Patient Age Associates with Tumor-Inltrating Lymphocytes Density in Breast Cancer

Background: Lymphocytes surrounding the cancer participate in tumor-related immune responses, and are called tumor-inltrating lymphocytes (TILs). Several recent reports suggest TILs to index the tumor-microenvironment (TME), and predict the therapeutic effect of chemotherapy. However, only few studies have studied the relationship between age and TILs. Aging reduces host immunity, and we predict that it may also affect TILs. Thus, we hypothesized that older breast cancer (BC) patients may have low TILs density than younger BC patients. Here, we retrospectively analyzed the differences in TILs by age and the therapeutic effects of pre-operative chemotherapy (POC) in BC patients aged less than 45 years and more than 60 years. Methods: POC was administered to 356 patients. We conrmed and compared TILs density and therapeutic effects in patients aged < 45 years (n=75) and those aged >61 years (n=116). TIL density was evaluated using pre-treatment needle biopsy specimens. Denition and evaluation of TILs was based on the International TILs Working Group 2014. Results: Based on subtype, younger patients showed signicantly higher pathological complete response rates than older patients with hormone receptor (HR)+ human epidermal growth factor receptor 2 (HER2)+ and HER2-enriched BC. In HR+HER2+BC, the objective response rate was signicantly high in younger than in older patients. Further, a signicant difference was observed for overall survival between these patients with triple-negative BC. Conclusions: Our study suggests that younger BC patients possess signicantly high TILs density than older patients. These differences may inuence the therapeutic ecacy in highly immunogenic subtypes.


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TILs density was evaluated using pretreatment specimens obtained by CNB or VAB. The TILs were de ned and evaluated based on the International TILs Working Group 2014 [1] as the average of the in ltrating lymphocytes within the tumor stroma at ve randomly selected elds. Next, the results were classi ed into four class (3: >50%; 2: >10-50%; 1: ≤10%; or 0: absent) ( Supplementary Fig. S1). Further, we de ned the scores 2 and 3 as "High", and scores 1 and 0 as "Low" according to previous reports [26,27]. Thus, in brief, the cut-off value of TILs density was set to 10%.

Statistical analysis
All statistical analyses were performed using the JMP software package (SAS, Tokyo, Japan). The distribution of TILs density by age was evaluated using Student's t-test. The Pearson's chi-square test was used to evaluate the relationship between each factor. Prognostic analyses, such as DFS or OS, were examined using the Kaplan-Meier method and log-rank test. The hazard ratio (HR) and 95% con dence interval (CIs) were calculated using the Cox proportional hazards model. Multivariable analysis was performed using the Cox regression model. A P-value < 0.05 was considered to be statistically signi cant.

Clinicopathological features of BC patients
The clinicopathological features of patients (n=356) treated with POC have been summarized in Table 1. The patients were operated at median age of 55 years (range, 24-78 years) and the median tumor diameter 28.7 mm (range, 9.2-119.8 mm). Skin in ltration was observed in 58 patients (16.3%). Further, imaging methods of diagnosis did not indicate lymph node metastasis in 121 patients (34.0 %). The number of ER-negative, PgR-negative, and HER2-positive patients was 187 (52.5 %), 242 (68.0 %), and 125 ( Further, while most of the clinicopathological factors were not signi cantly different, the rate of skin in ltration and PgR-negative status were signi cantly higher in the older that in the younger patients (P=0.002 and P=0.003, respectively) ( Table 2). Moreover, the ORR, although statistically insigni cant, was found to be higher in the younger than in the older patients (P=0.091). Correlation of TILs density with clinicopathological features and prognosis of patients First, the 356 patients were divided into high and low TILs density groups, and their correlation with clinicopathological factors was examined (Supplementary  Table S1). Following characteristics were observed in the low TILs than the high TILs group: ≥ 45 years (P = 0.008), skin invasion (P = 0.001), ER-positive (P <0.001), PgR-positive (P <0.001), HER2-negative (P = 0.011), Ki67-high (P <0.001), low ORR (P = 0.001), and low pCR rate (P <0.001).
Further, the patients were classi ed based on age as < 45 years, 46-60 years, and ≥ 61 years, and the distribution of TILs density was analyzed using t-test ( Fig. 1). Our analysis did not indicate signi cant difference in HR+ BC for any of the age groups. However, in HER2-enriched BC, the patients aged < 45 years had signi cantly higher TILs density than patients in other age groups (vs. 46-60 years: P = 0.002, and vs. ≥ 61 years: P = 0.018). Furthermore, in the TNBC category, the patients aged ≥ 61 years had signi cantly higher TILs density than patients in other age groups (vs. ≤ 40 years: P = 0.035, and vs. 46-60 years: P = 0.047).

Examination of clinicopathological factors and prognosis in the younger and older BC patients
First, we studied the correlation between TILs density and clinicopathological factors in the younger and older patients ( Table 2). Although patients aged 46-60 years were excluded from the analysis, the characteristics of the high TILs density group were similar to those for all patients: > 60 years (P = 0.047), skin in ltration (P = 0.001), ER-positive (P <0.001), PgR-positive (P = 0.001), HER2-negative (P = 0.002), lower ORR (P = 0.002), and lower pCR rate (P <0.001).
Further, younger patients showed signi cantly higher pCR rates than older patients in the HR+HER2-and HER2-enriched BC category (P = 0.021 and P = 0.048, respectively) (Table 3). Moreover, in HR+HER2+BC, the responder rate for ORR was signi cantly higher in the younger patients than in older patients (P = 0.009). However, no signi cant difference was observed in the effect of POC on TNBC.   Next, when DFS was compared between the younger and older patients, no signi cant difference was found overall or in any subtype (Fig. 2). Moreover, our analysis indicated that age or TILs was not a predictor of DFS in the univariate analysis (P = 0.619 and P = 0.066, respectively) ( Table 4). Although upon comparison of OS, a signi cant difference was observed between younger and older patients with TNBC (P=0.039, log-rank) (Fig. 3), the results were contrasting and suggested better OS in older patients than in younger patients. Additionally, in univariate analysis with OS, no signi cant difference in age and TILs density was observed (P = 0.346 and P = 0.216, respectively) ( Table 5).

Discussion
The characteristics of BC in the older patients have been often reported. For example, large tumor size [13,[28][29][30], frequent skin in ltration [29,31], infrequent lymph node metastasis [28,30], high rate of HR positivity [13,28], and fewer HER2-positive tumors [28 -30] have been reported in older patients. The clinicopathological characteristics of older BC patients in our study shows strong correlation to the decision of administering POC or not, though some features similar to those reported by others were identi ed.
While age-related differences in pCR rates have not been reported in several clinical trials, a pooled analysis observed high pCR rate in younger BC patients [14]. Moreover, reports suggest that the pCR rate decreased with age [10,13]. Analysis of BC based on subtype in these studies suggested a strong correlation between HR+HER2-and TNBC, whereas no signi cant difference with age in HER2-positive BC, which differed in our study, and the exact reason remains to be identi ed. Further, there are various molecular subtypes of TNBC, and their age at onset and pCR rates have been observed to differ across studies [32][33][34].
We anticipate that our analysis may have been affected by differences in molecular subtypes of TNBC, or due to differences in the chemotherapy regimen.
Furthermore, reports suggest that the expression of androgen receptor (AR) increases with age in BC patients [35][36][37], and that the AR-positive cases show low pCR rate than the AR-negative cases [38]. Additionally, newer biomarkers may also affect these outcomes.
Moreover, von Waldenfels et al. have reported that prognosis worsens with age in BC patients [13]. However, their study observed signi cant differences in prognoses between patients aged ≥65 years and those aged 40-50 or 51-65 years, but no signi cant difference between patients aged ≥65 years and those aged <40 years. Furthermore, studies reporting higher pCR rate in younger patients did not observe a signi cant difference in prognosis in TNBC [14]. In contrast, studies reported more than 10 years back suggest poor prognosis [39][40][41], and aggressive cellular properties in the younger BC patients [39,[42][43][44].
AR expression also affects prognosis and may contribute [38]. Additionally, with advent of newer biological treatments, the number of clinical trials claiming prognosis to differ with age have decreased.
Here, when we studied TILs at all ages, we observed correlation between TILs and clinicopathological factors, treatment effects, and prognosis similar to those reported previously. Moreover, our analysis suggests that younger BC patients had signi cantly higher TILs density than older BC patients. Additionally, agerelated ORR and pCR rates differed in HER2-positive BC. Moreover, a pooled analysis for TNBC alone reported that the older patients had signi cantly lower TILs than in younger patients [45]. This result can be attributed to the decrease in host immunity due to aging, and to the inherent cellular characteristics of BC that vary with age.
However, this study has a limitation that the criteria for dividing patients into younger and older patients was not well-de ned, and that the clinicopathological factors, other than TILs density, differed with age. However, the change in TME with age suggests that it may have in uenced the therapeutic effect due to the characteristics of the host's immune system, and the differences in cancer itself depending on the age. Additionally, in lung cancer, it has been reported that the therapeutic effect of the immune checkpoint inhibitors (ICIs) decreases in the older patients [46-48]. Therefore, age may also serve as an important clinical factor in deciding the course of treatment of BC patients with ICIs.

Conclusions
The analysis presented in this study suggests that younger BC patients show signi cantly higher TILs density than older patients, along with differences in prognoses between the groups. Moreover, these differences may allow selection of better treatment modalities for the highly immunogenic subtypes of BC.

Declarations
Ethics approval and consent to participate A written informed consent to participate in the study was obtained from each subject in accordance with the declaration of Helsinki principles. Each patient or the patient's family was fully informed of the investigational nature of this study and provided their written, informed consent. The study protocol was approved by the Ethics Committee of Osaka City University (approve number #926).

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.

Funding
This study was supported in part by Grants-in-Aid for Scienti c Research (KAKENHI, Nos. 17K10559, 19K18067 and 20K08938) from the Ministry of Education, Science, Sports, Culture and Technology of Japan.

Authors' contributions
KT participated in the design of the study and drafted the manuscript. SK participated in the design of the study and manuscript editing. YA, WG, and TM helped with study data collection and manuscript preparation. MS, HT, KH and MO conceived the study, and participated in its design and coordination and helped to draft the manuscript. All authors have read and approved the nal manuscript. Figure 1 Correlation of TILs density with age of BC patients. Patients were grouped based on their BC subtype as: a) all cases, b) HR+HER2-, c) HR+HER2+, d) HER2enriched, and e) TNBC. The TILs density in each age-group in each subtype has been indicated using box-plot distribution analysis. P-values in the gure indicate statistical signi cance for each comparison obtained using t-test