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About 70% of breast cancers (BC) express estrogen receptor α (ER) and most of these are sensitive to endocrine therapies (ET). However, a significant fraction of patients with ER-positive (ER+) BC who benefit from adjuvant ET recur with metastatic disease after prolonged exposure to these therapies. Even patients with ER+ metastatic BC who respond to ET will eventually progress with antiestrogen-resistant hormone-independent disease. Activating mutations in the estrogen receptor gene ESR1 have been described in advanced ER+ BC resistant to ET, particularly in metastatic patients exposed to therapy with aromatase inhibitors (AI). ESR1 mutations frequently occur in a hotspot region within the ligand-binding domain of ER, constitutively activating the receptor in a ligand-independent manner and thus explaining the acquired resistance to hormonal therapy. Recent studies have reported the detection of ESR1 mutations in codons 536, 537, and 538 in circulating tumor DNA (ctDNA) of metastatic BC patients, ensuring the establishment of a causal relationship among ESR1 mutations and acquired resistance to AI therapy. Thereby we evaluated the presence of ESR1 mutations in plasma cell-free DNA (cfDNA) of metastatic ER-positive BC patients who progressed on AI therapy. Among patients with no ESR1 mutations detected, we questioned the presence of any other mutated gene that could be related to acquired resistance to ET. To answer these questions, we first investigated the presence of ESR1 recurrent mutations (L536R, Y537C/N/S, D538G) by Droplet Digital PCR (ddPCR) in ctDNA of 24 ER+ metastatic BC patients after acquired resistance to therapy with AI. ESR1 mutations were detected in 25% (6/24) of the patients, with ESR1 D538G and Y537N being the most frequent, and four patients showed polyclonal disease with more than one ESR1 mutation. The longest interval between the end of aromatase inhibitor treatment and the detection of ESR1 mutations was 2.3 years, corroborating with some studies that showed that these mutations remain readily detectable in ctDNA many months and years after stopping therapy. cfDNA from patients with no ESR1 mutations detected were analyzed by targeted cfDNA sequencing (cfDNA seq) of a 151-cancer gene panel. We were able to detect mutations in genes already known to be associated to ET resistance such as PIK3CA and AKT. We found one patient with AKT E17K mutation and four patients (4/12, 33%) with detectable PIK3CA-mutated ctDNA, with three patients with E545K mutation and one with H1047L mutation. Mutations in PIK3CA are frequently observed in BC and have been suggested as a predictive biomarker for PI3K-selective inhibitor treatment, alpelisib. Based in our results, we can conclude that detection of tumor genetic alterations in plasma cfDNA may have important clinical implications for personalized medicine, including monitoring of recurrence and therapy resistance and therapeutic guidance. Citation Format: Cibele Masotti, Franciele H. Knebel, Rudinei D. M. Linck, Julianne Lima, Andrea K. Shimada, João Victor Alessi, Bruna M. Zucchetti, Dimitrios Kleftogiannis, Louise Barber, Marco Gerlinger, Artur Katz, Max S. Mano, Anamaria A. Camargo, Fabiana Bettoni. Detection of ESR1 mutations in plasma cell-free DNA from metastatic ER-positive breast cancer patients resistant to hormone therapy [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A19.

Intratumoral heterogeneity (ITH) is a phenomenon where a tumor, over space and time, branches into a set of genetically distinct clones, which have unique somatic branch mutations, although sharing several somatic trunk mutations. Clonal populations expand or regress in response to selective pressures exerted by the tumor microenvironment. Therefore, ITH has already been linked to several cancer features such as the development of metastases and resistance against a broad range of therapies. Through whole exome sequencing, we can access the number of reads covering the alleles of somatic mutations in tumor samples. Thus, mutant-allele frequency (MAF), i.e., the proportion of reads reporting the mutant allele over all reads mapped to the locus, is informative about the number of clones within a tumor bearing this genetic variant. However, MAF estimations can be impaired by tumor purity, copy number alteration, or sampling approaches. It is proposed that the analysis of multiple samples of a tumor improve accuracy in quantifying clone populations, but single sample is cheaper and more feasible for the clinicians. Mutant-Allele Tumor Heterogeneity (MATH) is a method capable of trustworthily scoring ITH in single samples, as it is based on the range of the MAF distribution of a tumor. The higher the MATH score, the more heterogeneous is a tumor. The goal of this project is to evaluate the ITH levels of 6,390 tumor samples of 20 distinct tumor types, for which genomic data is publicly available through GDAC Firehose repository. We accessed the MAF files and filtered somatic variants: (i) selected only single nucleotide variants, (ii) mutant alleles reported by at least three reads, (iii) total coverage equal to or higher than 30 reads. We estimated MATH scores using R (v3.2.4). The distribution of MATH scores across distinct tumor types exhibited a wide variability (ranging from 12 to 128). Uterine corpus endometrial carcinoma had the lowest MATH scores, whereas prostate adenocarcinoma and testicular germ cell tumors presented the highest MATH scores. The wide ITH variability among nine distinct tumor types has already been assessed in previous studies by using another scoring methodology. High MATH score was significantly associated with decreased overall survival in head and neck squamous cell carcinoma and predicted the development of metastasis in individuals with colorectal cancer. We will further explore the correlation between MATH scores and clinical data to all the 20 tumors here described by exploring the corresponding public clinical data from the cBioPortal for Cancer Genomics. Should MATH score prove to be an optimal scoring method for single biopsies, capable of predicting patient's prognosis, drug response, or disease outcome, it will be a valuable tool for clinicians for a broad spectrum of cancer treatments. Citation Format: Cibele Masotti, Filipe F. Santos, Anamaria A. Camargo, Pedro A.F. Galante. Measuring intratumoral heterogeneity with a mutant-allele frequency score across distinct cancer types [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B65.

Response to neoadjuvant chemoradiotherapy (nCRT) varies substantially among patients with locally advanced rectal cancer, but key determinants of treatment response are not fully understood. Intratumoral genetic heterogeneity (ITGH) is a common feature of solid tumors and has been proposed to drive disease progression and treatment response. Others and we have recently demonstrated that primary nontreated rectal tumors exhibit significant ITGH, but the effect of nCRT in shaping the clonal architecture of rectal cancer has not yet been addressed. In the present work, we further expanded the analysis of ITGH in rectal cancer by analyzing whole-exome sequencing (WES) and clinical data from 79 primary nontreated rectal cancers obtained from The Cancer Genome Atlas. We show that primary rectal tumors exhibit a remarkable variability in the degree of ITH. We also analyzed the effect of nCRT on ITGH and monitored tumor cell subpopulation dynamics after treatment through WES of paired tumor samples collected at diagnosis (pretreatment) and at surgery following nCRT (post-treatment) from seven patients. We show that treatment exposure does not introduce novel somatic mutations or significantly alter tissue genetic heterogeneity. Instead, nCRT acts as a potent selective pressure, shaping the mutational landscape of rectal tumors and allowing the selection and expansion of tumor cell subpopulations, which are more prone to resist nCRT. Our results shed light on the highly heterogeneous and dynamic mutational landscape of rectal tumors and indicate that ITGH may directly affect response to nCRT. Citation Format: Cibele Masotti, Fabiana Bettoni, Bruna R. Correa, Angelita Habr-Gama, Joaquim Gama-Rodrigues, Maria Viana, Bruna B. Vailati, Guilherme P. Sao Juliao, Laura M. Fernandez, Pedro A. F. Galante, Rodrigo O. Perez, Anamaria A. Camargo. Intratumoral genetic heterogeneity in rectal cancer and response to neoadjuvant chemoradiotherapy [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B62.