BRAF mutation analysis in cell free tumoral DNA (cfDNA) of melanoma patients: preliminary results from the Spanish Melanoma Group prospective study GEM1304
Maria Gonzalez Cao1, Virtudes Soriano2, Delvys Rodríguez3, Teresa Puertolas4, , Eva Muñoz5, Ainara Soria6, Clara Mayo de las Casas1, Miguel Angel Molina-Vila1, Elizabeth Perez7, Margarita Magem8, Almudena Garcia9, Jose Luis Manzano10, Javier Cortes5, Rafael Rosell1,10 on behalf of the Spanish Melanoma Group
1Instituto Oncológico Dr Rosell, Hospital Universitario Quirón Dexeus, Barcelona, Spain; 2Fundación IVO. Valencia, Spain; 3Hospital Insular de Gran Canaria, Gran Canaria, Spain ; 4Hospital Miguel Servet, Zaragoza, Spain; 5Hospital Vall d’Hebron, Barcelona, Spain; 6Hospital Ramon y Cajal, Madrid, Spain; 7Hospital Costa del Sol, Malaga, Spain; 8Hospital Sant Pau, Barcelona, Spain; 9Hospital Marques de Valdecilla, Santander, Spain ; 10Catalan Institute of Oncology, Hospital Germans Trias i Pujol, Badalona, Barcelona, Spain.
References:1.G. Cao et al. AACR-EORTC-NCI Meeting 2013
2. BRAF on ctDNA from basal samples
No pt 35
Only Serum (s) n (%)
Only Plasma (p) n (%)
Bothn (%)
s or pn (%)
BRAF+ ctDNABRAF- ctDNA
3 (8.6%) 3 (8.6%) 18 (51.4%) 24 (68.6%)11 (31.4%)
No pt 20
Post-treatment BRAF+
Post-treatment BRAF-
Pre-treatment BRAF - 0 4 (100%)
Pre-treatment BRAF+ 6 (37%) 10 (63%)
3. Serial BRAF ctDNA analysis
5. PFS by BRAF status in first ctDNA evaluation
Background: One potential source of molecular information from the tumor is the presence of circulating tumor DNA fragments (ctDNA), that can be found in plasma and serum of cancer patients. Our previous data in BRAFV600 mutant melanoma patients treated with BRAF inhibitors showed a significant difference in median PFS by cfDNA status (3.5 months vs 13.57 months for BRAFV600 positive and negative, respectively p=0.026)1. In the ongoing prospective study, we studied the prognostic value for survival of basal and postreatment BRAF mutation on cfDNA and their correlation with clinical response along treatment
Abstract: Results:
Conclusions:
Significant difference in median PFS was found between
patients with BRAF mutation negativization versus
persistance of mutation at first ctDNA evaluation. Patients
with BRAF mutation on basal ctDNA that achieve an early
negativization could have as good prognosis as patients
without detectable BRAF mutation on basal ctDNA. Longer
follow up is needed to asses the prognostic value of BRAF
mutation on basal ctDNA.
Methods: BRAF mutations were determined using a 5’-nuclease PCR (Taqman®) assay in the presence of a peptide nucleic acid (PNA) clamp (Eurogentec, Belgium), designed to inhibit amplification of the wild-type (wt) DNA allele.
1. Clinical characteristics of patients Backgroud: Our previous data showed a prognostic value for BRAF mutation in basal ctDNA from BRAFV600 mutant melanoma patients treated with BRAF inhibitors (median PFS 3.5 months vs 13.57 months for BRAFV600 positive and negative, respectively p=0.026)1. The Spanish Melanoma Group (GEM) initiated a prospective translational study to assess the prognostic value of BRAFV600 mutation in ctDNA of stage IV melanoma patients and its role for monitoring disease evolution (GEM1304) (ClinicalTrials.gov Id: NCT01960634).Methods: A quantitative 5’-nuclease PCR based assay for determination of BRAFV600 mutation in ctDNA was developed and validated in 92 previously genotyped cancer patients. The assay detected 2.5 pg mutated DNA/µL and a ratio of V600E versus wild type allele of 1:20000, with clinical sensitivity of 58% and specificity of 100%. Planned accrual for ongoing GEM1304 study is 58 patients. Serum and plasma samples are collected before and during treatment until disease progression.Results: 35 patients, 76 samples, have been included. Pretreatment BRAF analysis is positive in serum and/or plasma in 24 (68.6 %) patients. Progression free survival at 12 months for patients with positive and negative BRAF mutation on basal ctDNA was 46.9% vs 66.7% (p=.09). Twenty patients have more than one sample; BRAFV600 was detected in pretreatment samples in 16/20 patients: in 10 responding patients BRAFV600 mutation was not detectable in subsequent analysis (13m+,10m+,9m+,6m+, 5m+,4m+,3m+,3m+,3m+,2m+). In 6 primary resistant patients, BRAFV600 persisted in follow up samples and their time to progression was less than 4 months for all of them. Progression free survival at 6 m according to ctDNA BRAF status on first sample evaluation was for positive and negative cases 16.7% and 76% (p=.0003). Discordant results between clinical response and BRAF status on ctDNA were found in one single patient: a radiologic partial response was observed but BRAFV600 mutation did not disappear from ctDNA and the patient died after 3 months on treatment. Re-evaluation of the performed scan showed new bone metastasis that had not been detected.Conclusions: Noninvasive genotyping of ctDNA by PCR assay could allow effective translation to the clinical setting. BRAFV600 mutation in pretreatment and postreatment samples could be a prognostic factor of survival.
Time (months)
20100
PF
S (
pro
port
ion)
1,0
,8
,6
,4
,2
0,0
BRAF Serum/Plasma
POSITIVE
NEGATIVE
Mutated DNA is “enriched” during the amplification
Clinical characteristics
Pt. with clinical data, n (%) 14 (40%)
Age, years Median (range) 52 (31-79)
Male, n(%) 5 (14%)
Stage, n (%)
M1a
M1b
M1c
1 (7%)
2 (14%)
11 (79%)
No. Metast sites, Median (range) 2 (1-6)
Metastatic sites, n (%)
CNS
Liver
Lung
Bones
Skin or nodes
5 (36%)
4 (29%)
8 (57%)
6 (43%)
8 (57%)
PFS6m 76% vs 16.67%
Median PFS not reached vs 3 m
p=0.0003
4. PFS by BRAF status in basal ctDNA
PFS1y 66.67% vs 46.88%
Median PFS not reached vs 4 m
p=0.09
Basal BRAF- (bB-) 62.50%BRAF- negativization (eB-) 100%
BRAF+ persistance (eB+) 16.67%
PFS6m (%)
eB- vs eB+ p=0.001
bB- vs eB- p=0.155
bB- vs eB+ p=0.010