International Tumor-infiltrating Lymphocytes Working Group Research Articles

Abstract PO1-01-03: Pathological response according to early metabolic remission in an interim FDG-PET scan and to tumor infiltrating lymphocytes - A secondary analysis of the phase II trial ABCSG 52/ATHENE investigating atezolizumab in early HER2+ BC

Abstract Background: ABCSG-52/ATHENE investigated a neoadjuvant chemotherapy de-escalation/immunotherapy escalation approach with trastuzumab, pertuzumab, atezolizumab and epirubicin for early-stage HER2-positive breast cancer. The pathological complete response rate (pCR; ypT0/Tis, ypN0) - the primary endpoint of the trial - was 60.3% (95% CI 47.5% - 71.9%), and was higher than the predefined threshold for positivity (≥ 40%). Interestingly, higher pCR rates were observed in PD-L1 immune cell (IC) negative tumors (pCR 69.2%) as compared to PD-L1 IC positive tumors (pCR 55.2%). No new safety concerns were observed. Primary outcome data were reported at the ESMO Breast Cancer Conference 2023. Here, we present the correlation of pathological outcome according to early metabolic response in an interim fluorodeoxyglucose F18 positron emission tomography (FDG-PET) scan, and according to stromal tumor infiltrating lymphocytes (TILs) at baseline. Methods: Patients (pts) with previously untreated, histologically confirmed HER2-positive early breast cancer (EBC; clinical prognostic stage cT1c-4a-d, N0–3, M0) were randomized 1:1 to two 3-weekly cycles of a chemotherapy-free induction phase (Part 1) with trastuzumab and pertuzumab (TP) plus 1200 mg atezolizumab (TP+A), or TP alone. Subsequently, all pts received 4 cycles of TP+A in combination with epirubicin 90 mg/m² (Part 2). Randomization was stratified according to baseline TILs (< 5% vs ≥ 5%), hormone receptor status (positive vs. negative), and prognostic stage (≤ IIA vs. ≥ IIB; AJCC v.8.0). An FDG-PET assessment was performed at baseline and after 2 cycles of study treatment. According to PERCIST, early metabolic responses were classified as metabolic complete response (mCR) and metabolic partial response (mPR) and association with outcome was tested with Fisher’s exact test. TIL assessment in pretreatment biopsy specimen were performed according to the recommendations from the International TILs Working Group. Lymphocytic predominant phenotype was defined as a TIL proportion of ≥ 50%. Correlation of TILs and pCR was modeled with logistic regression. Results: Overall, 58 pts were randomized to TP-A (n=29) or TP (n=29) at 9 sites. In 45 patients, a protocol predefined PERCIST assessment was available. An early mCR was achieved in 14 pts (n=14/45, 31.1%): 5 pts (n=5/21, 23.8%) in the TP group and 9 pts (n=9/24, 37.5%) in the TP-A group. Pts with and without an early mCR achieved a pCR in 92.9% (n=13/14) and 54.8% (n=17/31) of cases (p=0.016), respectively. An early mCR/mPR was achieved in 37 pts (n=37/45, 82.2%). Pts with and without an early mCR/mPR achieved a Residual Cancer Burden (RCB) class 0 or I in 89.2% (n=33/37) and 50.0% (n=4/8) of cases (p=0.024), respectively. The mean stromal TIL proportion was 23.9% in the overall population, with 22.8% in TP group and 25.0% in TP-A group. A lymphocytic predominant phenotype was seen in 10.3% of pts treated in the TP group, and 17.2% of pts treated in the TP-A group. No association between TIL proportion at baseline and pCR could be detected (OR for a 10%-point increase = 1.02, 95% CI 0.78-1.34). A moderate positive correlation between numeric values of TILs and PD-L1 ICs was observed (spearman r=0.57, P< 0.0001). Exploratory correlation analyses of PD-L1 status, TIL proportion and metabolic responses in interim FDG-PET scans will be presented at the meeting. Conclusions: In ABCSG-52, chemotherapy de-escalation with intensified immunotherapy was effective and safe. Early metabolic response assessment was feasible and predicted pathologic response; therefore, incorporation of interim FDG-PET results into de-escalation trials merits further investigation. In contrast to regimens without immune-checkpoint inhibitors, TIL proportion at baseline did not correlate with pathological outcome. Funding: Provision of IMP and financial support by Roche Austria GmbH Citation Format: Gabriel Rinnerthaler, Daniel Egle, Rupert Bartsch, Clemens Schmitt, Andreas Petzer, Marija Balic, Edgar Petru, Ursula Denison, Christian F. Singer, Vesna Bjelic-Radisic, Simon Gampenrieder, Michael Knauer, Karl Sotlar, Christine Brunner, Florian Posch, Dominik Hlauschek, Lidija Sölkner, Zsuzsanna Bago-Horvath, Martin Filipits, Manuela Gili, Michael Gnant, Richard Greil. Pathological response according to early metabolic remission in an interim FDG-PET scan and to tumor infiltrating lymphocytes - A secondary analysis of the phase II trial ABCSG 52/ATHENE investigating atezolizumab in early HER2+ BC [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 PO1-01-03.

Abstract PO4-16-04: Decoding the immune landscape of breast cancer (BC) during pregnancy (PrBC): Impact of hormone receptors (HR) and tumor-infiltrating lymphocytes (TILs) phenotype on gene expression signatures

Abstract Introduction: PrBC is an uncommon malignancy with aggressive behavior. Its pathogenesis involves distinct immune mechanisms associated with maternal-fetal tolerance and tumor-host immunoediting. PrBC displays specific patterns of TILs with increased CD8+ cells. Gaining a comprehensive understanding of the molecular processes underlying this immune synergy is crucial for enhancing PrBC patients’ clinical management. Here, we sought to identify dysregulated immune-related genes in PrBC and explore their association with HR status and TILs. Methods: A total of n=75 PrBC (age range 26-43 years) and n=67 age-matched early-onset breast cancer (EOBC) in non-pregnant women (controls; age range 28-43 years) were selected from our Institutional registry. For all cases TILs were quantified according to the International TILs Working Group recommendations and profiled by IHC for CD4 and CD8. RNA was extracted from representative FFPE tissue blocks to perform the expression analysis of 395 genes involved in tumor-immune interactions using a targeted NGS panel (Oncomine™ Immune Response Research Assay, Thermofisher). Samples with >1,000,000 mapped reads and >800,000 valid reads were considered adequate. R package DESeq2 software 1.38.3 was used for sequencing depth differences normalization and differential gene expression analysis. Differentially expressed genes (DEGs) were identified based on a significant p-value (p< 0.05). Results: The comparison between PrBC and EOBC revealed a total of n=7 DEGs. All of these genes were upregulated and belonged to distinct superfamilies, including Cancer/Testis (CT) Antigen (MAGEA1/3, XAGE1B), Interferons/Cytokines (IFNA17, IFNB1), Chemokines (CXCL13), and Immunoglobulin (PECAM1/CD31). Notably, the upregulation of Chemokines in respect to EOBC was observed exclusively in HR+ PrBC, whereas triple-negative (TN) PrBC did not exhibit this pattern compared to the control group. Hence, the upregulation of ALOX15B, an enzyme involved in fatty acid peroxidation, was specific to TN PrBC. The immune signatures showed significant variations between PrBC and EOBC also based on TILs density and subpopulations. Indeed, the upregulation of CT genes (MAGEA1, XAGE1B) was exclusively observed in PrBC cases characterized by low TILs levels and prevalence of CD8+ or CD4+ cells. Finally, CD4+ TILs were absent or low in PrBC with upregulated Interferons, Cytokines, Chemokines, and Immunoglobulin genes, but present in cases with increased expression of CT and KLRF1 (NK cells). Conclusion: These findings highlight the heterogeneity and distinct molecular characteristics of PrBC and EOBC. The upregulation of specific immune-related gene families, such as CT and Chemokines, in different PrBC subtypes may suggest their potential role as actionable biomarkers (e.g. MAGEA, a well-known oncogene and potential immunotherapy target). The differences in immune signatures and TILs subpopulations further emphasize the importance of the immune microenvironment in PrBC biology and behavior. Future studies could delve deeper into the clinical implications of these gene expression patterns and explore their relevance in personalized treatment strategies for PrBC. Citation Format: Konstantinos Venetis, Elham Sajjadi, Chiara Frascarelli, Mariia Ivanova, Marianna D'Ercole, Concetta Blundo, Massimo Giroda, Eugenia Di Loreto, Giovanna Scarfone, Stefano Ferrero, Paolo Veronesi, Viviana Galimberti, Fedro Alessandro Peccatori, Nicola Fusco, Elena Guerini-Rocco. Decoding the immune landscape of breast cancer (BC) during pregnancy (PrBC): Impact of hormone receptors (HR) and tumor-infiltrating lymphocytes (TILs) phenotype on gene expression signatures [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 PO4-16-04.

Abstract 3437: Stromal tumor-infiltrating lymphocytes in association with body size and composition among Black and White women with breast cancer

Abstract Background: Obesity is a major risk factor for breast cancer and negatively affects disease prognosis. Tumor-infiltrating lymphocytes (TILs) in the stroma of breast tissue play a major role in eliminating cancer cells and modulating immunotherapy efficacy. We aim to study the association between body size and composition with stromal TILs in breast tumor tissue by race. Methods: The study included 920 Black and 395 White women with newly diagnosed invasive breast cancer who have stromal TIL scores from the Women’s Circle of Health Study (WCHS). Body mass index (BMI), waist circumference (WC), and waist-to-hip ratio (WHR) were measured during the home interview. Pre-treatment H&E-stained tumor tissue sections were reviewed by our study pathologist and stromal TILs were scored on a scale of 0-100% at 10% increment according to the recommendations of the International TILs Working Group. Linear regressions adjusting for age at diagnosis, tumor grade, stage, and molecular subtype were used to test the differences in TILs across the categories of body size and composition indicators separately for Black and White women. Results: A higher stromal TILs score was associated with more aggressive breast cancer features, including higher grade, larger tumor size, more advanced stage, hormone receptor (HR) negativity, and human epidermal growth factor receptor 2 (HER2) positivity in both Black and White women (all P<0.05). Among Black women, WHR >0.90 vs ≤0.85 was associated with lower TILs overall (β = -3.5%, P = 0.045) and in triple-negative tumors (β = -12.3%, P = 0.003). Among White women, BMI 25.0 - 29.9 vs <25 was associated with higher TILs in HR-/HER2+ tumors (β = 28.1%, P = 0.031). A pattern suggested that BMI ≥30 vs <25 was associated with lower TILs in both HR-/HER2+ (β = -10.2, P=0.52) and triple-negative (β = -12.8, P=0.14) tumors, although the associations were not significant. No association was observed between TILs and any of the body composition parameters in women with HR+ tumors. Conclusions and Relevance: Body size and composition correlate with stromal TILs mainly in patients with HR- tumors, and this relation may be race-dependent. These findings suggest that body size and composition potentially modulate breast cancer treatment outcomes at least in part through TILs-dependent mechanisms. Funding: This work was in part supported by grants from the US National Institutes of Health (P01CA151135, R01CA100598, R01CA185623, P30CA016056, P30CA072720, K07CA201334, and R37CA248371), US Army Medical Research and Material Command (DAMD-17-01-1-0334), the Breast Cancer Research Foundation (CBA), and the Philip L. Hubbell family. Citation Format: Rand T. Akasheh, Thaer Khoury, Song Yao, Angela Omilian, Chi-Chen Hong, Elisa V. Bandera, Bonnie Qin, Nur Zeinomar, Christine Ambrosone, Ting-Yuan David Cheng. Stromal tumor-infiltrating lymphocytes in association with body size and composition among Black and White women with breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3437.

The Value of Tumor Infiltrating Lymphocytes (TIL) for Predicting the Response to Neoadjuvant Chemotherapy (NAC) in Breast Cancer according to the Molecular Subtypes.

The antitumor host immune response is an important factor in breast cancer, but its role is not fully established. The role of tumor infiltrating lymphocytes (TIL) as an immunological biomarker in breast cancer has been significantly explored in recent years. The number of patients treated with neoadjuvant chemotherapy (NAC) has increased and the identification of a biomarker to predict the probability of pCR (pathological complete response) is a high priority. We evaluated 334 cases of BC treated with NAC followed by surgical resection from 2020-2022 at the Ist Clinic of Oncological Surgery, Oncological Institute "Prof Dr I Chiricuta" Cluj Napoca. Of the above, 122 cases were available for histological evaluation both in pre-NAC biopsy and post-NAC resection tissue. Evaluation of biopsy fragments and resection parts were performed using hematoxylin eosin (H&E). The TIL evaluation took place according to the recommendations of the International TIL Working Group (ITILWG). There was a strong association between elevated levels of pre-NAC TIL. At the same time, there is a statistically significant correlation between stromal TIL and tumor grade, the number of lymph node metastases, the molecular subtype and the number of mitoses (p < 0.005). Intratumoral TIL showed a significant correlation with tumor size, distant metastasis, molecular subtype, number of mitosis, stage and lymph node metastasis (p < 0.005). We also demonstrated that high pre-NAC STIL represents a strong predictive marker for pCR. This study reveals the role of TIL as a predictive biomarker in breast cancer not only for the well-established TNBC (triple negative breast cancer) and HER2+ (Her2 overexpressed) subtypes but also in Luminal A and B molecular subtypes. In this scenario, the evaluation of sTIL as a novel predictive and therapy-predicting factor should become a routinely performed analysis that could guide clinicians when choosing the most appropriate therapy.

Immune microenvironment dynamics in breast cancer during pregnancy: impact of gestational age on tumor-infiltrating lymphocytes and prognosis.

Breast cancer during pregnancy (PrBC) is a rare condition known for its aggressive clinical behavior. The presence of tumor-infiltrating lymphocytes (TILs) has been shown to have a significant impact on the prognosis of these patients. Despite some biological characteristics of the tumor that may differ depending on the gestational age, little is known about the dynamics of the immune landscape within the tumor microenvironment (TME) in PrBC. Therefore, in this study, our objective was to gain comprehensive insights into the relationship between gestational age at breast cancer diagnosis and the composition of the TME. n = 108 PrBC were selected from our institutional registry and categorized based on the gestational age by trimester. For all cases, TILs were profiled according to the International TILs Working Group recommendations, and subtyped by CD4, CD8, and forkhead box P3 (FOXP3) immunohistochemistry. PD-L1 was tested according to the combined positive score (CPS) using the IHC 22C3 pharmDx assay, with a cutoff value of ≥10 for positivity. The statistical approach encompassed Fisher's and Chi-squared tests, with appropriate adjustments for multiple comparisons, logistic regression models, and survival analyses based on the Kaplan-Meier method. The proportion of patients with poorly differentiated (G3) neoplasms increased as the gestational age advanced (first trimester, n = 25, 56.8%; second trimester, n = 27, 69.2%; third trimester, n = 21, 87.5%; p = 0.03). The histologic subtypes as well as the hormone receptor (HR) and HER2 status did not show significant changes across different pregnancy trimesters. In the HR+/HER2- subtype, there was a higher proportion of tumors with high/moderate TILs in the early phases of pregnancy, similar to FOXP3 expression (TILs: first trimester, n = 10, 35.7%; second trimester, n = 2, 10.5%; third trimester, n = 0; p = 0.02; FOXP3: first trimester, n = 10, 40%; second trimester, n = 3, 15.8%; third trimester, n = 0; p= 0.03). The median follow-up for our cohort was 81 months. Patients who relapsed after a breast cancer diagnosis during the first trimester were more frequently PD-L1-negative, unlike those with no disease recurrence (n = 9, 100% vs. n = 9, 56.3%; p = 0.03; hormone therapy and n = 9, 100% vs. n = 7, 53.9%; p = 0.02; chemotherapy). No statistically significant differences were seen among the three trimesters in terms of survival outcome. The TME dynamics of HR+/HER2- PrBC vary based on gestational age, suggesting that immune tolerance expression during later gestational age could explain the increased aggressiveness of tumors diagnosed at that stage.

PD-L1 and TILs expression and their association with clinicopathological characteristics in Vietnamese women with primary invasive breast cancer.

Tumor-infiltrating lymphocytes (TILs) and programmed death ligand 1 (PD-L1) are promising new factors in the prognosis and prediction of breast cancer patients. Our study evaluated the prevalence of expression of TILs on hematoxylin and eosin (H&E) slides, PD-L1 expression on immunohistochemistry, and their association with clinicopathological characteristics in Vietnamese women with invasive breast cancer. This study was conducted on 216 women with primary invasive breast cancer. The evaluation of TILs on the HE slides was based on the International TILs Working Group 2014 recommendation. PD-L1 protein expression was determined using the Combined Positive Score, the number of tumor cells, lymphocytes, and macrophages stained by PD-L1 divided by the total viable tumor cells multiplied by 100. Based on the cutoff of 11%, the prevalence of TILs expression was 35.6%, of which highly expressed TILs (≥50%) accounted for 15.3%. Postmenopausal women and those who had a body mass index of 25 kg/m2 or greater had a higher odds of having TILs expression. However, patients who had the expression of Ki-67, HER-positive molecular subtype, and triple-negative subtype were more likely to have TILs expression. The prevalence of PD-L1 expression was 30.1%. A significantly higher odds of having PD-L1 was found in patients who had a history of benign breast disease, self-detected tumor and had TILs expression. The expression of TILs and PD-L1 is common in Vietnamese women with invasive breast cancer. Because of the importance of these expressions, routine evaluation to find women who had TILs and PD-L1 is needed so that treatment and prognosis can be optimized. Such routine evaluation can be targeted to those who had a high-risk profile found in this study.

Tumor-infiltrating lymphocytes (TILs) for prediction of response to platinum-based neoadjuvant chemotherapy (NACT) in triple-negative breast cancer (TNBC).

e12620 Background: According to several previous trials the rate of pathological complete response (pCR) for patients with TNBC is increased when carboplatin is added to NACT. However, it is largely unknown which group of patients would truly benefit from platinum-based NACT and for whom chemotherapy could be de-escalated. Several clinical trials have evaluated the stromal TILs as an important prognostic and predictive biomarker in breast cancer. We assessed the pCR rates and the outcomes in patients with TNBC randomized to receive NACT ± Carboplatin regarding TILs status. Methods: The clinical data were obtained from 132 patients diagnosed with TNBC (ER&amp;PgR &lt; 10%) and treated with either anthracycline-taxane NACT (n = 68) or сarboplatin-based chemotherapy (n = 64) from 2017 to 2022 at N.N. Petrov National Medicine Research Center of Oncology. TILs were evaluated in 132 formalin-fixed paraffin-embedded tumor tissue samples stained with H&amp;E at baseline biopsies according to guidelines from the International TILs Working Group. Classification of TILs into low ( &lt; 10%), intermediate (≤10% to &lt; 40%) and high (≥ 40%) levels was performed. Data are presented using the methods of descriptive statistics. The differences between groups were assessed using the Сhi-square test. P-values below 0.05 were considered statistically significant. Results: The proportion of patients who achieved a pCR was higher in the carboplatin group 66% than in patients without carboplatin 35% (OR 3,5; 95% CI 1,71-7,17; p = 0,0006). There were 25 (18.9%), 77 (58.3%) and 30 (22.7%) patients with low ( &lt; 10%), intermediate (≤10% to &lt; 40%) and high (≥ 40%) levels of TILs, respectively. In total, the tumors with high TILs had a pCR rate of 63.3%, compared with 46.1% for low TILs (OR 2,0; 95% CI 0,87-4,67; p = 0,097). No significant statistical difference in pCR rates was shown between carboplatin-based and standard NACT groups with high TILs - 71% and 54%, respectively (OR 2,06; 95% CI 0,45-9,30; p = 0,346). When analyzing a group with low TILs infiltration, the pCR was more frequent in the platinum-based 64% compared to 31% in the standard NACT group (OR 3,9; 95% CI 1,72-9,00; p = 0,0011). Conclusions: These data may support the de-escalation of carboplatin-based NACT in patients diagnosed with TNBC with high TILs infiltration. Further investigations are warranted to explore the predictive value of TILs in platinum-based NACT. [Table: see text]

Prognostic Value Of Tumor Infiltrating Lymphocytes Among HER-2+ Breast Cancer Patients Receiving Trastuzumab-Based Adjuvant Therapy

Background: Tumor Infiltrating Lymphocytes (TILs) have prognostic value on HER2 positive breast cancer (BC). The absence of standardized method for evaluating TILs causes variations in results of previous studies. This study is performed to evaluate the prognostic value of TILs in HER2 positive BC treated with trastuzumab-based adjuvant therapy using standardized method recommended by the International TILs Working Group.&#x0D; Aim: To analyze the prognostic value of TILs in HER2 positive BC patients receiving trastuzumab-based adjuvant therapy at Dr. Sardjito General Hospital, Yogyakarta and to analyze proportion differences between high TILs (≥30%) and low TILs (&lt;30%).&#x0D; Methods: This is a retropective cohort study on HER2 positive, stage 1-3 BC patients who received trastuzumab-based adjuvant therapy. Histopathology slides from 6 hospitals/laboratoriums were analyzed by two pathologists.&#x0D; Results: 73 data were available for analysis. TILs stroma &lt;30% was 65,8% and most patients received combination of anthracyclines, taxanes and trastuzumab (67,1%). There was no difference of overall survival between high and low TILs (p log rank: 0,331).&#x0D; Conclusion: HER2 positive BC with high TILs did not show better overall survival compared to those with low TILs. The proportion of HER2 positive breast cancer with high TILs was lower than those with low TILs.

Abstract P4-09-04: Correlation between histology and molecular subtypes in triple negative breast cancer

Abstract Correlation between histology and molecular subtypes in triple negative breast cancer Authors: Tabata Alves Domingos, MD; 1* Roberto Bonfim Pimenta Peixoto, MD; 1* Ashka Patel, BS1,2; Krishan Taneja, PhD; 1 Wendy Y. Chen, MD, MPH; 4,5,6 Elizabeth A. Mittendorf, MD, PhD; 5,6,7 Alexa Zimbalist, MS; 3 Elizabeth M. Cespedes Feliciano, ScD SM; 3 Deborah A. Dillon, MD 1,2 *equal contribution Author Affiliations 1. Department of Pathology, Brigham and Women’s Hospital, Boston, MA 02115 2. Breast Oncology, Dana-Farber Brigham Cancer Center, Boston, MA 02215 3. Division of Research, Kaiser Permanente Northern California, Oakland, CA 94612 4. Channing Division of Network Medicine, Brigham and Women’s Hospital, Boston, MA 02115 5. Department of Medical Oncology Dana Farber Cancer Institute, Boston, MA 02215 6. Harvard Medical School, Boston, MA 02215 7. Division of Breast Surgery, Brigham and Women’s Hospital, Boston, MA 02215 Background Recently defined molecular subtypes of triple negative breast cancer (TNBC) show distinct clinical outcomes and suggest new therapeutic targets but have not been integrated into current pathologic classification systems. Here, we describe the histopathologic features of TNBC according to four molecular subtypes: basal-like immune activated (BLIA), basal-like immune suppressed (BLIS), luminal androgen receptor (LAR) and mesenchymal (MES), classified using the NanoString BC360 gene expression assay. Methods Stage II and III invasive breast cancers were identified in the Kaiser Permanente (KP) clinical pathology archives (2005-2015) and triple negative status was determined from the KP Northern California Cancer Registry data on immunohistochemistry. Selected slides were reviewed by two pathologists who recorded key histopathologic features [histologic subtype, presence of apocrine, metaplastic or micropapillary features, nuclear grade, mitotic score, and Tumor Infiltrating Lymphocytes (TILs)] and marked the best tumor areas for molecular analysis. TILs were evaluated according to the guidelines of the International TILs Working Group. Cases were macrodissected and evaluated using the NanoString BC360 gene expression assay. Histologic features were then summarized according to molecular subtype. Results Of 72 TNBCs, 60 were classified as Basal-like (83.3%), 7 as HER2-enriched (9.7%) and 5 as Luminal A (6.9%). 41 cases were classified as BLIA (56.9%), 14 as BLIS (19.4%), 13 as LAR (18.0%) and 4 as MES (5.5%). Both BLIA and BLIS tumors showed uniformly high nuclear grade and high mitotic score but differed significantly in TILs (BLIA average 32% vs BLIS average 9%; p value&amp;lt; 0.001, t-test for mean difference in TILs). The majority of LAR cases (69%) showed apocrine differentiation, not present in any other molecular subtype (p value&amp;lt; 0.001, chi-square test for presence of apocrine differentiation). LAR cases showed high nuclear grade but a lower average mitotic score (average score of 2) compared with the basal-like subtypes. TILs in LAR tumors were intermediate (17%) between BLIA and BLIS tumors. Of the 4 MES cases, all showed high nuclear grade. TILs in the MES cases were also intermediate (14%) between BLIA and BLIS tumors. Other histologic features, including lobular subtype, metaplastic and micropapillary features were not associated with specific triple negative molecular subtypes. Conclusion TILs are high in the BLIA molecular subtype (average 32%), low in the BLIS subtype (average 9%) and intermediate in LAR (average 17%) and MES (average 14%) subtypes. Apocrine features, if present in a TNBC, are a strong predictor of LAR molecular subtype. The inclusion of TILs and apocrine features (both easily derived from H&amp;E slides) in routine pathology reporting could improve the classification of TNBC and aid in the identification of patients more likely to respond to specific therapies for the BLIA, BLIS and LAR subtypes, especially in resource-limited settings. Citation Format: Tabata Alves Domingos, Roberto Bonfim Pimenta Peixoto, Ashka Patel, Krishan Taneja, Wendy Y. Chen, Elizabeth A. Mittendorf, Alexa Zimbalist, Elizabeth M. Cespedes Feliciano, Deborah A. Dillon. Correlation between histology and molecular subtypes in triple negative breast cancer [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 P4-09-04.

Abstract P6-01-23: Role of tumor infiltrating lymphocytes and PD-L1 expression in the response to eribulin and pembrolizumab in metastatic triple negative breast cancer (mTNBC) on the ENHANCE1 trial

Abstract Background Treatment with combination chemo-immunotherapy has become the front-line standard for eligible patients with PD-L1 positive mTNBC. PD-L1 is the only approved biomarker for pembrolizumab in metastatic breast cancer for response to combination chemo-immunotherapy, however given that it is not predictive of response in all cases additional biomarkers are needed. Tumor infiltrating lymphocytes (TILs) have been shown to be both predictive and prognostic in operable TNBC, but there are fewer data regarding the role of TILs in mTNBC. In this study we report the associations between TILs and outcomes in patients treated prospectively on the ENHANCE-1 study with eribulin and pembrolizumab. Methods ENHANCE-1 was a single arm phase Ib/II trial which evaluated the efficacy and safety of eribulin and pembrolizumab in 167 patients (pts) with mTNBC who had received 0-2 prior lines of therapy (66 pts in the first line setting [stratum 1] and 101 pts with 1-2 prior lines of therapy [stratum 2]). Objective response rate (ORR) was defined as percentage of pts with either complete response (CR) or partial response (PR) by RECIST 1.1. The ORR was 25.8% in stratum 1 and 21.8% in stratum 2. Stromal TILs (sTIL) and intratumoral TILs were evaluated on whole slide H&amp;E sections from biopsy specimens used for enrollment on ENHANCE-1 by breast pathology according to the International TILs Working Group guidelines. PD-L1 positivity was determined via immunohistochemistry using the Agilent 22C3 antibody. We also assessed TIL density digitally using machine learning classifiers to identify tumor/stromal tissue areas and individual lymphocytes. Results We found that there was statistically significant increase in sTIL counts in responders compared to non-responders in stratum 1 (p=0.002) but not in stratum 2 (p=0.99). We did not find any associations between intratumoral TILs and response. Quantitative PD-L1 scoring via combined proportion score (CPS) was also positively associated with response in stratum 1 (p=0.01) but not in stratum 2 (p=0.34). We also find that sTIL counts are most correlated to CPS scores (continuous) for non-responders within stratum 1 (R2=+0.55, p&amp;lt; 0.01). Conclusion In this population of patients with mTNBC treated prospectively with eribulin and pembrolizumab, sTILs and PDL1 CPS were each individually associated with a positive response in patients treated with front-line combination chemo-immunotherapy. Neither was predictive for patient response in stratum 2. One important caveat is the biopsies were not required immediately prior to enrollment, possibly confounding the tumor microenvironment (TME) at the time of analysis. Our data contribute to emerging data that sTILs can act as a biomarker for response to immune checkpoint inhibition when utilized in the front-line setting for mTNBC. Further characterization of the TME via quantitative immunofluorescence is ongoing. This study was funded by Eisai IIS-E7389 Citation Format: Mathew Kearney, Hua Guo, Rami Vanguri, Qi Wang, Michelle Garlin, Courtney Connelly, Kevin Kalinsky, Eileen P. Connolly. Role of tumor infiltrating lymphocytes and PD-L1 expression in the response to eribulin and pembrolizumab in metastatic triple negative breast cancer (mTNBC) on the ENHANCE1 trial [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 P6-01-23.

Abstract P4-02-30: Association between tumor infiltrating lymphocytes (TILs) and the HER2DX assay in early-stage of HER2-positive (HER2+) breast cancer

Abstract Background: The HER2DX assay is a genomic test in early-stage HER2-positive (HER2+) breast cancer that provides prognostic and predictive information. HER2DX is a supervised learning algorithm incorporating tumor size, nodal staging, and 4 gene expression signature scores (immune/IGG, tumor cell proliferation, luminal differentiation and the expression of the HER2 amplicon). Among them, the IGG signature is associated with both overall survival and probability of achieving a pathologic complete response (pCR). Here, we studied the association of percentage (%) of tumor infiltrating lymphocytes (TILs) with HER2DX scores, immune genes and other breast cancer-related genes. Methods: HER2DX and %TILs were evaluated in 670 formalin-fixed paraffin-embedded (FFPE) samples of HER2+ breast cancer, including in 3 clinical studies SHORTHER (n=437), PAMELA (n=86), a cohort of patients treated with anti-HER2 therapy plus chemotherapy at Hospital Clínic of Barcelona (n=147). The %TILs were quantified by histological evaluation with hematoxylin eosin staining according to International TILs Working Group guidelines. The nCounter platform determined the expression of 192 genes and HER2DX scores. Pearson correlations (Cor) and Significance Analysis of Microarrays (SAM) with a false-discovery rate (FDR) &amp;lt; 5% assessed the association between %TILs and the expression of individual genes or HER2DX signature scores. Results: A moderate correlation was observed between %TILs and the immune IGG signature (Cor=0.56, p&amp;lt; 0.001). Of note, 171 (25.52%) cases had low %TILs (&amp;lt; 30%) and high IGG score, while 1 (0.15%) case had high %TILs (≥30%) and low IGG score. The %TILs were significantly associated with the expression of immune genes, ERBB2, IGG signature and HER2 amplicon score, and negatively associated with the expression of luminal genes (i.e., ESR1, PRG and BCL2). An unclear relationship between TILs and proliferation genes was observed. Finally, moderate correlations were observed between %TILs and HER2DX pCR score (Cor=0.48, p&amp;lt; 0.001) and between %TILs and HER2DX risk score (Cor=0.33, p&amp;lt; 0.001). Conclusions: Important differences exist between the %TILs and the HER2DX IGG signature in early-stage HER2+ breast cancer. The %TILs should not be used to predict the HER2DX scores. Biologically, a higher %TILs indirectly capture a higher ERBB2 expression and a lower expression of luminal genes, both associated with response to anti-HER2 treatment. Citation Format: Esther Sanfeliu, Fara Brasó-Maristany, Maria Vittoria Dieci, Mercedes Marín-Aguilera, Blanca González-Farré, Gaia Griguolo, Tomas Pascual, Patricia Galván, Laura Angelats, Oleguer Castillo, Paula Blasco, Valeria Sirenko, Pedro Jares, Joan Antón Puig-Butillé, Laia Paré, Antonio Martínez, Antonio Llombart-Cussac, Javier Cortés, Ana Vivancos, Patricia Villagrasa, Joel S Parker, Charles M Perou, Aleix Prat, PierFranco Conte, Valentina Guarneri. Association between tumor infiltrating lymphocytes (TILs) and the HER2DX assay in early-stage of HER2-positive (HER2+) breast cancer [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 P4-02-30.

Abstract P2-21-04: The tumor immune microenvironment composition and prognostic value in breast cancer during pregnancy is dynamic during the gestation period

Abstract Introduction: Breast cancer (BC) during pregnancy (PrBC) is an uncommon malignancy characterized by a more aggressive clinical course compared to pregnancy-unrelated BC. Specific patterns of tumor-infiltrating lymphocytes (TILs) subpopulations have been observed in these patients, with significant prognostic roles. Previous studies demonstrated the varying histopathologic and prognostic profiles of PrBC by gestational age. However, the underlying immune landscape dynamics has never been investigated. Here, we sought to provide comprehensive insights into the association between gestational age at breast cancer diagnosis and tumor immune microenvironment (TIME) composition. Materials and Methods: A total of 110 PrBC were selected from our Institutional registry and categorized based on the trimester in which they were diagnosed. All cases were subjected to TILs profiling according to the International TILs Working Group recommendations. Immunohistochemistry for CD4, CD8, forkhead box P3 (FOXP3), and PD-L1 (clone 22C3) on a Dako Omnis platform was performed. Fisher’s and Chi-squared tests, multinomial logistic regression models, ROC curve, and survival analyses were performed. Results: The proportion of patients with high histologic grades incremented with the increase in gestational age (1st, n=24, 53%; 2nd, n=27, 69.2%; 3rd trimester, n=20, 87.0%; p=0.02). Neither breast cancer subtypes nor the hormone receptor (HR) and HER2 status changed significantly according to the pregnancy trimester. In HR+/HER2- subtype, the proportion of TILs+ tumors were higher in the early phases of pregnancy (1st, n=29, 100%; 2nd, n=17, 89.5%; 3rd trimester, n=9, 81.8%; p=0.04) imprinted by FOXP3 positivity where more FOXP3+ TILs were seen in the first months and decreased progressively (1st, n=10, 55.6%; 2nd, n=2, 11.8%; 3rd trimester, n=0, 0%; p&amp;lt; 0.01)). While in the triple negative breast cancer (TNBC) population, the proportion of PD-L1+ tumors (i.e. CPS&amp;gt;1) was significantly higher in the later stages of pregnancy (1st, n=2, 16.7%; 2nd, n=2, 18.2%; 3rd trimester, n=5, 71.4%; p=0.03). Patients who relapsed after a BC diagnosis during the 1st and 2nd trimesters lacked more frequently FOXP3+ and CD8+ cells, unlike those with no disease recurrence (n=21, 77.8% vs. n=17, 48.6%; p=0.02 and n=18, 66.7% vs. n=10, 28.6%; p&amp;lt; 0.01, respectively). Conclusions: TIME dynamics of PrBC are different according to the gestational age in both HR+ and TNBC PrBC. Our results suggest that immune tolerance events are likely to involve PrBC at later gestational age. Specific escape mechanisms (i.e., TILs and FOXP3 decrease in HR+ and PD-L1 expression in TNBC) might explain the aggressiveness of PrBC diagnosed during the later gestational age. Citation Format: Elham Sajjadi, Konstantinos Venetis, Mariia Ivanova, Marianna Noale, Concetta Blundo, Giovanna Scarfone, Eugenia Di Loreto, Stefano Ferrero, Stefania Maggi, Paolo Veronesi, Viviana Enrica Galimbreti, Giuseppe Viale, Fedro Alessandro A. Peccatori, Elena Guerini-Rocco, Nicola Fusco. The tumor immune microenvironment composition and prognostic value in breast cancer during pregnancy is dynamic during the gestation period [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 P2-21-04.

THE PROGNOSTIC INDICATOR OF TUMOR INFILTRATING LYMPHOCYTES IN BREAST CARCINOMA

Introduction: Breast cancer is one of the common malignant tumors in women. The use of biomarkers ensures breast cancer patients receive optimal treatment. Tumor-infiltrating lymphocytes (TILs) are emerging as one of the key prognostic markers of breast cancer. Our study aims to evaluate percentage of TILs in Objectives And Methodology: tumor tissue, grading TILs based on the recommendations by an International TIL Working Group 2014 &amp; compare with ER &amp; PR status and in relation to concentration of TILs we assess prognosis of breast carcinoma for a period of 6 months. Results: Among 30 study subjects, TILs were of intermediate, high &amp; lower grade in 53.3%, 40% and 6.7% cases respectively. TILs expression was statistically significant in both ER and PR, positive &amp; negative groups (p&lt;0.05). TILs were statistically highly significant as a prognostic marker in hormone negative breast cancers (p&lt;0.05). Conclusion: Our study concludes that high TILs are correlated with favourable survival and response to treatment in breast cancer patients with ER, PR negative molecular subtype

Tumor-Infiltrating Lymphocytes in Localized Prostate Cancer: Do They Play an Important Role?

Background Localized prostate cancer is a heterogeneous entity, and new biomarkers are required for risk stratification. This study aimed to characterize tumor-infiltrating lymphocytes (TILs) in localized prostate cancer and assess their potential prognostic markers. Methodology Radical prostatectomy specimens were analyzed to determine infiltration levels of CD4+, CD8+, T cells, and B cells (characterized by CD20+ cells) in the tumor tissue using immunohistochemistry and the recommendations of the International TILs Working Group 2014.The clinical endpoint was biochemical recurrence (BCR), and the study sample was divided into two cohorts (cohort 1: without BCR; cohort 2: with BCR).Prognostic markers were assessed using Kaplan-Meier and univariate/multivariate Cox regression analysis using SPSS version 25 (IBM Corp., Armonk, NY, USA). Results We included 96 patients in this study. BCR occurred in 51% of the patients. Normal TILs infiltration was found in most of the patients (41/31, 87%/63%). T CD4+ infiltration was statistically superior in cohort 2. This enrichment was associated with BCR (p < 0.05; log-rank test). After adjustment for routine clinical variables and Gleason grade groups (grade group ≤2 and grade group ≥3), it remained an independent prognostic variable of early BCR (p < 0.05; multivariate Cox regression). Conclusions This study showed that immune cell infiltration appears to be an important prognostic variable for early recurrence in localized prostate cancer.

Abstract PR007: Tumoral infiltrating lymphocytes as a prognostic factor in triple negative breast cancer patients from Colombia

Abstract Introduction: Triple negative breast cancer (TNBC), occurs in 10%-20% of all breast cancer and presents a higher frequency in Latinas compared to non-Hispanic white women. It is highly immunogenic and high levels of tumor infiltrating lymphocytes (TILs) have been associated with a better overall survival and higher probability to achieve pathological complete response (pCR). Objective: This is the first study in Colombian women aimed to explore stromal TILs (sTILs) level and composition as a prognostic and predictive biomarker in TNBC. Methods: A total of 195 TNBC tumor biospecimens from patients diagnosed between 2008-2016 in 3 Colombian health institutions were included. Stromal TILs (sTILs) was evaluated following the recommendations of the International TILs Working Group 2014 in hematoxylin &amp; eosin slides from pre-treatment samples. The number of positive cells for CD4 and CD8 was evaluated by immunohistochemistry and digital image capture. Parametric and non-parametric tests were used to evaluate differences in clinic-pathological characteristics according to sTILs levels and composition. Differences in overall survival were analyzed using Kaplan-Meier curves and the log-rank test. Cox regression analysis was used to analyze sTILs as a prognostic marker for overall survival. A logistic regression model was applied to evaluate the association of sTILs with pCR. Results: Tumors with high sTILs levels were more likely to be early stage (64.4% stage I/II) compared to tumors with low sTILs levels (35.5%). Additionally, when compared patients with high sTILs vs. low sTILs, a higher percentage of patients with high sTILs didn’t receive neoadjuvant chemotherapy (NAC) (high:50% vs. low:32.7%, p=0.025) and received more conservative surgeries (high:60% vs. low:37.9%, p&amp;lt;0.05). Similar results were observed for patients with high CD4/8 infiltration. Longer overall survival times were observed in patients with high sTILs (84.9 mo vs. 41.4 mo, p&amp;lt;0.05), as well as in patients with high CD4+ infiltration (p&amp;lt;0.05) and CD8+ (p&amp;lt;0.015). In the multivariate analysis, low levels of sTILs was found to be independent prognostic factor associated with a higher risk of death (HR: 1.59, 95% CI 1.01-2.48). Regarding sTILs as a predictive biomarker, a higher number of patients with high sTILs and high CD4+ cells achieved clinical complete response to NAC (sTILs: 28.6% vs. 9.3%, p=0.022; CD4: 29% vs 9.4%, p=0.032) and pCR (sTILs: 42.9% vs. 15.8%, p=0.023; for CD4: 43.3% vs 16.3%, p=0.006) compared to patients with low infiltration. Likewise, a statistically significant association between sTILs and pCR was observed (OR: High sTILs 1.486, 95% CI 1.14 - 2.013). Similar results were observed for high CD4 and CD8 infiltration (OR: 1.262, 95% CI 1.061 - 1.536, OR: 1.337, 1.085 - 1.694, respectively). Conclusions: Our results suggest that sTILs levels are a prognostic marker for overall survival and a predictive marker for pCR in TNBC patients from Colombia as has been reported in previous studies including biospecimens from mostly European ancestry patients. Citation Format: Carlos A. Huertas-Caro, Mayra A. Ramirez, Diego Felipe Ballen, Juan Carlos Mejía, Luz Fernanda Sua-Villegas, Alicia Cock-Rada, Jovanny Zabaleta, Laura Fejerman, María Carolina Sanabria-Salas, Silvia J. Serrano-Gomez. Tumoral infiltrating lymphocytes as a prognostic factor in triple negative breast cancer patients from Colombia [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr PR007.

Stromal Tumor Infiltrating Lymphocytes (TIL) as a potential prognostic biomarker for recurrence in Locally Advanced Breast Cancer (LABC) patients

Background: Tumor-infiltrating lymphocytes (TIL) serves as the host adaptive immune response in breast cancer. TIL has the potential to be an independent prognostic factor in breast cancer patients. In this study, we aimed to examine the association between stromal TIL and recurrence in locally advanced breast cancer patients. Methods: A cohort retrospective study was conducted using medical records of female breast cancer patients with locally advanced breast cancer. We collected patients' data, including demographic data from the medical record. Stromal TIL was examined following the recommendations of the International TIL Working Group 2014. Results: 75 samples were included with an average age of 49.5±8.4. Ductal carcinoma was the most common type histologically (88.0%). Luminal B Her2-negative was the predominant breast cancer subtype (32.0%). There was a significant association between the breast cancer subtype and disease-free survival (P – 0.014). The optimal cut-off to determine the recurrence of breast cancer was 15%. The sensitivity, specificity, PPV, NPV, and accuracy of TIL to predict the 24-month disease-free survival (DFS) were 80.5%, 82.4%, 84.6%, 77.8%, and 81.3%, respectively. Every 10% increase in TIL percentage could raise the DFS by 5.45 months (p=0.001). Patients with high TIL values had higher survival than those with low TIL values. Conclusion: There was a significant correlation between the stromal TIL and the recurrence rate. The pre-therapy stromal TIL percentage can be employed as a potential biomarker to predict breast cancer recurrence, particularly in the first two years.

Tumor-infiltrating lymphocytes predict survival in ≥ pT2 urothelial bladder cancer

Tumor-infiltrating lymphocytes (TILs) are associated with improved survival in several types of cancers, including genitourinary cancers. However, multiple different scoring methods used to assess TILs complicate the comparison of different studies and are not always suitable for daily practice. In 2014, the International TILs Working Group (ITWG) proposed a simple and robust assessment method for a more standardized evaluation of TILs. Here, we validated this system in muscle-invasive urinary bladder cancer (MIBC). Patient history and histologic specimens from 203 patients with MIBC were retrospectively analyzed. The stromal TIL (sTIL) score was determined using the ITWG system and 3 groups were defined according to the degree of stromal lymphocytic infiltration: low (0–10%), intermediate (10–55%) and high (55–100%). Associations between sTIL score, clinicopathological variables, tumor-specific survival (TSS), overall survival (OS), and disease-free survival (DFS) were analyzed. High stromal lymphocytic infiltration was associated with significantly higher OS, TSS and DFS when compared to low grade sTILs. The survival benefit remained statistically significant in multivariate analyses, confirming that sTILs are a strong independent positive prognostic factor in patients with MIBC. In summary, the degree of sTILs as defined by the ITWG robustly predicts survival in MIBC patients. Prospective studies with larger case numbers are needed to determine whether sTILs should be included in staging guidelines and how they could aid in therapeutic decision making.

Combined assessment of metabolic response and tumor infiltrating lymphocytes as a predictor of outcomes following neoadjuvant therapy for HER2-positive breast cancer: Results from the randomized PREDIX HER2 trial.

593 Background: Abundance of tumor infiltrating lymphocytes (TIL) is prognostic in early HER2-positive breast cancer (BC). Response to neoadjuvant therapy (NAT) according to positron emission tomography combined with computed tomography (PET-CT) has been shown to predict pathologic complete response (pCR). There is paucity of data regarding long-term prognostication using PET-CT and the potential value of the combined assessment of both these biomarkers. Methods: PREDIX HER2 (NCT02568839) is a prospective randomized phase 2 trial that compared standard NAT (docetaxel, trastuzumab, pertuzumab) with trastuzumab emtansine, in patients with HER2-positive BC. Overall, 202 patients were included (197 evaluable) and the primary efficacy analysis showed no difference in pCR or event-free survival (EFS) between the two groups (Hatschek, JAMA Oncology 2021). Assessment with fluorine 18–labeled fluorodeoxyglucose PET-CT was performed at baseline and after 2 and 6 treatment cycles, and SUVmax was evaluated as a continuous variable. TILs were assessed at baseline biopsies according to guidelines from the International TIL Working Group (J.H.). The aim of this secondary analysis was to investigate the combined assessment of TIL and PET-CT as an early predictor of response to NAT. Results: Overall, 112 patients underwent baseline PET-CT and 109 after C2, whereas 173 had baseline TIL. In multivariable analysis, baseline SUVmax did not predict pCR (ORadj= 1.04, 95% CI 0.97-1.12, p = 0.259) or EFS (HRadj= 1.07, 95% CI 0.98-1.17, p = 0.117). In contrast, higher SUVmax at C2 predicted lower pCR (ORadj= 0.65, 95% CI 0.48-0.87, p = 0.005) and worse EFS (HRadj= 1.18, 95% CI 1.04-1.34, p = 0.01). Baseline TIL &gt; 10% (median cut-off) provided additional prognostic information to clinical parameters (stage and hormone receptor expression) and C2 SUVmax (LR-Δχ2 = 7.19, p = 0.007; ORadj= 3.52, 95% CI 1.37 – 9.06, p = 0.009). 75% of patients with high TIL and C2 SUVmax &lt; 2.49 achieved pCR, compared with 13.8% of those with low TIL and high C2 SUVmax and 39.1%-41.3% for the intermediate groups (p = 0.001). Conclusions: SUVmax after two cycles of NAT for HER2-positive BC is an independent predictor of both short- and long-term outcomes. A combined assessment with TIL may facilitate early selection of good responders for de-escalation and poor responders for alternative treatment strategies. Clinical trial information: NCT02568839.

Abstract P4-07-02: Tumor microenvironment characteristics and prognosis in breast cancer during pregnancy: The role of differentially expressed immune-related genes

Abstract Introduction: Breast cancer during pregnancy (PrBC) accounts for ~4% of breast cancer cases in young women and its intrinsic biology is still largely undetermined. Tumor microenvironment (TME) of PrBC has been recently characterized with low levels of stromal tumor-infiltrating lymphocytes (TILs) and high relative expression of programmed death-ligand 1 (PD-L1), suggesting an increased immune evasion. The underlying immune landscape, however, has not been unveiled. Given the significant alterations of the immune system during gestation, we hypothesized that the TME of PrBC might have distinct biological traits. Here, we sought to evaluate the outcome of PrBC according to the TME characteristics and to assess whether pathogenic mechanisms of immune evasion are involved. Methods: Representative formalin-fixed paraffin-embedded tissue blocks of 83 consecutive PrBC and 89 age-matched early-onset pregnancy-unrelated breast cancers (controls) were subjected to immunohistochemistry (IHC) using antibodies against CD4, CD8, forkhead box P3 (FOXP3), and PD-L1 (clone 22C3) on a Dako Omnis platform. For all cases, TILs were evaluated according to the International TILs Working Group recommendations. Next-generation sequencing gene expression of 395 genes involved in tumor-immune interactions (Oncomine™ Immune Response Research Assay) was performed on RNA extracted from PrBC (n=20) and controls (n=16). Samples with mapped reads &amp;gt;1,000,000 and valid reads &amp;gt;800,000 were considered adequate. Fisher’s and Chi-squared tests, multinomial logistic regression models, ROC curve, and survival analyses were performed. Results: The fraction of tumors with CD8+TILs was significantly higher in PrBC than in the controls (n=71(85.0%) vs. n=61(68.5%); p=0.02), being mirrored by less cases with CD4+TILs (n=27 (32.5%) vs. n=43 (48.3%); p=0.03). Even higher differences were observed in hormone receptor (HR)+/HER2-negative tumors (CD8: n=39 (88.6%) vs. n=39 (66.1%); p=0.01). After a median follow-up of 78 (range, 1-247), 66/83 women (79.5%) with PrBC were alive and 53/83 (63.8%) relapse-free. Overall, PrBC with CD8+TILs had a better outcome compared to CD8-negative PrBC (OS 81 vs 69 months p=0.05) and CD8 expression was associated with better outcomes in HR+/HER2-negative tumors (OS p=0.02; DFS p=0.04). The overall comparison of immune-related genes in the 34 cases (PrBC, n=18; controls, n=16) that reached the quality parameters revealed significant differences in the expression of 63 immune-related genes. Of these, 4 genes (IFNA17, IFNB1, FUT4, and PECAM1) were upregulated, while 59 genes were downregulated in PrBC compared to the controls. Interestingly, IFNA17, IFNB1, and FUT4 remained upregulated in HR+ PrBC, where a slightly reduced number of differentially expressed genes was observed (n=60). In HR-/HER2- PrBC, only 25 genes were differentially expressed, of which 9, including IFNA17 and PECAM1, were significantly upregulated. Discussion: These data have the potential of improving our knowledge of the immunobiology that characterizes PrBC, suggesting that in these tumors the higher frequency of CD8+TILs might be related to an enhanced anti-tumor immune response, as CD8 expression was associated with better outcomes in PrBC. On the other hand, given that interferons (IFNs) may also trigger immune suppressive mechanisms in cancer cells, the activation of type I IFNs encoded by IFNA17 and IFNB1 seen in our RNA-seq analysis, combined with the lower frequency of CD4+TILs observed, suggest CD4+ cell suppression as a possible mechanism of immune evasion. Conclusion: PrBC TME is characterized by specific patterns of TILs subpopulations due to the possible activation of type I IFNs and its assessment might help in identifying women at high risk of death and recurrence. Citation Format: Nicola Fusco, Elham Sajjadi, Konstantinos Venetis, Barbara Buonomo, Concetta Blundo, Massimo Giroda, Eugenia Di Loreto, Giovanna Scarfone, Stefano Ferrero, Paolo Veronesi, Viviana E. Galimberti, Massimo Barberis, Giuseppe Viale, Elena Guerini-Rocco, Fedro A. Peccatori. Tumor microenvironment characteristics and prognosis in breast cancer during pregnancy: The role of differentially expressed immune-related genes [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-07-02.

Factors Associated with Lymph Node Metastasis in Special Types of Invasive Breast Cancer at National Cancer Hospital in the Period of 2018 - 2021

This paper assesses the rate of lymph node metastasis and the correlation between lymph node metastasis and some pathological characteristics in special types of invasive breast cancer. In the paper, a descriptive cross‐sectional study was conducted on 200 patients with special types of invasive breast cancer at the Institute of Molecular Biology and Pathology - National Cancer Hospital from August 2020 to April 2021. The study results show that 32% of the 200 cases had nodal metastases. The factors associated with the increased risk of lymph node metastasis were of histological types (invasive lobular, invasive micropapillary carcinomas) (p = 0.000), high tumour grade (p = 0.000), the presence of lymphovascular invasion (p = 0.000), tumor-infiltrating lymphocytes (TILs) &lt; 10% (p = 0.000), the presence of tumor necrosis (p = 0.001), Her-2/neu overexpression (p = 0.01), Ki-67 ≥ 20% (p = 0.036), HER2-positive breast cancer (p = 0.015). Overall, the rate of lymph node metastasis was 32%. Histological type, tumour grade, the presence of lymphovascular invasion, TILs, tumor necrosis, Her-2/neu and Ki-67 expression, and molecular subtype were factors related to lymph node metastasis in special types of invasive breast cancer.&#x0D; Keywords:&#x0D; Lymph node metastasis, invasive breast cancer, special types.&#x0D; References&#x0D; [1] World Health Organization, Breast Tumors (5th ed), International Agency for Research on Cancer, Lyon, France, 2019.[2] K. M. Masters, S. L.Wong, R. C. Martin et al., Dermal Injection of Radioactive Colloid Is Superior to Peritumoral Injection for Breast Cancer Sentinel Lymph Node Biopsy: Results of A Multiinstitutional Study, Annals of Surgery, Vol. 233, No. 5, 2001, pp. 676, https://doi.org/10.1097/00000658-200105000-00012. [3] A. V. Ashturkar, G. S. Pathak, S. D. Deshmukh, H. T. Pandave, Factors Predicting the Axillary Lymph Node Metastasis in Breast Cancer: Is Axillary Node Clearance Indicated in Every Breast Cancer Patient? Indian Journal of Surgery, Vol. 73, No. 5,2011, pp. 331-335, https://doi.org/10.1007/s12262-011-0315-5. [4] M. R. Peter, D. L.Michelino, V. Tricia et al., Prediction of Axillary Lymph Node Status in Breast Cancer Patients by Use of Prognostic Indicators, JNCI: Journal of the National Cancer Institute, Vol. 86, No. 23, 1994, pp. 1771-1775, https://doi.org/10.1093/jnci/87.8.607. [5] R. Salgado, C. Denkert, S. Demaria et al., The Evaluation of Tumor-Infiltrating Lymphocytes (TILs) in Breast Cancer: Recommendations by An International TILs Working Group 2014, Annals of Oncology, Vol. 26, No. 2, 2015, pp. 259-271, https://doi.org/10.1093/annonc/mdu450. [6] K. H. Young, Statistical Notes for Clinical Researchers: Chi-Squared Test and Fisher's Exact Test, Restorative Dentistry &amp; Endodontics, Vol. 42, No. 2, 2017, pp. 152-155, https://doi.org/10.5395/rde.2017.42.2.152.[7] L. G. L. Tan, Y. Y. Tan, D. Heng, M. Y. Chan, Predictors of Axillary Lymph Node Metastases in Women with Early Breast Cancer in Singapore, Singapore Med J, Vol. 46, No. 12, 2005, pp. 693, https://doi.org/10.1016/s0959-8049(98)80134-2. [8] A. Barth, P. H. Craig, M. J. Silverstein, Predictors of Axillary Lymph Node Metastases in Patients with T1 Breast Carcinoma, Cancer, Vol. 79, No. 10, 1997, pp. 1918-1922, https://doi.org/10.1002/(SICI)10970142(19970515) 79:10&lt;1918::AID-CNCR12&gt;3.0.CO;2-Y. [9] Y. Iwasaki, Axillary Node Metastasis from T1N0M0 Breast Cancer: Possible Avoidance of Dissection in A Subgroup, Japanese Journal of Clinical Oncology, Vol. 28, No. 10, 1998, pp. 601-603, https://doi.org/10.1093/jjco/28.10.601. [10] G. Viale, S. Zurrida et al., Predicting the Status of Axillary Sentinel Lymph Nodes in 4351 Patients with Invasive Breast Carcinoma Treated in A Single Institution, Cancer, Vol. 103, No. 3, 2005, pp. 492-500, https://doi.org/10.1002/cncr.20809. [11] S. K. Çetintaş, M. Kurt et al., Factors Influencing Axillary Node Metastasis in Breast Cancer, Tumori Journal, Vol. 92, No. 5, 2006, pp. 416-422, https://doi.org/10.1177/030089160609200509. [12] T. V. To, A Study of Morphology, Immunohistochemistry and Their Prognostic Value in Breast Carcinoma, Ph.D Dissertation, Hanoi Medical University, Hanoi, 2004 (in Vietnamese).[13] R. Lauria, F. Perrone et al., The Prognostic Value of Lymphatic and Blood Vessel Invasion in Operable Breast Cancer, Cancer, Vol 76, No. 10, 1995, pp. 1772-1778, https://doi.org/10.1002/1097-0142(19951115)76 :10&lt;1772::AID-CNCR2820761014&gt;3.0.CO;2-O.[14] R. Yerushalmi, M. M. Hayes, K. A. Gelmon, Breast Carcinoma - Rare Types: Review of the Literature, Annals of Oncology, Vol. 20, No. 11, 2009, pp. 1763-1770, https://doi.org/10.1093/annonc/mdp245. [15] A. Caziuc, D. Schlanger, G. Amarinei, Can Tumor-Infiltrating Lymphocytes (TILs) Be a Predictive Factor for Lymph Nodes Status in Both Early Stage and Locally Advanced Breast Cancer?, Vol. 8, No. 5, 2019, pp. 545, https://doi.org/10.3390/jcm8040545. [16] K. Takada, S. Kashiwagi, Y. Asano, Prediction of Lymph Node Metastasis by Tumor-Infiltrating Lymphocytes in T1 Breast Cancer, BMC Cancer, Vol. 20, No. 1, 2020, pp. 598, https://doi.org/10.1186/s12885-020-07101-y.[17] N. T. Ha, Evaluation of Axillary Lymph Node Status and Vascular Infiltration by Molecular Type of Breast Carcinoma, Master Thesis, Hanoi Medical University, Hanoi, 2018 (in Vietnamese).[18] J. Makki, Diversity of Breast Carcinoma: Histological Subtypes and Clinical Relevance, Clinical Medicine Insights: Pathology, Vol. 8, 2015, pp. 23-31, https://doi.org/10.4137/cpath.s31563. [19] L. A. Carey et al, Breast Cancer Subtypes, and Survival in the Carolina Breast Cancer Study, JAMA, Vol. 295, No. 21, 2006, pp. 2492, https://doi.org/10.1001/jama.295.21.2492.