Background: Similar to poorly differentiated thyroid carcinoma (PDTC), papillary thyroid carcinoma with high-grade features (PTC HGF) demonstrates increased mitotic activity and/or necrosis; however, PTC HGF is excluded from the World Health Organization (WHO) definition of PDTC based on maintained nuclear features of PTC. Methods: Consecutive tumors that met criteria for PTC HGF, defined as tumors with maintained nuclear features of PTC and mitoses numbering 5 or more per 10 contiguous high-power fields and/or tumor necrosis, and PDTC (defined as per the WHO criteria) were identified. Clinicopathologic characteristics, follow-up data, and targeted next-generation sequencing results were compared between groups. Results: There were 15 PTC HGF and 47 PDTC. PTC HGF was associated with a higher rate of pT4 disease (53% vs. 13%, p = 0.0027) and lymph node metastases (73% vs. 38%, p = 0.049). The disease-specific survival was worse for patients with PTC HGF compared with those with PDTC using Kaplan–Meier estimation ( p < 0.001) and was worse in subgroup analysis evaluating patients with widely invasive PDTC (i.e., those with a similar rate of pT4 disease) and PTC HGF ( p = 0.040). PTC HGF had a higher BRAF ^V600E mutation rate (42% vs. 3%; p = 0.003), a trend toward more gene fusions (25% vs. 3%; p = 0.052), and a higher rate of relative gain of 1q (67% vs. 15%; p = 0.002) than PDTC. Conclusions: Our results demonstrate that PTC HGF are important to recognize based on their aggressive behavior. The molecular differences between PTC HGF and PDTC suggest that PTC HGF should be considered a distinct group from PDTC.

The lack of readily available, patient-derived materials for molecular genetic testing of many heterozygous or rare disorders creates a major impediment for laboratory proficiency and quality control procedures. The paucity of clinically derived mutation-positive samples could be surmounted if it were possible to construct artificial samples containing mutations of interest that would sufficiently resemble natural human samples. Such samples could then function as acceptable and realistic performance evaluation challenges and quality control reagents for recipient laboratories. Using the cystic fibrosis gene (CFTR) as a prototype, we have devised and executed experiments designed to generate unique DNA samples that could be used for these purposes. We used site-directed mutagenesis to generate mutations of interest in plasmid DNA derived from common bacterial artificial chromosome sources containing the cystic fibrosis transmembrane conductance receptor gene. CFTR mutations G85E and 1078delT were chosen to represent mutations in the original American College of Medical Genetics-recommended population-screening panel of 25 mutations. DNA samples containing predetermined concentrations and ratios of wild-type and mutated plasmids, bacterial artificial chromosomes of interest, and nonhuman genomic carrier DNA were characterized and tested in-house and in a group of nine pilot testing laboratories using a variety of technical platforms. The results indicate that these constructs, containing CFTR mutations in heterozygous and homozygous states, can serve as valid and accessible materials for quality assurance, including performance evaluation, proficiency testing, and assay quality control.

The accurate diagnosis and classification of myelodysplastic/myeloproliferative neoplasm (MDS/MPN) are challenging due to the overlapping pathological and molecular features of myelodysplastic syndrome (MDS) and myeloproliferative neoplasm (MPN). We investigated the genomic landscape in different MDS/MPN subtypes, including chronic myelomonocytic leukemia (CMML; n = 97), atypical chronic myeloid leukemia (aCML; n = 8), MDS/MPN-unclassified (MDS/MPN-U; n = 44), and MDS/MPN with ring sideroblasts and thrombocytosis (MDS/MPN-RS-T; n = 12). Our study indicated that MDS/MPN is characterized by mutations commonly identified in myeloid neoplasms, with TET2 (52%) being the most frequently mutated gene, followed by ASXL1 (38.7%), SRSF2 (34.7%), and JAK2 (19.7%), among others. However, the distribution of recurrent mutations differs across the MDS/MPN subtypes. We confirmed that specific gene combinations correlate with specific MDS/MPN subtypes (e.g., TET2/SRSF2 in CMML, ASXL1/SETBP1 in aCML, and SF3B1/JAK2 in MDS/MPN-RS-T), with MDS/MPN-U being the most heterogeneous. Furthermore, we found that older age (≥65 years) and mutations in RUNX1 and TP53 were associated with poorer clinical outcomes in CMML (p < 0.05) by multivariate analysis. In MDS/MPN-U, CBL mutations (p < 0.05) were the sole negative prognostic factors identified in our study by multivariate analysis (p < 0.05). Overall, our study provides genetic insights into various MDS/MPN subtypes, which may aid in diagnosis and clinical decision-making for patients with MDS/MPN.

BRAF mutations are rare in myeloid neoplasms and are reported to be associated with poor treatment outcomes. The purpose of our study is to characterize BRAF mutations in myeloid neoplasms using a next-generation sequencing (NGS) panel based on the experiences of a single cancer center. We conducted a retrospective review of patients with myeloid neoplasms who underwent the HopeSeq studies between January 2018 and September 2023. A total of 14 patients with myeloid neoplasms carrying BRAF mutations were included in our cohort. The clinical, pathological, and molecular features of these patients were investigated. Our study indicates that BRAF mutations are rare in myeloid neoplasms, constituting only 0.53% (14/2632) of all myeloid neoplasm cases, with the most common BRAF mutation being BRAF V600E (4/14; 28.6%). Interestingly, we observed that six out of seven patients with acute myeloid leukemia (AML) exhibited AML with monocytic differentiation, and all the patients with AML exhibited an extremely poor prognosis compared to those without BRAF mutations. TET2 (5/14; 35.7%), ASXL1 (4/14; 28.6%), and JAK2 (4/14; 28.6%) were the three most frequently co-mutated genes in these patients. Moreover, we noted concurrent KMT2A gene rearrangement with BRAF mutations in three patients with AML (3/7; 42.9%). Our study suggests that although BRAF mutations are rare in myeloid neoplasms, they play a crucial role in the pathogenesis of specific AML subtypes. Furthermore, RAS pathway alterations, including BRAF mutations, are associated with KMT2A gene rearrangement in AML. However, these findings warrant further validation in larger studies.

Recurrent fusions drive the pathogenesis of many hematological malignancies. Compared to routine cytogenetic/fluorescence in situ hybridization (FISH) studies, the RNA-based next-generation sequencing (NGS) fusion assay enables the identification of both known and novel fusions. In many cases, these recurrent fusions are crucial for diagnosis and are associated with prognosis, relapse prediction, and therapeutic options. The aim of this study is to investigate the application of the RNA-based NGS fusion assay in hematological malignancies. Our study included 3101 cases with available fusion results, and a fusion event was identified in 17.6% of cases. The discordant rate between the RNA-based NGS fusion assay and cytogenetic/FISH studies was 36.3%. Further analysis of discordant cases indicated that, compared to cytogenetic/FISH studies, the RNA-based NGS fusion assay significantly improved the identification of cryptic fusion genes, such as NUP98::NSD1, P2RY8::CRLF2, and KMT2A fusions involving different partners. Additionally, our study identified 24 novel fusions and 16 cases with the simultaneous presence of two fusions. These additional findings from the RNA-based NGS fusion assay resulted in improved risk stratification, disease targeting and monitoring. In conclusion, our study demonstrates the feasibility and utility of an RNA-based NGS fusion assay for patients with hematological malignancies, suggesting that it may be essential for the routine clinical workup of these patients.

Purpose: NCI selected a network of Clinical Laboratory Improvement Amendments–certified laboratories performing routine next-generation sequencing (NGS) tumor testing to identify patients for the NCI Molecular Analysis for Therapy Choice (NCI-MATCH) trial. This large network provided a unique opportunity to compare variant detection and reporting between a wide range of testing platforms. Experimental Design: Twenty-eight NGS assays from 26 laboratories within the NCI-MATCH Network, including the NCI-MATCH central laboratory (CL) and 11 commercial and 14 academic designated laboratories (DL), were used for this study. DNA from eight cell lines and two clinical samples were sequenced. Pairwise comparisons in variant detection and reporting between each DL and CL were performed for single-nucleotide variant, insertion and deletion, and copy-number variant classes. Results: We observed high concordance in variant detection between CL and DL for single-nucleotide variants and insertions and deletions [average positive agreement (APA) > 95.4% for all pairwise comparisons] but lower concordance for variant reporting after analysis pipeline filtering. We observed much higher agreement between CL and assays using amplification as the target enrichment method (84.2% < APA ≤ 95.7%, average APA = 88.7%) than other assays using hybridization capture (69.7% < APA ≤ 93.8%, average APA = 77.4%) due to blacklisting of actionable variants in low complexity regions. For copy-number variant reporting, we observed high agreement (APA > 82%) except between CL and two assays (APA = 76.9% and 71.4%) due to differences in estimation of copy numbers. Notably, for all variants, differences in variant interpretation also contributed to reporting discrepancies. Conclusions: This study indicates that different NGS tumor profiling tests currently in widespread clinical use achieve high concordance between assays in variant detection. For variant reporting, observed discrepancies are mainly introduced during the bioinformatic analysis.