Voss, Jesse S.

Abstract OBJECTIVES Paragangliomas have unique features in the mediastinum, in part due to their location. Because of their paucity, they have not been thoroughly investigated. We studied the clinical, pathological, immunohistochemical and molecular features of mediastinal paragangliomas. METHODS Immunohistochemistry, next-generation sequencing mutation panel and the Oncoscan assay were performed. RESULTS Twenty-four patients with mediastinal paraganglioma (7 men, 29.2%) had a median age of 45.5 years (19.8–72.2). Twenty-one (87.5%) paragangliomas were completely resected. Six (of 24, 25.0%) tumours were considered metastatic. Mitotic activity occurred in 11 (of 24, 45.8%) paragangliomas. Programmed death-ligand 1 (PD-L1) (n = 23) was expressed in 6 (26%) patients in 10% (n = 2) and 1% (n = 4) of tumour cells, respectively. SDHB expression was lost in 19 (of 22, 86.4%) cases. ATRX expression was lost in 11 (of 23, 47.8%) cases. Next-generation sequencing revealed a single pathogenic mutation in 10 (of 19) specimens including SDHB (n = 4), SDHD (n = 6), SDHC (n = 1), ATRX (n = 1), and ≥2 mutations in 2 cases [SDHC and TERT (n = 1); SDHB, ATRX and TP53 (n = 1)]. Germline mutation analysis revealed the same succinate dehydrogenase mutation (or lack thereof) as identified in the paraganglioma in 11 (of 12) cases. During a median follow-up (n = 21) of 4.8 years (0.8–14.9), 3 patients developed metastases; 4 patients died, at least 1 of disease. CONCLUSIONS Mediastinal paragangliomas can be associated with morbidity and mortality. Many mediastinal paragangliomas have been reported to be associated with syndromes such as multiple endocrine neoplasia, von Hippel-Lindau or succinate dehydrogenase syndrome with mutation profiles dominated by alterations in genes associated with these syndromes.

AbstractBACKGROUNDDroplet digital PCR (ddPCR) is an emerging technology for quantitative cell-free DNA oncology applications. However, assay performance criteria must be established in a standardized manner to harness this potential. We reasoned that standard protocols used in clinical chemistry assay validation should be able to fill this need.METHODSWe validated KRAS, EGFR, and BRAF quantitative ddPCR assays based on the Clinical Laboratory Improvement Act regulations for laboratory-developed tests in clinical chemistry and the matching Clinical and Laboratory Standards Institute guidelines. This included evaluation of limit of the blank (LOB), limit of detection (LOD), limit of quantification (LOQ), intraassay and interassay imprecision, analytical range, dilution linearity, accuracy (including comparison with orthogonal platforms), reference range study, interference, and stability studies.RESULTSFor the ddPCR assays, the LOB was 4 mutant copies, LODs were 12 to 22 copies, and LOQs were 35 to 64 copies. The upper limit of the dynamic range was 30000 copies, and dilutions were linear down to the LOQs with good accuracy of spike recovery of Horizon reference material. Method comparisons with next-generation sequencing and an alternative ddPCR platform showed complete qualitative agreement and quantitative concordance, with slopes of 0.73 to 0.97 and R2s of 0.83 to 0.99. No substantial interferences were discovered. Wild-type copy numbers in plasma ranged from 462 to 6169/mL in healthy individuals.CONCLUSIONSStandard clinical chemistry assay validation protocols can be applied to quantitative ddPCR assays. This should facilitate comparison of the performance of different assays and allow establishment of minimal significant change thresholds in monitoring applications.

e21057 Background: BRAF, NRAS, CDKN2A and P53 mutations are commonly observed pathogenic mutations in cutaneous melanoma. The influence of mutations such as BRAF, NRAS, CDKN2A and P53 on treatment response in the PD1 inhibitor era has not been well defined. Methods: This study included patients with melanoma with a cutaneous or unknown primary. All patients had genomic profiling with the Mayo Clinic 50-gene solid tumor mutation panel. Treatment response to first line immunotherapy with PD1, CTLA-4, or combination PD1/CTLA-4 inhibitors were assessed by response rate and time-to-progression (TTP) rate at 12 months. Each patient outcome was stratified by non-mutually exclusive gene mutation status into BRAF, NRAS, P53, CDKN2A, or quadruple negative (N4) groups. Results: Genomic profiling was performed on 208 patients, of whom 102 received first line immunotherapy. Most patients received a PD1 inhibitor (n = 62), followed by CTLA-4 inhibitor (n = 34), and combination therapy (n = 6). A trend towards improved outcomes was observed in CDKN2A and BRAF mutated patients with worse outcomes noted in P53, N4, and NRAS patients. The percent without progression at 12 months were BRAF 52.2%, CDKN2A 58.2%, NRAS 30.5%, P53 37.7%, and N4 26.7% (see table below). Response rates were BRAF 46.4%, CDKN2A 38.5%, NRAS 22.2%, P53 40.0%, and N4 33.3%. Conclusions: There is a trend towards improved outcomes with immunotherapy in BRAF and CDKN2A mutated patients while P53, NRAS and N4 patients have inferior outcomes. [Table: see text]

Purpose Molecular diagnostic testing has become an integral part of the evaluation of patients with metastatic colorectal cancer (CRC). Expanded mutational testing, such as next-generation sequencing (NGS), often identifies mutations with unclear clinical or prognostic implications. One such example is BRAF mutations that occur outside of codon 600 (non-V600 BRAF mutations). Methods We conducted this multicenter, retrospective cohort study to characterize the clinical, pathologic, and survival implications of non-V600 BRAF mutations in metastatic CRC. We pooled patients in whom non-V600 BRAF mutations were identified from NGS databases at three large molecular genetics reference laboratories. Results A total of 9,643 patients with metastatic CRC underwent NGS testing. We identified 208 patients with non-V600 BRAF mutations, which occurred in 2.2% of all patients tested and accounted for 22% of all BRAF mutations identified. Cancers with non-V600 BRAF mutations, compared with cancers with V600E BRAF (V600E BRAF) mutations, were found in patients who were significantly younger (58 v 68 years, respectively), fewer female patients (46% v 65%, respectively), and patients who had fewer high-grade tumors (13% v 64%, respectively) or right-sided primary tumors (36% v 81%, respectively). Median overall survival was significantly longer in patients with non-V600 BRAF-mutant metastatic CRC compared with those with both V600E BRAF-mutant and wild-type BRAF metastatic CRC (60.7 v 11.4 v 43.0 months, respectively; P < .001). In multivariable analysis, non-V600 BRAF mutation was independently associated with improved overall survival (hazard ratio, 0.18; P < .001). Conclusion Non-V600 BRAF mutations occur in approximately 2.2% of patients with metastatic CRC and define a clinically distinct subtype of CRC with an excellent prognosis.