Plasma ctdna RAS/RAF mutations analysis for monitoring overall survival (OS) and heterogeneity in metastatic colorectal cancer patients (mcrc)
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1 Plasma ctdna RAS/RAF mutations analysis for monitoring overall survival (OS) and heterogeneity in metastatic colorectal cancer patients (mcrc) Authors: Andrea Petricca Mancuso, Veronica Varchetta, Fabrizio Montagnese, Carla Campanella, Rita Brandi, Marco Vitale, Giuseppe Soda on behalf CATHEDRA Cancer Group Villa Margherita Cancer Center, Rome, Italy; University La Sapienza, Rome, Italy Abstract ID: 701 (202035) Poster Board #: G14 Date: Saturday, January 20, 2018 Time: 12:30 PM - 2:00 PM (Poster Session) Location: Level 1, West Hall
2 Background The study is a non-profit academic, open-label, single center study carried out by the CATHEDRA cooperative group. The study was designed to estimate the clinical usefulness of genetic analysis for circulating tumor DNA (ctdna) by digital polymerase chain reaction in patients with mcrc. Methods We compared B-RAF and K/N/H-RAS mutations detected in surgical or endoscopic ultrasound-guided fine-needle aspiration biopsy tissue DNA and in ctdna from 63 patients with mcrc. p53 mutational status and PD-L1 expression in the serum and in tissues respectively were also analyzed with a comparison with survival rates.
3 Methods Tumor samples were analyzed with the Ion AmpliSeqTM Colon and Lung Cancer Panel (Thermofisher, Milan, Italy) using Ion Torrent semiconductor sequencing. The sensitivity of the method was set at 2% allelic frequency. Deep-sequencing analysis was carried out using the Oncomine Comprehensive Tumor Panel (Thermofisher), with a coverage >2000x. Plasma samples were analyzed with BEAMing digital PCR technology using OncoBEAM KRAS/ NRAS/BRAF - Sysmex Inostics. The discordant cases between tissue and plasma testing were analyzed using the QX200 Droplet Digital PCR (ddpcr) System and the Bio-Plex Instruments. Tumor cell PD-L1 expression by concordance IHC and immunofluorescence (Ab214565) was evaluated using H-score (positivity H score > 55). P53 was analyzed on blood samples by next generation whole exon sequencing of P53.
4 Methods Statistical analysis The Kaplan Meier method was used to estimate median progression- free survival time (mpfs) and median overall survival (mos) time. P values were calculated using log-rank tests at a significance level of 5%. Differences between categorical data were measured using v2 and Fisher s exact test, when adequate. All statistical analyses were carried out using IBM-SPSS statistics version Differences between continuous variables were investigated by Mann Whitney U-test.
5 Tab 1: Patients Characteristic (PC) and Treatments n:63 WORST PLASMA PC (Tab 2) RAS WT RAS mut B-RAF mut n, (male/female) 26 (10/16) 37 (19/18) 6 (5/1) Age <65, % Location Descending-sigmoid Ascending Transverse Prior Adjuvant Therapies, n First Line Treatment, n Folfoxiri Folfoxiri + Bevacizumab Folfiri + Cetuximab 5 Folfiri + Avastin 6 31 Folfox + Panitumumab 5
6 Results The frequencies of K-RAS mutations in tissue samples at G12V, G12D, and G12R in codon 12 were 18 of 63 samples (28.57%), 5 of 63 samples (7.93%), and 6 of 63 samples (9.52%), respectively. Conversely, the rates of the mutations in ctdna were 22 of 63 samples (34.92%), 6 of 63 samples (9.52%), and 9 of 63 samples (14.28%), respectively. No H-RAS or N-RAS mutation were detected Overall, the K-RAS mutation rates in tissue and ctdna were 45.72% and 58.72%, respectively, and the concordance rate between them was 29 of 37 samples (78,37%). Tab 2 Tissue K-RAS Tissue B-RAF Plasma K-RAS Plasma B-RAF Mutations Detected Wild Type Total
7 Results 2 tissue samples harbored a BRAF mutation (3.17%) in a complete discordance with same mutation detected in 6 different serum ctdna (9.52%). Tab 3 Patients Tissue Evaluation Plasma ctdna Evaluation 1 B-RAF c.1799_t>a p.v600e B-RAF c.1801 A>G p.k601e 2 B-RAF c.1799_tg>aa p.v600e B-RAF c.1798_1799 GT>AA p.v600k 3 none B-RAF c.1799_t>a p.v600e 4 none B-RAF c.1799_t>a p.v600e 5 none B-RAF c.1406_g>c (G469A) 6 none B-RAF c.1799_t>a p.v600e
8 Results Fig 1 %OS Tissue RAS mutations %OS Plasma RAS mutations OS did not appear to differ by the presence of K-RAS mutations in tissue or in the serum DNA (figure 1), The OS of patients with B-RAF mutations in ctdna was significantly shorter than that of patients with tissue K-RAS/B-RAF mutations (11 vs 19 months, p < , figure 2). Months % Overall Survival Tissue K-RAS mut. mos: 23,37 months (C.I.95%: 13,0-34,0) Plasma K-RAS mut. mos: 22,08 months (C.I. 95%: 12,0-31,0) Log rank Test P=>0.05
9 Fig. 2 Overall Survival in patients with Tissue RAS and B-RAF mutations vs Plasma B-RAF mutations % Overall Survival Tissue RAS/B-RAF mut. mos: 19,2 months (C.I.95%: 11,0-27,0) Plasma B-RAF mut. mos: 11,16 months (C.I. 95%: 9,0-14,0) Log rank Test P=0,0001 Patients at Risk Months Presented by:
10 Results PD-L1 positivity was significantly associated with p53 mutational status (concordance: 87,5%) and with a worse outcome for OS. Fig.3 Tab 4 Patients % PDL-1 Expression p53 mutational status Codon Concordance (Y/N) Mutations OS (ms) N none Y K-RAS Y none Y K-RAS Y none Y none Y K-RAS Y B-RAF 8 PD-L1 expression evaluated by concordance IHC and immunofluorescence (Ab214565) using an H-score positivity H score > 55
11 Results Fig. 3: Overall Survival (OS) comparison between biological different classes of patients (pts) 1 Tissue/Plasma real WT pts 2 Tissue K-RAS mut pts 3 Plasma K-RAS mut/pts 4 Tissue K-RAS/B-RAF mut pts 5 Plasma B-RAF mut pts 6 PDL-1 +/p53 mutated Overall Survival (months)
12 Conclusions/Take-Away Points - Analysis of ctdna is a new useful procedure for detecting mutations in mcrc patients. In our series it appears to discover an higher number of mutations (10-15%) respect to same analysis in the tissue. - This non invasive method may have great potential to understand tumor heterogeneity. Some B-RAF mutations can remain undetected in high heterogeneity cancers using tissue analysis. Liquid biopsy appears a new strategy for the planning therapies especially in this particular class of patients. - Our Series identifies 4 prognostic classes of patients: 1) K-RAS WT 2) Tissue K-RAS mut/b-raf mut 3) Plasma B-RAF mut 4) PDL-1/p53 mut - Our Research confirm that p53 could regulate PDL1 (via mir-34, JNCI, 2015 Nov 17;108,1). Presented by:
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