A Rare MSH2 Variant as a Candidate Marker for Lynch Syndrome II Screening in Tunisia: A Case of Diffuse Gastric Carcinoma.

Several syndromic forms of digestive cancers are known to predispose to early-onset gastric tumors such as Hereditary Diffuse Gastric Cancer (HDGC) and Lynch Syndrome (LS). LSII is an extracolonic cancer syndrome characterized by a tumor spectrum including gastric cancer (GC). In the current work, our main aim was to identify the mutational spectrum underlying the genetic predisposition to diffuse gastric tumors occurring in a Tunisian family suspected of both HDGC and LS II syndromes. We selected the index case "JI-021", which was a woman diagnosed with a Diffuse Gastric Carcinoma and fulfilling the international guidelines for both HDGC and LSII syndromes. For DNA repair, a custom panel targeting 87 candidate genes recovering the four DNA repair pathways was used. Structural bioinformatics analysis was conducted to predict the effect of the revealed variants on the functional properties of the proteins. DNA repair genes panel screening identified two variants: a rare MSH2 c.728G>A classified as a variant with uncertain significance (VUS) and a novel FANCD2 variant c.1879G>T. The structural prediction model of the MSH2 variant and electrostatic potential calculation showed for the first time that MSH2 c.728G>A is likely pathogenic and is involved in the MSH2-MLH1 complex stability. It appears to affect the MSH2-MLH1 complex as well as DNA-complex stability. The c.1879G>T FANCD2 variant was predicted to destabilize the protein structure. Our results showed that the MSH2 p.R243Q variant is likely pathogenic and is involved in the MSH2-MLH1 complex stability, and molecular modeling analysis highlights a putative impact on the binding with MLH1 by disrupting the electrostatic potential, suggesting the revision of its status from VUS to likely pathogenic. This variant seems to be a shared variant in the Mediterranean region. These findings emphasize the importance of testing DNA repair genes for patients diagnosed with diffuse GC with suspicion of LSII and colorectal cancer allowing better clinical surveillance for more personalized medicine.

CDH1 Germline Variants in a Tunisian Cohort with Hereditary Diffuse Gastric Carcinoma.

Mutational screening of the CDH1 gene is a standard treatment for patients who fulfill Hereditary Diffuse Gastric Cancer (HDGC) testing criteria. In this framework, the classification of variants found in this gene is a crucial step for the clinical management of patients at high risk for HDGC. The aim of our study was to identify CDH1 as well as CTNNA1 mutational profiles predisposing to HDGC in Tunisia. Thirty-four cases were included for this purpose. We performed Sanger sequencing for the entire coding region of both genes and MLPA (Multiplex Ligation Probe Amplification) assays to investigate large rearrangements of the CDH1 gene. As a result, three cases, all with the HDGC inclusion criteria (8.82% of the entire cohort), carried pathogenic and likely pathogenic variants of the CDH1 gene. These variants involve a novel splicing alteration, a missense c.2281G > A detected by Sanger sequencing, and a large rearrangement detected by MLPA. No pathogenic CTNNA1 variants were found. The large rearrangement is clearly pathogenic, implicating a large deletion of two exons. The novel splicing variant creates a cryptic site. The missense variant is a VUS (Variant with Uncertain Significance). With ACMG (American College of Medical Genetics and Genomics) classification and the evidence available, we thus suggest a revision of its status to likely pathogenic. Further functional studies or cosegregation analysis should be performed to confirm its pathogenicity. In addition, molecular exploration will be needed to understand the etiology of the other CDH1- and CTNNA1-negative cases fulfilling the HDGC inclusion criteria.

New insights in the clinical implication of HOXA5 as prognostic biomarker in patients with colorectal cancer.

BACKGROUND: Homeobox A5 (HOXA5) is a member of the HOX protein family which is involved in several carcinogenesis pathways, and is dysregulated in many cancer types. However, its expression and function in human colorectal cancer (CRC) is still largely unknown. OBJECTIVE: This study aimed to evaluate HOXA5 expression in Tunisian patients with CRC in order to define new potential biomarker. METHODS: An immunohistochemical labeling using an HOXA5 antibody was performed on 85 formalin fixed paraffin embedded specimens from patients with CRC. Six normal colon mucosa cases were used as controls. RESULTS: HOXA5 expression showed a cytoplasmic staining in both tumor and stromal/endothelial cells. Loss or low HOXA5 expression was seen in tumor cells in 74/85 cases (87.06%) and in stromal/endothelial cells, in 77/85 (90.59%). In control group of normal colon mucosa HOXA5 was moderately expressed in all the cases. The abnormal expression, was significantly associated to lymph nodes metastasis in tumor cells (p= 0.043) and in stromal/endothelial cells (p= 0.024). CONCLUSION: HOXA5 immunostaining results suggest the valuable role of this protein in colorectal carcinogenesis. Moreover, the association of lymph node metastasis to HOXA5 abnormal expression underlies its crucial role in colorectal cancer dissemination and prognosis.

Dysregulated PDGFR alpha expression and novel somatic mutations in colorectal cancer: association to RAS wild type status and tumor size.

BACKGROUND: Platelet derived growth factor receptor alpha (PDGFRα) has been considered as a relevant factor in tumor proliferation, angiogenesis and metastatic dissemination. It was a target of tyrosine kinase (TK) inhibitors emerged in the therapy of diverse cancers. In colorectal cancer, the commonly used therapy is anti-epithelial growth factor receptor (EGFR). However, both RAS mutated and a subgroup of RAS wild type patients resist to such therapy. The aim of this study is to investigate PDGFRα protein expression and mutational status in colorectal adenocarcinoma and their association with clinicopathological features and molecular RAS status to provide useful information for the identification of an effective biomarker that might be implicated in prognosis and treatment prediction. METHODS: Our study enrolled 103 formalin fixed paraffin-embedded (FFPE) colorectal adenocarcinoma. PDGFRα expression was investigated by immunohistochemistry (IHC). Hotspot exon 18 of PDGFRA was studied by PCR followed by Sanger sequencing and RAS status was determined by real time quantitative PCR. Thirteen normal colon tissues were used as negative controls. RESULTS: PDGFRα staining was detected in the cytoplasm of all tissues. Low expression was observed in all normal colon mucosa. In adenocarcinoma, 45% (45/100) of cases showed PDGFRα overexpression. This overexpression was significantly associated with mutations in exon 18 (P = 0.024), RAS wild type status (P < 10-3), tumor diameter (P = 0.048), whereas there was no association with tumor side (P = 0.13) and other clinicopathological features. CONCLUSION: Overexpression of PDGFRα in adenocarcinoma suggests its potential role in tumor cells growth and invasion. The association between PDGFRα overexpression in both tumor and stromal adenocarcinoma cells with RAS wild type status suggests its potential role in anti-EGFR therapy resistance and the relevance of using it as specific or adjuvant therapeutic target.

Identification of novel pathogenic MSH2 mutation and new DNA repair genes variants: investigation of a Tunisian Lynch syndrome family with discordant twins.

BACKGROUND: Lynch syndrome (LS) is a highly penetrant inherited cancer predisposition syndrome, characterized by autosomal dominant inheritance and germline mutations in DNA mismatch repair genes. Despite several genetic variations that have been identified in various populations, the penetrance is highly variable and the reasons for this have not been fully elucidated. This study investigates whether, besides pathogenic mutations, environment and low penetrance genetic risk factors may result in phenotype modification in a Tunisian LS family. PATIENTS AND METHODS: A Tunisian family with strong colorectal cancer (CRC) history that fulfill the Amsterdam I criteria for the diagnosis of Lynch syndrome was proposed for oncogenetic counseling. The index case was a man, diagnosed at the age of 33 years with CRC. He has a monozygotic twin diagnosed at the age of 35 years with crohn disease. Forty-seven years-old was the onset age of his paternal uncle withCRC. An immunohistochemical (IHC) labeling for the four proteins (MLH1, MSH2, MSH6 and PMS2) of the MisMatchRepair (MMR) system was performed for the index case. A targeted sequencing of MSH2, MLH1 and a panel of 85 DNA repair genes was performed for the index case and for his unaffected father. RESULTS: The IHC results showed a loss of MSH2 but not MLH1, MSH6 and PMS2 proteins expression. Genomic DNA screening, by targeted DNA repair genes sequencing, revealed an MSH2 pathogenic mutation (c.1552C>T; p.Q518X), confirmed by Sanger sequencing. This mutation was suspected to be a causal mutation associated to the loss of MSH2 expression and it was found in first and second degree relatives. The index case has smoking and alcohol consumption habits. Moreover, he harbors extensive genetic variations in other DNA-repair genes not shared with his unaffected father. CONCLUSION: In our investigated Tunisian family, we confirmed the LS by IHC, molecular and in silico investigations. We identified a novel pathogenic mutation described for the first time in Tunisia. These results come enriching the previously reported pathogenic mutations in LS families. Our study brings new arguments to the interpretation of MMR expression pattern and highlights new risk modifiers genes eventually implicated in CRC. Twins discordance reported in this work underscore that disease penetrance could be influenced by both genetic background and environmental factors.