Abstract P3-05-09: LAG3+ Tumor Infiltrating Lymphocytes Predict Outcome in Treatment Naïve Triple Negative Breast Carcinoma
Authors: Bose Shikha , Wang Yizhou , Ramanujan V Krishnan , Walts Ann E. 
Published: March 2023

Significance: Triple negative breast carcinoma (TNBC), characterized by lack of estrogen (ER) and progesterone receptors (PR) and absence of Her2/neu (Her2) receptor amplification, typically follows an aggressive course that includes high relapse rates, rapid progression, and poor outcome. Advances in treatment have been limited by molecular heterogeneity within TNBCs and lack of effective molecular targets. Although a subset of TNBC associated with tumor infiltrating lymphocytes (TILS) is more immunogenic than other breast cancers, response to immunotherapy has been variable and reported in only 10-40% of cases. Identification of prognostic immune biomarkers will identify novel therapeutic targets and facilitate appropriate patient selection for therapy. Aim: To analyze the immune gene expression profile in TNBC and identify a prognostic marker. Methods: Targeted mRNA sequencing was performed on formalin fixed paraffin embedded whole sections from 30 treatment naïve TNBC with TILs (15 cases with a favorable outcome (recurrence free overall survival (OS) ≥5yrs) and 15 with unfavorable outcome (never disease free or dead of disease in &lt; 5yrs) using the HTG Edge Seq Assay (HTG Molecular Diagnostics, Inc. Tucson AZ) to analyze the differential expression of 1392 immune related genes in their Precision Immuno-Oncology Panel. The top 40 up- and down- regulated DE genes with adjusted p-value of &lt; 0.001 were then validated against the TNBC mRNA profiles in The Cancer Genome Atlas (TCGA) database. LAG3 was one of four genes that showed significant differential expression in both data sets. We used immunohistochemistry (IHC) (Rabbit monoclonal AntiLAG3 antibody, Abcam plc.) to analyze LAG3 expression in whole sections from 57 consecutive TNBCs where tumor excision had been the first line treatment. All patients were female, age 29-89 years (median 55 yrs). The infiltrating ductal carcinomas varied from 0.6 to 9.0 cm (median 2.6 cm). All tumors were ER-, PR-, Her2- with a median Ki67 of 42%. 26 patients had metastatic carcinoma in 1-17 lymph nodes (median 2). Two patients had metastatic disease at presentation. Disease free survival (DFS) varied from 0 to 114.8 months (median 81.4 mos.), and overall survival (OS) varied from 7.6 to 114.8 months (median 81.5 mos.). LAG3 expression was categorized as negative (0), low (1+) and high (2+). The student t-test was used to correlate LAG3 expression with DFS and OS. Results: Differential expression analysis from our data set yielded 222 statistically significant differentially expressed genes. Validation against the TCGA TNBC data set revealed 4 common genes, one of which is LAG3. Only TILS showed LAG3 expression by IHC: 10 cases no expression, 17 low expression and 29 high expression. Median DFS and OS in TNBC with no LAG3 expression was 32.3 and 55.2 months, in TNBC with low expression 83.3 and 81.1 and those with high expression was 82 and 82.1 month. Median DFS and OS in LAG3 negative cases was significantly different from those in LAG3 positive cases (p=0.038, p=0.013) and between LAG3 0 and LAG3 2+ positive cases (p=0.002, p=0.0018). No difference in either median DFS or OS was seen when LAG3 1+ and LAG3 2+ cases were compared. Conclusions: 1. LAG3 is a prognostic marker for TNBCs. 2. LAG3 is expressed only on TILS and shows heterogenous expression. 3. TNBC with no LAG3 expression had a median survival of 32 months, suggesting they might benefit from more aggressive therapy than TNBC that express LAG3. 4. Confirmation of these results requires a larger TNBC cohort.

Citation Format: Shikha Bose, Yizhou Wang, V Krishnan Ramanujan, Ann E. Walts. LAG3+ Tumor Infiltrating Lymphocytes Predict Outcome in Treatment Naïve Triple Negative Breast Carcinoma [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-05-09.