Implementation of an NGS panel for clinical practice in paraffin-embedded tissue samples from locally advanced and metastatic melanoma patients

Abstract

Aim: Single biomarker diagnostic test of BRAFV600 locus in metastatic melanoma is mandatory for treatment decision; however, multiple-gene based techniques, such as targeted next-generation sequencing (NGS) are being used to maximize the number of patients that can benefit from a targeted therapy. The main objective of this study is to investigate whether an NGS panel could be adopted in routine clinical care for advanced melanoma. Methods: Patients diagnosed with advanced melanoma at our center from 2017 to 2019 were included. Presence of genetic alterations was performed using two methods: real-time polymerase chain reaction-based Idylla test (Biocartis) and NGS with the oncomine solid tumor DNA kit (Thermo Fisher Scientific). Total genomic DNA was extracted from formalin-fixed and paraffin embedded samples for sequencing. Results: A total of 155 samples were evaluated for molecular analysis but 40 samples (25.8%) were inadequate for sequencing. The clinical utility of BRAFV600 real-time polymerase chain reaction and targeted-NGS was compared in 29 samples and a very good concordance was observed (Kappa = 0.89, 95% confidence interval 0.68 ± 1.05). An oncogenic mutation by NGS was found in 75 samples (65%) – 53% of whom were candidates for personalized therapies. The most prevalent mutated genes were BRAF (39%), TP53 (23%), and NRAS (14%). Other genes identified at lower incidence (< 5%) were: PIK3CA, ERBB4, CTNNB1, STK11, FGFR1, SMAD4, KRAS, FGFR3, PTEN and AKT. Co-occurrence of oncogenic mutations was detected in 40% of the samples. Among the mutations identified, TP53 was significantly more prevalent in men (men 31.8% versus women 12.2%, P = 0.03) and NRAS in women (men 9.1% versus women 24.4%, P = 0.03). Conclusions: Targeted-NGS testing is a feasible technique to implement in the routine clinical practice. Based on our results, NGS has provided more information on target-genes than RT-PCR technique, maximizing the benefit for patients with advanced melanoma.

EBUS-TBNA Cytological Samples for Comprehensive Molecular Testing in Non–Small Cell Lung Cancer

“…For controversial cases, a consensus was reached over a double headed microscope. References [ 18 , 20 , 21 , 22 , 23 ] are cited in the supplementary material .…”

Detection of mutations in the <i>BRAF</i> in patients with melanoma in routine clinical practice: Survey results

“…Интересно, например, что проведенное в Онкологическом центре принцессы Маргарет (г. Торонто, Канада) в 2022 г. ретроспективное исследование 66 случаев тестирования BRAF у взрослых пациентов с меланомой демонстрирует среднее время 12 дней (95% доверительный интервал -ДИ 8-15), когда морфолог направляет материал, по сравнению с 20 днями (95% ДИ 16-23), если тест был запрошен другим специалистом; p<0,001. Когда тест BRAF и биопсию проводили в одном и том же учреждении, среднее время выполнения BRAF составляло 13 дней (95% ДИ [6][7][8][9][10][11][12][13][14][15][16][17][18][19], тогда как если образец был передан из другого учреждения -19 (95% ДИ 16-21); p=0,02. В исследовании, опубликованном в 2022 г. в журнале Archives of Pathology & Laboratory Medicine, рассматривали сроки определения мутации в гене BRAF у пациентов с меланомой в случае, если тест заказывает морфолог или клиницист, до получения финального морфологического заключения у пациентов с впервые диагностированной НММ или с положительными результатами биопсии сторожевого лимфоузла (reflex testing).…”

“…Наиболее распространенным вариантом мутации в гене BRAF является V600E (замена валина на глутаминовую кислоту), которая встречается примерно в 80% случаев меланом с BRAF-мутацией, V600K обнаруживается в 15% случаев, а мутации V600R/M/D/G -примерно в 5% [10]. Некоторые крупные исследования, проведенные в Австралии [11], Техасе [12] и Флориде [13], показывают, что генотип V600E распространен не во всех популяциях, а V600K (превращение валина в лизин) может составлять 20% или более меланом с мутацией BRAF.…”

Molecular Markers and Targets in Melanoma

“…BRAF is the most important therapeutic target and the most frequent genetic alteration in cutaneous melanoma, affecting 40–60% of cases [ 20 ]. The most frequently found BRAF mutation is V600E, which affects about 80% of BRAF -mutated melanomas; V600K involves 15% of cases; and V600R/M/D/G mutations are found in about 5% of cases [ 45 ]. The BRAF V600E mutation is frequently associated with the superficial spreading subtype, younger patient age, and non-CSD skin sites, such as the trunk and proximal areas of the extremities.…”