR) antigens on tissue microarray or molecular biology using pentaplex PCR. The Mayo Clinic risk score of MTS was calculated from medical records. Sensibility and specificity of each test for the detection of MTS were determined. Results: We included 107 patients, 8 with multiple SNs, for a total of 123 SNs (43 sebaceous adenomas, 19 sebaceomas, and 61 sebaceous carcinomas (SC)). Loss of at least one MMR protein was observed in 70.7% of tumors, while 48% had a microsatellite instable phenotype. Concordance between both techniques was 92.9%, with a 0.85 Cohen’s kappa coefficient. Nineteen patients (20.2%) had a ≥2 points Mayo Clinic risk score, one having a pMMR SC. Among the 13 patients with confirmed MTS, 2 had a low Mayo Clinic risk score (1 point). IHC had the highest sensitivity for MTS screening (100%) with a specificity of 34.1%, while a >2-point Mayo Clinic risk score had a lower sensitivity (92%) but a higher specificity (89%). Conclusion: To detect MTS in SN patients, the first-line Mayo Clinic risk score followed by IHC appears to be the most accurate strategy with lower cost for society. This strategy should be adapted to the medico-economic resources of each country.

Exploring the Diagnostic Potential of IHC and Next-Generation Sequencing Approaches for Endometrial Carcinoma Detection and Classification

Background: Globally, in 2018 endometrial cancer (EC) regarded as the sixth most commonly diagnosed cancer and the fourteenth leading cause of cancer with more deaths in females. Nowadays classical and modern classifications can be performing for diagnosing various types of cancers including endometrial cancer. Based on this our Objective of study is to determine the effectiveness of immunohistochemical observation, a quick and easy method for determining MSI and/or other types of EC which based on modern classification. As molecular study takes more cost we want to compare the both methods and showing the possibility of doing IHC instead of molecular study. Methods: This study designed to establish a small gene panel of Next Generation Sequencing NGS of endometrial cancer patients targeting 12 genes; MSH6, MLH3, MSH3, CDH1, PTEN, MSH2, MLH1, PMS2, FGFR2, BRCA1, MUTYH and AREL1. By using DNBSEQ-G400 Platform, Human Core Exome kit and Python software for analysis is used. At the same time, Dako kit used to performing IHC for six primary antibodies used to detect each of MSH6, MSH2, MLH, PMS2, PTEN and CDH1. The primary antibodies were applied on 5 µm formalin- fixed paraffin- embedded (FFPE). Results; histopathological examinations showed that all patients were at stage II endometrioid endometrial carcinoma. The FIGO classification were Ia and Ib. Microsatellite instability (MSI), nuclear staining immunoreaction for PTEN antibodies and membranous E- cadherin immunoreactions observed through IHC study. The molecular studies detected several polymorphisms which have clinical significant and some of them have conflict interpretations. Conclusions; we considered that, simply immunoreaction staining procedure can be use as alternative method for MSI phenotype detection rather than any type of more expensive and complex method of NGS. Concerning PTEN and ECAD1 antibodies reactions, also could be dependable in diagnosis and treatment and performing them if molecular diagnosis not available.

Pathologic, immunologic, and clinical analysis of the microsatellite instability phenotype in endometrial carcinoma

The objective of this study was to determine if quantifying the microsatellite instability (MSI) phenotype could serve as a biomarker for clinical and immunologic features of deficient mismatch repair (dMMR) endometrial cancer (EC). Patients with EC undergoing hysterectomy whose tumors demonstrated dMMR were included. Immunohistochemistry (IHC) of mismatch repair proteins and polymerase chain reaction analysis of NR27, BAT25, BAT26, NR24, and NR21 microsatellite loci were performed on each case. The MSI phenotype was quantified by subtracting the number of nucleotides of each microsatellite in tumor tissue from the corresponding microsatellite in paired normal tissue and summing the absolute differences. This was termed marker sum (MS)and is a novel quantification. Tumor-infiltrating lymphocytes (TILs) were identified by IHC for CD3, CD4, and CD8 and quantified with digital image analysis. Tumor infiltration of lymphocytes and clinical characteristics were stratified by MS. Four hundred fifty-nine consecutive patients with dMMR EC were analyzed. MS ranged from 1 to 32. Post hoc, 2 cohorts were defined using receiver operating characteristic curves(MS less than 13 and MS greater than 12). With the exception of tumor grade, all clinical and pathologic features, all tumor characteristics, and the numbers of TILs were similar between cohorts. The MSI phenotype is highly variable in dMMR EC, and no correlation between the immune profile and the severity of the MSI phenotype was observed.

Serrated colonic lesions. Current review

Serrated lesions, as a rule, were considered benign and were likened to hyperplastic polyps by anatomists and gastroenterologists. These views persisted until about 2010. However, recent data showed that serrated lesions can potentially transform into colorectal cancer (CRC). The World Health Organization classification identifies 4 categories of serrated lesions: hyperplastic polyps, sessile serrated lesions, traditional serrated adenoma and unclassified serrated adenomas. Sessile serrated lesions with dysplasia and traditional serrated adenomas are the most common precursors of CRC. Development of CRC from serrated lesions occurs through two different molecular pathways, namely, sporadic microsatellite instability and CpG island methylator phenotype, and the latter is considered the main mechanism inactivating serrated CRC pathway. In contrast to adenoma–carcinoma pathway, APC-inactivating mutations are rare in serrated adenomas.

Genetic and epigenetic characteristics of patients with colorectal cancer from Uruguay.

Colorectal cancer (CRC), the 3rd most frequent cancer worldwide, affects both men and women. This pathology arises from the progressive accumulation of genetic and epigenetic alterations. In this study, KRAS, NRAS, PIK3CA, and BRAF gene mutations, mismatch repair (MMR) genes methylation profile, microsatellite instability (MSI) and CpG Island Methylator Phenotype (CIMP) status were assessed. The associations of these molecular features with clinicopathological data were also investigated. A hundred and eight unselected CRC samples and their histological and clinical data, were gathered between 2017 and 2020. The prevalence of KRAS, NRAS and BRAF gene mutations was similar to that described in other populations. 28.7% of tumors were KRAS-mutated, mostly in men, distal location, with a CIMP-negative status. BRAFV600E frequency was 6.5% and associated with MSI (p=0.048), MLH1-methylated (p<0.001) and CIMP-High (p<0.001) status. We also confirmed that BRAFV600E tumors were more prevalent in older women and proximal location. A striking different result was the lack of most common variants in the PIK3CA gene. A complete absence of PIK3CA-mutated tumors in a population has not been previously reported. Among MMR genes, the only with an aberrant methylation pattern was MLH1 gene. Its frequency was 9.25%, lower than previously reported. Methylated tumors were most frequent in patients older than 70 years old and proximal tumor location. Finally, CIMP-High status was mainly observed in moderately differentiated tumors with a rate of 15.7%. Our findings were consistent with previous reports in other populations, but also showed some features unique to our cohort. This study is the first to report the analysis of a large number molecular biomarkers of CRC in Uruguay and one of the few performed in Latin-America.

Non-classical phenotypes of mismatch repair deficiency and microsatellite instability in primary and metastatic tumors at different sites in Lynch syndrome.

Lynch syndrome is a genetic disease characterized by abnormal DNA replication caused by germline variation in the mismatch repair (MMR) gene. There are rare non-classical phenotypes with loss of MMR protein expression and inconsistent microsatellite stability (MSS) in Lynch syndrome-related colorectal cancers. However, the difference between microsatellite instability (MSI) of extraintestinal tumors in a patient with Lynch syndrome has been closely studied. Herein, we reported the non-classical phenotypes of mismatch repair deficiency (dMMR) and MSI in four cases of Lynch syndrome in patients with colorectal cancer and other primary and metastatic tumors. A retrospective analysis was conducted on four patients diagnosed with Lynch syndrome between 2018 and 2022 in the Department of Pathology of the Rocket Forces Specialized Medical Center. A one-step immunohistochemical (IHC) assay was employed to detect loss in the expression of Lynch syndrome-associated MMR proteins (MLH1, PMS2, MSH2, and MSH6). MSI detection was performed in both primary and metastatic tumors at different sites in the four patients using NCI 2B3D (BAT25, BAT26, D2S123, D17S250, and D5S346) and single nucleotide site (BAT25, BAT26, NR21, NR24, NR27, and MONO27) methods. In addition, related MMR gene germline variation, somatic mutations, and MLH1 gene promoter methylation were analyzed using next-generation sequencing and TaqMan probe-based methylation-specific polymerase chain reaction (MethyLight). Two of the four patients were heterozygous for MSH6 germline pathogenic variation, and the other two were heterozygous for MSH2 germline pathogenic variation. In all cases, IHC detection of protein expression of the MMR gene with germline variation was negative in all primary and metastatic tumors; non-classical phenotypes of dMMR and MSI were present between primary and metastatic tumors at different sites. dMMR in Lynch colorectal cancer demonstrated high MSI, whereas MSI in primary and metastatic tumors outside the intestine mostly exhibited MSS or low MSI. The non-classical dMMR and MSI phenotype are mostly observed in Lynch syndrome, even in the context of MMR protein expression loss. Extraintestinal tumors infrequently present with a high degree of MSI and often exhibit a stable or low degree of MSI.

The Role of Histology-Agnostic Drugs in the Treatment of Metastatic Castration-Resistant Prostate Cancer.

Precision medicine has opened up a new era in the development of anti-cancer agents that is focused on identifying biomarkers predictive of treatment response regardless of tumor histology. Since 2017, the Food and Drug Administration has approved six drugs with histology-agnostic indications: pembrolizumab (both for tumors with the mismatch-repair deficiency (dMMR)/high microsatellite instability (MSI-H) phenotype and for those with the high tumor mutational burden (TMB-H) phenotype), dostarlimab (for dMMR tumors), larotrectinib and entrectinib (for tumors harboring neurotrophic tyrosine receptor kinase (NTRK) fusions), and the combination of dabrafenib plus trametinib (for BRAF V600E-mutated tumors). The genomic alterations targeted by these antineoplastic agents are rare in metastatic castration-resistant prostate cancer (mCRPC). Furthermore, only a small number of mCRPC patients were enrolled in the clinical trials that led to the approval of the above-mentioned drugs. Therefore, we critically reviewed the literature on the efficacy of histology-agnostic drugs in mCRPC patients. Although the available evidence derives from retrospective studies and case reports, our results confirmed the efficacy of pembrolizumab in dMMR/MSI-H mCRPC. In contrast, few data are available for dostarlimab, larotrectinib, entrectinib, and dabrafenib-trametinib in this subset of patients. Large, multi-institutional registries aimed at collecting real-world data are needed to better comprehend the role of tissue-agnostic drugs in mCRPC patients.

Association between c-type lectin-like receptor 2 and microsatellite instability in colorectal cancer: a cross-sectional study

BackgroundAs a transmembrane protein, C-type lectin-like receptor 2 (CLEC-2) is mainly expressed on platelets and released into plasma after platelet activation. Activated platelets participate in the regulation of innate immune cells. Patients with different microsatellite statuses have distinct immune profiles. This study aimed to investigate the association of plasma CLEC-2 levels with microsatellite status among colorectal cancer (CRC) patients.MethodsA cross-sectional analysis of 430 CRC patients from Harbin Medical University Cancer Hospital was conducted. CLEC-2 levels were measured with fasting venous blood samples drawn from each participant before any treatment. The microsatellite status was evaluated with DNA obtained from fresh frozen tumor tissue samples. The other clinical data were collected and recorded based on the medical system records.ResultsCLEC-2 levels were significantly higher among patients with high microsatellite instability phenotype than the stable microsatellite group, adjusting for other confounding variables.ConclusionsThe increased CLEC-2 is associated with the high microsatellite instability subtype of CRC.

Abstract 5419: A unique pattern of heterogeneous MMR protein expression in colorectal cancer unveils different degrees of tumor mutational burden and distinct tumor microenvironment features

Abstract Introduction: Mismatch repair (MMR) protein expression in colorectal cancer (CRC) is usually homogeneously retained or lost. However, rare CRC lesions may show a heterogeneous pattern of MMR protein expression. We aimed to evaluate the molecular effect of a heterogeneous expression of the MMR proteins in a complex context of concomitant loss of one of the other effectors of the DNA repair mechanism. Experimental procedures: We evaluated MMR protein expression (by immunohistochemistry -IHC- for MLH1, MSH2, MSH6 and PMS2) in 200 CRCs, identifying 3 groups with proficient (MMRp), deficient (MMRd) and heterogeneous (MMRh) MMR. MMRh tumors were micro-dissected based on the expression of the heterogeneous marker, DNA was extracted and subjected to targeted sequencing (TSO500 panel, Illumina). RNA was purified from bulk tumors of all MMRh cases and in a control series of 10 MMRp and 10 MMRd CRCs and analyzed using the IO360 panel (NanoString). The degree of tumor infiltrating lymphocytes (TILs) was calculated on H&amp;E slides and on IHC sections stained with antibodies raised against CD8. Results: Twenty-nine out of 200 cases were MMRd (14.5%). Nine cases (4.5%) showed a heterogeneous pattern of MMR expression, with 6 tumors harboring concomitant loss of one of the other MMR proteins, resulting in a microsatellite instable (MSI) phenotype. Five out of the 9 cases were suitable for separate mutational analysis of IHC-positive and IHC-(double)negative components of the tumor. Both components of all the lesions exhibited a high tumor mutation burden (mean TMB: 59 mut/Mb) in line with the MSI status, nevertheless a significant increase in TMB in the double-negative components was observed (mean TMB: neg. 67 mut/Mb vs pos. 53 mut/Mb), due to a higher number of sub-clonal, non-synonymous variants compared to the positive component. Comparative gene expression analyses between MMRd, MMRp and MMRh CRCs highlighted differential gene expression patterns, with MMRh tumors displaying a strong activation of angiogenesis, MAPK and PI3K-AKT axes. Moreover, these tumors showed the highest number of TILs, characterized by a substantial population of exhausted CD8+ lymphocytes. Conclusions: We describe a unique subgroup of CRCs showing heterogeneous expression of MMR proteins, in a background of concomitant loss of one of the other markers. This heterogeneity is associated with i) an increase of TMB and of unstable loci in the negative component, with additional markedly sub-clonal variants, ii) a high number of TILs, mostly exhausted CD8+ lymphocytes, and iii) a differential activation of signaling pathways. Taken together these data provide indirect evidence that heterogeneous loss of MMR proteins may impact on the biology of CRCs. Whether this pattern may influence response to immune checkpoint inhibition remains to be determined. Citation Format: Enrico Berrino, Maria Costanza Aquilano, Emanuele Valtorta, Vito Amodio, Giovanni Germano, Marco Gusmini, Ivana Sarotto, Anna Sapino, Silvia Marsoni, Andrea Sartore-Bianchi, Alberto Bardelli, Salvatore Siena, Emanuela Bonoldi, Caterina Marchio. A unique pattern of heterogeneous MMR protein expression in colorectal cancer unveils different degrees of tumor mutational burden and distinct tumor microenvironment features [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 5419.

Abstract 1692: Single-cell DNA sequencing from frozen endometrial tumors to address clonal evolution of somatic mutations

Abstract Background: Single-cell DNA sequencing with droplet microfluidics has enabled the characterization of genetic heterogeneity in hematologic malignancies and fresh solid tumors through the direct DNA sequencing of thousands of single cells from individual samples without the need of whole-genome amplification. The application of this technology in banked solid tumor samples has been rather limited to date, however. Here, we describe and validate a method to characterize clonal evolution of somatic mutations from frozen tumor specimens, utilizing endometrial carcinomas (ECs) as an example. Methods: Tissue cores were obtained from frozen ECs and the extracellular matrix was enzymatically digested. A subset of extracted nuclei were used for bulk DNA extraction and targeted sequencing. The remaining nuclei were encapsulated with cell lysis buffer, and the DNA was primed and barcoded with hydrogel beads containing 317 cancer gene amplicons using a Tapestri instrument (Mission Bio). Following droplet PCR, Illumina libraries were generated and sequenced on a NovaSeq6000. Sequencing reads were trimmed and mapped to the reference genome. Barcode sequences were error-corrected and cells were detected from barcodes based on number of reads assigned to each barcode and amplicon read completeness. Genotyping was performed using GATK and the allele drop-out (ADO) rate was calculated. Non-neoplastic cells were defined by the absence of somatic mutations. Allele-specific copy number (ASCN) profiles for each tumor cell were estimated by normalizing mean reads per cell per amplicon to reads of non-neoplastic cells. Results: We performed single-cell genotyping and ASCN profiling on two ECs with matched bulk sequencing data. For EC1, 5,004 tumor and 132 non-neoplastic cells were sequenced, for a 97% tumor purity. For EC2, 2,981 tumor and 165 non-neoplastic cells were obtained, for a 95% tumor purity. The average number of reads per amplicon per cell for EC1 and EC2 was 116 and 146 with an ADO rate of 6% and 8%, respectively. The data obtained from single-cell analysis were concordant with the matched bulk sequencing data in terms of clonal and subclonal mutations, and their respective cancer cell fraction. Single-cell sequencing further revealed the presence of loss of heterozygosity and emergence of minor subclones as late events in tumor evolution, which could not be inferred by bulk sequencing. In EC2, branch mutations displayed a spectrum consistent with microsatellite instability (MSI), reflecting its acquired MSI phenotype. Conclusions: Here we describe a robust method to perform single-cell targeted DNA sequencing with droplet microfluidics of individual nuclei isolated from frozen tissue samples, providing the opportunity to determine the repertoire of somatic mutations at single cell resolution and to infer cancer clonal architecture and dynamics utilizing banked solid cancer tissue samples. Citation Format: Arnaud Da Cruz Paula, Yingjie Zhu, Shirin Issa Bhaloo, Fresia Pareja, Timothy Hoang, Pier Selenica, Etta Hanlon, Thais Basili, Higinio Dopeso, Nadeem Abu-Rustum, Jorge Sergio Reis-Filho, Britta Weigelt. Single-cell DNA sequencing from frozen endometrial tumors to address clonal evolution of somatic mutations [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 1692.

Diabetes mellitus in relation to colorectal tumor molecular subtypes: A pooled analysis of more than 9000 cases.

Diabetes is an established risk factor for colorectal cancer. However, colorectal cancer is a heterogeneous disease and it is not well understood whether diabetes is more strongly associated with some tumor molecular subtypes than others. A better understanding of the association between diabetes and colorectal cancer according to molecular subtypes could provide important insights into the biology of this association. We used data on lifestyle and clinical characteristics from the Colorectal Cancer Family Registry (CCFR) and the Genetics and Epidemiology of Colorectal Cancer Consortium (GECCO), including 9756 colorectal cancer cases (with tumor marker data) and 9985 controls, to evaluate associations between reported diabetes and risk of colorectal cancer according to molecular subtypes. Tumor markers included BRAF and KRAS mutations, microsatellite instability and CpG island methylator phenotype. In the multinomial logistic regression model, comparing colorectal cancer cases to cancer‐free controls, diabetes was positively associated with colorectal cancer regardless of subtype. The highest OR estimate was found for BRAF‐mutated colorectal cancer, n = 1086 (ORfully adj: 1.67, 95% confidence intervals [CI]: 1.36‐2.05), with an attenuated association observed between diabetes and colorectal cancer without BRAF‐mutations, n = 7959 (ORfully adj: 1.33, 95% CI: 1.19‐1.48). In the case only analysis, BRAF‐mutation was differentially associated with diabetes (P difference = .03). For the other markers, associations with diabetes were similar across tumor subtypes. In conclusion, our study confirms the established association between diabetes and colorectal cancer risk, and suggests that it particularly increases the risk of BRAF‐mutated tumors.

Microsatellite Instability Analysis and Its Prognostic Value in Invasive Nonampullary Duodenal Adenocarcinoma

Purpose: Nonampullary duodenal adenocarcinoma (NADA) is a rare disease. Although several prognostic factors have been reported for this disease, they remain controversial due to their rarity. In this study, we retrospectively analyzed 54 cases of invasive NADA, focusing on the microsatellite instability (MSI) phenotype, programmed cell death-ligand 1 (PD-L1) expression, and prognostic factors. Methods: Expression of the PD-L1 protein and cell differentiation markers in tumors was detected by immunohistochemistry. Microsatellite markers (NR-21, NR-22, NR-24, BAT-25, and BAT-26) were amplified for MSI assessment by PCR. Results: The incidence of MSI in invasive NADA was 35.2%. No significant correlation between the MSI phenotype and clinicopathological factors was observed. Positive expression of PD-L1 by immune cells was common in advanced-stage disease (p = 0.054), and positive expression of PD-L1 in cancer cells correlated significantly with the histologically undifferentiated type (p = 0.016). Kaplan-Meier survival analysis demonstrated a significantly better overall survival (OS) in patients with MSI (p = 0.013) and at early-stage disease (p = 0.000) than in those with microsatellite-stable or at late tumor stages. Univariate and multivariate analyses showed that MSI (hazard ratio [HR]: 0.282, 95% confidence interval [CI]: 0.106–0.751, p = 0.011) and early tumor stage (stage I-II) (HR: 8.81, 95% CI: 2.545–30.500, p = 0.001) were independent better prognostic factors of OS. Conclusions: MSI and early tumor stage (stage I-II) were independent better prognostic factors of OS. A high proportion of MSI phenotypes and positive PD-L1 expression may be helpful for identifying immune checkpoint inhibitors as a novel therapeutic strategy.

Микросателлитная нестабильность в нейроэндокринных новообразованиях толстой кишки

Microsatellite instability (MSI) characterizes a special molecular genetic subtype of malignancies and is associated with the deficiency of mismatched DNA repair. There are no reliable data on the frequency of MSI in colorectal neuroendocrine neoplasms due to the relative rarity of this cancer type. The prognostic significance of MSI is debatable. The aim of this study was to investigate the frequency of the MSI phenotype among colorectal neuroendocrine neoplasms (NENs) with different primary location, grade and stage. Twenty-nine patients (15 men and 14 women, mean age: 62.5 years) included in the study underwent surgery for colorectal neuroendocrine tumors between 2015 and 2018. The mean follow-up period was 3.8 years. Colorectal NENs were grouped by primary location and stage. The majority of the patients (52%) had stage III cancer at diagnosis. The microsatellite stability (MSS) phenotype was confirmed in 24 patients (83%), whereas the MSI phenotype was observed in 5 patients (17%). All MSI-positive tumors were stage I well-differentiated grade G1 or G2 neuroendocrine tumors (NETs) of the rectum. Overall survival was 50% for patients with stage II MSS-positive NENs of the colon and rectum, 33% for stage III and 0% for stage IV. For patients with stage I MSI-positive NENs of the rectum, overall survival was 100%. Thus, the frequency of MSI-positive colorectal NENs was estimated.