Abstract P3-04-04: Germline and somatic mutation status in tissues from BRCA1/2 carriers
Authors: Kotoula V , Demiri E , Fostira F , Vrettou E , Papadopoulou K , Tikas I , Papazisis K , Zaramboukas T , Asimaki-Vlachopoulou A , Miliaras S , Fountzilas E , Ananiadis A , Chrisafi S , Poulios C , Natsiopoulos I , Tsiftsoglou A , Fountzilas G 
Published: February 2018

Abstract
Background – aim: In carriers of BRCA1/2 pathogenic mutations (mut), it is expected that the germline mut is present in all tissues, particularly in normal; the somatic mut status in normal tissues from these patients is usually not addressed. We investigated the mut status in normal and tumor tissues in a real-life cohort of BRCA1/2 carriers who underwent prophylactic surgery.
Methods: All 53 women had known BRCA1/2 germline mut that had been assessed independently; 42 had previous cancer manifestation (PCM); all had prophylactic mastectomy; 22 had prophylactic hystero-salpingo-oophorectomy. By using a 60-gene NGS panel, we examined the mut status of 231 samples, 39 peripheral blood and 192 paraffin tissues (FFPE: 46 tumors, out of which 43 breast; 97 normal breast [NB]; 49 normal ovary and salpinx [NGYN]). Germline mut status was interrogated in tissues with the above panel, Sanger sequencing and a multiplex PCR protocol for large exonic deletions, along with extensive FFPE DNA quality control (QC) to exclude false negatives.
Results: Eight patients carried germline BRCA2 and 45 BRCA1 mut (29 in the BRCT-domain; 31 substitutions/indels). We identified somatic mut in 85% of the tumors and in 64% of the normal samples; mut were found significantly more often (p=0.003) and in higher numbers (p&lt;0.001) in NGYN than in NB. In NB and NGYN, top 3 genes with somatic mut were BRCA2 (28%), BRCA1 (17%), TP53 (7%). In tumors, somatic mut were most frequent in TP53 (49%; p&lt;0.001) and BRCA1 (38%; p=0.039). Among all tissue types, the 5 tumors post-neoadjuvant treatment had the highest and NB the lowest mut load (p=0.001). In NB and NGYN, mut load was not affected by PCM or BRCA1 mut domain but it was higher in BRCA1 vs. BRCA2 carriers (p=0.027) and in those with BRCA1 substitutions/indels vs. exon deleting and skipping mut (p&lt;0.001). In tumors, germline BRCA1 substitutions/indels were associated with higher mut load (p=0.014). We validated germline mut status in all blood samples and in 111 tissue samples that passed FFPE DNA QC from 40 patients. The germline mut was not found in 14 samples (4 breast tumors; 3 NB; 7 NGYN) from 10 (25%) patients, all BRCA1 carriers, 9 with germline mut in the BRCT-domain. The only non-BRCT domain germline mut that was lost in one breast tumor, p.V1234fs, was replaced by the R1751* (validated), again in the BRCT domain. In normal tissues, those with lost germline mut had significantly less somatic mut compared to those with preserved germline mut (p&lt;0.001).
Conclusions: In BRCA1/2 carriers, somatic mut in BRCA genes and TP53 are present in normal breast and GYN tissues, more frequently in the latter, and seem associated with the mutated gene and with the type of mut in the germline. The mut status of normal breast tissue does not seem to be affected by neoadjuvant chemotherapy for breast cancer. The observed BRCA1 germline mut loss, particularly in normal tissues, may be approached as a negative selection for the inherited mut; similarly to the described germline mut reversion after chemotherapy, tissues may react to deleterious effects of haploinsufficiency, which needs functional validation.
Citation Format: Kotoula V, Demiri E, Fostira F, Vrettou E, Papadopoulou K, Tikas I, Papazisis K, Zaramboukas T, Asimaki-Vlachopoulou A, Miliaras S, Fountzilas E, Ananiadis A, Chrisafi S, Poulios C, Natsiopoulos I, Tsiftsoglou A, Fountzilas G. Germline and somatic mutation status in tissues from BRCA1/2 carriers [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-04-04.