Abstract P2-07-02: Tumor infiltrating lymphocytes density and coding mutations effects on the outcome of operable triple negative breast cancer patients
Authors: Kotoula V , Fountzilas E , Chatzopoulos K , Alexopoulou Z , Timotheadou E , Xanthakis I , Gogas H , Skondra M , Christodoulou C , Papadopoulou K , Chrisafi S , Koutras A , Xepapadakis G , Venizelos V , Efstratiou I , Patsea H , Kalogeras KT , Lakis S , Fountzilas G 
Published: May 2016

Abstract
Background-Aim: Neoantigens are considered to trigger host immune responses against tumors, which may be reflected by tumor infiltrating lymphocytes (TILs) density within the tumor stroma. High TILs levels have been associated with favorable triple-negative breast cancer (TNBC) patient outcome. Herein we evaluated the presence of coding mutations and TILs density with regard to outcome in a cohort of TNBC patients treated with anthracycline-based adjuvant chemotherapy.
Patients and Methods: Paraffin TNBC tissues from 242 patients treated in the context of four prospective clinical trials were histologically reviewed and submitted to massively parallel semiconductor sequencing with a custom panel targeting 57 breast cancer (BC)-related genes. Mutations (mut) were evaluated in 210 informative samples as missense/nonsense amino acid changing variants, with minor allele frequency &lt;1% in the case of single nucleotide polymorphisms. TILs density was morphologically evaluated as percent of the stromal area in 197 tumors; lymphocyte predominant (LP) BC tumors were called for TILs &gt;50%. Disease-free survival (DFS) was used as the endpoint for the present analysis.
Results: 426 Mut were observed for 40 genes in 147 TNBC patients (70%). Among mutated genes, ranging from 1 in 97 tumors up to &gt;10 in 8 tumors, the most frequently affected were TP53 (102 tumors, 69%) and PIK3CA (40 tumors, 27%). Intriguingly, mut rate (p=0.042) and number of mut genes (p=0.018) per tumor were inversely associated with TILs density. Nineteen tumors (10%) were LP-TNBC, carrying TP53 and PIK3CA mut as the only coding alterations in 10 and 3 cases, respectively. LP-TNBC patients did not experience any relapses during a follow-up period of 46-152 months (mean 66 months). For the 90% of non-LP-TNBC, the previously reported outcome benefit for 10% increments of TILs density was only demonstrated for tumors with 31-50% TILs. In non-LP-TNBC, upon adjustment for standard clinicopathological parameters, PIK3CA mut, TP53 mut and TILs density as a continuous variable, TP53 mut and nodal status independently conferred unfavorable DFS (HR=1.89, 95% CI 1.03-3.47, p=0.040 and HR=2.89, 95% CI 1.59-5.24, p=0.001, respectively). When continuous TILs density was added in the multivariate models in the entire cohort, 10% increments significantly predicted favorable DFS (HR=0.73, 95% CI 0.59-0.91, p=0.006), while high nodal status predicted unfavorable DFS (HR=2.75, 95% CI 1.51-4.99, p&lt;0.0001).
Conclusions: In the present study, tumors with higher TILs density, including LP-TNBC, were not characterized by multiple mutations or mutated genes with the panel tested. In TNBC, increasing TILs density is a strong favorable and high nodal status a strong unfavorable prognosticator. Importantly, LP-TNBC may be regarded as a distinct subgroup with excellent prognosis concerning 10% of TNBC. In non-LP-TNBC, TP53 mut and nodal status were significant unfavorable prognosticators. These data may suggest that the level of morphologically assessed TILs density does not necessarily correspond to the tumoral mutational load and merit validation in larger cohorts.
Citation Format: Kotoula V, Fountzilas E, Chatzopoulos K, Alexopoulou Z, Timotheadou E, Xanthakis I, Gogas H, Skondra M, Christodoulou C, Papadopoulou K, Chrisafi S, Koutras A, Xepapadakis G, Venizelos V, Efstratiou I, Patsea H, Kalogeras KT, Lakis S, Fountzilas G. Tumor infiltrating lymphocytes density and coding mutations effects on the outcome of operable triple negative breast cancer patients. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-07-02.