Racial/Ethnic Differences Among Tumor Inltrating Lymphocytes in Breast Cancer Tumors

Purpose Tumor inltrating lymphocytes (TILs) have emerged as a predictor of cancer treatment response and patient outcomes, including for breast cancer. Current studies investigating racial/ethnic differences in TILs and immune proles in breast cancer offer varying results. Our study hopes to address the paucity of data in breast cancer tumor microenvironment from different racial/ethnic groups not well represented in the literature. Methods We reviewed 183 cases of women diagnosed with early stage breast cancer who received neoadjuvant treatment at two large health systems in Hawaii between 2008 and 2020. We evaluated clinical and demographic information including: age at diagnosis, race/ethnicity, tumor stage, tumor subtype according to ER, PR, and HER2 receptor status and compared them with obtaining a pathologic complete response (pCR) and amount of stromal TILs (sTILs). We found a signicantly greater amount of sTILs in Asians (37.7%, p=0.01) and Native Hawaiian/Pacic Islander (NHPI) (37.2%, p=0.02) patients compared to White patients on multivariate analysis. We found no signicant differences in pCR among the different racial/ethnic groups.


Introduction
The tumor microenvironment, speci cally tumor in ltrating lymphocytes (TILs) in breast cancer, have been gaining more attention. TILs incorporate a heterogeneous composition of T-cells, B-cells, and other lymphocytes, and it is an emerging biomarker that is both predictive of cancer treatment response and prognostic of cancer outcomes [1]. Certain breast cancer subtypes--triple negative (TN, ER-/PR-/HER2-) and HER2+--which are known to have higher stromal TIL (sTIL) amounts, have also been associated with improved prognosis and response to chemotherapy [2] which can aid clinicians in predicting response to neoadjuvant chemotherapy. Speci c TILs, namely CD8+ T cells, are associated with an increase in obtaining a pathologic complete response (pCR) after neoadjuvant therapy, regardless of breast cancer subtype [3].
Differences in TILs among various racial/ethnic groups have been evaluated. The Women's Circle of Health Study examined differences in TILs, particularly CD8+ T cells, in breast tumors between Black and White women and found that Black women with TN breast cancer and high CD8+ T cell density were found to have better overall survival than patients with low CD8+ T cell density [4]. Another study in HER2+ breast tumors showed the presence of CD8+ T cells was associated with a decrease in mortality [5]. Chen  We examined sTILs in early stage breast tumors who had received neoadjuvant treatment from two major health systems in Hawaii from 2008-2020. Our study hopes to address the paucity of data in breast cancer tumor microenvironment from different racial/ethnic groups not well represented in the literature.

Methods
This study evaluated 183 cases of early stage breast cancer who received neoadjuvant treatment from two large health systems from 2008-2020. A retrospective chart review was performed along with pathological evaluation from core biopsies and subsequent surgical excisions. sTILs were determined according to recommendations proposed by the International Immuno-Oncology Biomarker Working Group in 2017, separating the percent of stromal and intratumoral TILs [7,8]. Diagnostic core biopsies stained with hematoxylin and eosin were evaluated for stromal lymphocyte in ltration. Areas evaluated include central tumor stroma and stroma at the invasive margin. Tumor cell areas, areas of necrosis and scarring were not included. Lymphocytes and plasma cells were included while neutrophils were excluded. Cases were reported as "percent stromal lymphocytes".
A clinical chart review was performed on the neoadjuvant breast cancer cases and included demographic and clinical information: age at diagnosis, self-reported ethnicity/race, tumor subtype as well as treatment history: neoadjuvant/adjuvant therapies, including chemotherapy, HER2 directed therapy, endocrine therapy, surgery, radiation. Patient charts were also reviewed for recurrence of breast cancer and death.

This study was reviewed and approved by the Hawaii Paci c Health and Queens Medical Center
Institutional Review Boards for human subjects research. Statistical analysis was performed using the SAS 9.4 (SAS Institute Inc., Cary, NC) software. The REG procedure applied linear regression analyses on the continuous TILs (percentage tumor in ltrating lymphocytes) outcome. The predictor variables were race, age, subtype and health system. For the HPH subset analysis the variables included were: race, age, BMI, stage, chemotherapy, subtype and pCR. We ran both univariable (unadjusted) and multivariable (adjusted for other predictors) models.

Results
A total of 183 cases of early stage breast cancer from patients who received neoadjuvant treatment were evaluated. The most commonly represented ethnicity was Asian with 72 cases (39.3%), followed by Native Hawaiian/Paci c Islander (NHPI) with 53 cases (29%), White with 48 cases (26.2%), and Other with 10 cases (5.5%) (Table1). The most common subtype was ER-/PR-/HER2-with 62 cases (33.9%), followed by ER-/PR-/HER2+ with 56 cases (30.6%), ER+ or PR+/HER2+ with 50 cases (27.3%), and ER+ or PR+/HER2-with 15 cases (8.2%). The ethnicity with the most predominant proportion of cases with a pCR was Other with 5 cases (50.0%), followed by NHPI with 26 cases (49.1%), Asian with 29 cases (40.3%), and White with 18 cases (37.5%) ( Table 1).  Table 2). On univariable analysis, signi cantly lesser percentages of sTILs were seen among women between the ages of 60-69 years, however on multivariable analysis there were no signi cant differences found according to patient age ( Table 2). With both univariable and multivariable analysis, there were signi cantly greater percentages of sTILs found among women who had ER-/PR-/HER2+ or TN breast cancer subtypes ( Table  2).  Percentage of pCR in breast cancer tumors of all patients based on age at diagnosis, race, and breast cancer subtype were evaluated (Table 4). In both univariable and multivariable analyses, we did not see signi cant differences in pCR among racial/ethnic groups studied (Asian, NHPI and Other) compared to White women. On univariable analysis, there was a signi cantly less likelihood of a pCR among women between the ages of 60-69 years compared to other age groups; however there were no signi cant differences found among all ages on multivariable analysis. In both univariable and multivariable, there was a signi cantly higher likelihood of obtaining a pCR among women who had ER-/PR-/HER2+ breast cancer (3.02 and 3.06 vs 0.6 and 0.59, p=0.01 and p=0.02 respectively) compared to ER+/PR+/HER2breast cancer (Table 4).  [12]. In contrast, Abdou et al. found breast cancer tumors from Black patients had higher CD8+ T cell tumor in ltrating densities than in White patients [4]. In previous studies, Black breast cancer patients had a higher amount of macrophage in ltrating tumors with a high number of CD163+ macrophages and increased macrophage proliferation [13]. Carrio et al. showed CD68+ macrophages were signi cantly more abundant in tumors from Black and Latina patients but showed no differences in overall survival when compared to White patients [14]. Another study found higher levels of Proliferating Cell Nuclear Antigen positive (PCNA+) tumor-associated macrophages and shorter disease-free survival in Latina patients compared to White patients [15]. Given the poor prognosis seen among the NHPI breast cancer patients [16], characterizing these TILs is an important future step to better understand how the tumor microenvironment may contribute to these disparities.
Some studies have also noticed differences in TIL amount when strati ed for variables other than patient race. In Black women, CD8+ T cell density was seen to be higher in patients who were younger or with hormone-negative high grade tumors [4]. However, in western Kenyan patients, immune cell in ltration density was signi cantly associated with increased tumor proliferation index but not signi cantly correlated with molecular subtype or tumor grade [17]. In another study, no differences in sTIL percentages were observed to be associated with patient age or BMI at diagnosis or tumor stage [9].
Similarly, the literature shows inconsistencies with regards to relationships between TIL count/density and grade/stage of breast tumors across racial groups. O'Meara et al. showed no signi cant difference in TIL amount between Black and White breast cancer patients [12]. However, Abdou et al. showed higher TILs amount can be found in tumors of younger patients, hormone-negative tumors, and high grade tumors in both Black and White patients, irrespective of race [4].
We saw signi cantly lower rates of pCR among women between the ages of 60-69 years compared to other age groups. Although we did not see differences in sTILs according to age in our study, prior data show lower percentages of sTILs among women in this patient age. There are known lower TIL percentages in tumors of older patients [4]. Our ndings support data showing older cancer patients receiving treatment are less likely to have a pathologic complete response irrespective of race/ethnicity. In our subset of patients being analyzed for additional variables, we did not see any differences in sTILs according to BMI at diagnosis, stage or treatment regimen.
Limitations to this study include incomplete patient medical records with variable neoadjuvant treatment data. Although we were able to obtain a subset of cases with complete medical data, we did not see differences when accounting for those additional variables. Although the pathologic TILs evaluations were performed according to the International Immuno-Oncology Biomarker Working Group, the cases were evaluated by two sets of different pathologists, introducing possible variability. This approach however, provides an avenue to broadly incorporate TILs measurement in pathology reports. Our medical center has already routinely reported on sTILs percent and volume for all neoadjuvant TN and HER2+ breast cancer cases. Finally, neoadjuvant cases prior to 2015 are sparse as TN and HER2+ historic treatment strategies did not routinely focus on the neoadjuvant approach.

Conclusions
Racial/ethnic differences in the amount of sTILs in breast cancer tumors suggest that higher sTIL percentages alone do not predict for pCR. Increases in sTILs in Asian and NHPI patients suggest differences in immune cell pro les in the breast tumor microenvironment. This may in part contribute to known outcome disparities in these populations.

Declarations
Author's contributions All authors made substantial contributions to conception and design, analysis and interpretation of data, and critical review of the manuscript. JF conceived of the study, coordinated the data acquisition and analysis, generated tables and gures and helped to draft the manuscript. IP contributed to the statistical methods, data analysis, generated tables and gures and helped to draft the manuscript. AS and SB contributed to the data analysis and helped to draft the manuscript. AT, KV, CW and MF contributed to the data acquisition and analysis and helped to draft the manuscript. All authors read and approved the nal manuscript.

Funding
This work was completed as part of the funding from the University of Hawaii Cancer Center P30 CA071789 Grant from the National Institutes of Health (J. Fukui, I. Pagano). The funding body had no role in the design; in the collection, analysis, or interpretation of data; in writing of the manuscript; or in the decision to submit the manuscript for publication.

Con icts of interest/Competing interest
The authors declare that they have no competing interests.

Ethics approval and Consent to participate
The Institutional Review Boards at the University of Hawaii, Hawaii Paci c Health and Queens Medical Center approved this study. All aspects of the investigation compiled with current rules for research involving Human Subjects.
Availability of data and material The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.