Expanding the genotype-phenotype spectrum in hereditary colorectal cancer by gene panel testing

Hereditary syndromes causing colorectal cancer include both polyposis and non-polyposis syndromes. Overlapping phenotypes between the syndromes have been recognized and this make targeted molecular testing for single genes less favorable, instead there is a gaining interest for multi-gene panel-based approaches detecting both SNVs, indels and CNVs in the same assay. We applied a panel including 19 CRC susceptibility genes to 91 individuals of six phenotypic subgroups. Targeted NGS-based sequencing of the whole gene regions including introns of the 19 genes was used. The individuals had a family history of CRC or had a phenotype consistent with a known CRC syndrome. The purpose of the study was to demonstrate the diagnostic difficulties linked to genotype-phenotype diversity and the benefits of using a gene panel. Pathogenicity classification was carried out on 46 detected variants. In total we detected sixteen pathogenic or likely pathogenic variants and 30 variants of unknown clinical significance. Four of the pathogenic or likely pathogenic variants were found in BMPR1A in patients with unexplained familial adenomatous polyposis or atypical adenomatous polyposis, which extends the genotype-phenotype spectrum for this gene. Nine patients had more than one variant remaining after the filtration, including three with truncating mutations in BMPR1A, PMS2 and AXIN2. CNVs were found in three patients, in upstream regions of SMAD4, MSH3 and CTNNB1, and one additional individual harbored a 24.2 kb duplication in CDH1 intron1

Broad phenotypic spectrum in familial adenomatous polyposis; from early onset and severe phenotypes to late onset of attenuated polyposis with the first manifestation at age 72

<p>Abstract</p> <p>Background</p> <p>Familial adenomatous polyposis (FAP) is typically characterized by multiple colonic polyps and frequent extracolonic features. Whereas the number of colonic polyps has been linked to the <it>APC </it>gene mutation, possible genotype-phenotype correlations largely remain to be defined for the extracolonic manifestations.</p> <p>Methods</p> <p>Full genomic sequencing combined with multiplex ligation-dependent probe amplification was used to identify <it>APC </it>gene mutations, which were correlated to the clinical presentations.</p> <p>Results</p> <p>10 novel <it>APC </it>gene mutations were identified in 11 families. A broad spectrum of extracolonic manifestations was identified in most of these individuals. Two sisters with an insertion in codon 528 (c.1582_1583insGC) both showed severe phenotypes with classical polyposis, upper gastrointestinal polyps and thyroid cancer. A woman with a 3'<it>APC </it>mutation (c.5030_5031insAA) developed colon cancer at age 72 as the first manifestation of attenuated FAP.</p> <p>Conclusion</p> <p>With an increasing number of FAP families diagnosed, a broad and variable tumor spectrum and a high frequency of extracolonic manifestations are gradually recognized. We report novel <it>APC </it>mutations and present two FAP cases that suggest familial aggregation of thyroid cancer and demonstrate the need to consider attenuated FAP also among elderly patients with colon cancer.</p

Sinonasal T-cell lymphoma in the differential diagnosis of lethal midline granuloma using in situ hybridization for Epstein-Barr virus RNA

"Lethal midline granuloma" of the upper airways generally encompasses T-cell lymphoma and Wegener's granulomatosis in Western populations. Treatment and outcome for each is different, but their pathological distinction may not always be possible on routine biopsy specimens. Within a defined population between 1947 and 1994, we found 12 cases of primary sinonasal T-cell lymphoma, all with a CD20-, CD3+ immunophenotype in paraffin sections. We studied the occurrence of the Epstein-Barr virus RNA EBER1 using colorimetric in situ hybridization (ISH) with an oligoprobe. All available biopsy specimens from each patient were hybridized to detect the presence of EBER1 in relation to the phase of lymphoma progression. In addition, ISH was performed on 23 cases of nonspecific rhinitis and 10 cases of Wegener's granulomatosis to determine the specificity of the method in the differential diagnosis of inflammatory/ulcerative lesions. In ten cases of lymphoma, initial biopsy specimens showed the early phase with minimal lymphocytic atypia ("polymorphic reticulosis"). Four of these (including one recurrence) had been missed by experienced pathologists, resulting in a diagnostic delay of 2 to 8 yr. The remaining two cases were in the late phase, i.e., malignant grade atypia was apparent in the initial biopsy specimen, and neither was misdiagnosed as being benign. All hybridizable lymphoma sections, regardless of phase of development, gave a strong ISH signal easily detected at low magnification in 50 to 100% of tumor cells. Scattered positive cells were usually present even in necrotic areas. In contrast, no case of Wegener's granulomatosis or nonspecific rhinitis produced a true hybridization signal. We conclude that a negative EBER1 ISH provides strong evidence against T-cell lymphoma in the differential diagnosis of lethal midline granuloma in our population. Conversely, a strong ISH signal for EBER1 in immunohistochemically determined T-cell infiltrates within sinonasal tissues provides strong support for the presence of lymphoma

Ezrin expression in rectal cancer predicts time to development of local recurrence

Improved outcome after rectal cancer surgery requires identification of novel risk factors of tumour recurrence in order to personalise therapy, that is, enhanced selection of high-risk patients to additional radiochemotherapy or intensified follow-up. In several tumour types, including colorectal cancer, high expression of the membrane-cytoskeleton linker ezrin has been suggested to impair prognosis but has not yet reached clinical application. We evaluated the expression of ezrin in rectal cancer with a focus on the identification of a marker for local tumour recurrence. Immunohistochemical expression of ezrin was analysed in 104 primary rectal cancers from patients who developed local recurrences despite being treated with R0 major abdominal surgery. Time to local recurrence and distant metastasis as well as 5-year overall and cancer-specific survival were used as end points. Ezrin expression was weak in 17% of the tumours, moderate in 62%, and intense in 21%. The time to local recurrence was significantly shorter (p = 0.0004) for patients with tumours showing high ezrin expression. No correlation between ezrin expression and time to distant metastasis was identified. Survival data were similar between groups irrespective of ezrin expression in the primary tumours. Our findings suggest that increased expression of ezrin may represent a marker of aggressive biological behaviour in rectal cancer. Although further validation is needed, ezrin may represent a relevant marker for personalised treatment of rectal cancer with respect to risk of local recurrence after R0 surgery

Efficient and reproducible identification of mismatch repair deficient colon cancer : Validation of the MMR index and comparison with other predictive models

Background: The identification of mismatch-repair (MMR) defective colon cancer is clinically relevant for diagnostic, prognostic and potentially also for treatment predictive purposes. Preselection of tumors for MMR analysis can be obtained with predictive models, which need to demonstrate ease of application and favorable reproducibility. Methods. We validated the MMR index for the identification of prognostically favorable MMR deficient colon cancers and compared performance to 5 other prediction models. In total, 474 colon cancers diagnosed ≥ age 50 were evaluated with correlation between clinicopathologic variables and immunohistochemical MMR protein expression. Results: Female sex, age ≥60 years, proximal tumor location, expanding growth pattern, lack of dirty necrosis, mucinous differentiation and presence of tumor-infiltrating lymphocytes significantly correlated with MMR deficiency. Presence of at least 4 of the MMR index factors identified MMR deficient tumors with 93% sensitivity and 76% specificity and showed favorable reproducibility with a kappa value of 0.88. The MMR index also performed favorably when compared to 5 other predictive models. Conclusions: The MMR index is easy to apply and efficiently identifies MMR defective colon cancers with high sensitivity and specificity. The model shows stable performance with low inter-observer variability and favorable performance when compared to other MMR predictive models

The added value of PMS2 immunostaining in the diagnosis of hereditary nonpolyposis colorectal cancer.

dentification and characterization of the genetic background in patients with the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome is important since control programmes can in a cost-effective manner prevent cancer development in high-risk individuals. HNPCC is caused by germline mismatch repair (MMR) gene mutations and the genetic analysis of HNPCC therefore includes assessment of microsatellite instability (MSI) and immunohistochemical MMR protein expression in the tumor tissue. MSI is found in >95% of the HNPCC-associated tumors and immunostaining using antibodies against the MMR proteins MLH1, MSH2, and MSH6 has been found to correctly pinpoint the affected gene in about 90% of the cases. The PMS2 antibody was the most recently developed and we have in a clinical material assessed the added value of PMS2 immunostaining in 213 patients with suspected hereditary colorectal cancer. All 119 MSS tumors showed retained expression for all four antibodies and PMS2 did thus not identify any underlying MMR defect in these cases. However, PMS2 immunostaining contributed to the characterization of the MMR defect in a subset of the MSI tumors. Concomitant loss of MLH1 and PMS2, which functionally interact in the MutL alpha complex, was found in 98% of the tumors from patients with germline MLH1 mutations. Among the 12 MSI-high tumors with retained expression of MLH1, MSH2 and MSH6, 8 tumors showed loss of PMS2 staining, and mutations in MLH1 were identified in 2 and mutations in PMS2 in 3 of these individuals. In summary, isolated loss of PMS2 was found in 8% of the MSI-high tumors in our series, including 8/12 previously unexplained MSI-high tumors, in which mutations either in MLH1 or in PMS2 were identified in five cases