Annamalai, Lakshmanan

Abstract Introduction: Striking clinical responses to immunotherapies in subsets of patients with a variety of solid tumors have prompted the search for predictive biomarkers. Recent studies have proposed that gene specific and genome wide mutation and copy number signatures in tumor cells may be predictive of responses to immune checkpoint blockade. Notably high levels of PD-L1 on tumor cells, a context associated with an adaptive immune response, has been linked to specific oncogenic driver lesions including loss of PTEN, activating KRAS mutation, and MYC amplification, and to the total burden of copy number variants (CNVs) in aneuploid tumors. Triple negative breast cancers (TNBCs) typically have multiple driver mutations and high levels of CNVs in their genomes. Thus there is significant interest in exploiting genomic data for the development of prognostic immunotherapy biomarkers for patients with TNBC. Study Design: We interrogated 62 well annotated surgical resections from patients with TNBC and assessed the associations of genomic lesions with expression of PD-1 and PDL-1 in tumor and non-tumor cells in each sample. We applied a systematic approach to rigorously interrogate the genomes of each TNBC sample. Tumor ploidy was initially measured with DNA content flow cytometry followed by sorting the nuclei of distinct diploid, tetraploid, and aneuploid cell populations from each TNBC. The next level of analysis measured genome wide copy number variants (CNV) with oligonucleotide arrays designed for CNV detection using purified (>95%) tumor populations. This enabled the discrimination and mapping of CNVs including single copy losses and gains, focal amplifications, and homozygous deletions across each TNBC genome. Finally we generated whole exome data in a subset of samples to increase the resolution within loci of interest and to incorporate mutations of individual genes into our genomic signatures. This combined approach provides high resolution measures of TNBC genomes from ploidy, whole chromosome and chromosome arm level CNVs, focal amplicons, breakpoints and homozygous deletions, to the level of gene specific indels and mutations. In parallel whole tissue samples were screened by IHC for PD-1 and PD-L1 using validated antibodies and established scoring methods for staining of tumor and non-tumor cells. Results and Conclusions: High levels of PD-1 and PD-L1 were detected in 5/60 (8.3%) and 16/60 (26.7%) evaluable cases with staining of PD-1 exclusively on non tumor cells, while PD-L1 was primarily on tumor cells; 15/16 (93.8%) cases. These data were then used to test the associations of individual recurring genomic lesions, and the extent and nature of chromosome aberrations on the expression patterns of PD-1 and PD-L1. Homozygous deletion of PTEN or activating mutation in PIK3CA did not correlate with increased expression of either immune checkpoint regulator in TNBC cells. Furthermore tumors with highly aberrant aneuploid genomes and distinct oncogenic drivers frequently express relatively low levels of both proteins suggesting an intrinsic escape from immunosurveillance. Citation Format: Barrett MT, Lenkiewicz E, Malasi S, Yearley JH, Annamalai L, McCullough AE, Anderson KS, Pockaj BA. Genomic lesions and PD-1/PD-L1 expression in resected triple negative breast cancers [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 P2-07-05.

Abstract Introduction: The programmed cell death-1 (PD-1) immune checkpoint is a normal regulator of the autoimmune response to inflammatory stimuli. Several cancers have taken advantage of this mechanism by harboring its ligand (PD-L1), thus activating the checkpoint to evade immune destruction. Increased expression of PD-L1 on tumor cells has been linked with worse outcomes in several cancers, including breast cancer. In triple-negative breast cancer (TNBC), expression of PD-L1 is increased compared to luminal cancers and as such, has become a favorable target for anti-tumor therapies. However, few studies evaluate the impact of PD-1 and PD-L1 expression on overall survival (OS) in TNBC. The aim of this study was to assess OS based on PD-1 and PD-L1 positivity and distribution in the tumor microenvironment. Methods: All patients with newly diagnosed TNBC from 1999 to 2015 with adequate pathologic specimen were identified for evaluation. Surgical pathology specimens were stained by immunohistochemistry (IHC) for PD-1 and PD-L1. Those considered PD-1+ or PD-L1+ had a staining score of 3, 4, or 5. Patient and clinical characteristics of the adjuvant group were compared by PD-1 and PD-L1 status with Chi-Square and ANOVA, as appropriate; characteristics of the neoadjuvant population were described. OS was calculated using Kaplan-Meier method and compared by Log Rank test for PD-1, PD-L1 positivity and tumor microenvironment status. Results: Fifty-two patients met inclusion; 38 had surgery prior to chemotherapy while 14 had neoadjuvant chemotherapy. There were 15 PD-1+ patients (39%) and 21 PD-L1+ (55%) patients in the treatment naive cohort. No significant difference was found in the treatment naive group between age, race, postmenopausal status, final stage, or pathologic characteristics (e.g. tumor size, mitotic rate, and grade) for all PD-1+ and PD-L1+ patients. Of the 21 PD-L1+ patients, staining was present in the tumor, tumor/non-tumor (mixed), and non-tumor microenvironment 42.9%, 33.3%, and 23.8% of the time, respectively (p=0.008). No significant difference was found in OS between PD-1+ and PD-1- patients (p=0.509) or PD-L1+ and PD-L1- patients (p=0.240). However, distribution of PD-L1 staining significantly associated with OS. Those demonstrating PD-L1 staining in the tumor had a significantly higher OS than those with PD-L1 staining in the non-tumor microenvironment (p=0.008). In the subset of PD-L1+ patients, those with staining in the tumor show a 10-year OS of 100% versus 60% for those with staining in non-tumor (p=0.006). In the neoadjuvant cohort, 14.3% of patients expressed PD-1 positivity, while 42.9% had PD-L1 positivity; the distribution of the PD-L1 staining in tumor 14%, mixed 29%, and non-tumor 50%. No difference in OS was found based on PD-1 or PD-L1 (p=0.660) positivity in the neoadjuvant cohort. Conclusion: A large number of TNBC stain for PD-1 and/or PD-L1. The implications of this are yet to be determined. In this small cohort, pattern of staining had a significant impact on overall survival in treatment naïve patients. This finding will need to be validated on a larger cohort but may have important implications for treatment by defining patients who may benefit by checkpoint inhibitor therapy. Citation Format: Linnaus ME, Kosiorek H, Barrett MT, Dueck A, Anderson KS, Ocal IT, McCullough AE, Annamalai L, Yearley JH, Pockaj BA. Effect of PD-1 and PD-L1 in the tumor microenvironment on overall survival of triple-negative breast cancer patients [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr PD5-03.