Fabienne Grieu-Iacopetta

Optimizing the multimodal approach to pancreatic cyst fluid diagnosis developing a volume-based triage protocol

The objective of this study was to develop a triage algorithm to optimize diagnostic yield from cytology, carcinoembryonic antigen (CEA), and v‐Ki‐ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) testing on different components of a single pancreatic cyst fluid specimen. The authors also sought to determine whether cell block supernatant was suitable for CEA and KRAS testing.

Human papillomavirus and gene mutations in head and neck squamous carcinomas

Background:  Human papillomavirus (HPV) infection is implicated as an aetiological factor in head and neck squamous carcinomas (HNSCC), especially in the tonsils of the oropharyngeal region. This study investigates the frequency of HPV infection, p16 and p53 tumour profile and mutations in epidermal growth factor receptor (EGFR), Kirsten RNA Associated Rat Sarcoma 2 Virus (KRAS) and B‐Raf proto‐oncogene serine/threonine protein kinase (BRAF) genes in tonsillar and non‐tonsillar HNSCCs and correlates with clinical outcome and histopathological parameters in previously unstudied cohort of patients.

BRAF p.Val600Glu (V600E) mutation detection in thyroid fine needle aspiration cell block samples: a feasibility study

Summary Assessing BRAF mutation status in thyroid fine needle aspiration (FNA) cytology samples by both immunohistochemistry (IHC) and molecular methods has been documented in recent literature. We aim to highlight issues relating to quality and quantity of cellular material and DNA extracted from cell block samples. BRAF mutation status was assessed by both molecular and IHC methods in cell block material from thyroid FNA samples over a range of diagnostic categories, and correlated with available follow-up resection specimens. Of 39 samples there were 14 cases with ‘inconclusive’ cytology (Bethesda diagnostic categories 3, 4 or 5) and 25 cases with malignant cytology. Follow-up information was available in 38 of 39 cases and resection material for comparison in 18 of 39 case. Detection of BRAF mutation in cell block samples by combined molecular and IHC methods showed 100% specificity and 71.4% sensitivity compared to subsequent histologically confirmed BRAF mutated papillary thyroid carcinoma. IHC detected BRAF mutation in two (8.2%) cases which were negative by molecular methods and confirmed mutation positive by IHC and molecular methods on subsequent histology. Low extracted DNA concentration did not appear to preclude detection of BRAF mutation, although cell blocks with lower tumour cell content were over-represented in cases that were wild-type on FNA material and BRAF mutant on subsequent histology. BRAF mutation detection in cell block material is feasible and highly specific for papillary thyroid carcinoma. Best results are obtained by a combination of molecular and IHC methods.

Clinical and therapeutic implications of BRAF mutation heterogeneity in metastatic melanoma

Heterogeneity of BRAF mutation in melanoma has been a controversial subject. Quantitative data on BRAF allele frequency (AF) are sparse, and the potential relationship with response to BRAF inhibitors (BRAFi) in patients with metastatic melanoma is unknown. We quantitatively measured BRAF AF in a cohort of treatment naïve metastatic melanoma samples by pyrosequencing and correlated with survival data in patients treated with BRAFi as part of their clinical care. Fifty‐two samples from 50 patients were analysed. BRAF V600E mutations were detected in 71.1% of samples followed by V600K (25%) and V600R (3.9%). There was a wide range of AF from 3.9% to 80.3% (median 41.3%). In 33 patients treated with BRAFi, there was no difference in overall or progression‐free survival when the patients were categorized into high or low AF groups. There was no correlation between AF and degree of response, and no difference in survival based on genotype.

FOXP3+ T regulatory lymphocytes in primary melanoma are associated with BRAF mutation but not with response to BRAF inhibitor

Summary Tumour infiltrating lymphocytes in primary melanoma have been found to correlate with patient outcomes. A subpopulation of tumour infiltrating lymphocytes expresses the transcription factor forkhead box protein 3 (FOXP3). These are known as FOXP3+ T-regulatory cells (Tregs) and are thought to play an immune suppressive role in tumourigenesis. In most tumours, including melanoma, a high density of intratumoural FOXP3+ Tregs has been associated with poor prognosis. It is not known whether these cells also influence the response to BRAF inhibition therapy in metastatic melanoma. In the present study we retrospectively investigated the density of FOXP3+ Tregs in primary melanomas, with known subsequent metastasis, in relation to various clinicopathological parameters including BRAF and NRAS mutation status, and response to BRAF inhibitor therapy. The intratumoural density of FOXP3+ Tregs was two-fold higher in melanomas with mutant BRAF compared to those with wild type BRAF status (p = 0.03). In patients treated with BRAF kinase inhibitors FOXP3+ Treg density in the primary tumour was not predictive of treatment response (p = 0.38).

BRAF mutation detection in hairy cell leukaemia from archival haematolymphoid specimens

Summary Hairy cell leukaemia (HCL) is a rare, indolent chronic B-cell leukaemia accounting for approximately 2% of all adult leukaemias. The recent association of the BRAF p.Val600Glu (V600E) mutation in HCL makes it a valuable molecular diagnostic marker. We compared the ability of Sanger sequencing, fluorescent single-strand conformational polymorphism (F-SSCP) and high resolution melting (HRM) analysis to detect BRAF mutations in 20 cases of HCL consisting of four archival Romanowsky stained air-dried peripheral blood and bone marrow aspirate smears, 12 mercury fixed decalcified bone marrow trephine biopsies, three formalin fixed, paraffin embedded (FFPE) splenectomy samples and one fresh peripheral blood sample. DNA was amplified and BRAF mutation status determined by the three methods above. V600E mutation was identified in 94%, 89% and 72% of HCL cases by F-SSCP, HRM and Sanger sequencing, respectively. In one case, in addition to the p.Val600Glu mutation, a p.Lys601Thr (K601T) mutation was identified. DNA from archival slide scrapings, mercury-fixed and FFPE tissue can be used to identify BRAF mutations with high sensitivity, especially using HRM/F-SSCP. The V600E mutation can be used as a supplementary molecular marker to aid in the diagnosis of HCL and the presence of the mutation may provide a target for therapy.

Molecular profiling study of a recurrent ovarian mucinous tumour with a mural nodule of anaplastic carcinoma, providing supportive evidence of dedifferentiation

S S143 directional sequencing (SBS) has been used as the gold standard for EGFR mutational analysis, however there are emerging new assays utilizing targeted real time PCR technology. In this study we compared the performance of ‘Cobas 4800’ (COBAS) against Sanger sequencing. Methods: 480 consecutive formalin fixed paraffin embedded samples of lung adenocarcinoma were simultaneously tested for EGFR mutations by SBS and COBAS. Mutational results were catogorised as positive, negative and invalid. Unweighted Kappa test was utilsed to compare the concordance between two assays. Results: After exclusion of invalid results (n=16), 477 samples from 458 patients (47.2% male, 52.8% female) underwent statistical analysis. There was an excellent observed percentage agreement of 98.3% (kappa value 0.944, SE 0.0194) between the two methods. The combined mutation detection rate (19%) was superior to either SBS (18.4%) or COBAS (18%). EGFR mutation frequency was significantly higher in women (23%) compare to men (12%). Discussion: COBAS assay is a diagnostically robust platform comparable with Sanger with very high analytical sensitivity and short turns around time. COBAS failure is due to lower sensitivity to samples with low DNA quality and its limited primer detection range, while Sanger is mostly affected by its lower analytic sensitivity related to low volume of diagnostic material. Consequently, the higher combined mutation detection rate necessitates a dual testing strategy to guarantee detection of novel mutations and mutations outside the COBAS detection range, and avoiding false negative results due to lower analytical sensitivity of SBS. 78. MOLECULAR PROFILING STUDY OF A RECURRENT OVARIAN MUCINOUS TUMOUR WITH A MURAL NODULE OF ANAPLASTIC CARCINOMA, PROVIDING SUPPORTIVE EVIDENCE OF DEDIFFERENTIATION Nima Mesbah Ardakani, Anup Naran, Fabienne Grieu-Iacopetta, Colin Stewart 1Anatomical Pathology Department, PathWest, QEII, and 2Anatomical Pathology Department, PathWest, KEMH, Perth,

An immunohistochemical and molecular analysis of papillary proliferation of the endometrium

Papillary proliferations of the endometrium (PPEs) are uncommon lesions that are often associated with endometrial polyps. PPEs occasionally precede or co-exist with atypical endometrial hyperplasia or adenocarcinoma, but their pathogenesis and relationship to endometrial neoplasia is uncertain. In the present study 11 PPEs, including eight benign papillary proliferations (BPPs) and three complex papillary hyperplasias (CPHs) were examined immunohistochemically for expression of PAX2, BAF250a, p16, β-catenin and DNA mismatch repair (MMR) proteins. Molecular analysis was also performed on the CPHs using targeted next generation sequencing (NGS). All PPEs demonstrated at least one immunohistochemical abnormality with altered expression of p16 and PAX2 in nine and seven cases, respectively, and β-catenin in one case. However, none of the cases showed loss of BAF250a or MMR protein staining. All CPHs showed KRAS mutations with additional mutations in AKT1 and FBXW7 in one case each, and both PIK3CA and CTNNB1 in the remaining case. Therefore, PPEs demonstrate immunophenotypical and molecular overlap with endometrial endometrioid neoplasia, although loss of BAF250a and MMR protein function do not appear to contribute significantly to these lesions. KRAS mutations may be important drivers in CPHs but this finding needs to be confirmed in larger studies.

34. BRAF V600E mutation detection in cell blocks of thyroid fine needle aspiration cytology samples

Introduction: BRAF mutation c.1799T>A, p.Val600Glu (or V600E) in thyroid neoplasms is exclusively associated with papillary carcinoma (PTC) or anaplastic thyroid carcinoma. Demonstration of BRAF mutation has been suggested as a supplement to confirmation of PTC in cytology material. Recently a V600E mutation specific antibody has been developed. We aim to assess the feasibility of testing for BRAF V600E mutational status in cytology (cell block) material. Methods: 39 adequate cell blocks (CBs) and 18 available follow-up histology cases were tested by immunohistochemistry (IHC) (clone VE1, 1:400, Spring Biosciences, Pleasanton, California, using Optiview amplification kit) and molecular methods (castPCR: Competitive Allele Specific Taqman PCR, Life Technologies, CA, USA), followed by single-strand confirmation polymorphism (SSCP) if required. All cell blocks tested included H+E sections to confirm adequacy of abnormal cells. Any fine granular cytoplasmic staining in abnormal cells was considered as a positive IHC result. DNA content on CBs ranged from 2.4–54.1 ng/&mgr;L and was considered adequate for both molecular methods. Results: Table shows test results for BRAF mutation in cytology and histology for IHC or molecular methods. Table. No title available. Conclusions:BRAF V600E mutation assessment in thyroid cell block cytology material is feasible although the role in diagnostic algorithm requires further investigation.Specificity of CB BRAF positivity by both IHC and molecular methods was 100% (no false positive findings).Discrepancies noted between IHC and molecular methods requires further investigation.