A tumor focused approach to resolving the etiology of DNA mismatch repair deficient tumors classified as suspected Lynch syndrome.

Routine screening of tumors for DNA mismatch repair (MMR) deficiency (dMMR) in colorectal (CRC), endometrial (EC) and sebaceous skin (SST) tumors leads to a significant proportion of unresolved cases classified as suspected Lynch syndrome (SLS). SLS cases (n = 135) were recruited from Family Cancer Clinics across Australia and New Zealand. Targeted panel sequencing was performed on tumor (n = 137; 80×CRCs, 33×ECs and 24xSSTs) and matched blood-derived DNA to assess for microsatellite instability status, tumor mutation burden, COSMIC tumor mutational signatures and to identify germline and somatic MMR gene variants. MMR immunohistochemistry (IHC) and MLH1 promoter methylation were repeated. In total, 86.9% of the 137 SLS tumors could be resolved into established subtypes. For 22.6% of these resolved SLS cases, primary MLH1 epimutations (2.2%) as well as previously undetected germline MMR pathogenic variants (1.5%), tumor MLH1 methylation (13.1%) or false positive dMMR IHC (5.8%) results were identified. Double somatic MMR gene mutations were the major cause of dMMR identified across each tumor type (73.9% of resolved cases, 64.2% overall, 70% of CRC, 45.5% of ECs and 70.8% of SSTs). The unresolved SLS tumors (13.1%) comprised tumors with only a single somatic (7.3%) or no somatic (5.8%) MMR gene mutations. A tumor-focused testing approach reclassified 86.9% of SLS into Lynch syndrome, sporadic dMMR or MMR-proficient cases. These findings support the incorporation of tumor sequencing and alternate MLH1 methylation assays into clinical diagnostics to reduce the number of SLS patients and provide more appropriate surveillance and screening recommendations.

A tumor focused approach to resolving the etiology of DNA mismatch repair deficient tumors classified as suspected Lynch syndrome.

Routine screening of tumors for DNA mismatch repair (MMR) deficiency (dMMR) in colorectal (CRC), endometrial (EC) and sebaceous skin (SST) tumors leads to a significant proportion of unresolved cases classified as suspected Lynch syndrome (SLS). SLS cases (n=135) were recruited from Family Cancer Clinics across Australia and New Zealand. Targeted panel sequencing was performed on tumor (n=137; 80xCRCs, 33xECs and 24xSSTs) and matched blood-derived DNA to assess for microsatellite instability status, tumor mutation burden, COSMIC tumor mutational signatures and to identify germline and somatic MMR gene variants. MMR immunohistochemistry (IHC) and MLH1 promoter methylation were repeated. In total, 86.9% of the 137 SLS tumors could be resolved into established subtypes. For 22.6% of these resolved SLS cases, primary MLH1 epimutations (2.2%) as well as previously undetected germline MMR pathogenic variants (1.5%), tumor MLH1 methylation (13.1%) or false positive dMMR IHC (5.8%) results were identified. Double somatic MMR gene mutations were the major cause of dMMR identified across each tumor type (73.9% of resolved cases, 64.2% overall, 70% of CRC, 45.5% of ECs and 70.8% of SSTs). The unresolved SLS tumors (13.1%) comprised tumors with only a single somatic (7.3%) or no somatic (5.8%) MMR gene mutations. A tumor-focused testing approach reclassified 86.9% of SLS into Lynch syndrome, sporadic dMMR or MMR-proficient cases. These findings support the incorporation of tumor sequencing and alternate MLH1 methylation assays into clinical diagnostics to reduce the number of SLS patients and provide more appropriate surveillance and screening recommendations.

Evaluating Multiple Next-Generation Sequencing-Derived Tumor Features to Accurately Predict DNA Mismatch Repair Status.

Identifying tumor DNA mismatch repair deficiency (dMMR) is important for precision medicine. Tumor features, individually and in combination, derived from whole-exome sequenced (WES) colorectal cancers (CRCs) and panel-sequenced CRCs, endometrial cancers (ECs), and sebaceous skin tumors (SSTs) were assessed for their accuracy in detecting dMMR. CRCs (n = 300) with WES, where mismatch repair status was determined by immunohistochemistry, were assessed for microsatellite instability (MSMuTect, MANTIS, MSIseq, and MSISensor), Catalogue of Somatic Mutations in Cancer tumor mutational signatures, and somatic mutation counts. A 10-fold cross-validation approach (100 repeats) evaluated the dMMR prediction accuracy for i) individual features, ii) Lasso statistical model, and iii) an additive feature combination approach. Panel-sequenced tumors (29 CRCs, 22 ECs, and 20 SSTs) were assessed for the top performing dMMR predicting features/models using these three approaches. For WES CRCs, 10 features provided >80% dMMR prediction accuracy, with MSMuTect, MSIseq, and MANTIS achieving ≥99% accuracy. The Lasso model achieved 98.3% accuracy. The additive feature approach, with three or more of six of MSMuTect, MANTIS, MSIseq, MSISensor, insertion-deletion count, or tumor mutational signature small insertion/deletion 2 + small insertion/deletion 7 achieved 99.7% accuracy. For the panel-sequenced tumors, the additive feature combination approach of three or more of six achieved accuracies of 100%, 95.5%, and 100% for CRCs, ECs, and SSTs, respectively. The microsatellite instability calling tools performed well in WES CRCs; however, an approach combining tumor features may improve dMMR prediction in both WES and panel-sequenced data across tissue types.

Immunohistochemistry for Protein Detection in Esophageal Squamous Cell Carcinoma.

Immunohistochemistry is the identification of a cell protein by a specific antibody targeting that protein. It is the most common ancillary test to study the pathology of cancer. Immunohistochemical protein markers are used to differentiate poorly differentiated squamous cell carcinoma from poorly differentiated adenocarcinoma or neuroendocrine carcinomas. They could be used to identify and type the carcinoma in metastatic locations. Importantly, immunodetection of markers also helps in prediction of response to therapies as well as assessing the different biomarkers related to the pathogenesis and clinical behavior of esophageal squamous cell carcinoma. Successful application of the immunochemistry depends on understanding the mechanisms and principles as well as the limitations of the procedure. Automation of the procedure by different models of automatic stainers is widely used in diagnostic laboratories. The use of autostainers streamlines the workflows and certainly reduces the labor, time, and cost of using immunohistochemistry in clinical and research settings.

Evaluation of multidisciplinary strategies and traditional approaches in teaching pathology in medical students.

This study aims to evaluate the impact on the implementation of multiple strategies to improve medical student's pathology learning experience. In two consecutive years, medical students after a whole year of enrolling in pathology teaching, were invited to complete questionnaires rating and commenting on the personal learning experience of multiple teaching resources delivered in pathology. In both years, the overall score was high (mean score = 4.57 ± 0.63 /5) for the newly introduced sessions, namely histology lectures, clinical integrations and virtual microscopy pre-practical sessions. However, this was only marginally different from that of traditional practical (mean = 4.37 ± 0.68/5) and pathology lecture sessions (mean = 4.42 ± 0.61 /5). In addition, 53% positive correlation was noted for the overall responses between virtual microscopy guided pathology modules and practical sessions indicating the benefit of virtual microscopy in better preparing students for these sessions (P < 0.001). Qualitative comments suggested that the virtual microscopy sessions along with clinical scenario based learning were extremely useful for students' learning in pathology. To conclude, a multidisciplinary approach by clinical integration and flexibility in the mode of delivery by the use of virtual microscopy has the potential to better engage students to the learning of pathology.

Promoter hypermethylation inactivate tumor suppressor FAM134B and is associated with poor prognosis in colorectal cancer.

The present study aims to examine promoter methylation status of FAM134B in a large cohort of patients with colorectal adenocarcinomas. The clinical significances and correlations of FAM134B promoter methylation with its expression are also analysed. Methylation-specific high-resolution melt-curve analysis followed by sequencing was used to identify FAM134B promoter methylation in colorectal adenomas (N = 32), colorectal adenocarcinomas (N = 164), matched adjacent non-neoplastic colorectal mucosae (N = 83) and colon cancer cell lines (N = 4). FAM134B expression was studied by real-time quantitative polymerase chain reaction, immunohistochemistry, and Western blots. FAM134B promoter methylation was more frequent in adenocarcinomas (52%; 85/164) when compared to that of adenomas (28%; 9/32) and non-neoplastic mucosae (35%; 29/83). Cancer cells exhibited higher methylation when compared to non-neoplastic cells. FAM134B promoter methylation was inversely correlated with low FAM134B copy number and mRNA/protein expressions, whereas in-vitro demethylation has restored FAM134B expression in colon cancer cells. FAM134B promoter methylation was associated with high histological grade (P = .025), presence of peri-neural infiltration (P = .012), lymphovascular invasion (P = .021), lymph node metastasis (P = .0001), distant metastasis (P = .0001) and advanced pathological stages (P = .0001). In addition, FAM134B promoter methylation correlated with cancer recurrence and poor survival rates of patients with colorectal adenocarcinomas. To conclude, FAM134B promoter methylation plays a key role in regulating FAM134B expression in vitro and in vivo, which in turn contributes to the prediction of the biological aggressiveness of colorectal adenocarcinomas. Furthermore, FAM134B methylation might act as a marker in predicting clinical prognosis in patients with colorectal adenocarcinomas.

MicroRNA-186-5p overexpression modulates colon cancer growth by repressing the expression of the FAM134B tumour inhibitor.

OBJECTIVES: The role and underlying mechanism of miR-186-5p in colorectal cancer remain unknown. The present study aims to examine the various cellular effects of miR-186-5p in the carcinogenesis of colorectal cancer. Also, the interacting targets and association of clinicopathological factors with miR-186-5p expression in patients with colorectal cancer were analysed. METHODS: The miR-186-5p expression levels in colorectal cancer tissues (n=126) and colon cancer cell lines (n=3) were analysed by real-time PCR. Matched non-neoplastic colorectal tissues and a non-neoplastic colonic epithelial cell line were used as controls. Various in vitro assays such as cell proliferation, wound healing and colony formation assays were performed to examine the miR-186-5p specific cellular effects. Western blots and immunohistochemistry analysis were performed to examine the modulation of FAM134B, PARP9 and KLF7 proteins expression. RESULTS: Significant high expression of miR-186-5p was noted in cancer tissues (p< 0.001) and cell lines (p<0.05) when compared to control tissues and cells. The majority of the patients with colorectal cancer (88/126) had shown overexpression of miR-186-5p. This miR-186-5p overexpression was predominantly noted with in cancer with distant metastasis (p=0.001), lymphovascular permeation (p=0.037), microsatellite instability (MSI) stable (p=0.015), in distal colorectum (p=0.043) and with associated adenomas (p=0.047). Overexpression of miR-186-5p resulted in increased cell proliferation, colony formation, wound healing capacities and induced alteration of cell cycle kinetics in colon cancer cells. On the other hand, inhibition of endogenous miR-186-5p reduced the cancer growth properties. miR-186-5p overexpression reduced FAM134B expression significantly in the cancer cells (p<0.01). Also, FAM134B and miR-186-5p expressions are inversely correlated in colorectal cancer tissues and cells. CONCLUSION: The miR-186-5p expression promotes colorectal cancer pathogenesis by regulating tumour suppressor FAM134B. Reduced cancer cells growth followed by inhibition of miR-186-5p highlights the potential of miR-186-5p inhibitor as a novel strategy for targeting colorectal cancer initiation and progression.

Adrenal oncocytic phaeochromocytoma with putative adverse histologic features: a unique case report and review of the literature.

Oncocytic phaeochromocytomas are exceedingly rare tumours. To date, there are three reported cases in the literature. This report describes a case of adrenal oncocytic phaeochromocytoma with unique features and malignant potential in a 68-year-old man. The patient presented with an incidental non-functional mass discovered on routine radiological investigation, which was subsequently excised. Histologically, the tumour cells showed oncocytic features with high-grade nuclear abnormalities and foci of extension to the peri-adrenal fat. Immunohistochemistry performed was positive for chromogranin, CD56, S-100 and p53 and negative for inhibin, HMB-45, EMA, AE1/AE3, Cam 5.2 and calretinin. Electron microscopy showed electron dense granules of neurosecretory type, which confirmed the diagnosis. The malignant potential of the tumour was assessed on available histologic scoring systems, which demonstrated a high malignant potential. However, no recurrence was detected after 5 years of follow-up. Compared to all the previously reported cases of oncocytic phaeochromocytoma, this patient was the oldest on presentation, was the only case with identified high malignant potential and has the longest follow-up. A review of the literature showed that all the oncocytic phaeochromocytomas reported were non-functional, non-metastasizing and were described in women. To conclude, oncocytic phaeochromocytoma should be in the differential diagnoses of oncocytic tumours of the adrenal gland. Additional studies are needed to predict the behaviour of this entity.

The roles of JK-1 (FAM134B) expressions in colorectal cancer.

The aims of the present study are to investigate the clinicopathological correlations of JK-1(FAM134B) expression and its relationship to carcinogenesis in a colorectal adenoma-adenocarcinoma model. JK-1(FAM134B) protein expression was studied in a colon cancer cell line by Western blot and immunocytochemistry. JK-1(FAM134B) expression profiles at mRNA and protein levels were investigated in cancer tissues from 236 patients with colorectal adenocarcinoma and 32 patients with colorectal adenoma using real-time polymerase chain reaction and immunohistochemistry. The findings were then correlated with the clinicopathological features of these tumours. JK-1(FAM134B) protein was demonstrated in the colon cancer cells by Western blot. The protein was located in the nuclei of the tumour cells at both cellular and tissue levels. In colorectal adenocarcinomas, lower levels of JK-1(FAM134B) protein expression were associated with younger age (p=0.032), larger tumour size (p=0.004), advanced cancer stages (p=0.016) and higher rates of cancer recurrence (p=0.04). Also, lower levels of JK-1(FAM134B) mRNA expression were associated with advanced cancer stages (p=0.02) and presence of lymphovascular invasion (p=0.014). Higher JK-1(FAM134B) mRNA and protein expression levels were identified in adenomas and non-neoplastic mucosae, compared to carcinomas (p=0.005). To conclude, JK-1(FAM134B) mRNA expression and JK1 (FAM134B) protein levels varied with the different stages of progression of colorectal tumours. The expression levels of the gene were associated with clinicopathological features in patients with colorectal adenocarcinoma suggesting that JK-1(FAM134B) gene has roles in controlling some steps in the development of the invasive phenotypes from colorectal adenoma to early staged as well as advanced staged colorectal adenocarcinomas.

JK1 (FAM134B) represses cell migration in colon cancer: a functional study of a novel gene.

BACKGROUND: JK1 is a novel cancer-related gene with unknown functional role in carcinogenesis. The aim of this study is to investigate the role of JK1 gene in carcinogenesis in an in vitro cell proliferation and migration analysis model. METHODS: Small hairpin RNAs (shRNA) were designed to knock-down JK1 expression in colon cancer cell line (SW480) using transduction ready lentiviral particles. Cell proliferation and cell migration assays were performed on multiple extracellular matrices to investigate the cellular effects of JK1 in colon cancer cells. A non-cancer colonic epithelial cell line (FHC) was used to compare the expression of JK1 in cancer cell line. RESULTS: JK1 knock-down did not affect cellular proliferation or survival in colon cancer. However, the manipulation increased cancer cell migration rates on collagen and fibronectin substrates. CONCLUSIONS: JK1 was shown for the first time to have a functional role in the pathogenesis of colon cancer. The results imply that JK1 represses the capacity of cancer cells to migrate within their tissue. They also concurred with the previous findings of JK1 activity correlations with clinical and pathological features in colon cancer. The capacity may have utility as a means to prevent cancer cells forming metastases.

JK1 (FAM134B) gene and colorectal cancer: a pilot study on the gene copy number alterations and correlations with clinicopathological parameters.

AIMS: The aims of the study are to characterize changes in JK-1 (FAM134B) at the DNA level in colorectal adenocarcinoma and adenoma and exploring the possible correlations with clinical and pathological features. METHOD: JK-1 gene DNA copy number changes were studied in 211 colorectal carcinomas, 32 colorectal adenoma and 20 colorectal non-cancer colorectal tissue samples by real-time quantitative polymerase chain reaction. The results were correlated with clinical and pathological parameters. RESULTS: Colorectal adenomas were more likely to be amplified than deleted with regard to JK-1 (FAM134B) DNA copy number change. The copy number level of JK-1 (FAM134B) DNA in colorectal adenocarcinomas was significantly lower in comparison to colorectal adenomas. Changes in JK-1 (FAM134B) DNA copy number were associated with histological subtypes, and cancer stage. Lower copy numbers were associated with higher tumor stage, lymph node stage and overall pathological stage of cancer. Conversely, higher DNA copy numbers were detected more often in the mucinous adenocarcinoma. CONCLUSIONS: This is the first study showing significant correlations of the JK-1 (FAM134B) gene copy number alterations with clinical and pathological features in a large cohort of pre-invasive and invasive colorectal malignancies. The changes in DNA copy number associated with progression of colorectal malignancies reflect that JK-1 (FAM134B) gene could play a role in controlling some steps in development of the invasive phenotypes.