The EGFRvIII transcriptome in glioblastoma, a meta-omics analysis.

BACKGROUND: EGFR is among the genes most frequently altered in glioblastoma, with exons 2-7 deletions (EGFRvIII) being amongst its most common genomic mutations. There are conflicting reports about its prognostic role and it remains unclear whether and how it differs in signalling compared with wildtype EGFR. METHODS: To better understand the oncogenic role of EGFRvIII, we leveraged four large datasets into one large glioblastoma transcriptome dataset (n=741) alongside 81 whole-genome samples from two datasets. RESULTS: The EGFRvIII/EGFR expression ratios differ strongly between tumours and ranges from 1% to 95%. Interestingly, the slope of relative EGFRvIII expression is near-linear, which argues against a more positive selection pressure than EGFR wildtype. An absence of selection pressure is also suggested by the similar survival between EGFRvIII positive and negative glioblastoma patients. EGFRvIII levels are inversely correlated with pan-EGFR (all wildtype and mutant variants) expression, which indicates that EGFRvIII has a higher potency in downstream pathway activation. EGFRvIII-positive glioblastomas have a lower CDK4 or MDM2 amplification incidence than EGFRvIII-negative (p=0.007), which may point towards crosstalk between these pathways. EGFRvIII-expressing tumours have an upregulation of 'classical' subtype genes compared to those with EGFR-amplification only (p=3.873e-6). Genomic breakpoints of the EGFRvIII deletions have a preference towards the 3' end of the large intron-1. These preferred breakpoints preserve a cryptic exon resulting in a novel EGFRvIII variant and preserve an intronic enhancer. CONCLUSIONS: These data provide deeper insights into the complex EGFRvIII biology and provide new insights for targeting EGFRvIII mutated tumours

Contribution of molecular genetics to the morphological characterization of breast cancer

Breast cancer is the most frequent malignancy among western women. One of the major challenges in the management of breast cancer is its heterogeneity. Breast cancer patients at the same stage of disease may have markedly different life expectancies and responsiveness to therapy. This heterogeneity is not always evident from tumour morphology alone but may reflect the presence of complex molecular genetic alterations in breast cancer requiring additional analysis. Morphological classification systems of breast cancer provide important but as yet insufficient prognostic information. Although several histological subtypes have been described, most invasive breast carcinomas are classified as either lobular (15%) or ductal (80%) carcinoma. The most obvious difference between lobular and ductal tumours is the invasive growth pattern: the latter grow in nests and may still show glandular differentiation, while lobular carcinomas have a diffuse and noncohesive infiltration pattern which might be related to loss of E-cadherin expression. At present, the clinical and biological relevance of discriminating lobular from ductal histology remains controversial. Breast cancers are further subdivided by tumour grade which is based on 3 morphological parameters: glandular differentiation, nuclear atypia and mitotic activity. However, this grading system is most appropriate for subclassifying ductal carcinomas since lobular tumours are devoid of glandular differentiation. Moreover, more than 40% of newly diagnosed breast cancers are classified as high-grade invasive ductal carcinoma and show variable disease outcomes. Our current understanding of breast cancer has substantially improved with the introduction of molecular genetics. Genomic studies show similar genetic alterations in lobular and low-grade ductal carcinomas. By contrast, different and highly complex genetic alterations may be involved in the pathogenesis of high-grade ductal carcinomas. Gene expression profiling studies led to the identification of 4 “new” molecular breast cancer subtypes: the luminal A, luminal B,Summary General Introduction: breast cancer classification Hypothesis & Aims Materials & Methods Study 1: Expression differences underlying morphological breast cancer subtypes Study 2: Evaluation of different techniques to determine the HER2 status in breast cancer Study 3: Significance of polysomy 17 in breast cancer Study 4: Checkpoint kinase 1 is highly expressed in triple negative breast carcinomas Discussion & Perspectivesstatus: publishe

Next generation sequencing for GNAS uncovers CD34 as a sensitive marker for intramuscular myxoma

BACKGROUND: Intramuscular myxoma is a soft tissue myxoid tumor with a broad morphological differential diagnosis and recent developments have led to the identification of markers that can exclude some, but not all, differential diagnostic entities. However, a sensitive confirmatory marker for intramuscular myxoma has not been clearly identified. Since there is some evidence that mutations in the GNAS gene could be such a marker, we evaluated our results of next-generation sequencing testing for GNAS mutations performed in recent years on our series of intramuscular myxoma. MATERIALS AND METHODS: Next-generation sequencing was performed on 10 cases of intramuscular myxoma diagnosed between 2015 and 2019, using either the TruSight Tumor 26 panel or an in-house developed 97 cancer gene panel. Additionally, immunohistochemistry for CD34 was performed on all cases. RESULTS: All intramuscular myxomas showed a diffuse and strong expression of CD34 and a GNAS mutation was found in 88% of cases, making this a very sensitive positive test for the diagnosis of intramuscular myxoma. CONCLUSIONS: Under the condition that contemporary next-generation sequencing is applied as testing method, searching for GNAS mutations is a very sensitive confirmatory test for the diagnosis of intramuscular myxoma, obviating the necessity to perform tests that exclude other entities by the virtue of their negative result. The molecular tests results also identified strong and diffuse CD34 expression as a sensitive, albeit non-specific, marker for intramuscular myxoma.status: publishe

Next generation sequencing for GNAS uncovers CD34 as a sensitive marker for intramuscular myxoma.

BACKGROUND: Intramuscular myxoma is a soft tissue myxoid tumor with a broad morphological differential diagnosis and recent developments have led to the identification of markers that can exclude some, but not all, differential diagnostic entities. However, a sensitive confirmatory marker for intramuscular myxoma has not been clearly identified. Since there is some evidence that mutations in the GNAS gene could be such a marker, we evaluated our results of next-generation sequencing testing for GNAS mutations performed in recent years on our series of intramuscular myxoma. MATERIALS AND METHODS: Next-generation sequencing was performed on 10 cases of intramuscular myxoma diagnosed between 2015 and 2019, using either the TruSight Tumor 26 panel or an in-house developed 97 cancer gene panel. Additionally, immunohistochemistry for CD34 was performed on all cases. RESULTS: All intramuscular myxomas showed a diffuse and strong expression of CD34 and a GNAS mutation was found in 88% of cases, making this a very sensitive positive test for the diagnosis of intramuscular myxoma. CONCLUSIONS: Under the condition that contemporary next-generation sequencing is applied as testing method, searching for GNAS mutations is a very sensitive confirmatory test for the diagnosis of intramuscular myxoma, obviating the necessity to perform tests that exclude other entities by the virtue of their negative result. The molecular tests results also identified strong and diffuse CD34 expression as a sensitive, albeit non-specific, marker for intramuscular myxoma

An Unexpected Response to Imatinib in a "Wild-Type" Gastrointestinal Stromal Tumor

INTRODUCTION: Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract and the most frequent sarcomas in some geographic regions. In patients with metastatic GIST, the tyrosine kinase inhibitor imatinib is the first-line standard of care. Mutations in KIT or specific platelet-derived growth factor receptor alpha (PDGFRA) gene aberrations in the tumor cells predict a favorable response to this agent, while tumors without KIT or PDGFRA mutations ("wild-type" GISTs) are usually resistant to such treatment. Next-generation sequencing (NGS) is commonly used for mutational analysis of GISTs. CASE PRESENTATION: We present a case of an unexpected response to imatinib treatment in a GIST that was initially called "wild-type" based on routine NGS. A spectacular response to empirical imatinib treatment triggered further genetic analysis and led to the identification of a 45-bp duplication in KIT exon 11 undetectable by routine NGS. CONCLUSION: Negative findings on routine NGS testing for KIT alterations do not exclude the presence of actionable drug targets, as in the case of larger or complex gene insertions or deletions. Updating the NGS bioinformatics pipeline to ensure identification of larger deletions or insertions or additional Sanger sequencing is warranted in NGS driver-negative GISTs in order to allow accurate detection of actionable mutations.status: publishe

Fibromatosis-like metaplastic carcinoma: a case report and review of the literature

BACKGROUND: We report an unusual case of low-grade fibromatosis-like metaplastic carcinoma (LG-FLMC) of the breast. This exceedingly rare epithelial breast malignancy has been reported only 68 times in the past 20 years, and is classified as a subtype of metaplastic breast carcinoma (MBC). It is a locally aggressive tumor with a low potential for lymph node and distant metastases, but with a tendency to recur after excision. Here we describe a less common presentation of LG-FLMC, provide its molecular characterization, discuss the major differential diagnosis and bring a short review of the literature. CASE PRESENTATION: A 65-year-old woman presented with a self-palpated breast lump that had discordant radio-pathological features. While imaging results were compatible with an infiltrative malignancy, on core needle biopsy (CNB) a sharply delineated lesion composed by a bland-looking population of spindle cells was observed; excision was recommended for final diagnosis. Histology of the resection specimen showed small areas of epithelial differentiation and foci of peripheral invasion. Immunohistochemical analysis revealed a co-immunoreactivity for epithelial and myoepithelial markers in the spindle cell component. Mutation analysis with a capture-based next generation sequencing method revealed pathogenic mutations in GNAS, TERT-promotor and PIK3R1 genes. A diagnosis of LG-FLMC was rendered. CONCLUSION: This case highlights the importance of a broad differential diagnosis, exhaustive sampling and the use of a broad immunohistochemical panel whenever dealing with a low-grade spindle cell lesion in the breast, and provides further insights into the molecular background of LG-FLMC.status: publishe

Actinobaculum schaalii causing Fournier's gangrene

Actinobaculum schaalii, which belongs to the group of Gram-positive rods, is difficult to culture. Using molecular genetics, Actinobaculum schaalii could be identified as a causing microorganism in a case of Fournier's gangrene

Polysomy 17 in Breast Cancer: Clinicopathologic Significance and Impact on HER-2 Testing

PURPOSE: Polysomy 17 is frequently found in breast cancer and may complicate the interpretation of HER-2 testing results. We investigated the impact of polysomy 17 on HER-2 testing and studied its clinicopathologic significance in relation to HER2 gene amplification. PATIENTS AND METHODS: In 226 patients with primary invasive breast carcinoma, HER2 gene and chromosome 17 copy numbers were determined by dual-color fluorescent in situ hybridization (FISH). The interpretation of FISH results was based on either absolute HER2 gene copy number or the ratio HER2/chromosome 17. Results were correlated with HER-2 protein expression on immunohistochemistry (IHC), HER2 mRNA expression by reverse transcriptase polymerase chain reaction (RT-PCR), and with various clinicopathologic parameters. RESULTS: All cases with an equivocal HER-2 result by FISH, either by absolute HER2 copy number (44 of 226 patients; 19.5%) or by the ratio HER2/chromosome 17 (three of 226 patients; 1.3%), displayed polysomy 17. On its own, polysomy 17 was not associated with HER-2 overexpression on IHC or increased HER2 mRNA levels by RT-PCR. Moreover, and in contrast with HER2 gene amplification, polysomy 17 was not associated with high tumor grade, hormone receptor negativity, or reduced disease-free survival. CONCLUSION: Polysomy 17 affects HER-2 testing in breast cancer and is a major cause of equivocal results by FISH. We show that tumors displaying polysomy 17 in the absence of HER2 gene amplification resemble more HER-2-negative than HER-2-positive tumors. These findings highlight the need for clinical trials to investigative whether polysomy 17 tumors benefit from HER-2-targeted therapy.status: publishe