Evaluating morphological features for predicting microsatellite instability status in colorectal cancer

11093 Background: Defective DNA mismatch repair (MMR) results in microsatellite instability (MSI) and is detected in approximately 15% of sporadic colon cancers. MMR status has been shown to provide prognostic and predictive information in primary colon cancers. We sought to develop a model to predict MMR deficiency using clinically available data, and thereby facilitate patient selection for MMR or MSI testing. Methods: TNM stage II and III colon carcinomas (n= 982) were studied from six 5- fluorouracil-based adjuvant therapy trials conducted by the North Central Cancer Treatment Group. MMR status in tumors had been analyzed by MSI (using mono- and dinucleotide markers) or by immunohistochemistry for MMR proteins (hMLH1 and hMSH2). Logistic regression and a recursive partitioning and amalgamation (RPA) analysis was used to identify important predictive factors of MMR status. Factors explored included age, gender, histologic grade, tumor site, stage, lymph node metastases, and T-stage. Results: Defective MMR was found in147 (15%) cancers. Tumor site was the most important predictor of MMR status followed by histologic grade. Distal tumors had a low likelihood of defective MMR (3% rate overall; 13/468), whereas proximal tumors had a greater likelihood of defective MMR (26%; 130/506). For patients with proximal tumors, the addition of histologic grade and gender increased the prediction of defective MMR ( Table ). Using tumor site, histologic grade, and gender, the logistic regression model showed excellent discrimination (c- statistic = 0.81). Conclusions: Tumor site is an important predictor of defective MMR that is rare in distal and increased in proximal tumors. The combination of proximal site, poor differentiation, and female gender resulted in a 51% likelihood of defective MMR. Therefore, this model can facilitate the selection of sporadic colon cancers for MMR or MSI testing to enable its use in clinical decision-making. [Table: see text] No significant financial relationships to disclose.

See Article on Page 28-34 Colorectal cancer (CRC) is a heterogeneous tumor in terms of different types of genetic instabilities and molecular pathogeneses. Through a molecular analysis, CRC can be classified into several groups based on genomic status (microsatellite instability and chromosomal instability) and epigenomic status (CpG island methylator phenotype) [1]. These molecular characteristics are usually more useful than histopathologic features in not only establishing the pathogenesis of a particular colon cancer but also predicting patient prognosis. A microsatellite instability (MSI) is a phenomenon that occurs in a subset of CRC (15%-20%) and manifests itself as an expansion or a deletion of a microsatellite repeat in the patient's tumor DNA, when compared to the patient's normal DNA. The DNA mismatch repair (MMR) gene normally corrects replicative errors. The DNA MMR system consists of several proteins, such as MLH1, MSH2, MSH6, and PMS2, which detect and correct these errors. A defective DNA MMR and subsequent MSI result either from a MMR gene germline or somatic mutation or from methylation of a MMR promoter gene (usually MLH1), resulting in the loss of protein function. A MSI is generally due to a defective MMR. A defective MMR is found in most of the patients with clinically-diagnosed hereditary nonpolyposis colorectal cancer (HNPCC) and in approximately 13%-20% of those diagnosed with sporadic CRC. MSI-H cancers generally show a more favorable prognosis than MSS/MSS-L cancers [2]. Cancers with or without instability at 40% [3]. A meta-analysis of CRC patients including 1,277 MSI-H patients showed that MSI-H cancers were associated with a better prognosis than MSS cancers [4]. In addition, MSI-H cancers have a lower incidence of lymph-node or systemic metastasis compared to MSS cancers. Furthermore, adjuvant 5-Fluorouracil systemic chemotherapy has been revealed to have no oncologic benefits in the treatment of stages II and III colon cancer [5]. This study analyzed retrospectively the clinicopathologic features and prognoses in patients with stages II and III MSI CRCs [6]. Surprisingly, current study showed that stage II MSI-H CRC had a significantly unfavorable trend for 3-year disease free survival compared to MSI-L/MSS CRC, even though the study demonstrated that the clinicopathologic features in MSI-H cancer were similar to those reported in other studies. Furthermore, interestingly, the MSI-H phenotype was a significantly independent poor prognostic factor in the multivariate analysis. A previous study had demonstrated that fluorouracil and leucovorin (FL) adjuvant chemotherapy was associated with a good prognosis for patients with stage II and stage II MSI colorectal cancers [7] while another study, in contrast, reported conflicting outcomes [8]. This study included only 20 MSI-H patients, which number of patients seems to be too small to evaluate any effect of FL adjuvant therapy in stage II MSI-H patients. These observations indicate that MSI status still remains controversial in terms of predicting outcomes and personalized therapy. Although the benefit of adjuvant chemotherapy has been well established in patients with stage III CRC, whether FL adjuvant therapy is effective for treating for stage II MSI CRC is still not clear. Therefore, studies with larger numbers of high-risk stage II patients are necessary to accurately evaluate the true benefit of adjuvant therapy. Until these issues are addressed, concluding that MSI-H patients with stage II cancer do not benefit from FL adjuvant therapy may be premature. However, MMR status may be especially useful in the identification of patients who have Lynch syndrome and may influence the decision as to the need for further adjuvant treatment of CRC patients.

Performance of Next-Generation Sequencing for the Detection of Microsatellite Instability in Colorectal Cancer With Deficient DNA Mismatch Repair

Microsatellite instability (MSI) is associated with defective DNA mismatch repair in various human malignancies. Using a unique fluorescent technique, we have observed two distinct modes of dinucleotide microsatellite alterations in human colorectal cancer. Type A alterations are defined as length changes of ≤6 bp. Type B changes are more drastic and involve modifications of ≥8 bp. We show here that defective mismatch repair is necessary and sufficient for Type A changes. These changes were observed in cell lines and in tumours from mismatch repair gene-knockout mice. No Type B instability was seen in these cells or tumours. In a panel of human colorectal tumours, both Type A MSI and Type B instability were observed. Both types of MSI were associated with hMSH2 or hMLH1 mismatch repair gene alterations. Intriguingly, p53 mutations, which are generally regarded as uncommon in human tumours of the MSI+ phenotype, were frequently associated with Type A instability, whereas none was found in tumours with Type B instability, reflecting the prevailing viewpoint. Inspection of published data reveals that the microsatellite instability that has been observed in various malignancies, including those associated with Hereditary Non-Polyposis Colorectal Cancer (HNPCC), is predominantly Type B. Our findings indicate that Type B instability is not a simple reflection of a repair defect. We suggest that there are at least two qualitatively distinct modes of dinucleotide MSI in human colorectal cancer, and that different molecular mechanisms may underlie these modes of MSI. The relationship between MSI and defective mismatch repair may be more complex than hitherto suspected.

Colorectal cancer (CRC) is one of the major causes of cancer-related morbidity and mortality worldwide. Despite of recent advances of variable therapeutic techniques in CRC, cancer progression and metastasis remain the major contributors to cancer-related morbidity and mortality.1 Cancer progression and metastasis are complex processes and arise through the accumulation of multiple genetic and epigenetic alterations.2 Therefore, advances in our understanding of these molecular alterations of CRC enable us to predict outcomes, personalize medicine and improve the care of CRC patients. Over the past years, meticulous morphologic and molecular features of CRC have led to the development of three major pathways including chromosomal instability (CIN), microsatellite instability (MSI), and cytosine-phospho-guanine (CpG) island methylator phenotype (CIMP).3,4 Most CRCs develop from conventional adenoma through the traditional adenoma-carcinoma pathway characterized by CIN involving KRAS, C-MYC, APC, and p53 mutations.3,4 About 15% to 20% of CRCs develop through the MSI arising from defects in the DNA mismatch repair (MMR) system to correct errors that often occur during DNA replication, and associated with Lynch syndrome. MSI is controlled by MMR genes including MLH1, MSH2, MSH6, and PMS2.3,4 CIMP, characterized by the hypermethylation of CpG islands has been identified as an important third major pathway, which accounts for almost 40% of CRCs. Especially, hypermethylation of CpG islands located in the promoter region leads to transcriptional silencing of tumor suppressor genes and other tumor-related genes.4–6 Therefore, CIMP status can influence the prognosis of CRC patients. Previous studies investigated clinicopathological and molecular features of CRC according to the CIMP status, and focused on the association between CIMP status and CRC prognosis. CIMP-high CRCs present with distinct clinicopathological and molecular features such as older age, female gender, proximal tumor location, poor differentiation, and high rate of MSI, KRAS and BRAF mutation, compared to CIMP-low/negative CRCs.5–7 However, the association between CIMP status and CRC prognosis are inconsistent in previous published reports.5–7 In this issue of Gut and Liver, Kang et al.8, investigated the MSI and CIMP status in Korean colon cancer patients except many rectal cancer patients, and examined their correlation with clinicopathological features including survival. The authors showed that regardless of the MSI status, CIMP-high colon cancer was associated with a poor survival outcome, and colon cancer with CIMP-high/MSI-negative in subgroup analysis showed a poor survival outcome compared to CIMP-low and negative/MSI-negative colon cancers. However, as the authors described, this study population was relatively small and had low incidence of MSI-high or CIMP-high cancers, which might influence some results, especially the association of CIMP status for CRC prognosis.8 Other Korean studies also showed that CIMP-high CRCs were significantly associated with female gender, proximal tumor location, poor differentiation, nodal metastasis, more advanced cancer, BRAF mutations, MSI, and poor prognosis.9,10 According to the combination of CIMP and MSI status, the CIMP-high/MSI-negative subtype showed the worst clinical outcome due to BRAF mutation.9,10 The clinicopathological and molecular features of CIMP-high CRCs in Korean studies8–10 were similar with those of previous published reports.5–7 However, although CIMP-high CRC patients were related to poor prognosis in many studies including Korean studies, other studies reported no association between CIMP-high CRC patient and prognosis or even noted a better survival benefit from adjuvant chemotherapy.5–7 There are several explanations for this discrepancy. First, results are inconsistent, perhaps due to variable factors including MSI, KRAS and BRAF mutations, variations in use of adjuvant chemotherapy, the ambiguous pathophysiolgy of CIMP, methodologic differences such as differences in the selected methylation markers and definitions of CIMP.5,6 Actually, specific methylated loci using for definition of CIMP and prevalence of CIMP status are different among studies. It is the major concern in studying CIMP status in variable cancers. Second, CIMP is an event that predominantly occurs in early stage of carcinogenesis and may less involve to cancer progression. Therefore, although much effort has been made to identify the impact of CIMP status as prognostic biomarkers in clinical practice, it still cannot be concluded whether association between CIMP status and CRC prognosis exist. In future, large-scale well-designed studies should be considered in order to clarify the impact of CIMP on the prognostic significance in CRC patients.

Su1923 Immune Microenvironment in Colonic Carciongenesis: Sporadic Mismatch Repair Genes Defects Are Associated to Hicd80+ Lamina Propria Monuclear Cells Infiltration

The microsatellite instability (MSI) pathway is one of the important mutational pathways that play a critical role in colorectal carcinogenesis. About 15% of colorectal cancers (CRCs) are characterized by MSI. MSI tumors usually arise because of a genetic defect in mismatch repair (MMR) genes, one of the main DNA-repairing systems. MMR is a highly conserved biological pathway that plays a key role in maintaining genomic stability by correcting the base-base mismatches and insertion/deletion mispairs generated during DNA replication and recombination. Escherichia coli MutS and MutL and their eukaryotic homologs, MutSα and MutLα, respectively, are key players in MMR-associated genome maintenance. Mutations in at least five pivotal genes of MMR, namely, in those encoding mutS homolog 2 (MSH2), mutL homolog 1 (MLH1), mutS homolog 6 (MSH6), postmeiotic segregation increased 1 (PMS1), and postmeiotic segregation, increased 2 (PMS2) have been found in CRC, highlighting the importance of understanding the basic structure and functions of the essential molecules that make up the MMR system. In this review, we have attempted to focus on this aspect, that is, the role that MMR molecules play in CRC carcinogenesis.

Background/Objectives: In metastatic colorectal cancer (mCRC) the evaluation of mismatch repair (MMR) and microsatellite instability (MSI) status is essential to identify patients eligible for treatment with immune-checkpoint inhibitors (ICI). This study aims to evaluate the potential utility of Comprehensive Genomic Profiling (CGP) in assessing MSI status, in addition to other immunotherapy-predictive biomarkers such as high tumor molecular burden (TMB) and the POLE and POLD1 mutations. Methods: A total of 138 mCRC tumor samples underwent a first-level molecular test (MMR status by immunohistochemistry, MSI by a melting-based PCR approach and RAS/BRAF mutational status by a small next-generation sequencing (NGS) panel) and second-level CGP analysis by the FoundationOne CDx assay. The prevalence of dMMR and MSI tumors was reported. Moreover, the concordance between the MMR and MSI status was determined, and discordant cases were discussed. Results: Twelve cases (8.7%) were MMR-deficient (dMMR); 10 showed high MSI and TMB (>10 mut/Mb). MSI status assessed by CGP and PCR was concordant in all cases except one MSH6-deficient tumor. Two dMMR cases were stable with low TMB. Moreover, in two MLH1/PMS2-deficient cases CGP revealed pathogenic alterations in the MSH2 and MSH6 genes; in both cases, the MLH1 promoter was hypermethylated. A high TMB was the only positive biomarker in 11 cases with a proficient MMR system and no MSI. Conclusions: MSI assessment by CGP analysis showed high concordance (98%) with MMR and was helpful in evaluating ICI eligibility in three out of twelve dMMR cases. Overall, compared to standard methods, analyzing a broader range of microsatellite loci and the simultaneous assessment of multiple predictive biomarkers by CGP may increase diagnostic accuracy and improve therapeutic assessment.

Objective: Fusobacterium nucleatum (F. nucleatum) is a microbial pathogen that contributes to the initiation and progression of colorectal cancer. Although evidence points to its immunosuppressive effects in colorectal cancer, F. nucleatum has been associated with high-level microsatellite instability (MSI) which is characterized by intense immune response. We hypothesized that the association of F. nucleatum with local immunosuppression might be stronger in tumors with high-level MSI or a high neoantigen load, which is a direct consequence of MSI that influences tumor-immune interactions. Design: We utilized molecular pathological epidemiology database of 1,027 colon and rectal cancer cases in the Nurses’ Health Study and the Health Professionals Follow-up Study. We assessed the association of F. nucleatum (negative, low, or high) with tumor immunity status in strata of MSl status. We measured F. nucleatum DNA in tumor tissue using quantitative PCR. As immunity status, we evaluated hystopathological lymphocytic reaction (tumor-infiltrating lymphocytes [TIL], intratumoral periglandular reaction, peritumoral lymphocytic reaction, or Crohn's-like lymphoid reaction) and measured T cell densities (CD3+, CD8+, CD45RO [PTPRC]+ or FOXP3+ cells) using immunohistochemistry and computer-assisted image analysis. Logistic regression model was used to adjust for potential confounders, including CpG island methylator phenotype, LINE-1 methylation level, and KRAS, BRAF, and PIK3CA mutations. Using whole-exome sequencing data (N = 586), we conducted secondary analyses to assess the interaction of F. nucleatum with a neoantigen load. Results: The association between F. nucleatum and TIL differed by MSI status (P for interaction = 0.0017; with the adjusted α level of 0.006). Compared with F. nucleaum-negative cases, F. nucleatum-positive cases were associated with low-level TIL in MSI-high tumors (odds ratio, 0.40 [95% confidence interval, 0.14-1.08] and 0.40 [95% confidence interval, 0.17-0.97] for F. nucleaum low and high cases, respectively), but not in microsatellite stable/MSI-low tumors. We did not find any interaction between F. nucleatum and MSI status in T cell subsets. The association between F. nucleatum and TIL did not differ significantly by levels of a neoantigen load (P for interaction = 0.015). Conclusions: The association of F. nucleatum with the immunosuppressive microenvironment may be stronger in colorectal cancer with high-level MSI. Our findings suggest the interplay between F. nucleatum and MSI status in host immune response to the tumor. Citation Format: Kosuke Mima, Tsuyoshi Hamada, Reiko Nishihara, Jonathan Nowak, Zhi Rong Qian, Keisuke Kosumi, Marios Giannakis, Susan Bullman, Hideo Baba, Curtis Huttenhower, Wendy Garrett, Andrew Chan, Matthew Meyerson, Charles Fuchs, Shuji Ogino. Fusobacterium nucleatum and tumor immunity status according to microsatellite instability in colorectal cancer [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 4560. doi:10.1158/1538-7445.AM2017-4560

A departure from the concept of simple adenoma to carcinoma sequence is the realization that colorectal cancer (CRC) develops through at least two major molecular pathogenic pathways, namely chromosomal instability (CIN) and microsatellite instability (MSI) [1,2]. The mechanisms underlying CIN might have been the first to be deciphered but they are just being understood in detail. It is proposed that CIN may be associated with defects in mitotic spindle apparatus hence tumors arising through this pathway are usually characterized by widespread chromosomal deletions, translocations and loss of heterozygosity at the molecular level. On the other hand, MSI pathway is responsible for up to 15% of all CRCs that also include a number of familial syndromes [3,4]. Hereditary non-polyposis colorectal cancer (HNPCC also known as Lynch syndrome) is the prototype for the tumors characterized by MSI. Tumors developing through this pathway have alterations in the length of short, repeated (usully mononucleotide or dinucleotide) sequences of DNA, i.e., microsatellites [5–10]. This phenomenon reflects the underlying defect in the DNA mismatch repair (MMR) system which is a multi-protein complex responsible for correction of errors arising during normal cell division [5,7,11,12]. In 1997, the National Cancer Institute Workshop on Hereditary Non-polyposis Colorectal Cancer Syndrome proposed specific markers for MSI assessment which included BAT25, BAT26, D5S346, D2S123 and D17S250, known as Bethesda panel [13]. If no alterations are found the tumor is classified as microsatellite stable (MSS) and if two or more loci are abnormal the tumor is classified as showing a high level of microsatellite instability (MSI-H). In cases showing abnormalities at one locus, testing of an additional five loci is recommended. If abnormal loci constitute less than 40% of the tested markers, the tumor is classified as showing a low level of microsatellite instability (MSIL). MSS cancers account for up to 85% of all CRCs and occur in sporadic and hereditary settings such as familial adenomatous polyposis (FAP) and attenuated FAP. The biological significance and underlying genetic abnormalities in cancers with the MSI-L phenotype are debated and remain to be fully characterised. It has been reported that on morphological grounds, MSS and MSI-L cancers are indistinguishable. MSI-H cancers constitute up to 15% of all CRCs. MSI-H cancers (sporadic and in hereditary non-polyposis colorectal cancer (HNPCC) patients) have characteristic anatomical and histological features. Proximal colon incidence is seen in the majority of cases. Most of the tumors are polypoid masses and some may display a mucinous cut surface [14,15]. Stricture formation due to a diffuse growth pattern is uncommon; however, extensive necrosis is often observed. The tumors usually have a well-defined margin. A significant fraction of tumors are also classified as poorly differentiated. A characteristic feature of diagnostic importance is the presence of tumor infiltrating lymphocytes (TIL), most of which are cytotoxic T cells. B lymphocytes’ aggregates are

The microsatellite instability (MSI) status of colorectal cancer is relevant to the prognosis and therapy of sporadic cancer and to the relative likelihood of the inherited syndrome hereditary nonpolyposis colon cancer (HNPCC). Immunohistochemistry (IHC) for mismatch repair proteins has been proposed as a surrogate marker for MSI. IHC for mismatch repair proteins hMLHl and hMSH2 was performed on 17 right-sided colorectal cancers whose MSI status had been previously determined by polymerase chain reaction (PCR) amplification of the Bethesda consensus panel of microsatellite repeats. Thirteen of the 17 colon cancers showed IHC positivity with both hMLHl and hMSH2 antibodies and were stable by PCR MSI testing. Four of the tumors were negative by hMLHl and positive by hMSH2; all four showed microsatellite instability by PCR MSI testing. There was therefore a perfect concordance between negative IHC for one of the mismatch repair proteins and MSI as determined by PCR. IHC testing of mismatch repair proteins is an excellent predictor of colorectal cancer MSI status. This rapid and inexpensive test also provides information not available from conventional MSI testing, namely the identity of the faulty mismatch repair gene. IHC results can therefore be used to guide the subsequent mutational analysis of mismatch repair genes in cases of HNPCC. (The J Histotechnol 27:000, 2004)Submitted: January 27, 2004 Accepted: with revisions August 2, 2004

149 Background: Microsatellite instability (MSI) plays a crucial role as a cancer immunotherapy and prognosis biomarker, making accurate MSI detection essential. However, currently, there is still a lack of available data on the impact of neoadjuvant therapy on MSI status, as well as validated data on standardized methods for MSI testing in small biopsy samples. The study aimed to investigate the concordance of MSI status between paired biopsy and surgical samples, as well as the impact of neoadjuvant therapy on MSI status using a novel MSI next-generation sequencing (MSI-NGS) detection panel. Methods: A total of 137 colorectal cancer (CRC) patients were enrolled for this study, of whom 116 with paired biopsy and surgical samples were analyzed. A custom MSI-NGS panel was employed and its performance was compared to MSI polymerase chain reaction (MSI-PCR), which served as the gold standard. Results: Out of the 116 cases, 112 patients showed consistent MSI status between biopsy and surgical samples, with an overall accuracy of 97% regardless of the detection method used. In the cases with MSI discrepancy (6 cases), 83% (5/6) of the patients showed a transition from MSS to MSI-H from biopsy to surgery. Interestingly, all eight patients who received neoadjuvant chemotherapy maintained an unchanged MSI status. However, in one patient who received adjuvant treatment and underwent a repeat biopsy after surgery, the MSI status exhibited alterations. Further analysis of clonal evolution revealed that this heterogeneity stemmed from the disappearance of the original clones and the emergence of new clones. Additionally, the NGS panel exhibited strong concordance with MSI-PCR and high accuracy, sensitivity and specificity with an AUC of 0.942 after rigorous validation. Conclusions: The study revealed a high concordance in MSI status between paired biopsy and surgical samples by employing a custom MSI-NGS panel for MSI status detection, providing reliable basis for further research in this field. Moreover, neoadjuvant chemotherapy seemed to have no impact on MSI status in our study, whereas postoperative adjuvant chemotherapy may influence changes in MSI status, highlighting the necessity of reevaluating MSI status after surgery. The findings also underscore the potential of NGS-based MSI detection as a valuable tool for clinical decision-making.

Background: Metaplastic breast cancers (MPBC) are rare, typically triple negative aggressive tumors composed of both, adenocarcinoma and metaplastic elements. Recent evidence that TNBC and MPBC can respond vigorously to immune checkpoint inhibitor therapy (Adams et al, ASCO 2017 and npj Breast Cancer 2017) has prompted the following comprehensive genomic profiling (CGP) and histopathologic assessment of tumor infiltrating lymphocytes (TIL) designed to uncover potential biomarkers of immunotherapy response for MPBC, including mutational burden, Microsatellite Instability (MSI) status and gene amplification of 9p21.4 (or CD274, which includes the PD-L1 locus). Methods: 12,214 locally aggressive, relapsed and metastatic breast malignancies (mBM) were subjected to CGP using DNA extracted from 40 µm of FFPE sections and adaptor ligation-based libraries to a mean coverage depth >650X for up to 315 cancer-related genes. The results were analyzed for all classes of genomic alterations (GA) including base substitutions, insertions and deletions, select rearrangements, and copy number changes. Tumor mutational burden (TMB) was determined on 1.1 Mbp of sequenced DNA. MSI status was determined by an algorithm based on the sequencing results. TIL were assessed on archived H&E tumor sections and enumerated per guidelines established by the TIL Working Group (Salgado, Ann Oncol 2015) in a subset of MPBC with the highest TMB and compared with low TMBC cases. Results: 165 of mBM cases were MPBC (1.4%) and are included in this study. All patients were female with a median age of 60 (range 24-86). 165 of the MPBC cases (100%) harbored a wide variety of GA involving more than 100 individual genes. The most common GA were identified in TP53 (65%), followed by PIK3CA (37%). No cases of MSI hi status (0/103) and only one case with amplification of 9p21.4 (1/165, 0.6%) were observed. Most MPBC had a low mutational burden, with a median TMB of 2.7 mutations/Mb (range 0-39.6). Only 11/165 tumors (6.7%) were found to have a TMB over 10 mutations/Mb, including 3 cases (1.8%) with TMB >20. Tumor sections were available for TIL review from 9/11 cases with highest TMB, as well as 11 control cases with lowest TMB. TIL were more frequently observed in high versus low TMB MPBC, with median TIL percentage of 40 and 20 (range 10-80 and range 10-60), respectively, although this difference was not statistically significant (Wilcoxon rank-sum test, p=0.15). Conclusions: Genomic profiling in the largest cohort of MPBC reported to date confirms that MPBC is enriched for TP53 and PIK3CA mutations and many tumors harbor targetable GA. The frequently observed tumoral PD-L1 expression in MPBC is not based on gene amplification as amplification of 9p21.4 is rare. Most tumors had a low mutation burden, and no significant association of TIL with TMB was observed, suggesting additional processes underlying MPBC immunogenicity. Citation Format: Taff J, Suh J, Singh B, Denkert C, Troxel AB, Ross JS, Adams S. Metaplastic breast cancers: Genomic profile, mutational burden and TILs [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-05-03.

BackgroundWith the recent emergence of immune checkpoint inhibitors, microsatellite instability (MSI) status has become an important biomarker for immune checkpoint blockade therapy. There are growing technical demands for the integration of different genomic alterations profiling including MSI analysis in a single assay for full use of the limited tissues.MethodsTumor and paired control samples from 64 patients with primary colorectal cancer were enrolled in this study, including 14 MSI-high (MSI-H) cases and 50 microsatellite stable (MSS) cases determined by MSI-PCR. All the samples were sequenced by a customized NGS panel covering 2.2 MB. A training dataset of 28 samples was used for selection of microsatellite loci and a novel NGS-based MSI status classifier, USCI-msi, was developed. NGS-based MSI status, single nucleotide variant (SNV) and tumor mutation burden (TMB) were detected for all patients. Most of the patients were also independently detected by immunohistochemistry (IHC) staining.ResultsA 9-loci model for detecting microsatellite instability was able to correctly predict MSI status with 100% sensitivity and specificity compared with MSI-PCR, and 84.3% overall concordance with IHC staining. Mutations in cancer driver genes (APC, TP53, and KRAS) were dispersed in MSI-H and MSS cases, while BRAF p.V600E and frameshifts in TCF7L2 gene occurred only in MSI-H cases. Mismatch repair (MMR)-related genes are highly mutated in MSI-H samples.ConclusionWe established a new NGS-based MSI classifier, USCI-msi, with as few as 9 microsatellite loci for detecting MSI status in CRC cases. This approach possesses 100% sensitivity and specificity, and performed robustly in samples with low tumor purity.

Microsatellite instability (MSI) is a genetic feature of colorectal cancer (CRC) associated with peculiar clinicopathological features and improved prognosis. It is demonstrated in >90% of patients with hereditary non-polyposis colorectal cancer (HNPCC) in which DNA mismatch repair (MMR) defects are the cause of MSI. HNPCC develop at a young age, and patients are at increased risk for additional cancers (e.g. endometrial, stomach, or biliary tract cancer). MSI is found in 15–20% of sporadic CRC, often based on epigenetic silencing (i.e. MLH1). The prognostic and predictive value of MSI is not yet fully elucidated, although improved survival in MSI is suggested. Distinguishing sporadic MSI from HNPCC is at present guided by the combination of familial history, immunohistochemistry, and genotyping of MMR genes (MLH1, MSH2, MSH6, and PMS2). The efficiency and accuracy of MSI testing is evolving, with more knowledge achieved concerning multipopulation polymorphisms, selection of markers (e.g. quasimonomorphic vs. dinucleotides), and more time-efficient panels (e.g. not requiring normal/germline DNA match). The role of distinct genetic features, such as BRAF V600E mutations often found in sporadic MSI but not in HNPCC, may further improve future test algorithms. The present knowledge and controversies pertaining to molecular testing of MSI and MMR defects in CRC are presented.