Connull Leslie

Anaplastic large-cell lymphoma associated with breast implants: A unique entity within the spectrum of peri-implant effusions

Anaplastic large‐cell lymphoma (ALCL) is a rare and newly described complication associated with breast implants. Patients often present with a peri‐implant effusion, which is amenable to fine‐needle aspiration. The laboratory handling of peri‐implant effusions for cytology and ancillary studies is as crucial as recognizing the characteristic cytology of ALCL.

Detection of EGFR mutational profile by direct dideoxy sequencing in cytology and non-cytology biopsy samples

Summary Epidermal growth factor receptor (EGFR) mutational analysis is recommended in the diagnostic work-up of non-small cell lung carcinoma. The first diagnostic biopsy is usually obtained by a minimally invasive procedure, especially in patients with unresectable disease. This paper aims to compare the types of somatic EGFR mutations detected by cytology and non-cytology samples by direct dideoxy sequencing and propose practical guidelines for handling such material. Only samples with sufficient polymerase chain reaction (PCR) product were considered, a total 310 samples (302 patients), of which 168 samples were cytology material and 142 samples were non-cytology biopsy material. All samples were assessed for tumour content and bidirectional direct sequencing was performed on exons 18, 19, 20 and 21. There were 49 cases with EGFR mutation detected (16.2%), without a significant difference in the detection of mutations between either cytology or non-cytology material. EGFR mutation was detected in most sample types including endoscopic ultrasound guided FNA, bronchial washings/brushings and pleural/peritoneal fluid samples. Cytology material can provide an adequate source of material for EGFR mutational analysis, with coordinated effort between clinicians and pathologists critical for best outcome.

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).

T-lymphoblastic proliferation and florid multifocal follicular dendritic cell proliferation occurring in hyaline-vascular Castleman disease in a patient with a possible familial predisposition

Hyaline-vascular Castleman disease (HVCD) often presents as unicentric disease which is usually cured by surgical resection. Here, we report a case of a 23-year-old man with HVCD in the mediastinum, in which an apparently indolent T-lymphoblastic proliferation and extensive organoid proliferation of follicular dendritic cells were also present. The patient had no systemic involvement and no further disease at follow-up. In addition, there is a possible unique family history of HVCD. This case highlights an interesting combination of elements, mostly likely benign in nature.

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.

30. Genome-Wide Micro-RNA profiling distinguishes diffuse large B-Cell lymphoma of the central nervous system from systemic DLBCL

Introduction: Primary central nervous system lymphomas (PCNSL) are diffuse large B-cell lymphomas (DLBCL) with clinical and prognostic features distinct from systemic diffuse large B-cell lymphomas. Limited previous studies suggest that PCNSL in the immunocompetent patient shows differential expression of micro-RNAs (miRNAs) distinct from systemic DLBCL, suggesting different pathogenesis. MiRNAs are short non-coding single stranded RNAs with a role in post-transcriptional regulation of protein expression, some shown to be associated with cancer development and progression. Methods: The expression of a panel of miRNAs was measured by quantitative real-time PCR after isolating RNA from formalin fixed paraffin embedded (FFPE) tissue of primary CNS lymphoma (n = 57), controlled by normalising to normal brain samples (n = 12) and endogenous miRNA controls (hsa-SNORD48, hsa-SNORD47, hsa-SNORD44 and hsa-U6). The expression profile was compared to that of systemic DLBCL (n = 30), controlled by normalising to benign tonsil tissue (n = 12) and the same endogenous miRNA controls. Results: Hierarchical clustering revealed a distinct expression profile in PCNSL compared to systemic DLBCL. The most significantly overexpressed miRNAs in PCNSL compared to DLBCL were hsa-miR-155, hsa-miR-146a, hsa-miR-1321 and hsa-miR-200c. The most underexpressed miRNA in PCNSL compared to DLBCL were hsa-miR-145, hsa-miR-122, hsa-miR-497 and hsa-miR-579. Discussion: Primary CNS lymphoma is usually associated with a dismal prognosis. The expression of B-cell related miRNAs in PCNSL is distinct from that of systemic DLBCL, suggesting a different biological entity. To our knowledge this is the first genome-wide microRNA profiling conducted to distinguish systemic DLBCL from PCNSL. We have identified a number of miRNAs which appear to differ in expression between the two entities and these findings could provide insights into the clinical behaviour and pathogenesis of PCNSL and aid discovery of potential therapeutic targets.

21. Comparative study between immunohistochemistry and microsatellite instability for screening of hereditary non-polyposis colorectal cancer syndrome

Background and Aim Screening for hereditary non-polyposis colorectal carcinoma (HNPCC) includes molecular methods to assess microsatellite instability (MSI) and immunohistochem-ical (IHC) analysis of DNA mismatch repair (MMR) proteins. We aimed to compare these approaches as screening tools in a general laboratory setting. Method All cases of colorectal carcinoma resection reported from 2006 to mid-2008 in patients aged 55 years or less were reviewed. Immunohistochemical slides for MLH1, PMS2, MSH2 and MSH6 were scored. MSI status of all tumours was determined using FSCCP for deletions in BAT26 and NR24 mononucleotide repeats. Results Significant loss of staining, complete loss of nuclear staining in 450% but 5100% of nuclei, for one or more MMR proteins was detected in 25 (36.2%) cases. Six (8.7%) cases had 100% loss for one or more MMR proteins and all (100%) were MSI positive. BAT26 and NR24 deletions were detected in three additional cases that had only 50–80% loss by IHC, therefore 33.3% of cases would have been missed by MMR IHC. Conclusion Screening for HNPCC by IHC alone will lead to interpretation difficulties often requiring MSI testing for confirmation. In this study the sensitivity of MSI was higher than IHC, and unlike MMR IHC, MSI testing interpretation is non-ambiguous and easy to interpret. Although IHC and MSI are complementary to each other, we recommend MSI molecular testing to be used as the preferable first line screening method of HNPCC. Depending on the availability of resources, IHC can either be performed in parallel with MSI or only on MSI positive cases.