Tumor Infiltrating Lymphocytes Predicting Long -Term Outcomes in HER2-Negative Breast Cancer Patients with Visceral Metastases

Background: Tumor-infiltrating lymphocytes (TILs) were found to be associated with a better clinical outcome in specific subtypes of breast cancer. Aim: To study the association between TILs and the prognosis of Egyptian patients with HER2-negative breast cancer metastatic to the viscera. Methods: This prospective study included 100 patients with HER2-negative metastatic breast cancer. Intratumoral TILs, stromal TILs, and CD4 and CD8 were examined in the pathological specimens and their relationship with survival and response to treatment was studied. Results: At a median follow-up period of 43 months, the median overall survival was 44.7 months (95%CI: 39.2-50.2) and the 5-year overall survival rate was 28%. A high level of CD8+ve TILs was associated with significantly longer overall survival (p<0.001) and progression-free survival (p=0.043). There was no significant correlation between intratumoral TILs, stromal TILs, or CD4+ve and overall survival. Conclusions: A higher level of CD8+ve TILs is associated with better overall as well as progression-free survival in HER2-negative breast cancer with visceral metastases.


Introduction
Breast cancer is a significant public health issue expected to cause more than one million new cases and nearly half a million fatalities annually. It is the most prevalent cancer among women in Egypt, accounting for one-third of all cancer cases in this group. 1 Mortality because of breast cancer is thought to be over 11%, according to the Egyptian registry, making it the second leading cause of cancer-related death after liver cancer. 2 Although therapeutic approaches for breast cancer have improved, there is still a need for new biomarkers to direct treatment in the metastatic scenario. In primary breast cancer, tumorinfiltrating lymphocytes (TILs) were found to be related to prognosis for many years. 3 In a process known as the elimination phase of immune recognition and eradication of malignant cells, CD8+ T cells and CD4+ T cells were discovered by Dunn et al. in 2004 to be the primary components of immune-surveillance related lymphocytes and to be capable of recognizing and eliminating malignant cells. 4 Although it is generally acknowledged that having a high TIL infiltration is associated with a better prognosis, there is much debate over the TIL subpopulations and the observed response. 5 TILs 2 are determined to be T cells in around 75% of cases, and CD8+ T cells are associated with better clinical outcomes. 6 Specifically, type 1 T helper (Th-1) cells from CD4+ T cells are a favorable prognostic indicator and are frequently linked to improved overall clinical outcomes. 6,7 According to Denkert et al., a high number of TILs has drawn significant interest as a biomarker for predicting pathological complete response (pCR) following neoadjuvant chemotherapy. The proportion of intratumoral TILs (iTILs) as a continuous variable was shown to correlate independently with pCR by the same research group. 8 While TILs have a prognostic utility in the adjuvant breast cancer treatment setting, there is a dearth of information in the metastatic scenario. 9 Although iTILs and stromal TILs (sTILs) often correlate, certain studies have discovered a greater connection with one or the other. 8,11 This study aimed to examine the relationship between the infiltration by TILs and the treatment outcome in Egyptian patients with metastatic HER2negative breast cancer.

Methods
This was a prospective observational study that included 100 patients with breast cancer and was conducted at Assuit University Hospitals between January 2015 and January 2021.

Patients
The inclusion criteria were as follows: age between 18 and 70 years, Eastern Cooperative Oncology Group (ECOG) performance scale of 0 to 2, pathologically proven malignant breast cancer positive for HER2, visceral metastases, and scheduling for chemotherapy. Patients with bone as the only metastatic sites were excluded as well as those expected to be non-compliant.

Evaluation of tumor-infiltrating lymphocytes (TILs)
The Pathology Department of Assiut University Hospital provided the paraffin blocks. Histological analysis was done on the hematoxylin and eosinstained slides and the HER2, estrogen receptors (ER), and progesterone receptors (PR) immunostained slides. HER2 stain scores of 0 or +1 were regarded as negative. If ER / PR expression was more than 10%, it was regarded as positive. 10 Sections of 3-4 m thick formalin-fixed paraffinembedded tissue blocks were cut and mounted on positively charged slides. Utilizing antigen retrieval in Tris EDTA, a DAKO automatic immunostainer was employed (pH9). A second kit for envision detection was used. Mayer's hematoxylin was used as a counterstain for the tissue slices.
Stromal TILs were recognized as lymphocytes dispersed throughout the tumoral stroma and do not directly interact with carcinoma cells while Intratumoral TILs (iTILs) are those in tumor nests that have cell-to-cell contact with carcinoma cells. Infiltrating lymphocytes were evaluated and calculated under a microscope at five high-power fields. The median lymphocyte percentages within tumor cell clusters (iTILs) and within the stroma (sTILs) were calculated. Then it was determined how many CD4-positive and CD8-positive lymphocytes were present in iTILs and sTILs.

Treatment and follow up
Patients who had never had chemotherapy were treated with an anthracycline/taxane regimen. Those who already received an anthracycline/taxane regimen before were treated with other protocols. Subtypes of luminal breast cancer were treated with vinorelbine and capecitabine, whereas those with the triple-negative disease were treated with gemcitabine and platinum. Following the completion of systemic chemotherapy, patients with ER/PR-positive disease were offered hormonal therapy. Patients with the triple-negative disease were kept under follow-up

End Point Definition
The primary endpoint was progression-free survival (PFS), which was correlated with the proportion of iTILs and other evaluated factors (sTILs, and CD4 and CD8 positivity). Progression-free survival was defined as the duration from the commencement of treatment until the first sign of a progressing, relapse, or death from any cause.
Secondary end-points included the response to 1 st line chemotherapy for the metastatic disease and overall survival (OS) which was defined as the duration from commencing treatment to death from any cause. The relationship between iTILs and other variables and response to treatment and OS was studied as well.

Statistical Methods
Using IBM SPSS Statistics for Windows, version 22.0. (Armonk, NY: IBM Corp), all data were gathered, tabulated, and statistically analyzed. Continuous variables were expressed as the mean and SD or median and range, and categorical variables as number and percentage. When appropriate, the Pearson Chi-square test or Fisher's exact test was used to compare the distribution of categorical variables. The Kaplan-Meier method was used to estimate time-to-event distributions, and distributions were compared using the two-sided exact log-rank test. All tests were two-sided.
Statistical significance was defined as p-value <0.05.

Results
Ninety-three out of the 100 enrolled patients (93%) met the inclusion criteria and seven patients were excluded because of the inadequate tumor tissue available to complete the full pathological investigations. Patient and tumor characteristics and the results of immunohistochemistry (IHC) studies are illustrated in Table 1.

Discussion
To the best of our knowledge, this study is among the first conducted in Egypt to evaluate the prognostic significance of TILs in patients with metastatic HER2-negative breast cancer.
In this group, a high level of iTILs and sTILs was linked with a better PFS. This finding is consistent with the results of Luen et al. who suggested a link between TILs and survival in cases of advanced HER2-positive breast cancer. 12 Additionally, in the BIG 02-98 adjuvant study, Loi et al. found a relationship between TILs and better OS in patients with triple-negative breast cancer. 13 The CD8 and CD4 positivity of lymphocytes was examined in this study and we found a significant correlation between CD4 and CD8 positivity and PFS. CD8+ve lymphocytes were positively correlated with OS which is concordant with Ma et al. data. 14 Furthermore, our results matched those of Song et al. who suggested that elevated levels of CD8+ve T lymphocytes represent a novel independent predictor of PFS. 15 Mahmoud et al. found that tumor-infiltrating CD8+ve T lymphocytes have antitumor activity as judged by their favorable effect on patients' survival and could potentially be exploited in the treatment of breast cancer. 16 The nodal status and tumor size correlated significantly with CD8+ve cells in our study. This is consistent with the findings of Mahmoud et al. 16 We also found that CD4+ve lymphocytes correlated significantly with age, tumor grade, and PR status. This is supported by the findings of Matkowski et al., who reported a strong association between CD4 expression and lymph node status and lymphatic infiltration intensity. 17 Our findings show a very strong correlation between sTILs and lymphovascular invasion. Additionally, we found a strong association between iTILs infiltration and the presentation of metastatic disease from the start. This is supported by the findings of Miyoshi et al. who described a substantial correlation between TILs and nodal metastasis, ER status, PR status, tumor grade, and Ki67 labeling index. 18 In the current study, there was a significant correlation between iTILs IHC staining and the first site of metastasis, specifically lung metastasis. This is consistent with the finding of Takada et al. that higher iTILs density was associated with lung metastasis, which was the most frequent site of distant metastasis in their study that included ERpositive/HER2-negative patients. 19 This study has limitations that include the small number of patients recruited which is related mainly to the challenges in retrieving the paraffin blocks. Also, it the difficulty in taking a biopsy from distant metastases like small pulmonary nodules to assess the level of TILs in the metastatic site and to compare it with TILs in the primary tumor. This can be assessed in a larger study comparing the differences in the percentage of TILs in the primary and metastatic sites.
CD8+ve lymphocytes positive were linked to increased OS. Given that they are straightforward and affordable markers, we propose that iTILs, sTILs, CD4, and CD8 lymphocytes be used as prognostic tools in the metastatic phase of breast cancer and later may be useful as predictive markers for various types of treatment to provide a better service to our patients despite the country's limited resources.