Adequacy of cytology and small biopsy samples obtained with rapid onsite evaluation (ROSE) for predictive biomarker testing in non-small cell lung cancer.

Complete biomarker workup of non-small cell lung cancer (NSCLC) specimens is essential for appropriate and timely clinical management decisions. This can be challenging to achieve from small cytology and histology specimens, with increasing numbers of molecular and immunohistochemical biomarkers required. We conducted a 5 year retrospective audit of cases at our institution to assess the diagnostic and biomarker testing adequacy rates, particularly those specimens obtained with rapid onsite evaluation (ROSE), performed by a cytopathologist and a cytology scientist or pathology trainee, including all endobronchial ultrasound guided transbronchial needle aspirations (EBUS-TBNA), CT guided lung fine needle aspirations (FNA) and CT guided lung core biopsies. A total of 5,354 cases were identified, of which 92.2% had sufficient material for diagnosis. Of the 1506 cases identified with a recorded diagnosis of lung adenocarcinoma or NSCLC, not otherwise specified, 1001 (66.5%) had biomarker testing requested. Sufficient material was available in 89.5% of cases for a complete biomarker workup which included EGFR and KRAS mutational testing (all cases), ALK, ROS1 and PD-L1 immunohistochemistry (all cases), and ALK and ROS1 FISH (as required). For EGFR and KRAS mutational testing across both cytology and histology specimens, 99% of cases were sufficient. Of the samples in which a complete biomarker workup was unable to be performed, approximately half were only insufficient due to inadequate numbers of tumour cells for PD-L1 immunohistochemistry. Excluding PD-L1 IHC, 952 (95.1%) of samples obtained with ROSE were sufficient for the remainder of the testing requirements. Next generation sequencing using a 33 gene custom AmpliSeq panel was achieved in up to 72% of cases. In conclusion, small cytology and histology specimens obtained with ROSE are suitable for predictive biomarker testing in NSCLC, although attention needs to be paid to obtaining sufficient cells (>100) for PD-L1 immunohistochemistry.

Evaluation of molecular analysis in challenging ovarian sex cord-stromal tumours: a review of 50 cases.

Molecular profiling was performed in 50 problematic ovarian sex cord-stromal tumours (SCSTs) most of which were seen in consultation. Following analysis, 17 were classified as adult granulosa cell tumour (AGCT), 16 of which showed a FOXL2 sequence variant (mutation); the initial favoured diagnosis in five of the cases was benign thecoma/fibrothecoma. Thirteen tumours ultimately classified as cellular fibroma or thecoma were FOXL2 sequence variant negative which was helpful in excluding AGCT. All six Sertoli-Leydig cell tumours (SLCTs) demonstrated DICER1 'hot spot' sequence variants, and one case each of AGCT and SLCT showed high grade histological transformation associated with a concurrent TP53 sequence variant. All eight unclassified SCSTs were negative for FOXL2 mutations and the six tested cases were DICER1 wild type; however, three tumours demonstrated MET, CTNNB1 or TP53 sequence variants. Four cases were classified as juvenile granulosa cell tumour, and one of these harboured a GNAS sequence variant. The single gynandroblastoma and microcystic stromal tumours in the series demonstrated FOXL2 and CTNNB1 alterations, respectively. In summary, molecular analysis aids in accurate classification of challenging ovarian SCSTs and sometimes leads to revision of the favoured provisional diagnosis. TP53 sequence variants may be associated with dedifferentiation in both SLCTs and AGCTs.

Implementation of next generation sequencing technology for somatic mutation detection in routine laboratory practice.

The introduction of next generation sequencing (NGS) in the routine diagnostic setting is still in the development phase and has been limited by its complexity. Targeted NGS offers an attractive alternative to performing multiple single target assays and is very useful in meeting the increasing clinical demand for testing of multiple genetic aberrations in cancer specimens. To this end, we carried out a blinded validation study on 113 tumours in a diagnostic laboratory and compared mutation results from targeted NGS with those from Sanger sequencing, pyrosequencing, competitive allele specific TaqMan polymerase chain reaction (CAST PCR) and Cobas assays. DNA was extracted from formalin fixed, paraffin embedded (FFPE) tissue samples that included core biopsies, resections and cytology samples from three common and one rare cancer types [non-small cell lung cancer (NSCLC), colorectal cancer (CRC), malignant melanoma (MM) and gastrointestinal stromal tumour (GIST)]. Libraries were prepared using the TruSight Tumour 26 gene panel and NGS was carried out on the MiSeq instrument. Results from NGS were concordant with the mutational status determined by other platforms in 107 of the 113 cases tested (94.7%). The sequencing quality for NGS failed in four of the six false negative cases, while a further two samples gave false negative results because the c-KIT mutations were located outside the range of the NGS panel. One NSCLC sample contained an EGFR mutation previously detected by the Cobas assay. Reanalysis of the NGS data for this sample using a cut-off allele frequency of 1% revealed the mutation had an allele frequency of 2%, which was below the recommended software-determined threshold of 3%. NGS detected 113 additional mutations that were not previously known from analysis by the conventional methods. Twenty-six of these have known clinical importance, 37 have potential clinical significance, while 50 were novel mutations with unknown clinical significance. NGS detected variants using inputs of 10-20 ng of FFPE extracted DNA and from specimens with a tumour cell content less than 50%, for which when possible we recommend microdissection. We conclude that results from targeted NGS are highly concordant with those from other mutation testing platforms. Targeted NGS is suitable for a range of sample types received in the diagnostic pathology laboratory, including those with limited material or with low tumour cell content (TCC). This work has allowed us to determine the quality parameter settings required in order to obtain robust mutation data by NGS.

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, ß-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 ß-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.

Droplet Digital PCR for Mutation Detection in Formalin-Fixed, Paraffin-Embedded Melanoma Tissues: A Comparison with Sanger Sequencing and Pyrosequencing.

The identification of somatic mutations is crucial for guiding therapeutic decisions about personalized melanoma treatment. However, genetic analysis of tumors is usually performed on limited and often low-quality DNA from tumors with low tumor cellularity and high tumor heterogeneity. Different mutation-detection platforms exist, with varying analytical sensitivities. Here we evaluated the detection of common mutations in BRAF, NRAS, and TERT promoter in 40 melanoma FFPE tissues using Droplet Digital (dd)PCR, and compared the results to the detection rates obtained by Sanger sequencing and pyrosequencing. The cellularity of tumors analyzed ranged from 5% to 50% (n = 28) and 50% to 90% (n = 12). Overall, droplet digital (dd)PCR was more sensitive, detecting mutations in 12.5% and 23% of tumors deemed as wild-type by pyrosequencing and Sanger sequencing, respectively. The increased sensitivity of ddPCR was more apparent among tumors with <50% tumor cellularity. Implementation of ddPCR-based assays may facilitate analysis of early-stage tumors and support research into improving outcomes in melanoma patients.

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.

Detection of Epidermal Growth Factor Receptor Mutations in Lung Adenocarcinoma: Comparing Cobas 4800 EGFR Assay With Sanger Bidirectional Sequencing.

INTRODUCTION: Accurate detection of epidermal growth factor receptor (EGFR) mutations has a crucial role in the current treatment of patients with lung adenocarcinoma, and identification of clinically relevant mutations would qualify patients for treatment with tyrosine kinase inhibitors. Historically, Sanger sequencing has been used as the reference standard assay for EGFR mutational analysis; however, Cobas 4800 is a relatively new method. In the present study, we compared the performance of the Cobas assay against that of Sanger sequencing. MATERIALS AND METHODS: A total of 493 consecutive formalin-fixed paraffin-embedded samples of lung adenocarcinoma were simultaneously tested for EGFR mutations using both methods. RESULTS: After exclusion of the invalid results (n = 19), 474 samples from 455 patients were analyzed. The Cobas assay showed a mutation detection rate comparable to that of Sanger sequencing (18.1% vs. 17.9%, respectively; P < .05). Excellent agreement of 98.9% (κ, 0.964) was observed between the 2 methods. CONCLUSION: The Cobas assay is a fast and diagnostically robust platform with high analytical sensitivity; however, it is limited by its detection range and low tolerance to low DNA quality. Sanger sequencing is mostly affected by its lower analytic sensitivity. Ultimately, a dual testing strategy will be justified to increase the detection of novel mutations and reduce the false-negative results within an acceptable turnaround time.

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

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

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.

BRAF mutation detection in hairy cell leukaemia from archival haematolymphoid specimens.

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.

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

BACKGROUND: 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. METHODS: Fifty-four pancreatic cysts were triaged according to a volume-dependent protocol to generate fluid (neat and supernatant) and cell block specimens for cytology, comparative CEA, and KRAS testing. Follow-up histology, diagnostic cytology, or a combined clinicopathologic interpretation was recorded as the final diagnosis. RESULTS: There were 26 mucinous cystic lesions and 28 nonmucinous cystic lesions with volumes ranging from 0.3 mL to 55 mL. Testing different components of the specimens (cell block, neat, and/or supernatant) enabled all laboratory investigations to be performed on 50 of 54 cyst fluids (92.6%). Interpretive concordance was observed in 17 of 17 cases (100%) and in 35 of 40 cases (87.5%) that had multiple components tested for CEA and KRAS mutations, respectively. An elevated CEA level (>192 ng/mL) was the most sensitive test for the detection of a mucinous cystic lesion (62.5%) versus KRAS mutation (56%) and "positive" cytology (61.5%). KRAS mutations were identified in 2 of 25 mucinous cystic lesions (8%) in which cytology and CEA levels were not contributory. CONCLUSIONS: A volume-based protocol using different components of the specimen was able to optimize diagnostic yield in pancreatic cyst fluids. KRAS mutation testing increased diagnostic yield when combined with cytology and CEA analysis. The current results demonstrated that supernatant is comparable to neat fluid and cell block material 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. METHODS: A retrospective clinical study was performed utilising the demographic data and pathological specimens from 60 out of 726 head and neck cancer patients. Smoking and alcohol history, tumour staging, treatment and outcomes were recorded. Histopathology and immunochemistry for p16 and p53 was performed and HPV DNA was detected with polymerase chain reaction. Genomic DNA from all cancers were analysed for somatic mutations of EGFR, BRAF and KRAS genes. RESULTS: 20 (33%) of 60 cases were tonsillar squamous carcinomas and 38 (66%) were non-tonsillar. 19 (95%) of the 20 tonsillar cancers and three (8%) of 38 non-tonsillar patients were patients who were HPV 16-positive. Nine (47%) of the 19 HPV 16-positive tonsillar cases were p16 positive. Gene mutations were rare. There was a statistically significant (P < 0.05) improved survival of patients with HPV positive tonsillar tumours, younger age and non-smokers. CONCLUSION: Although limited in numbers, this study reinforces the role of HPV infection in HNSCC and its association with a more favourable clinical course in younger non-smokers worldwide. Gene mutation frequencies were low in all cancers tested and routine testing not recommended.