Microsatellite Instability Status In Colorectal Cancer 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.

Prediction of Microsatellite Instability in Colorectal Cancer Using a Machine Learning Model Based on PET/CT Radiomics.

We investigated the feasibility of preoperative 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET)/computed tomography (CT) radiomics with machine learning to predict microsatellite instability (MSI) status in colorectal cancer (CRC) patients. Altogether, 233 patients with CRC who underwent preoperative FDG PET/CT were enrolled and divided into training (n=139) and test (n=94) sets. A PET-based radiomics signature (rad_score) was established to predict the MSI status in patients with CRC. The predictive ability of the rad_score was evaluated using the area under the receiver operating characteristic curve (AUROC) in the test set. A logistic regression model was used to determine whether the rad_score was an independent predictor of MSI status in CRC. The predictive performance of rad_score was compared with conventional PET parameters. The incidence of MSI-high was 15 (10.8%) and 10 (10.6%) in the training and test sets, respectively. The rad_score was constructed based on the two radiomic features and showed similar AUROC values for predicting MSI status in the training and test sets (0.815 and 0.867, respectively; p=0.490). Logistic regression analysis revealed that the rad_score was an independent predictor of MSI status in the training set. The rad_score performed better than metabolic tumor volume when assessed using the AUROC (0.867 vs. 0.794, p=0.015). Our predictive model incorporating PET radiomic features successfully identified the MSI status of CRC, and it also showed better performance than the conventional PET image parameters.

Evaluating the Microsatellite Instability of Colorectal Cancer Based on Multimodal Deep Learning Integrating Histopathological and Molecular Data.

Microsatellite instability (MSI), an important biomarker for immunotherapy and the diagnosis of Lynch syndrome, refers to the change of microsatellite (MS) sequence length caused by insertion or deletion during DNA replication. However, traditional wet-lab experiment-based MSI detection is time-consuming and relies on experimental conditions. In addition, a comprehensive study on the associations between MSI status and various molecules like mRNA and miRNA has not been performed. In this study, we first studied the association between MSI status and several molecules including mRNA, miRNA, lncRNA, DNA methylation, and copy number variation (CNV) using colorectal cancer data from The Cancer Genome Atlas (TCGA). Then, we developed a novel deep learning framework to predict MSI status based solely on hematoxylin and eosin (H&E) staining images, and combined the H&E image with the above-mentioned molecules by multimodal compact bilinear pooling. Our results showed that there were significant differences in mRNA, miRNA, and lncRNA between the high microsatellite instability (MSI-H) patient group and the low microsatellite instability or microsatellite stability (MSI-L/MSS) patient group. By using the H&E image alone, one can predict MSI status with an acceptable prediction area under the curve (AUC) of 0.809 in 5-fold cross-validation. The fusion models integrating H&E image with a single type of molecule have higher prediction accuracies than that using H&E image alone, with the highest AUC of 0.952 achieved when combining H&E image with DNA methylation data. However, prediction accuracy will decrease when combining H&E image with all types of molecular data. In conclusion, combining H&E image with deep learning can predict the MSI status of colorectal cancer, the accuracy of which can further be improved by integrating appropriate molecular data. This study may have clinical significance in practice.

Development and validation of a radiomics-based nomogram for the preoperative prediction of microsatellite instability in colorectal cancer

BackgroundPreoperative prediction of microsatellite instability (MSI) status in colorectal cancer (CRC) patients is of great significance for clinicians to perform further treatment strategies and prognostic evaluation. Our aims were to develop and validate a non-invasive, cost-effective reproducible and individualized clinic-radiomics nomogram method for preoperative MSI status prediction based on contrast-enhanced CT (CECT)images.MethodsA total of 76 MSI CRC patients and 200 microsatellite stability (MSS) CRC patients with pathologically confirmed (194 in the training set and 82 in the validation set) were identified and enrolled in our retrospective study. We included six significant clinical risk factors and four qualitative imaging data extracted from CECT images to build the clinics model. We applied the intra-and inter-class correlation coefficient (ICC), minimal-redundancy-maximal-relevance (mRMR) and the least absolute shrinkage and selection operator (LASSO) for feature reduction and selection. The selected independent prediction clinical risk factors, qualitative imaging data and radiomics features were performed to develop a predictive nomogram model for MSI status on the basis of multivariable logistic regression by tenfold cross-validation. The area under the receiver operating characteristic (ROC) curve (AUC), calibration plots and Hosmer-Lemeshow test were performed to assess the nomogram model. Finally, decision curve analysis (DCA) was performed to determine the clinical utility of the nomogram model by quantifying the net benefits of threshold probabilities.ResultsTwelve top-ranked radiomics features, three clinical risk factors (location, WBC and histological grade) and CT-reported IFS were finally selected to construct the radiomics, clinics and combined clinic-radiomics nomogram model. The clinic-radiomics nomogram model with the highest AUC value of 0.87 (95% CI, 0.81–0.93) and 0.90 (95% CI, 0.83–0.96), as well as good calibration and clinical utility observed using the calibration plots and DCA in the training and validation sets respectively, was regarded as the candidate model for identification of MSI status in CRC patients.ConclusionThe proposed clinic-radiomics nomogram model with a combination of clinical risk factors, qualitative imaging data and radiomics features can potentially be effective in the individualized preoperative prediction of MSI status in CRC patients and may help performing further treatment strategies.

Expression of Thomsen-Friedenreich Antigen in Colorectal Cancer and Association with Microsatellite Instability.

Microsatellite instability (MSI) is a molecular phenotype due to a deficient DNA mismatch repair (dMMR). In colorectal cancer (CRC), dMMR/MSI is associated with several clinical and histopathological features, influences prognosis, and is a predictive factor of response to therapy. In daily practice, dMMR/MSI profiles are identified by immunohistochemistry and/or multiplex PCR. The Thomsen–Friedenreich (TF) antigen was previously found to be a potential single marker to identify MSI-high gastric cancers. Therefore, in this study, we aimed to disclose a possible association between TF expression and MSI status in CRC. Furthermore, we evaluated the relationship between TF expression and other clinicopathological features, including patient survival. We evaluated the expression of the TF antigen in a cohort of 25 MSI-high and 71 microsatellite stable (MSS) CRCs. No association was observed between the expression of the TF antigen and MSI-high status in CRC. The survival analysis revealed that patients with MSI-high CRC showed improved survival when the TF antigen was expressed. This finding holds promise as it indicates the potential use of the TF antigen as a biomarker of better prognosis in MSI-high CRCs that should be validated in an independent and larger CRC cohort.

Impact of Pre-Analytical Factors on MSI Test Accuracy in Mucinous Colorectal Adenocarcinoma: A Multi-Assay Concordance Study.

Immunohistochemistry (IHC) and polymerase chain reaction (PCR) and fragment separation by capillary electrophoresis represent the current clinical laboratory standard for the evaluation of microsatellite instability (MSI) status. The importance of reporting MSI status in colorectal cancer is based on its potential for guiding treatment and as a prognostic indicator. It is also used to identify patients for Lynch syndrome testing. Our aim was to evaluate pre-analytical factors, such as age of formalin-fixed and paraffin-embedded (FFPE) block, neoplastic cell percentage, mucinous component, and DNA integrity, that may influence the accuracy of MSI testing and assess the concordance between three different MSI evaluation approaches. We selected the mucinous colorectal cancer (CRC) histotype for this study as it may possibly represent an intrinsic diagnostic issue due to its low tumor cellularity. Seventy-five cases of mucinous CRC and corresponding normal colon tissue samples were retrospectively selected. MMR proteins were evaluated by IHC. After DNA quality and quantity evaluation, the Idylla™ and TapeStation 4200 platforms were adopted for the evaluation of MSI status. Seventy-three (97.3%) cases were successfully analyzed by the three methodologies. Overall, the Idylla™ platform showed a concordance rate with IHC of 98.0% for microsatellite stable (MSS)/proficient MMR (pMMR) cases and 81.8% for MSI/deficient MMR (dMMR) cases. The TapeStation 4200 system showed a concordance rate with IHC of 96.0% for MSS/pMMR cases and 45.4% for MSI/dMMR cases. The concordance rates of the TapeStation 4200 system with respect to the Idylla™ platform were 98.1% for MSS profile and 57.8% for MSI profile. Discordant cases were analyzed using the Titano MSI kit. Considering pre-analytical factors, no significant variation in concordance rate among IHC analyses and molecular systems was observed by considering the presence of an acellular mucus cut-off >50% of the tumor area, FFPE year preparation, and DNA concentration. Conversely, the Idylla™ platform showed a significant variation in concordance rate with the IHC approach by considering a neoplastic cell percentage >50% (p-value = 0.002), and the TapeStation 4200 system showed a significant variation in concordance rate with the IHC approach by considering a DNA integrity number (DIN) ≥4 as cut-off (p-value = 0.009). Our data pinpoint a central role of the pre-analytical phase in the diagnostic outcome of MSI testing in CRC.

The landscape of DNA damage response (DDR) pathway in colorectal cancer (CRC).

4064 Background: Abnormal DDR is a hallmark of cancer, relating to genome instability, anti-tumor immunity, and sensitivity to chemotherapeutic agents and radiation. We conducted a large-scale investigation to clarify the alteration of DDR pathway in CRC. Methods: Tumor samples from 9321 CRC patients were retrospectively reviewed. Next-Generation Sequencing (NGS) on a custom-designed panel enriching 592 gene targets was performed. Samples with mutations detected in any of 29 DDR-related genes were deemed DDR-mutant (DDR-MT); the rest DDR-wild type (DDR-WT). Microsatellite instability (MSI) status was tested with a combination of immunohistochemistry (IHC), fragment analysis and NGS. Tumor mutational burden (TMB) was calculated based on somatic nonsynonymous missense mutations. PD-L1 was tested by IHC (SP142). Consensus molecular subtype (CMS) was developed using RNA sequencing data. Results: Of 9321 cases, 1290 (13.8%) were DDR-MT. DDR-MT frequency was higher in right vs. left sided (20.9% vs 10.8%, p < 0.001) and MSI-H vs. MSS (76.4% vs 9.5%, p < 0.001) cases. In the MSS cases, right-sided had marginally higher frequency of DDR-MT than left-sided (10.6% vs 9.1%, p = 0.055), with much higher frequency of Fanconi anemia pathway alteration in right-sided (1.5% vs 0.7%, p < 0.01). CMS1 subtype had the highest frequency of DDR-MT (34.8%); CMS2 had the lowest (7.1%). DDR-MT cases (vs. DDR-WT) had higher mutation rate of ARID1A (55.0% vs 19.1%, p < 0.0001), PIK3CA (22.6% vs 15.8%, p < 0.0001) and BRAF (20.4% vs 7.3%, p < 0.0001), and lower mutation rate of TP53 (48.2% vs 76.1%, p < 0.0001), APC (60.5% vs 74.5%, p < 0.0001) and KRAS (44.0% vs 49.8%, p < 0.001). Mean TMB was much greater in DDR-MT than DDR-WT (All: 20.9/Mb vs 7.7/Mb, p < 0.0001; MSS: 13.7/Mb vs 7.6/Mb, p < 0.05). PD-L1 positivity was also higher in DDR-MT compared to DDR-WT (All: 10.1% vs 2.7%, p < 0.0001; MSS: 4.8% vs 2.4%, p < 0.0001). Conclusions: Alteration of the DDR pathway was strongly associated with MSI status in CRC. The primary tumor sidedness might also be related, as DDR-MT was more prevalent in right-sided tumors. Elevated TMB and PD-L1 expression in DDR-MT CRC indicate more activated anti-tumor immune profiles compared to DDR-WT, regardless of MSI status, suggesting possible therapeutic benefit from immune checkpoint inhibitors in DDR-MT CRC.

Abstract 1712: Assessing tumor mutation load using an NGS-based, routine-friendly target gene panel

Abstract In several cancer models, it was shown that tumor mutation load correlates with response to immune checkpoint blockade therapy. However, whole exome sequencing of FFPE samples is not ideal for implementation on molecular pathology laboratories. In colorectal cancer (CRC), mutation load is correlated with microsatellite instability (MSI), which can therefore be used as surrogate marker for mutation load. In this study, we aimed at determining whether a target NGS-based mutation panel can be used for routine testing of mutation load in cancer, by assessing its performance in identifying MSI+ and MSI- CRC samples. The Oncomine Tumor Mutation Load Assay, which targets 409 genes corresponding to 1.7Mb of sequencing data, was used. It is a two-pool panel, optimized for FFPE samples, and only requires 20 ng of DNA in total. A cohort of 16 CRC samples was used. These included six cases MSI+, known to present a hypermutable phenotype caused by the loss of DNA mismatch repair activity, and 10 MSI- samples, which typically have a lower rate of somatic mutation. DNA was isolated from 10 uM slide cuts using the MagMAX FFPE DNA/RNA Ultra Kit. A multiplex of eight samples per 540 sequencing chip was sequenced using an Ion S5 XL system. In our data set, we observed an average of 67.2 mutations per Mb for the MSI+ samples and an average of 29.7 mutations per Mb for the MSI- samples (p=0.004). Using a cut-off of 40.0 mutations per Mb, we were able to classify correctly every sample as MSI+ or MSI-. Interestingly, we observed a tri-modal distribution of the samples, suggesting there might be a sub-group of MSI+ CRC cases that show a level of mutation load comparable to that of MSI- CRC cases. It remains to be seen whether these MSI+, low mutation load CRC cases are non-responders to treatment with immunotherapy. A validation cohort of CRC cases is currently being analyzed to validate our findings. In this study, we demonstrate it is viable to use a target and routine-amenable NGS-based sequencing gene panel to identify CRC samples with different levels of mutation load. It remains to be seen whether mutation load is a better predictor of response to immunotherapy than MSI status in CRC. This approach also needs to be validated in different tumor types. Citation Format: Jose Costa, Joana Reis, Margarida Fernandes, Rafaela Silva, Luis Cirnes, Ruchi Chaudhary, Fatima Carneiro, Jose C. Machado. Assessing tumor mutation load using an NGS-based, routine-friendly target gene panel [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 1712.

Comprehensive Analysis of the Relationship Between RAS and RAF Mutations and MSI Status of Colorectal Cancer in Northeastern China

Background/Aims: Colorectal cancer (CRC) is mainly caused by chromosomal instability (CIN) and microsatellite instability (MSI). The RAS and RAF genes are essential components of the CIN pathway, and several studies have found that RAS and RAF mutations are associated with MSI status in CRC. Here, we examined these three factors in CRC in Northeast China and aimed to reveal new details of the relationship between these mutations and MSI status. Methods: This study involved 290 patients with CRC who had RAS or RAF gene mutation detected using fluorescence-based allele-specific polymerase chain reaction or Sanger sequencing. The majority of the identified patients were found to harbor MSI (MSI status). Accurate molecular detection was carried out using formalin-fixed paraffin-embedded tissue or blood samples. Results: The rates of RAS and RAF mutations were 58.5% and 4.1%, respectively. The prevalence of RAS mutation in CRC was clearly higher and that of RAF mutation was lower in Northeast China compared with previously reported cohorts in other locations. High MSI level (MSI-H status) was more complex, at around 10%. This was consistent with previous data from China. However, compared with data reported from other continents, MSI-H was higher than that of Japan or South Korea in Asia, and lower than that of Europe or the United States. Conclusion: RAS/RAF mutations and MSI status in CRC are closely associated with tumor location and ethnicity. Further studies investigating the relationship between these three factors can help in the development of treatment strategies for patients with CRC.

138P - Detection of microsatellite instability (MSI) in colorectal cancer samples with the automated Idylla MSI Test

Background: Detection of microsatellite instability (MSI) is recommended for all colorectal cancer (CRC) patients. Current clinical reference methods to detect MSI stain for mismatch repair proteins or analyze frequently mutated DNA repeat regions. The Idylla MSI Test is being developed using a unique set of novel biomarkers (Zhao et al. 2014; eLife) capable of faster detection with greater specificity and selectivity compared to current methods. Methods: To assess the suitability of the novel marker set to detect MSI status in CRC, we profiled 8 or more markers in 870 CRC samples. Several clinical sites and different ethnic groups (Afro American, Caucasian, East-Asian, Hispanic and Indian) were included to assess robustness of marker selection. Repeat length was determined on FFPE DNA by PCR followed by melting curve analysis. Two-hundred and one samples were additionally screened with a reference methodology for MSI detection (Promega MSI analysis system). Results: Hundred fifty-three samples (17.6%) were classified as MSI-H and 693 samples (79.7%) as MSS with the novel set of biomarkers, while 24 samples (2.8%) could not be classified. Concordance analysis was performed on 201 samples. The overall percent agreement with results available for both methods (173/201) was 93.6%. 11/173 (6.4%) were scored MSI-H for Idylla and MSS for the reference method; conversely no MSI-H cases for the reference method and MSS for Idylla were detected. 24/201 (11.9%) samples failed with the reference method, even after repeat testing, while only 8/201 (4.0%) samples failed with the Idylla methodology. Conclusions: This study on a diverse set of CRC samples successfully demonstrated the validity of the novel MSI biomarkers to discriminate between MSI-H and MSS status. The Idylla MSI Test is currently under development on the most performant markers. Compatibility with the fully integrated Idylla platform will allow providing accurate and reliable results, with actionable results generated within 150 minutes from just one tumor FFPE slice (no reference sample required). Legal entity responsible for the study: Biocartis NV Funding: None Disclosure: G. Maertens, B. De Craene, E. Rondelez, K. Peeters, L. Vandenbroeck, L. Van Puymbroeck, G. Vandercruyssen, E. Sablon: Biocartis Employee.

Genomic profiling of colorectal cancers and the future of personalized treatment

New technologies have enabled faster, cheaper and more accurate genomic and other types of profiling. Therefore, treatment has become more customized according to molecular subtype. Here, we summarize the current status of genomic profiling for colorectal cancer (CRC) and discuss future directions. Recently, the CRC Subtyping Consortium classified CRC into four subtypes: CMS1, microsatellite instability immune (14%); CMS2, canonical (37%); CMS3, metabolic (13%); and CMS4, mesenchymal (23%). Testing for KRAS, NRAS and BRAF mutations, and microsatellite instability status in CRC has proven essential for treatment decisions. Tumor heterogeneity and the evolution of drug-resistant subclones after therapy should be further assessed and pursued. Patient-derived xenografts and liquid biopsies might facilitate the development of optimum and accurate personalized therapy regimens.

Relationships between p14ARF Gene Methylation and Clinicopathological Features of Colorectal Cancer: A Meta-Analysis.

We conducted a meta-analysis to explore the relationships between p14ARF gene methylation and clinicopathological features of colorectal cancer (CRC). Databases, including Pubmed, Embase and Cochrane Library, were searched and, finally, a total of 18 eligible researches encompassing 1988 CRC patients were selected. Combined odds ratios (ORs) with 95% confidence intervals (95% CIs) were evaluated under a fixed effects model for absence of heterogeneity. Significant associations were observed between p14ARF gene methylation and tumor location (OR = 2.35, 95% CI: 1.55–3.55, P = 0.001), microsatellite instability (MSI) status (OR = 3.28, 95% CI: 2.12–5.07, P<0.0001). However, there were no significant associations between p14ARF gene methylation and tumor stage, tumor differentiation. We concluded that p14ARF gene methylation may be significantly associated with tumor location, and MSI status of CRC.

Low Level of Microsatellite Instability Correlates with Poor Clinical Prognosis in Stage II Colorectal Cancer Patients.

The influence of microsatellite instability (MSI) on the prognosis of colorectal cancer (CRC) requires more investigation. We assessed the role of MSI status in survival of individuals diagnosed with primary colorectal cancer. In this retrospective cross-sectional study the MSI status was determined in 158 formalin-fixed paraffin-embedded tumors and their matched normal tissues from patients who underwent curative surgery. Cox proportional hazard modeling was performed to assess the clinical prognostic significance. In this study we found that MSI-H tumors were predominantly located in the colon versus rectum (p = 0.03), associated with poorer differentiation (p = 0.003) and TNM stage II/III of tumors (p = 0.02). In CRC patients with stage II, MSI-L cases showed significantly poorer survival compared with patients who had MSI-H or MSS tumors (p = 0.04). This study indicates that MSI-L tumors correlate with poorer clinical outcome in patients with stage II tumors (p = 0.04) or in tumors located in the colon (p = 0.02). MSI-L characterizes a distinct subgroup of CRC patients who have a poorer outcome. This study suggests that MSI status in CRC, as a clinical prognostic marker, is dependent on other factors, such as tumor stage and location.

144 Association between interleukin 17/interleukin 17 receptor gene polymorphism and papillary thyroid cancer

Material and Methods: To evaluate the extent of ITH in colorectal cancer (CRC) with microsatellite instability (MSI), we obtained 4−7 biopsies from 39 CRCs followed by MSI analysis either using the Bethesda MSI evaluation system or Promega system with 5 mononucleotide markers. Results: We found decreased prevalence of MSI (+) by the Promega system compared to the Bethesda system. The overall discordance between the two systems was 54%. In contrast to the previous studies that had shown discordance only in low MSI (MSI-L), our results showed the discordance not only in MSI-L, but also in high MSI (MSI-H) cases. Among the MSI (+) CRCs, ITH of MSI status was identified in 41.7% of CRC by the Bethesda system and 22.2% by the Promega system. In terms of MSI markers, the ITH originated from dinucleotide markers in most cases (69%), but it originated from mononucleotide markers (31%) as well. Pooling of DNA from a regional biopsy with MSI (+) with additional biopsies from stable MSI (MSS) showed that this approach was beneficial to increase the sensitivity of MSI detection. Conclusions: Our results indicate that ITH of MSI phenotype by the Bethesda system is more overestimated than previously identified. However, because there was considerable ITH of MSI subtypes and markers even by the Promega system, our data suggest that analysis of MSI status in multiple regional biopsies is needed for a better evaluation of MSI status in CRC.

Regional bias of intratumoral genetic heterogeneity of nucleotide repeats in colon cancers with microsatellite instability.

Intratumoral heterogeneity (ITH) may produce regional biases in genotype and phenotype evaluation in a single tumor and may impede proper cancer diagnosis. To evaluate the extent of ITH in colorectal cancer (CRC) with microsatellite instability (MSI), we obtained 4-7 biopsies from 39 CRCs followed by MSI analysis either using the Bethesda MSI evaluation system or Promega system with 5 mononucleotide markers. We found decreased prevalence of MSI (+) by the Promega system compared to the Bethesda system. The overall discordance between the two systems was 54 %. In contrast to the previous studies that had shown discordance only in low MSI (MSI-L), our results showed the discordance not only in MSI-L, but also in high MSI (MSI-H) cases. Among the MSI (+) CRCs, ITH of MSI status was identified in 41.7 % of CRC by the Bethesda system and 22.2 % by the Promega system. In terms of MSI markers, the ITH originated from dinucleotide markers in most cases (69 %), but it originated from mononucleotide markers (31 %) as well. Pooling of DNA from a regional biopsy with MSI (+) with additional biopsies from stable MSI (MSS) showed that this approach was beneficial to increase the sensitivity of MSI detection. Our results indicate that ITH of MSI phenotype by the Bethesda system is more overestimated than previously identified. However, because there was considerable ITH of MSI subtypes and markers even by the Promega system, our data suggest that analysis of MSI status in multiple regional biopsies is needed for a better evaluation of MSI status in CRC.

Clinicopathologic characteristics of colorectal cancer with microsatellite instability

Colorectal cancer (CRC) can be classified according to the level of microsatellite instability (MSI) exhibited by the tumor. The aim of this study was to determine MSI status in CRC from Tunisia and to identify clinical and pathological characteristics of MSI-H tumors.Microsatellite status was determined by polymerase chain reaction amplification using standard markers (BAT25, BAT26, D2S123, D5S346 and D17S250, the Bethesda panel) in 44 CRC cases. Molecular results were correlated with pathological and clinical features.Six CRC cases (13.8%) showed high-level instability (MSI-H), 14 cases had low level instability (MSI-L), and the remainders were stable (MSS). Immunohistochemical analysis showed loss of MSH2 protein in 3 cases among the 6 MSI-H tumors, whereas no silencing of MLH1 or MSH6 was found in any case. Significant differences in age and family history of cancers were observed between MSI-H and MSS/MSI-L groups (p=0.01 and p=0.002). However, statistical analysis showed that there were no significant differences between MSI-H and MSS/MSI-L tumors in terms of tumor location, lymph node involvement and stage of disease. Regarding histological features, MSI-H tumors were more likely to be poorly differentiated (p=0.003), to have a medullary pattern (p=0.005), and to harbor increased numbers of peritumoral lymphocytes (p=0.001).These findings indicate that careful observation of the tumor morphology can assist in the identification of unstable colorectal cancers requiring molecular investigations.

Microsatellite instability testing in Korean patients with colorectal cancer

Microsatellite instability (MSI) testing is useful for identifying patients with hereditary nonpolyposis colorectal cancer and detecting sporadic colorectal cancer that develops through replication error pathways. A pentaplex panel is recommended by the National Cancer Institute for MSI testing, but simplified mononucleotide panels and immunohistochemistry of mismatch repair proteins are widely employed for convenience. This study was to evaluate the MSI status of colorectal cancer in Korean patients. This study included 1,435 patients with colorectal adenocarcinoma subjected to surgical resection. The pentaplex Bethesda panel was used for MSI testing. Seventy nine (5.5%) carcinomas were classified as MSI-high (MSI-H) and 95 (6.6%) as MSI-low (MSI-L). BAT-26 and BAT-25 were unstable in 73 and 75 of 79 MSI-H carcinomas, respectively. With the panel comprising these 2 mononucleotide markers, 72 carcinomas were diagnosed as MSI-H, compared to the Bethesda panel data (72/79, 91.1%). In contrast, BAT-26 or BAT-25 were unstable in only 7 (7.4%) of the 95 MSI-L tumors. In the panel with 2 dinucleotide markers, D17250 linked to p53 and D2S123 to hMSH2, detection rates were 89.9% (71/79) for MSI-H and 80.0% (76/95) for MSI-L carcinomas, compared to the Bethesda panel. Moreover, we compared the frequency of MSI tumor in our patients with those reported previously from Western countries. In conclusion, the frequency of MSI-H appears lower in colorectal cancer patients in Korea. A simplified panel for MSI testing with BAT-26 and BAT-25 seems not effective for the accurate evaluation of MSI status, particularly in MSI-L colorectal carcinomas, in our patients.

High-frequency microsatellite instability and BRAF mutation (V600E) in unselected Serbian patients with colorectal cancer

Microsatellite instability (MSI) is a genetic consequence of a MisMatch Repair defect in colorectal cancer (CRC). We compared clinicopathohistological features with MSI status of CRC and evaluated prognostic significance of MSI status and BRAF mutation in the group of MSI-H tumors. 155 primary CRCs were excised surgically, 2006-2008. MSI analysis was carried out using a fluorescence-based pentaplex polymerase chain reaction technique. BRAF mutation (V600E) was analyzed by direct sequencing in MSI-H tumors. For all patients were evaluated: age, gender, localization, tumor cell type, tumor differentiation, mucin production, lymphocytic infiltration (TILs) and TNM stage. Patients' disease-free survival (DFS) was compared according to MSI and BRAF status using Kaplan-Meier test. Of the 155 CRCs, 19 (12.3%) were MSI-H, and 136 (87.7%) were MSS/L. BRAF mutations were found in 4 of the MSI-H tumors. Patients with MSI-H CRC had lower recurrence rate (log rank test; P=0.04) than MSS/L group. Patients with MSI-H tumor and BRAF mutation had worse DFS than MSI-H tumors without this mutation (log rank test; P=0.01). Most of the clinicopathologic characteristics of MSI-H CRC in Serbian patients are similar to those reported in previous studies. Patients with MSI tumor phenotype had favourable prognosis, but in those with BRAF mutation higher recurrence rate was observed.

Microsatellite instability in colorectal cancer

Approximately 20 percent of right-sided colon cancers and 5 percent of left-sided colon and rectal cancers have a deficient DNA mismatch repair system. This results in the widespread accumulation of mutations to nucleotide repeats, some of which occur within the coding regions of cancer-related genes such as TGFβRII and BAX. A standardized definition for microsatellite instability (MSI) based on the presence of deletions to mononucleotide repeats is gaining widespread acceptance in both research and the clinic. Colorectal cancer (CRC) with MSI are characterized histologically by an abundance of tumor-infiltrating lymphocytes, poor differentiation and a signet ring or mucinous phenotype. In younger patients these tumors usually develop along the chromosomal instability pathway, in which case the mismatch repair genes are inactivated by germline mutation, somatic mutation and loss of heterozygosity. In older patients MSI CRC usually develops against a background of widespread hypermethylation that includes methylation-induced silencing of the mismatch repair gene MLH1. The overall biological and clinical phenotype of MSI CRC that arise in these two pathways is likely to be different and may account for some of the discordant results reported in the literature relating to the clinical properties of these tumors. The available evidence indicates that MSI is unlikely to be a clinically useful marker for the prognostic stratification of early-stage CRC. The predictive value of MSI for response to 5-fluorouracil-based chemotherapy remains controversial, while for other agents the predictive value is difficult to assess because they are used in combination regimens. The MSI phenotype is being actively investigated for novel therapeutic approaches based on the principle of synthetic lethality. Finally, the MSI status of CRC is an extremely useful marker for population-based screening programs that aim to identify individuals and families with the hereditary cancer condition known as Lynch syndrome.

A Novel Logistic Model Based on Clinicopathological Features Predicts Microsatellite Instability in Colorectal Carcinomas

High-frequency microsatellite instability has been reported to be associated with good prognosis in colorectal adenocarcinoma. However, methods to assess microsatellite instability (MIN) are based on genetic assays and are not ideally suited to most histopathology laboratories. The aim of the present study was to develop a model for prediction of MIN status in colorectal cancer based on phenotypic characteristics. Clinicopathological features of a cohort of 204 patients with primary colon cancer were retrospectively reviewed following predetermined criteria. Genetic assessment of MIN status was performed on DNA extracted from sections of formalin-fixed, paraffin-embedded specimens by testing a panel of 11 microsatellite markers. Logistic regression analysis generated a mathematical tool capable of identifying colorectal tumors displaying MIN status with a sensitivity of 77.8% and a specificity of 96.8%. Features associated with instability included the proximal location of the lesions, occurrence of solid and/or mucinous differentiation, absence of cribriform structures, presence of peritumoral Crohn-like reaction, expansive growth pattern, high Ki67 proliferative index, and p53-negative phenotype. This approach predicts microsatellite instability in colorectal carcinoma with an overall assigned accuracy of 95.1% and a negative predictive value of 97.8%. Implementation of this tool to routine histopathological studies could improve the management of patients with colorectal cancer, especially those presenting with stage II and III of the disease. It will also assist in identifying a subset of patients likely to benefit from adjuvant chemotherapy.