Shachner, Tracy R.

Abstract Background: Tertiary lymphoid structure (TLS) is an organized form of ectopic lymphoid aggregates (LA) with secondary lymphoid organ structure. Several studies showed that the presence of TLS associates with improved outcomes in multiple cancers when treated with immune checkpoint inhibitors. However, data is currently limited regarding TLS and outcomes in HER2+ breast cancer patients (pts) treated with adjuvant trastuzumab. Furthermore, distinguishing TLS and simple LA is challenging in standard hematoxylin and eosin (H&E) staining, particularly when the germinal center is absent. Emerging studies also showed the prognostic value of MHC expression in breast cancer but mainly in triple-negative breast cancer. In this study, we evaluated integrated pathological quantification and genomic data to assess functional TLS in association with MHC expression and outcomes in the N9831 trial. Methods: Pathological evaluation of LA in H&E slides from pts treated in Arm A (chemotherapy alone) and Arm C (chemotherapy with concurrent trastuzumab) in N9831 was performed. NanoString was used to quantify mRNA of MHC class I and II expression as well as TLS-related immune genes, including IFNG, ICOSLG, CXCL13, CXCR3, BCL6, IL21R, ICOS, PDCD1, CXCR5, CXCL9, TBX21, CD38, CXCL10, CLXCL11, IL21, CD200, CD19, MS4S1. Wilcoxon rank sum test, Chi-squared test, Kaplan-Meier method, and Cox regression model were used to evaluate the association between LA and baseline characteristics, MHC expression, and outcomes. Results: LA was evaluated in 526 pts in Arm A and 485 in Arm C. Greater number of LA was significantly associated with stromal tumor-infiltrating lymphocytes (sTILs), hormone receptor negative, and higher tumor grade, but not stage and age. Using multivariable Cox regression analysis, increasing numbers of LA were associated with improved recurrence-free survival in both arms combined (RFS, p 0.028). However, when evaluating each arm separately, LA was associated with improved RFS only in Arm A (HR 0.6, 95%CI 0.43-0.84, p 0.003) but not in Arm C (HR 0.72, 95%CI 0.47-1.1, p 0.134). We further evaluated TLS-related immune genes in 252 pts in Arm C with LA ≥ 1. As a continuous variable, higher expression of BCL6 (HR 0.61, 95%CI 0.41-0.92, p 0.019) and IL21R (HR 0.78, 95%CI 0.62-0.98, p 0.03) were associated with improved RFS in Arm C pts with LA ≥ 1. However, these genes were not significantly associated with outcomes in Arm C pts without LA with BCL6 (HR1.07, 95%CI 0.67-1.71, p 0.776) and IL21R (HR 0.96, 95%CI 0.71-1.29, p 0.775). Moreover, we evaluated differential gene expression between tumors with LA ≥ 1 with high BCL6 vs. LA 0. All MHC class I and II (HLA A, B, C, E, DQ, DM, DO, DP, and DQ) were significantly higher in tumors with LA ≥ 1 with high BCL6 (p < 0.001). Conclusion: A greater number of LA was associated with improved outcomes in pts with early-stage HER2+ breast cancer, particularly when treated with chemotherapy alone. Using histologic and genomic integration with the combination of pathological LA and TLS-related immune genes, we identified that pts with functional TLS with LA ≥ 1 and higher expression of BCL6 or IL21R had significantly improved outcomes when treated with trastuzumab. High MHC class I and II expressions are associated with the presence of functional TLS, underscoring the crucial role of antigen presentation in the generation of an effective adaptive immune response. Support: U10 CA180821, U10 CA180882, U24 CA196171, https://acknowledgments.alliancefound.org; Genentech; Clinicaltrials.gov Id: NCT00005970 Citation Format: Saranya Chumsri, Tracy Shachner, Zhuo Li, Nadine Norton, Alvaro Moreno-Aspitia, Gerardo Colon-Otero, Edith Perez, Aziza Nassar, E. Thompson, Keith Knutson. MHC Class I and II Expression, Functional Tertiary Lymphoid Structure (TLS), and Outcomes in Early-Stage HER2-Positive (HER2+) Breast Cancer in NCCTG N9831 (Alliance) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-25-04.

AbstractBackgroundCutaneous metastases from non‐cutaneous neuroendocrine neoplasms are rare; however, distinction from primary neuroendocrine carcinomas of the skin (Merkel cell carcinoma) guides clinical management.MethodsWe performed a retrospective review from September 1, 2010 to September 30, 2020 of the histopathologic, immunohistochemical, and clinical characteristics of metastatic neuroendocrine neoplasms to the skin from non‐cutaneous primaries.ResultsFourteen patients were identified for the study (nine males and five females; mean age of 59.5 years). Fifteen skin specimens from 14 patients were available for review. At the time of skin biopsy, a known non‐cutaneous neuroendocrine neoplasm was present in 50% of patients. Primary sites of neuroendocrine carcinoma included lung (n = 5), terminal ileum (n = 2), and one each from prostate, breast, rectum, uterus, esophagus, and sinus, with one unknown (suspected bladder malignancy). Eleven of fourteen patients are dead of disease; one was lost to follow‐up. All 15 specimens showed subcutaneous/deep dermal involvement with six involving the papillary dermis and one involving the epidermis. The tumors ranged from well to poorly differentiated. Two of fifteen specimens showed focal CK20 positivity (one metastatic uterine small cell carcinoma and one metastatic ileal carcinoid). TTF‐1 was performed in 13 specimens and was positive in six, of which two were of non‐pulmonary origin.ConclusionsWhile immunohistochemical stains, in particular CK20, CK7, and TTF‐1, are integral in the workup of confirming the origin of neuroendocrine tumors found in the skin, results vary and are often non‐specific for a single primary site. Therefore, complete radiologic imaging as well as clinical correlation should be recommended to further aid in the identification of a non‐cutaneous primary neoplasm.

Abstract Background: Previous studies showed conflicting results regarding association between stromal tumor infiltrating lymphocytes (sTIL) and outcome in HER2+ breast cancer. While sTIL were not associated with outcome in patients (pts) treated with trastuzumab in N9831, immune functions genes are linked to outcome in these pts. TLS is an organized form of ectopic lymphoid aggregates (LA) with secondary lymphoid organ structure. Several studies showed that TLS associates with improved outcome in multiple cancers. However, distinguishing TLS and simple LA is challenging, particularly when germinal center is absent. In this study, we evaluated integrated pathological quantification and genomic data to assess functional TLS. Methods: Pathological evaluation of LA in H&E slides from pts treated in Arm A (chemotherapy alone) and Arm C (chemotherapy with concurrent trastuzumab) in N9831 was performed. NanoString was used to quantify mRNA. Wilcoxon rank sum test, Chi squared test, Kaplan-Meier method and Cox regression model were used to evaluate association between LA and baseline characteristics as well as outcomes. Results: LA was quantified in 1011 pts (526 Arm A, 485 Arm C). Greater number of LA was significantly associated with higher tumor grade, ER/PR negativity and increasing sTIL, but not age, tumor size or lymph node status. Increasing numbers of LA were associated with improved RFS in both arms combined (p 0.028). However, using multivariable Cox regression analysis in each treatment arm, LA (≥ 1 vs 0) was associated with improved RFS only in Arm A (HR 0.6, 95%CI 0.43-0.84, p 0.003) but not Arm C (HR 0.72, 95%CI 0.47-1.1, p 0.134). Further evaluation of expression of TLS-related immune genes including IFNG, ICOSLG, CXCL13, CXCR3, BCL6, IL21R, ICOS, PDCD1, CXCR5, CXCL9, TBX21, CD38, CXCL10, CLXCL11, IL21, CD200 was carried out among 252 pts in Arm C with LA ≥ 1. Among these 16 TLS-related immune genes, as a continuous variable, higher expression of BCL6 (HR 0.61, 95%CI 0.41-0.92, p 0.019) and IL21R (HR 0.78, 95%CI 0.62-0.98, p 0.03) were associated with improved RFS in Arm C pts with LA ≥ 1. However, these genes were not significantly associated with outcome in Arm C pts without LA with BCL6 HR1.07 (95%CI 0.67-1.71, p 0.776) and IL21R HR 0.96 (95%CI 0.71-1.29, p 0.775). Conclusion: Similar to sTIL, greater number of LA was associated with improved outcome in HER2+ pts treated with chemotherapy alone but not chemotherapy in combination with trastuzumab. Using histogenomic integration with the combination of pathological LA and TLS-related immune genes, we identified that pts with functional TLS with LA ≥ 1 and higher expression of BCL6 or IL21R had significantly improved outcome when treated with trastuzumab. Future studies are needed to further confirm these findings. Support: U10 CA180821, U24 CA196171, https://acknowledgments.alliancefound.org; Genentech; Clinicaltrials.gov Id: NCT00005970 Citation Format: Saranya Chumsri, Tracy R. Shachner, Zhuo Li, Nadine Norton, Alvaro Moreno-Aspitia, Gerardo Colon-Otero, Edith A. Perez, E. A. Thompson, Aziza Nassar, Keith L. Knutson. Functional tertiary lymphoid structure (TLS) and outcome in HER2-positive (HER2+) breast cancer in NCCTG N9831 (Alliance) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5627.

535 Background: Invasive lobular carcinoma (ILC) is a rarer form of breast cancer, accounting for 10% of the disease cases. HER2 overexpression in ILC is infrequent and limited data exist regarding clinical characteristics and outcome of HER2-positive (HER2+) ILC patients (pts) treated with adjuvant trastuzumab. Methods: Patient characteristics were compared between ILC and invasive ductal carcinoma (IDC) using Wilcoxon rank sum test for continuous variables and Chi-square test for categorical variables. Kaplan-Meier (KM) method was used to estimate the freedom from mortality and recurrence. Cox regression model was used to evaluate the association between ILC and outcomes adjusted for other characteristics. NanoString technology was used to quantify mRNA to develop immune-related gene signatures. Results: From a total of 3,304 pts, 122 (3.7%) pts had ILC. Pts with ILC were significantly older (median age 54 vs. 49 years), had larger tumors, lower grade, more ER and PR positive tumors, and more lymph node involvement (25.4% had N3 disease compared to 12.7% in IDC). Overall, with KM analysis, pts with ILC had significantly worse overall survival (OS, p = 0.005) and recurrence-free survival (RFS, p = 0.046) compared to IDC. The 15-year freedom from recurrence was merely 57.67% in ILC compared to 72.68% in IDC. A significant number of hormone receptor-positive (HR+) ILC pts developed late recurrence with cumulative event rates increasing from 23% at 5 years to 42% at 15 years. Nevertheless, in multivariate Cox regression analysis adjusting for other clinical characteristics, including age, tumor size, grade, ER/PR, and lymph node status, lobular histology was not significantly associated with worse outcome for OS (HR = 1.19, 95%CI 0.67-2.1, p = 0.55) and RFS (HR = 1.5, 95%CI 0.9-2.5, p = 0.12), as compared with IDC. However, ILC pts appeared to have similar degree of benefit from trastuzumab, with RFS HR = 0.58 compared to HR = 0.67 in the entire population. For immune landscape, there was no significant difference in gene signatures related to CD45, CD8, B cells, or cytotoxic cells. However, ILC had more enrichment in mast cell gene signature and fewer macrophage, NK CD56dim, and regulatory T cell signatures compared to IDC (p < 0.05). Conclusions: HER2+ ILC has distinct clinical characteristics and immune landscape compared to IDC. ILC pts appeared to have worse outcome compared to IDC likely because ILC pts often presented with more locally advanced disease. However, similar benefit of trastuzumab was observed in ILC pts. Due to high risk of late relapse in HR+ HER2+ ILC, extended adjuvant endocrine therapy should be considered in this group of high-risk pts. Clinical trial information: NCT00005970.