An effective approach for BRAF V600E mutation analysis of routine thyroid fine needle aspirates.

INTRODUCTION: Molecular testing for genetic alterations in thyroid neoplasms, including BRAF V600E (BRAF) mutation, are often applied to thyroid aspirates falling into the Bethesda System for Reporting Thyroid Cytopathology indeterminate categories. Current methods typically use dedicated aspirated material, without morphological determination of containing the cells of interest and may be of elevated cost. We describe our experience with BRAF mutation analysis on material obtained from Papanicolaou (PAP)-stained ThinPrep® (TP) slides. METHODS: Eighty-three cases collected between 2012 and 2019 with more than 100 cells were selected. An electronic record of a whole slide scan was made for each case before testing. The coverslips were removed, and DNA was extracted from material scraped from each slide using the Qiagen QIAamp DNA FFPE Tissue Kit. BRAF testing was performed using a highly sensitive mutation detection assay, either COLD-PCR, castPCR, or droplet digital PCR. RESULTS: Fourteen out of 83 cases had a BRAF mutation. Of these, 8 were classified as atypia of undetermined significance or suspicious for malignancy in which follow-up showed conventional papillary thyroid carcinoma in 5 out of 6 cases. The specificity and positive predictive value were 97% and 91%, respectively. CONCLUSIONS: BRAF mutation analysis can be performed on material obtained from routine clinical PAP-stained TP slides. As a first step, this unconventional effective approach may reduce costs related to the molecular evaluation of thyroid nodule aspirates and provides the opportunity for cytomorphological confirmation that the cells of interest are present in material submitted for BRAF mutation analysis.

Recommendations for Specimen and Therapy Selection in Colorectal Cancer.

INTRODUCTION: Next-generation sequencing has emerged as a clinical tool for the identification of actionable mutations to triage advanced colorectal cancer patients for targeted therapies. The literature is conflicted as to whether primaries or their metastases should be selected for sequencing. Some authors suggest that either site may be sequenced, whereas others recommend sequencing the primary, the metastasis, or even both tumors. Here, we address this issue head on with a meta-analysis and provide for the first time a set of sensible recommendations to make this determination. METHODS: From our own series, we include 43 tumors from 13 patients including 14 primaries, 10 regional lymph node metastases, 17 distant metastases, and two anastomotic recurrences sequenced using the 50 gene Ion AmpliSeq cancer NGS panel v2. RESULTS: Based on our new cohort and a meta-analysis, we found that ~ 77% of patient-matched primary-metastatic pairs have identical alterations in these 50 cancer-associated genes. CONCLUSIONS: Low tumor cellularity, tumor heterogeneity, clonal evolution, treatment status, sample quality, and/or size of the sequencing panel accounted for a proportion of the differential detection of mutations at primary and metastatic sites. The therapeutic implications of the most frequently discordant alterations (TP53, APC, PIK3CA, and SMAD4) are discussed. Our meta-analysis indicates that a subset of patients who fail initial therapy may benefit from sequencing of additional sites to identify new actionable genomic abnormalities not present in the initial analysis. Evidence-based recommendations are proposed.

Non-fusion mutations in endometrial stromal sarcomas: what is the potential impact on tumourigenesis through cell cycle dysregulation?

Targeted next-generation sequencing using the 50-gene Ion AmpliSeq Cancer Hotspot Panel v2 identified two significant point mutations in endometrial stromal sarcomas (ESS). Case 1 is a uterine mass from a quadragenarian woman with a karyotype lacking any known ESS rearrangements but demonstrated to have a CTNNB1-activating mutation (c.133T>C, p.[Ser45Pro]). Analysis of a uterine mass from case 2, a sexagenarian woman, revealed biallelic CDKN2A-inactivating mutations (c.172C>T, p.[Arg58Ter] and a deletion). Break-apart studies to identify YWHAE, JAZF1 and PHF1 rearrangements were negative in both tumours. We propose a model in which these point mutations may affect cell proliferation, converging at Wnt signalling and G1-S checkpoint control, that independently or in concert with a rare gene fusion result in ESS tumour development or progression.

A Novel Risk Stratification System for Thyroid Nodules With Indeterminate Cytology-A Pilot Cohort Study.

Background: Thyroid ultrasound (US), fine needle aspiration biopsy (FNAB), and molecular testing have been widely used to stratify the risk of malignancy in thyroid nodules. The goal of this study was to investigate a novel diagnostic approach for cytologically indeterminate thyroid nodules (ITN) based upon a combination of US features and genetic alterations. Methods: We performed a pilot cohort study of patients with ITN (Bethesda III/IV), who underwent surgical treatment. Based on standardized sonographic patterns established by the American Thyroid Association (ATA), each ITN received an US score (XUS), ranging between 0 and 0.9 according to its risk of thyroid cancer (TC). DNA and RNA were extracted from pathologic material, available for all patients, and subjected to Oncomine™ Comprehensive Assay v2 (OCAv2) next-generation sequencing. Each genetic alteration was annotated based on its strength of association with TC and its sum served as the genomic classifier score (XGC). The total risk score (TRS) was the sum of XUS and XGC. ROC curves were generated to assess the diagnostic accuracy of XUS, XGC, and TRS. Results: The study cohort consisted of 50 patients (39 females and 11 males), aged 47.5 ± 14.8 years. Three patients were excluded due to molecular testing failure. Among the remaining 47 patients, 28 (59.6%) were diagnosed with TC. BRAFV600E was the most common mutation in papillary TC, PAX8-PPARG fusion was present in NIFTP, pathogenic variants of SLX4, ATM, and NRAS were found in Hürthle cell TC and RET mutations in medullary TC. The diagnostic accuracy of XGC and TRS was significantly higher compared with XUS (88 vs. 62.5%, p < 0.001; 85.2 vs. 62.5%, p < 0.001, respectively). However, this increased accuracy was due to significantly better sensitivity (80.7 vs. 34.6%, p < 0.001; 84.6 vs. 34.6%, p < 0.001, respectively) without improved specificity (94.7 vs. 90%, p = 0.55; 85.7 vs. 90%, p = 0.63, respectively). Conclusion: Molecular testing might not be necessary in ITN with high-risk US pattern (XUS = 0.9), as specificity of TC diagnosis based on Xus alone is sufficient and not improved with molecular testing. OCAv2 is useful in guiding the management of ITN with low-to-intermediate risk US features (XUS < 0.9), as it increases the accuracy of TC diagnosis.

Next-Generation Sequencing: A Novel Approach to Distinguish Multifocal Primary Lung Adenocarcinomas from Intrapulmonary Metastases.

Distinguishing between multiple lung primary tumors and intrapulmonary metastases is imperative for accurate staging. The American Joint Committee on Cancer (AJCC) criteria are routinely used for this purpose but can yield equivocal conclusions. This study evaluated whether next-generation sequencing (NGS) using the 50-gene AmpliSeq Cancer Hotspot Panel version 2 can help facilitate this distinction. NGS was performed on known primary-metastatic pairs (8 patients) and multiple lung adenocarcinomas (11 patients). Primary-metastatic pairs had high mutational concordance. Seven pairs shared mutations, and 1 was concordant for having no mutations. Driver mutations in KRAS (n = 4), EGFR (n = 2), and BRAF (n = 1) were always concordant. Multiple lung tumors from 3 patients were completely concordant and predicted by NGS to be intrapulmonary metastases, whereas 8 had completely discordant mutations and were predicted to be independent primary tumors. The NGS prediction correlated with the AJCC (eighth edition) prediction in all patients for whom the latter was unequivocal (8 of 11). Furthermore, it separated patients by overall survival. Patients with predicted multiple independent primary tumors by NGS had better survival than those with distant metastases (P = 0.016, log-rank test), whereas those with predicted intrapulmonary metastases had no difference (P = 0.527). With further validation, the 50-gene panel has the potential to serve as an adjunct to the AJCC criteria.