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. 2021 Jun;6(3):100133.
doi: 10.1016/j.esmoop.2021.100133. Epub 2021 May 10.

High BRAF variant allele frequencies are associated with distinct pathological features and responsiveness to target therapy in melanoma patients

E Berrino  1 , A Balsamo  2 , A Pisacane  3 , S Gallo  4 , P Becco  4 , U Miglio  3 , D Caravelli  4 , S Poletto  5 , L Paruzzo  5 , C Debernardi  3 , C Piccinelli  3 , A Zaccagna  6 , P Rescigno  7 , M Aglietta  5 , A Sapino  8 , F Carnevale-Schianca  4 , T Venesio  9
Affiliations

High BRAF variant allele frequencies are associated with distinct pathological features and responsiveness to target therapy in melanoma patients

E Berrino et al. ESMO Open. 2021 Jun.

Abstract

Background: BRAF mutant melanoma patients are commonly treated with anti-BRAF therapeutic strategies. However, many factors, including the percentage of BRAF-mutated cells, may contribute to the great variability in patient outcomes.

Patients and methods: The BRAF variant allele frequency (VAF; defined as the percentage of mutated alleles) of primary and secondary melanoma lesions, obtained from 327 patients with different disease stages, was assessed by pyrosequencing. The BRAF mutation rate and VAF were then correlated with melanoma pathological features and patients' clinical characteristics. Kaplan-Meier curves were used to study the correlations between BRAF VAF, overall survival (OS), and progression-free survival (PFS) in a subset of 62 patients treated by anti-BRAF/anti-MEK therapy after metastatic progression.

Results: A highly heterogeneous BRAF VAF was identified (3%-90%). Besides being correlated with age, a higher BRAF VAF level was related to moderate lymphocytic infiltration (P = 0.017), to melanoma thickness according to Clark levels, (level V versus III, P = 0.004; level V versus IV, P = 0.04), to lymph node metastases rather than cutaneous (P = 0.04) or visceral (P = 0.03) secondary lesions. In particular, a BRAF VAF >25% was significantly associated with a favorable outcome in patients treated with the combination of anti-BRAF/anti-MEK drug (OS P = 0.04; PFS P = 0.019), retaining a significant value as an independent factor for the OS and the PFS in the multivariate analysis (P = 0.014 and P = 0.003, respectively).

Conclusion: These results definitively support the role of the BRAF VAF as a potential prognostic and predictive biomarker in melanoma patients in the context of BRAF inhibition.

Keywords: BRAF mutations; increased survival; melanoma; variant allele frequency.

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Conflict of interest statement

Disclosure The authors have declared no conflicts of interest. Data sharing All data relevant to the study are included in the article or uploaded as online supplemental information. Individual patient data cannot be shared. Disclaimer The views expressed are those of the author(s) and not necessarily those of the Candiolo Cancer Institute or the University of Turin. Ethics approval Clinical-pathological data were recovered after obtaining the informed consent from all the patients, approved by the medical ethical committee of the FPO-IRCCS, and carried out according to the principles of the Declaration of Helsinki.

Figures

Figure 1
Figure 1
BRAF MR and VAF correlations with the primary site of melanomas. (A) Bar stacked chart of the MR (frequency) for each sample, clustered for the primary site. (B) Box and whisker plot defining the VAF distribution for each primary site; the absence of visceral samples in the box plot is due to the too low number of mutated patients. (C) Double Y-axis box and whisker plot for the MR and the VAF (only for metastatic samples) grouped for the site of the metastasis; on the left Y-axis is reported the BRAF VAF, whereas on the right axis the MR; the MR was defined as the average MR by the rhombus symbol. OPM, occult primary melanoma; MR, mutation rate; VAF, variant allele frequency; WT, wild type.
Figure 2
Figure 2
BRAF MR and VAF correlations with both primary and metastatic features. (A) Box and whisker plot reporting the VAF (only for primary melanoma cases) of the groups defined for the TIL. (B) Box and whisker plot reporting the VAF (only for primary melanoma cases) of the groups defined by Clark's level. MR, mutation rate; VAF, variant allele frequency; TIL, tumor-infiltrating lymphocyte.
Figure 3
Figure 3
Patient response to treatment and the VAF distribution. (A) Box and whisker plot defining the BRAF VAF in the followed-up cohort grouped for the best response rate. The thick line represents the average value for each group. (B) Box and whisker plot defining the BRAF VAF in the followed-up cohort clustered for the response rate and for the therapy type; the thick line denotes the average VAF value for each group. CR, complete response; PD, progression disease; PR, partial response; SD, stable disease.
Figure 4
Figure 4
Survival data and the BRAF VAF. (A) OSt survival curve for low and high VAF patients (cut-off 25%). High VAF curve shows a statistically higher survival compared with low VAF curve. (B) PFS survival curve for low and high VAF patients (cut-off 25%). High VAF curve indicates a statistically higher time to progression compared with the low VAF curve. (C) OSt survival curve for low VAF (<25%), high VAF (>25%), MONO- and COMBO-treated patients; high VAF-COMBO curve shows a statistically higher survival compared with the low VAF-COMBO curve, high VAF-COMBO dotted curve, and low VAF-MONO dotted curve. (D) PFS survival curve for low VAF (<25%), high VAF (>25%), MONO- and COMBO-treated patients. High VAF-COMBO solid curve shows a statistically higher survival compared with the low VAF-COMBO solid curve, high VAF-COMBO dotted curve, and low VAF-MONO dotted curve. CR, complete response; Cum survival, cumulative survival; OSt, overall survival treatment onset; PFS, progression-free survival; PR, partial response; SD, stable disease; VAF, variant allele frequency.
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