Microsatellite Instability Phenotype Research Articles

The Developing Story of Predictive Biomarkers in Colorectal Cancer.

Colorectal cancer (CRC) is the third most common malignancy worldwide. Surgery remains the most important treatment for non-metastatic CRC, and the administration of adjuvant chemotherapy depends mainly on the disease stage, which is still the strongest prognostic factor. A refined understanding of the genomics of CRC has recently been achieved thanks to the widespread use of next generation sequencing with potential future therapeutic implications. Microsatellite instability (MSI) has been suggested as a predictive marker for response to anti-programmed-cell-death protein 1 (PD-1) therapy in solid tumors, including CRC. It should be noted that not all cancers with MSI phenotype respond to anti-PD-1 immunotherapy, highlighting the urgent need for even better predictive biomarkers. Mitogen-Activated Protein Kinase (MAPK) pathway genes KRAS, NRAS, and BRAF represent important molecular targets and could serve as independent prognostic biomarkers in CRC, and identify those who potentially benefit from anti-epidermal growth factor receptor (EGFR) treatment. Emerging evidence has attributed a significant role to inflammatory markers including blood cell ratios in the prognosis and survival of CRC patients; these biomarkers can be easily assessed in routine blood exams and be used to identify high-risk patients or those more likely to benefit from chemotherapy, targeted therapies and potentially immunotherapy. Analysis of cell-free DNA (cfDNA), circulating tumor cells (CTC) and/or micro RNAs (miRNAs) could provide useful information for the early diagnosis of CRC, the identification of minimal residual disease and, the evaluation of the risk of recurrence in early CRC patients. Even the selection of patients suitable for the new targeted therapy is becoming possible with the use of predictive miRNA biomarkers. Finally, the development of treatment resistance with the emergence of chemo-resistance clones after treatment remains the most important challenge in the clinical practice. In this context it is crucial to identify potential biomarkers and therapeutic targets which could lead to development of new and more effective treatments.

Synchronous endometrioid endometrial and ovarian carcinomas are biologically related: A clinico-pathological and molecular (next generation sequencing) study of 22 cases.

The criteria for distinction between independent primary tumors and metastasis from one site to the other in synchronous endometrioid endometrial and ovarian carcinoma (SEO) has been a matter of dispute for a long time. In our study we performed a comprehensive clinico-pathological and molecular analysis of 22 cases of SEO. Based on conventional clinico-pathological criteria the cases were classified as independent primary tumors (10 cases) and metastasis from one location to the other (12 cases). All tumors were analyzed by NGS with a panel of 73 genes (219 kbp). Clonal origin was confirmed in all cases by at least one shared mutation in PTEN, AKT1, PIK3CA, KRAS, TP53 and ARID1A. Two patients carried germline pathogenic mutation in cancer-predisposing genes BRCA1 or BARD1. Microsatellite instable phenotype was detected in 5/22 (22.7%) SEO, but in one case only in the endometrial tumor. In conclusion, our results showed that all 22 SEOs were clonally related, irrespectively of their clinico-pathological features. Even low grade and low stage tumors classified as independent primaries, according to the conventional morphological criteria, have a clonal origin. From the practical point of view, only the conventional morphological criteria should be used for the classification (staging) of these tumors. However, molecular profiling of these tumors may have prognostic and predictive meaning.

From Tumor Immunology to Immunotherapy in Gastric and Esophageal Cancer.

Esophageal and gastric cancers represent tumors with poor prognosis. Unfortunately, radiotherapy, chemotherapy, and targeted therapy have made only limited progress in recent years in improving the generally disappointing outcome. Immunotherapy with checkpoint inhibitors is a novel treatment approach that quickly entered clinical practice in malignant melanoma and renal cell cancer, but the role in esophageal and gastric cancer is still poorly defined. The principal prognostic/predictive biomarkers for immunotherapy efficacy currently considered are PD-L1 expression along with defects in mismatch repair genes resulting in microsatellite instability (MSI-H) phenotype. The new molecular classification of gastric cancer also takes these factors into consideration. Available reports regarding PD-1, PD-L1, PD-L2 expression and MSI status in gastric and esophageal cancer are reviewed to summarize the clinical prognostic and predictive role together with potential clinical implications. The most important recently published clinical trials evaluating checkpoint inhibitor efficacy in these tumors are also summarized.

Molecular and Computational Methods for the Detection of Microsatellite Instability in Cancer.

Microsatellite instability (MSI) is a genomic alteration in which microsatellites, usually of one to four nucleotide repeats, accumulate mutations corresponding to deletions/insertions of a few nucleotides. The MSI phenotype has been extensively characterized in colorectal cancer and is due to a deficiency of the DNA mismatch repair system. MSI has recently been shown to be present in most types of cancer with variable frequencies (from <1 to 30%). It correlates positively to survival outcome and predicts the response to immune checkpoint blockade therapy. The different methods developed for MSI detection in cancer require taking into consideration two critical parameters which influence method performance. First, the microsatellite markers used should be chosen carefully to ensure they are highly sensitive and specific for MSI detection. Second, the analytical method used should be highly resolute to allow clear identification of MSI and of the mutant allele genotype, and should present the lowest limit of detection possible for application in samples with low mutant allele frequency. In this review, we describe all the different molecular and computational methods developed to date for the detection of MSI in cancer, how they have evolved and improved over the years, and their advantages and drawbacks.

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

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

Cell-free DNA profiling of metastatic prostate cancer reveals microsatellite instability, structural rearrangements and clonal hematopoiesis

BackgroundThere are multiple existing and emerging therapeutic avenues for metastatic prostate cancer, with a common denominator, which is the need for predictive biomarkers. Circulating tumor DNA (ctDNA) has the potential to cost-efficiently accelerate precision medicine trials to improve clinical efficacy and diminish costs and toxicity. However, comprehensive ctDNA profiling in metastatic prostate cancer to date has been limited.MethodsA combination of targeted and low-pass whole genome sequencing was performed on plasma cell-free DNA and matched white blood cell germline DNA in 364 blood samples from 217 metastatic prostate cancer patients.ResultsctDNA was detected in 85.9% of baseline samples, correlated to line of therapy and was mirrored by circulating tumor cell enumeration of synchronous blood samples. Comprehensive profiling of the androgen receptor (AR) revealed a continuous increase in the fraction of patients with intra-AR structural variation, from 15.4% during first-line metastatic castration-resistant prostate cancer therapy to 45.2% in fourth line, indicating a continuous evolution of AR during the course of the disease. Patients displayed frequent alterations in DNA repair deficiency genes (18.0%). Additionally, the microsatellite instability phenotype was identified in 3.81% of eligible samples (≥ 0.1 ctDNA fraction). Sequencing of non-repetitive intronic and exonic regions of PTEN, RB1, and TP53 detected biallelic inactivation in 47.5%, 20.3%, and 44.1% of samples with ≥ 0.2 ctDNA fraction, respectively. Only one patient carried a clonal high-impact variant without a detectable second hit. Intronic high-impact structural variation was twice as common as exonic mutations in PTEN and RB1. Finally, 14.6% of patients presented false positive variants due to clonal hematopoiesis, commonly ignored in commercially available assays.ConclusionsctDNA profiles appear to mirror the genomic landscape of metastatic prostate cancer tissue and may cost-efficiently provide somatic information in clinical trials designed to identify predictive biomarkers. However, intronic sequencing of the interrogated tumor suppressors challenges the ubiquitous focus on coding regions and is vital, together with profiling of synchronous white blood cells, to minimize erroneous assignments which in turn may confound results and impede true associations in clinical trials.

Multiplicity of Advanced T Category-Tumors Is a Risk Factor for Survival in Patients with Colorectal Carcinoma.

BackgroundPrevious studies on synchronous colorectal carcinoma (SCRC) have reported inconsistent results about its clinicopathologic and molecular features and prognostic significance.MethodsForty-six patients with multiple advanced tumors (T2 or higher category) who did not receive neoadjuvant chemotherapy and/or radiotherapy and who are not associated with familial adenomatous polyposis were selected and 99 tumors from them were subjected to clinicopathologic and molecular analysis. Ninety-two cases of solitary colorectal carcinoma (CRC) were selected as a control considering the distributions of types of surgeries performed on patients with SCRC and T categories of individual tumors from SCRC.ResultsSCRC with multiple advanced tumors was significantly associated with more frequent nodal metastasis (p = .003) and distant metastasis (p = .001) than solitary CRC. KRAS mutation, microsatellite instability, and CpG island methylator phenotype statuses were not different between SCRC and solitary CRC groups. In univariate survival analysis, overall and recurrence-free survival were significantly lower in patients with SCRC than in patients with solitary CRC, even after adjusting for the extensiveness of surgical procedure, adjuvant chemotherapy, or staging. Multivariate Cox regression analysis revealed that tumor multiplicity was an independent prognostic factor for overall survival (hazard ratio, 4.618; 95% confidence interval, 2.126 to 10.030; p < .001), but not for recurrence-free survival (p = .151).ConclusionsFindings suggested that multiplicity of advanced T category–tumors might be associated with an increased risk of nodal metastasis and a risk factor for poor survival, which raises a concern about the guideline of American Joint Committee on Cancer’s tumor-node-metastasis staging that T staging of an index tumor determines T staging of SCRC.

Clinical Targeted Next-Generation Sequencing Shows Increased Mutational Load in Endometrioid-type Endometrial Adenocarcinoma With Deficient DNA Mismatch Repair.

A subset of endometrial adenocarcinomas (EACs) exhibit microsatellite instability and have deficient DNA mismatch repair (dMMR). The overall aim of the study was to compare the spectrum of mutations in endometrioid-type EAC with and without dMMR by using a clinically validated next-generation sequencing assay. We retrospectively identified 19 EACs with known mismatch repair status that had undergone targeted sequencing of a panel of cancer-related genes. The mismatch repair status was ascertained by immunohistochemistry against MLH1, PMS2, MSH2, and MSH6 mismatch proteins. Somatic mutations in EAC with dMMR were compared against those in cases with proficient MMR (pMMR). The dMMR EAC showed a normalized mean of 66.6 mutations/Mb per case compared with pMMR EAC with a mean of 26.2 (P<0.05). The most commonly mutated genes were PTEN (89% of dMMR, 50% of pMMR), PIK3CA (67% vs. 40%), ATM (89% vs. 40%), and FLT3 (67% vs. 50%). The transition/transversion ratio was 4.7 versus 2.8 for the dMMR and pMMR cohorts, respectively (P<0.05). Copy number variant analysis did not demonstrate significant differences between the dMMR and pMMR cohorts and was not correlated with histologic grade of EAC. In conclusion, tumorigenesis of EAC in the context of dMMR demonstrated heavier mutational burdens and higher transition/transversion ratio. The spectrum of genetic alterations can potentially help identify cases with microsatellite instability phenotype using next-generation sequencing data from a targeted cancer gene panel.

Characterisation of mesenchymal colon tumour-derived cells in tumourspheres as a model for colorectal cancer progression

Cellular plasticity, the ability of cells to switch from an epitheial phenotype to a mesenchymal one and vice versa, plays a crucial role in tumour progression and metastases development. In 20-25% of patients with colon cancer and in 18% of patients with rectal cancer, metastases are present at the time of the first diagnosis. They are the first cause of colorectal cancer (CRC)-related mortality, defining stage IV CRC, which is characterized by a relatively short overall survival. We previously isolated two primary colon adenocarcinoma cell cultures that had undergone epithelial-mesenchymal transition (EMT), one with a high microsatellite instability phenotype (T88) and one with a chromosomal instability phenotype (T93). The aim of this study was to establish a model with which to study EMT, stemness features and cell plasticity in cancer progression and to examine the effects of incubation with lithium chloride (LiCl), a specific glycogen synthase kinase 3 β (GSK-3β) inhibitor, on these cellular processes. Indeed, GSK3β is an important regulator of cell survival, which promotes tumourigenesis in colon cells by facilitating the crosstalk between colorectal cancer pathways. Thus, we further characterized our system of adherent primary mesenchymal colon cancer cells and their paired tumourspheres by examining the expression and localisation of a panel of markers, including E- and N-cadherin, CD133, CD44v6, aldehyde dehydrogenase 1 (ALDH1) and leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5). We also characterised the molecular features of these tumourspheres and examined their response to LiCl. Furthermore, we explored the effects of LiCl on cell motility and plasticity. We demonstrated that LiCl reduced cell migration, stemness features and cell plasticity. We also observed the atypical nuclear localisation of membrane proteins, including N-cadherin, CD133 and CD44v6 in mesenchymal tumour cells. Of note, CD133 and CD44v6 appeared to localise at the plasma membrane in cells with a more epithelial phenotype, suggesting that the cytoplasmic/nuclear localisation of these proteins could favour and characterize cell plasticity in colorectal cancer progression.

Improved Microsatellite Instability Detection and Identification by Nuclease-Assisted Microsatellite Instability Enrichment Using HSP110 T17

To the Editor: Microsatellite instability (MSI)1 is characterized by the accumulation of mutations (insertions or deletions of several nucleotides) in microsatellites, caused by DNA mismatch repair system deficiency. Approximately 15% to 20% of colorectal cancers (CRCs) present with MSI, the origin of which can be sporadic or owing to genetic predisposition to Lynch syndrome/hereditary nonpolyposis CRC or constitutional mismatch repair deficiency syndrome. The MSI phenotype is associated with a better survival rate for patients with stage II/III CRC and is also a major predictive biomarker for the efficacy of immune checkpoint blockade therapy in metastatic CRC. The gold standard method for MSI detection includes standard PCR followed by fragment analysis using the Bethesda or the Pentaplex panel, each composed of 5 microsatellite markers. However, this approach can lack analytical sensitivity, thus limiting potential clinical applications. Recent attempts to improve the analytical sensitivity of MSI detection include a method based on mutation enrichment during E- ice -COLD-PCR. This method uses HSP110 T17 (HT17), which, compared with the Pentaplex panel, is an improved microsatellite biomarker for the diagnosis of MSI/MSI-High in CRC and also bears prognostic and predictive information …

Expression Profile of LGR5 and Its Prognostic Significance in Colorectal Cancer Progression

We investigated the expression profile of leucine-rich, repeat-containing, G-protein-coupled receptor 5 (LGR5) during colorectal cancer (CRC) progression and determined the prognostic impact of LGR5 in a large cohort of CRC samples. LGR5 expression was higher in CRCs than in normal mucosa, and was not associated with other cancer stem cell markers. LGR5 positivity was observed in 68% of 788 CRCs and was positively correlated with older age, moderately to well-differentiated cells, and nuclear β-catenin expression. Enhanced LGR5 expression remained persistent during the adenoma-carcinoma transition, but markedly declined in the budding cancer cells at the invasive fronts, which was not due to altered wingless-type mouse mammary tumor virus integration site family (Wnt) or epithelial-mesenchymal transition signaling. LGR5 showed negative correlations with microsatellite instability and CpG island methylator phenotype, and was not associated with KRAS or BRAF mutation. Notably, LGR5 positivity was an independent prognostic marker for better clinical outcomes in CRC patients. LGR5 overexpression attenuated tumor growth by decreasing ERK phosphorylation along with decreased colony formation and migration abilities in DLD1 cells. Likewise, knockdown of LGR5 expression resulted in a decline in the colony-forming and migration capacities in LoVo cells. Taken together, our data suggest a suppressive role of LGR5 in CRC progression.

Abstract 4599: Detecting MSI phenotype in circulating blood DNA

Abstract Background: Microsatellite instability (MSI) is a hypermutator phenotype occurring in gastrointestinal, endometrial and colorectal tumors, and more rarely, in urinary tract, ovarian, breast, prostate, lung, head and neck, liver and glioblastoma tumors. The diagnosis of MSI phenotype has recently emerged as the first pan-tumor biomarker likely to predict clinical benefit from immune-checkpoint blockade therapy, making its precise identification primordial for treatment decisions and disease monitoring. Current diagnosis of MSI is performed by multiplex PCR of five microsatellite markers, followed by capillary electrophoresis to detect shifts in allele size. An important limitation of this method is its low sensitivity preventing its use when tumor content is below 10%. Methods: We designed ddPCR assays examining 3 microsatellites. Analytical performance was assessed in vitro. Clinical performance was evaluated in a series of FFPE and body fluid DNA samples obtained from patients with Stage IV colorectal or endometrial carcinomas, previously classified as MSI-H or MSS using the standard pentaplex PCR method. Mutant allele frequencies (MAF) quantified with the ddPCR MSI assay were also compared with the MAFs reported by ddPCR assays targeting specific BRAF or PIK3CA mutations. Results: The ddPCR assays for the 3 microsatellites were found to have high specificity and reached a limit of detection of &amp;lt;0.1%. A perfect concordance with the MSI status determined by the pentaplex assay was observed in the 50 tumor samples tested (25 MSI-H and 25 MSS). Importantly, the ddPCR assays diagnosed the MSI phenotype in the plasma of all patients with MSI-H tumors, without false positives in MSS cases. Furthermore, an excellent correlation was observed in the 6 FFPE (R2:0.81) and 8 body fluid (R2:0.99) MSI-H samples were the MAFs measured by our assay could be compared with the ones obtained by ddPCR assays targeting specific mutations in BRAF or PIK3CA genes. Conclusion: This technique allows a cost-effective, sensitive and large scale screening of the MSI phenotype, as well as the follow-up of MSI patients using liquid biopsies. Citation Format: Marc-Henri Stern, Amanda B. Silveira, Ivan Bieche, Samia Melaabi, Luc Cabel, Bruno Buecher, Jean-Yves Pierga, Francois-Clement Bidard, Charlotte Proudhon. Detecting MSI phenotype in circulating blood DNA [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 4599.

Abstract 3305: Cell-of-origin differentiation stages define methylation-based subtypes of human colorectal adenomas and carcinomas

Abstract Introduction: Colorectal adenomas are precursor lesions of colorectal cancers and represent clonal amplifications of single cells from colonic crypts. DNA methylation patterns specify cell-type identity during cellular differentiation and therefore provide novel opportunities for tumor subclassification and patient stratification. Methods: Infinium 450k and EPIC data from of non-malignant colorectal adenomas was applied to a consensus clustering algorithm. This defined epigenetic subtypes that were subsequently tested for clinico-pathological features, such as morphology and mutation status. Additionally, the subtypes were compared with Infinium 450k data from colonic crypt sections and also colorectal cancer samples provided by The Cancer Genome Atlas. Finally, clinical significance was addressed by testing for subtype specific overall survival rates. Results: The analysis of the adenoma dataset defined 3 distinct epigenetic subtypes of human premalignant lesions. These 3 subtypes also reflect 3 of 4 epigenetic subtypes identified in colorectal cancer. The fourth, cancer-specific subtype represents the previously known subclass defined by microsatellite instability phenotype and the CpG island methylator phenotype. Comparisons to Crypt section methylomes and to other reference methylomes revealed that the other 3 subtypes represent epigenetic programs reflecting various intestinal crypt cell differentiation stages. Patient survival correlated with the differentiation stages with a particular poor prognosis for patients with stem cell-related epigenetic signatures. Conclusion: Our results establish a novel and clinically relevant approach for colorectal adenoma and cancer classification and illustrate how differences in the cell-type of origin shape the tumor methylome. Citation Format: Felix Bormann, Manuel Rodríguez-Paredes, Felix Lasitschka, Dominic Edelmann, Axel Benner, Yehudit Bergman, Claudia R. Ball, Hanno Glimm, Heinz G. Linhart, Frank Lyko. Cell-of-origin differentiation stages define methylation-based subtypes of human colorectal adenomas and carcinomas [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 3305.

PO-276 Prognostic significance of LGR5, an intestinal stem cell marker, in colorectal cancers. (running head: LGR5 in colorectal cancers)

IntroductionLeucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) has been suggested as a promising cancer stem cell marker in colorectal cancers (CRCs). However, the clinical significance of LGR5 expression in CRCs remains controversial. Here, we investigated the expression profile of LGR5 and determined the prognostic impact of LGR5 in a large cohort of CRC samples.Material and methodsLGR5 expression was determined with fresh-frozen CRC tissues by real time-PCR analysis. Tissue microarrays (TMAs) containing 1133 CRC samples were constructed, on which RNA in situ hybridization (ISH) for LGR5 was performed. The functional effects of LGR5 expression on cancer cell proliferation and migration was assessed by in vitro transfection technique.Results and discussionsLGR5 expression was higher in CRCs than in normal mucosa, and was not associated with other cancer stem cell markers. LGR5 positivity was observed in 43% of CRCs and positively correlated with old age, well to moderate differentiation, and nuclear b-catenin expression. Enhanced LGR5 expression remained during the adenoma-carcinoma transition, but substantially declined in the budding cells. Because nuclear β-catenin expression was consistently observed in the budding cells and TGFβ1 treatment or SNAIL overexpression did not result in a decrease in the LGR5 expression in LOVO cells, Wnt or EMT pathway are not likely to be responsible for LGR5 suppression. LGR5 expression showed negative correlations with microsatellite instability and CpG island methylator phenotype, and was not associated with KRAS and BRAF mutations, suggesting an implication of LGR5 in chromosomal instability (CIN) pathway in CRC carcinogenesis. Notably, LGR5 positivity was an independent prognostic marker for better prognosis. LGR5 overexpression led to reduced tumour growth by decreasing ERK phosphorylation along with decreased colony forming and migration abilities in DLD1 cells. Likewise, knockdown of LGR5 expression resulted in a decline in the colony forming and migration capacities in LOVO cells. These data suggest the suppressive role of LGR5 in CRC progression.ConclusionEnhanced LGR5 expression remains persistent during the adenoma to carcinoma progression, and is associated with CIN pathway of colon carcinogenesis. Abrupt LGR5 down-regulation observed in the budding cancer cells at the invasive fronts is less likely due to altered Wnt or EMT signalling pathways. LGR5 was an independent prognostic marker for better clinical outcomes in CRCs.

Pathogenic somatic mutation (SM) of mismatch repair (MMR) genes and associations with microsatellite instability (MSI), tumor mutational burden (TMB) and SM in other DNA repair pathways in 24,223 tumor genomic profiles.

1505Background: SM disabling DNA repair mechanisms is common in cancer. SM in MMR genes (MLH1, MSH2, MSH6, PMS2) contribute to MMR deficiency and the MSI phenotype. Biallelic SM (bSM) of an MMR gen...

Clinicopathological and molecular differences between right-sided and left-sided colorectal cancer in Japanese patients.

The aim of this study was to clarify clinicopathological features, frequencies of molecular biomarkers, and prognoses in Japanese colorectal cancer patients and compare them with right-sided colon cancer (RCC) and left-sided colorectal cancer (LCRC). We consecutively selected 575 colorectal cancer patients who underwent surgical resection from 2008 to 2011. RCC was located from the cecum to the transverse colon, and LCRC was located from the splenic flexure to the rectum. Frequencies of KRAS gene mutation, BRAF gene mutation, microsatellite instability (MSI), l18qLOH and CpG island methylator phenotype (CIMP) were statistically analyzed between groups. Tumors were located in the RCC in 26.3% of patients and in the LCRC in 73.7%. Elderly patients, females and advanced diseases were significantly more frequent in the RCC group than in the LCRC group. However, venous invasion was significantly more frequent in LCRC than in RCC. Between groups, BRAF mutant type, KRAS mutant type, MSI and CIMP+ were significantly more frequent in RCC, whereas 18qLOH was significantly more frequent in LCRC. In overall survival, RCC demonstrated poor prognosis compared with LCRC; however, age, gender, stage, lymphatic invasion, KRAS status and BRAF status rather than tumor location were independent prognostic factors. In addition, the independent prognostic factors in RCC were different from those in LCRC in each stage. However, the consistency between OS and DFS was not observed in this study, excluding lymphatic invasion in LCRC. Comparing RCC with LCRC, RCC is different from LCRC in clinicopathological features, molecular biomarkers and prognostic factors in Japanese colorectal cancer patients. Since the proportions of molecular biomarkers of CRC in this study are different from Western CRCs, further studies are required to clarify the clinicopathological differences between Japanese CRCs and Western CRCs.

The molecular characteristics of colorectal cancer: Implications for diagnosis and therapy.

Colorectal cancer (CRC) results from the progressive accumulation of multiple genetic and epigenetic aberrations within cells. The progression from colorectal adenoma to carcinoma is caused by three major pathways: Microsatellite instability, chromosomal instability and CpG island methylator phenotype. A growing body of scientific evidences suggests that CRC is a heterogeneous disease, and genetic characteristics of the tumors determine their prognostic outcome and response to targeted therapies. Early diagnosis and effective targeted therapies based on a current knowledge of the molecular characteristics of CRC are essential to the successful treatment of CRC. Therefore, the present review summarized the current understanding of the molecular characteristics of CRC, and discussed its implications for diagnosis and targeted therapy.

Mismatch repair deficiency is associated with MSI phenotype, increased tumor-infiltrating lymphocytes and PD-L1 expression in immune cells in ovarian cancer

ObjectiveThe role of mismatch repair (MMR) deficiency in ovarian cancer (OC) pathogenesis and its association with other clinicopathologic features, such as microsatellite instability (MSI) and expression of checkpoint proteins, remain largely elusive. MethodsWe performed Immunohistochemistry (IHC) for MLH1, MSH2, MSH6 and PMS2 on full-section slides from 419 OCs to assess the MMR status. The clinical relevance of MMR deficiency was analyzed in combination with clinical data. The MSI status (by MSI assay) and expression of CD3, CD8, PD-1 and PD-L1 (by IHC) were compared in OCs with different MMR status. ResultsWe found that 2.6% OCs were MMR-negative, 4.3% OCs were MMR-low, and 63.6% of MMR-negative OCs were of endometrioid subtype. A significantly higher proportion of MMR-negative OCs were diagnosed at stage I or II compared to MMR-proficient OCs (p=0.0041). MSI was observed in all tested MMR-negative OCs, 14.3% of tested MMR-low OCs and 3.2% of tested MMR-proficient OCs. In addition, MMR-negative OCs had better progression free survival compared to MMR-proficient and MMR-low OCs (p=0.0046). Furthermore, the majority of OCs were PD-1-positive in intratumoral lymphocytes regardless of MMR status; while MMR-negative OCs exhibited significantly increased CD3+ and CD8+ tumor-infiltrating lymphocytes, and PD-L1+ intratumoral immune cells compared to MMR-proficient OCs. ConclusionOur data suggests that MMR deficient OC is a unique molecular subgroup, characterized by early stage of diagnosis, MSI phenotype, and increased tumor-infiltrating lymphocytes. These patients may be good candidates for anti-PD-1/PD-L1 therapy.

Oesophago-gastric cancer: One disease or separate entities?: Translating molecular subtyping into clinical practice

The comprehensive molecular analysis of esophageal adenocarcinomas (EAC), gastroesophageal junction (GEJ) carcinomas and gastric adenocarcinomas showed several resemblances and a continuum of molecular profile can be described (1). EAC are molecularly closer to gastric cancer than to esophageal squamous cell carcinoma (ESCC), which are in turn close to head and neck cell carcinoma. Among the 4 four described molecular groups of gastric cancer (Epstein-Barr virus (EBV) infection, microsatellite instability (MSI), chromosomal instability (CIN) and genomic stability (GS), EAC and gastric cancer group with a CIN phenotype form a distinct group from the others 3 gastric cancer groups. For the GEJ clearly derived for esophagus the only phenotype was the CIN. On the contrary, the MSI phenotype and the phenotype linked to EBV was observed only in GEJ for which the origin is uncertain or gastric. These results suggest that there is no specific anatomic entity but rather molecular entities with a large predominance of gastric CIN phenotype in the GEJ carcinomas, the others phenotypes show be considered as gastric cancers. Nevertheless, among the EAC and GEJ with CIN phenotype there is an inverse gradient of methylation from the esophagus to the stomach. Four clusters (C1 to C4) can be identified based on the abundance of CpG methylation. Several genes are specifically methylated according to the cluster of methylation and could explain a difference of sensitivity regarding alkylating agents. Several other differences in the CIN subgroup can be reported in terms of frequency of somatic genetic alteration in APC, SMARC4 or CDKN2A accordingly to the tumor location.

A novel DNA methylation panel accurately detects colorectal cancer independently of molecular pathway

BackgroundColorectal cancer (CRC) is one of the most incident cancers, associated with significant morbidity and mortality, and usually classified into three main molecular pathways: chromosomal instability, microsatellite instability (MSI) and CpG island methylator phenotype (CIMP). Currently, available screening methods are either costly or of limited specificity, impairing global implementation. More cost-effective strategies, including DNA methylation-based tests, might prove advantageous. Although some are already available, its performance is suboptimal, entailing the need for better candidate biomarkers. Herein, we tested whether combined use of APC, IGF2, MGMT, RASSF1A, and SEPT9 promoter methylation might accurately detect CRC irrespective of molecular subtype.MethodsSelected genes were validated using formalin-fixed paraffin-embedded tissues from 214 CRC and 50 non-malignant colorectal mucosae (CRN). Promoter methylation levels were assessed using real-time quantitative methylation-specific PCR. MSI and CIMP status were determined. Molecular data were correlated with standard clinicopathological features. Diagnostic and prognostic performances were evaluated by receiver operator characteristics curve and survival analyses, respectively.ResultsExcept for IGF2, promoter methylation levels were significantly higher in CRC compared to CRN. A three-gene panel (MGMT, RASSF1A, SEPT9) identified malignancy with 96.6% sensitivity, 74.0% specificity and 91.5 positive predictive value (area under the curve: 0.97), independently of tumor location, stage, and molecular pathway.ConclusionsCombined promoter methylation analysis of MGMT/RASSF1A/SEPT9 displays a better performance than currently available epigenetic-based biomarkers for CRC, providing the basis for the development of a non-invasive assay to detect CRC irrespective of the molecular pathway.