Abstract P5-01-19: Circulating tumor DNA (ctDNA) analysis for investigating resistance to chemotherapy with DNA-damage agents in patients with hereditary or sporadic triple-negative breast cancer
Authors: Carraro Dirce M , Brianese Rafael C , Torrezan Giovana T , de Brot Marina , de Paula Claudia AA , Formiga Maria NC , Sanches Solange M , de Lima Vladmir CC , Makdissi Fabiana BA 
Published: March 2020

Background: Triple-negative breast cancer (TNBC), a very aggressive breast cancer subtype, exhibits high variability at both molecular and clinical aspects. Loss of function (LOF) germline mutation in BRCA1 has a very close association with this subtype of breast cancer. BRCA1 impairment is associated with homologous recombination deficiency (HRD) and tumor with HRD may benefit from treatment with drugs that induces DNA damage and also with PARP1 inhibitors. We previously detected that a significant fraction of TNBC diagnosed in young Brazilian women exhibits BRCA1 impairment by mechanisms including germline pathogenic mutation and promoter hypermethylation and that this group of tumors presented better overall survival. In the current study our aim is to comprehensively characterize the resistance to chemotherapy (DNA-damage agents) in patients with Hereditary or Sporadic TNBC by investigating somatic mutations in circulating tumor DNA (ctDNA). Methods: A series of 34 TNBC patients were subjected to germline genetic testing using a 26- or 96-gene panel, including homologous recombination-related genes, for classifying the TNBC as Hereditary or Sporadic. Tumor mutation burden (TMB) analysis was assessed in 20 cases using a panel containing 409 cancer-associated genes. Tumor somatic mutations were detected by evaluating DNA from tumor biopsies (Formalin-fixed, paraffin-embedded tissue) and matched leucocyte and were screened in cell-free DNA from 6 serial plasma samples during neoadjuvant treatment and for 6 months after surgery. Results: Pathogenic germline mutations were identified in BRCA1 (17.6% - 6/34), BRCA2 (2.9% -1/34) and TP53 (2.9% - 1/34) genes. Additionally, variants of uncertain significance (VUS) were identified in 21 patients (61.7% - 21/34), being ATM, BUB1B and PMS2 the most affected genes by VUS. Based on the 34 patients that underwent pre-operative chemotherapy and surgery, 47% exhibited pathological complete response (pCR), 50% in hereditary and 48% in sporadic. In terms of somatic variants, tumor mutation burden (TMB) analysis showed that 20% had high and 80% low TMB, with no association with hereditary or sporadic status. Also, we found, on average, three somatic variants per tumor (range 0-7) and used them as tumor marks in the screening of ctDNA in plasma. Somatic mutations in TP53 were identified in 80% (16 of 21) tumor biopsy samples investigated. In DNA from plasma before treatment, confident detection of at least one tumor mutation (ctDNA) was observed in 60% of patients (10 of 16). Persistent ctDNA detection in plasma during treatment was observed in 6 out 10 (60%) patients with residual disease after surgery and only in 1 out of 6 (16%) in patients with pCR. For 3 patients persistent ctDNA was detected after surgery at increasing levels showing clear anticipation of disease progression or metastasis. Serial plasma ctDNA samples showed great association with the clinical response data suggesting that the chemotherapy-resistance mechanisms can be investigated by ctDNA in TNBC. Conclusions: Germline and somatic evaluation of TNBC can provide valuable information for improving the clinical management of patients. For somatic evaluation, development of gene panels optimized for FFPE samples and low DNA input is enabling a comprehensive detection of potentially clinically-actionable somatic variants with better success rate in cancer treatment. Although still challenging, investigation of somatic variants in ctDNA from plasma samples is feasible and presents huge potential in monitoring patients during chemotherapy, especially regarding both the presence of residual disease and monitoring of disease progression.

Citation Format: Dirce M Carraro, Rafael C Brianese, Giovana T Torrezan, Marina de Brot, Claudia AA de Paula, Maria NC Formiga, Solange M Sanches, Vladmir CC de Lima, Fabiana BA Makdissi. Circulating tumor DNA (ctDNA) analysis for investigating resistance to chemotherapy with DNA-damage agents in patients with hereditary or sporadic triple-negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-01-19.