Lymphocyte-predominant Breast Cancer Research Articles

Abstract PS18-10: Tumor-Infiltrating Lymphocytes and Breast Cancer Mortality in Racially and Ethnically Diverse Participants of the Northern California Breast Cancer Family Registry

Abstract Purpose: Greater stromal tumor-infiltrating lymphocyte (sTIL) percentage in pre-treatment breast tumor specimens is associated with superior response to neoadjuvant chemotherapy and survival. We hypothesized that sTIL-survival associations vary by race and ethnicity and mediate survival disparities. Patients and Methods: We evaluated pre-treatment percentages of sTILs in deciles for 284 women enrolled in the Northern California Breast Cancer Family Registry and diagnosed with a first primary invasive breast cancer from 1995-2005. We assessed associations of continuous sTIL scores and lymphocyte-predominant breast cancer (LPBC, defined as ≥50% lymphocytic infiltration of tumor stroma or cell nests) with clinical and epidemiologic characteristics using regression analysis and with breast cancer-specific mortality (BCM) and overall mortality (OM) using Cox proportional hazards regression. Results: The cohort was diverse (64% from racially and ethnically minoritized populations) with near-complete (98%) germline BRCA1/2 testing and long-term follow-up (average 16.2 years, range: 1.3-26.7). In multivariable analyses, higher sTIL score was associated with reduced BCM [LPBC versus non-LPBC: HR=0.44 (95% confidence interval 0.20-0.98); and per decile increase in sTIL score: HR=0.80 (0.69-0.93)], with the strongest association in the subset of patients with hormone receptor-negative disease [HR=0.71 (0.57-0.88) and HR=0.37 (0.14-0.94), respectively]. While there was no significant difference in sTIL score between racial and ethnic groups, the continuous sTIL-survival association was statistically significant among non-Hispanic White [HR=0.73 (0.57-0.94)] and Asian American [HR=0.56 (0.35-0.89)] women but was not seen among African American and Hispanic women. Conclusion: We identified novel sTIL-BCM differences by race and ethnicity, with better survival associated with sTIL enrichment among non-Hispanic White and Asian American but not among African American or Hispanic women. Additional studies are needed to determine whether survival and particularly the response to immunotherapy are differentially mediated by immune factors between racial and ethnic groups. Multivariable Cox Proportional Hazards Regression Models of Breast Cancer-Specific and Overall Mortality. Citation Format: Julia Ransohoff, Iain Miller, Jocelym Koo, Vishal Joshi, Allison Kurian, Kimberly Allison, Esther John, Melinda Telli. Tumor-Infiltrating Lymphocytes and Breast Cancer Mortality in Racially and Ethnically Diverse Participants of the Northern California Breast Cancer Family Registry [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 PS18-10.

Prognostic Value of "Basal-like" Morphology, Tumor-Infiltrating Lymphocytes and Multi-MAGE-A Expression in Triple-Negative Breast Cancer.

"Basal-like" (BL) morphology and the expression of cancer testis antigens (CTA) in breast cancer still have unclear prognostic significance. The aim of our research was to explore correlations of the morphological characteristics and tumor microenvironment in triple-negative breast carcinomas (TNBCs) with multi-MAGE-A CTA expression and to determine their prognostic significance. Clinical records of breast cancer patients who underwent surgery between January 2017 and December 2018 in four major Croatian clinical centers were analyzed. A total of 97 non-metastatic TNBCs with available tissue samples and treatment information were identified. Cancer tissue sections were additionally stained with programmed death-ligand 1 (PD-L1) Ventana (SP142) and multi-MAGE-A (mAb 57B). BL morphology was detected in 47 (49%) TNBCs and was associated with a higher Ki-67 proliferation index and histologic grade. Expression of multi-MAGE-A was observed in 77 (79%) TNBCs and was significantly associated with BL morphology. Lymphocyte-predominant breast cancer (LPBC) status was detected in 11 cases (11.3%) and significantly correlated with the Ki-67 proliferation index, increased number of intratumoral lymphocytes (itTIL), and PD-L1 expression. No impact of BL morphology, multi-MAGE-A expression, histologic type, or LPBC status on disease-free survival was observed. Our data suggest that tumor morphology could help identify patients with potential benefits from CTA-targeting immunotherapy.

Effect of Lipid Levels on Tumor-Infiltrating Lymphocytes and Prognosis in Patients with Triple-Negative Breast Cancer

Objective: Dyslipidemia can promote cell proliferation, malignant transformation, metastasis, and cancer recurrence. Moreover, it could also affect immune infiltration in the tumor microenvironment. Therefore, we aimed to explore the effects of lipid levels on tumor-infiltrating lymphocytes (TILs) and prognosis in patients with triple-negative breast cancer (TNBC). Methods: Samples from 222 patients with TNBC from July 2007 to December 2019 were obtained from the tissue specimen banks in 3 hospitals. The blood samples were used to detect the levels of lipid levels such as apolipoprotein B (Apo B), triglyceride (TG), and low-density lipoprotein cholesterol (LDL-C). The TILs in the 222 TNBC tissues were detected using hematoxylin and eosin (H&E) staining, and the relationship between the lipid levels, clinical characteristics, and prognosis was analyzed. Results: Among TNBC patients, the overall survival (OS) time and disease-free survival (DFS) time were lower in patients with high LDL-C levels than those with low LDL-C levels (p < 0.01, respectively). The DFS was shorter in patients with low stromal TIL (STIL) levels than those with moderate or high STIL levels (p = 0.023). Multifactor Cox regression analysis showed that LDL-C level, Apo B level, and lymphocyte-predominant breast cancer were independent risk factors for OS in TNBC patients. The number of positive lymph nodes, postoperative staging, and total amount of TILs were independent risk factors for DFS in TNBC patients. Conclusion: The LDL-C and STIL levels were correlated with survival and prognosis in patients with TNBC.

Abstract P3-04-10: The TILs-US score adding vascularity assessment based on ultrasonography for predicting tumor-infiltrating lymphocytes in human epidermal growth factor receptor 2-positive and triple-negative breast cancer

Abstract Background: Tumor-infiltrating lymphocytes (TILs) are a useful prognostic factor and predictive biomarker of neoadjuvant chemotherapy treatment response for breast cancer, especially in human epidermal growth factor receptor type 2 (HER2)-positive breast cancer and triple-negative breast cancer (TNBC). However, due to heterogeneity of TIL expression and distribution in the tissues, accurately predicting TIL expression, especially using limited core-needle biopsy specimens, is difficult. Therefore, an accurate and simple preoperative evaluation method is needed. We have reported that the TILs-ultrasonography (US) score determined by characteristic US findings has a predictive performance for lymphocyte-predominant breast cancer (LPBC). This study aimed to investigate whether the TILs-US score with added vascularity assessment has a better predictive performance for LPBC. Methods: This multicenter, retrospective study investigated the validation and scoring of the LPBC characteristic imaging findings and applied it for LPBC and non-LPBC prediction. A total of 100 patients with HER2-positive breast cancer (n = 59) and TNBC (n = 41) treated by curative surgery between January 2014 and December 2021 were evaluated. Stromal lymphocytes in surgical pathological specimens were evaluated; the cutoff value for predicting LPBC was defined as ≥50% stromal TILs. Preoperative US was examined for TIL indicators. The US images with characteristic TILs were scored for LPBC prediction. Univariate and multivariate logistic regression analyses were employed for each potential predictor variable of LPBC. Results: A total of 40 patients with ≥50% stromal TILs were defined as having LPBC. The examined characteristic US findings for predicting LPBC, shape (more lobulated), internal echo level (weaker), posterior echoes (stronger), and vascularity assessment (hypervascularity), were significantly associated with LPBC and used to assign the scoring for predicting LPBC. As previously reported, the TILs-US score ranged from 0–7 points based on three ultrasonic tissue characteristics: shape (round, oval, and polygonal or irregular, 0 points; lobulated, 1 point; and small lobulated, 2 points), internal echo level (high or equal, 0 points; low, 1 point; and extremely low, 2 points), and posterior echoes (shadowing or attenuating, 0 points; no change, 1 point; accentuated, 2 points; and extremely accentuated, 3 points). We further added vascularity assessment (avascular or hypovascular, 0 point; moderately vascular, 1 point; and hypervascular, 2 points) to this scoring system. Based on the receiver operating characteristics (ROC) curves (AUC [Area Under the Curve] 0.77), the score cutoff for predicting LPBC was 4 points for TILs-US score (sensitivity, 0.83; specificity, 0.55; and accuracy, 0.66). Multivariate logistic regression analysis revealed that cT (&amp;lt; T2), estrogen receptor (ER) negativity, and a TILs-US score of ≥4 points were significant LPBC predictors (odds ratio [OR] 3.60; p = 0.028; OR 8.68; p = 0.020; OR 5.99; p = 0.005). Conversely, based on the ROC curves (AUC 0.78), the score cutoff for predicting LPBC was 5 points after adding vascularity assessment (sensitivity, 0.93; specificity, 0.57; and accuracy, 0.71). Multivariate logistic regression analysis revealed that cT (&amp;lt; T2), ER negativity, and a TILs-US score adding vascularity assessment of ≥5 points were significant LPBC predictors (OR 5.12; p = 0.010; OR 10.3; p = 0.019; OR 20.1; p &amp;lt; 0.001). Conclusions: Including the vascularity assessment to the TILs-US score, which can be noninvasively obtained using US, is a more accurate preoperative predictor of LPBC. Vascularity assessment may be an auxiliary factor in predicting LPBC. Univariate and multivariate logistic analysis of significant clinicopathological factors predicting lymphocyte-predominant breast cancer. Citation Format: Yuri Kimura, Norio Masumoto, Sadako Akashi-Tanaka, Kayo Fukui, Midori Noma, Aya Nagata, Takashi Nakamura, Hiroaki Shima, Toshitaka Okuno, Akari Murakami, Yoshiaki Kamei, Shogo Nakano, Koji Arihiro. The TILs-US score adding vascularity assessment based on ultrasonography for predicting tumor-infiltrating lymphocytes in human epidermal growth factor receptor 2-positive and 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 P3-04-10.

The tumor-infiltrating lymphocyte ultrasonography score can provide a diagnostic prediction of lymphocyte-predominant breast cancer preoperatively.

Tumor-infiltrating lymphocytes (TILs) are known to predict the therapeutic effect in breast cancer. Although a preoperative tissue biopsy can be used to evaluate TILs, TILs that are heterogeneously distributed might require examination of all preoperative tissue biopsy samples. We have recently reported that the TIL ultrasonography (US) score, as determined by characteristic US findings, provides excellent predictive performance for lymphocyte predominant breast cancer (LPBC). We herein aimed to determine whether the preoperative TIL-US score can more accurately predict LPBC than preoperative tissue biopsy. We assessed 161 patients with invasive breast cancer that were treated with curative surgery between January 2014 and December 2017. Stromal lymphocytes were examined on preoperative tissue biopsy tissues and surgical pathological specimens. Breast cancer samples with ≥ 50% stromal TILs were defined as pre-LPBC (preoperative tissue biopsy) and LPBC (surgical pathological specimens). Useful factors for predicting LPBC were searched among clinicopathological factors. The TIL-US score cutoff value for predicting LPBC was 4 points based on the receiver operating characteristic curves (area under the curve: 0.88). Several significant predictors for LPBC were revealed by the undertaken multivariate logistic regression analysis (odds ratios: TIL-US score, 26.8; pre-LPBC, 18.6; HER2, 9.2; all, p < 0.05). The sensitivity, specificity, accuracy, positive predictive value, and negative predictive value were 0.74, 0.89, 0.85, 0.67, and 0.92 for the TIL-US score, respectively, and 0.51, 0.98, 0.87, 0.91, and 0.86 for the pre-LPBC, respectively. TIL-US scores can predict LPBC preoperatively and are characterized by a significantly high sensitivity and negative predictive value.

The TILs-US score on ultrasonography can predict the pathological response to neoadjuvant chemotherapy for human epidermal growth factor receptor 2-positive and triple-negative breast cancer

BackgroundThe tumor-infiltrating lymphocyte (TIL)-ultrasonography (US) score determined through characteristic US findings has predictive performance of lymphocyte-predominant breast cancer (LPBC). This study aimed to investigate whether the TIL-US score before neoadjuvant chemotherapy (NAC) can predict the pathological complete response (pCR). MethodsWe evaluated patients with human epidermal growth factor receptor type 2 (HER2)-positive breast cancer (n = 55) and triple-negative breast cancer (TNBC) (n = 24) who underwent surgery after NAC from November 2012 to April 2019. Pre-NAC biopsy examination was performed to examine stromal TILs; the cutoff value for predicting pCR was defined as ≥50% stromal TILs. The TILs-US score was calculated from the pre-NAC US findings. Based on previous data, the cutoff value for predicting pCR was set at ≥4 points. ResultsForty-six patients achieved pCR. The TIL-US score was significantly associated with the pCR rate (p = 0.003) but not the pre-NAC biopsy findings (p = 0.055). Multivariate logistic regression analysis revealed that a TILs-US score ≥4 and HER2 positivity were significant pCR predictors (odds ratio [OR] 3.69; p = 0.031; OR 8.74; p = 0.025). ConclusionsTILs-US scores can be used to evaluate the therapeutic effect of NAC, and may be used as a non-invasive, convenient, and an alternative method to assess stromal TILs in pre-treatment biopsies.

Clinico-pathological relationship between androgen receptor and tumour infiltrating lymphocytes in triple negative breast cancer.

BackgroundTriple negative breast cancer (TNBC) is an aggressive subtype of breast cancer (BC) with ill-defined therapeutic targets. Androgen receptor (AR) and tumour-infiltrating lymphocytes (TILs) had a prognostic and predictive value in TNBC. The relationship between AR, TILs and clinical behaviour is still not fully understood.MethodsThirty-six TNBC patients were evaluated for AR (positive if ≥1% expression), CD3, CD4, CD8 and CD20 by immunohistochemistry. Stromal TILs were quantified following TILs Working Group recommendations. Lymphocyte-predominant breast cancer (LPBC) was defined as stromal TILs ≥ 50%, whereas lymphocyte-deficient breast cancer (LDBC) was defined as <50%.ResultsThe mean age was 52.5 years and 27.8% were ≥60 years. Seven patients (21.2%) were AR+. All AR+ cases were postmenopausal (≥50 years old). LPBC was 32.2% of the whole cohort. Median TILs were 37.5% and 10% (p = 0.1) and median CD20 was 20% and 7.5% (p = 0.008) in AR− and AR+, respectively. Mean CD3 was 80.7% and 93.3% (p = 0.007) and CD8 was 75% and 80.8% (p= 0.41) in AR− and AR+, respectively. All patients who were ≥60 years old expressed CD20. LDBC was found to be significantly higher in N+ versus N− patients (p = 0.03) with median TILs of 20% versus 50% in N+ versus N−, respectively (p = 0.03). LDBC was associated with higher risk of lymph node (LN) involvement (odds ratio = 6; 95% CI = 1.05–34.21; p = 0.04).ConclusionsAR expression was evident in older age (≥50 years). Median CD20 was higher in AR− TNBC, while mean CD3 was higher in AR+ tumours. LDBC was associated with higher risk of LN involvement. Larger studies are needed to focus on the clinical impact of the relation between AR and TILs in TNBC.

Tumor-Infiltrating Lymphocytes and Prognosis in Stage I-III Triple-Negative Breast Cancer: A Retrospective Analysis of 258 Patients Treated Without Neoadjuvant Therapy.

<h3>Purpose/Objective(s)</h3> To investigate the prognostic value of tumor-infiltrating lymphocytes (TIL) in patients with triple-negative breast cancer (TNBC). <h3>Materials/Methods</h3> The clinicopathologic data of 258 patients with I-III stage TNBC treated in one institution from 2010 to 2013 were collected. Two-hundred and twelve patients (82.2%) received mastectomy and 46 (17.8%) received breast-conserving surgery. Eighty-four patients (32.6%) had stage I, 138 (53.5%) had stage II and 36 (14.0%) had stage III disease. All had received adjuvant chemotherapy without neoadjuvant therapy, and 96 (37.2%) received postoperative radiotherapy. The density of TILs with its expression of stromal (s) and intratumoral (i) CD8, FOXP3 (sCD8, iCD8, sFOXP3, iFOXP3) were evaluated by hematoxylin and eosin and immunohistochemical (IHC) staining. An IHC staining for PD-1 and PD-L1 (22C3) were also conducted. Locoregional recurrence (LRR), distant metastasis (DM) and disease-free survival (DFS) and overall survival (OS) rates were calculated by using the Kaplan-Meier method and compared by log-rank test. The association of relapse and survival outcomes with TILs concentration was evaluated using multivariable proportional hazards regression. Optimum cutoff values for sCD8, iCD8, sFOXP3 and iFOXP3 were identified using the maxstat package in R. <h3>Results</h3> At a median follow-up of 80 months (range, 9-111 months), patients with lymphocyte-predominant breast cancer (sTIL ≥60%) had significantly lower 5-year LRR (3.6% vs 15.6%; <i>P</i> = 0.047) and higher DFS (96.4% vs 84.5%; <i>P</i> = 0.025) compared to those with < 60% sTIL. Patients with sCD8 > 10% had significantly lower 5-year LRR (4.2% vs 15.7%, <i>P</i> = 0.010) and DM (2.8% vs 14.6%; <i>P</i> = 006) and higher 5-year DFS (95.8% vs 83.9%, <i>P</i> = 0.015) and OS (97.1% vs 91.2%, <i>P</i> = 0.044) compared to patients with sCD8 ≤10%. Patients with iCD8 > 5% had significantly lower 5-year LRR (2.2% vs 14.8%, <i>P</i> = 0.013) and DM (13.4%, <i>P</i> = 0.021) and higher 5-year DFS (97.8% vs 84.8%, <i>P</i> = 0.008) and OS (97.7 vs 91.7%, <i>P</i> = 0.030) compared to patients with iCD8 ≤5%. Patients with sFOXP3 > 3/HPF also had significantly lower 5-year LRR (4.5% vs 15.0%, <i>P</i> = 0.012) and DM (5.9% vs 13.5%, <i>P</i> = 0.043) and higher 5-year DFS (92.7% vs 85.0%, <i>P</i> = 0.034) and OS (95.6% vs 91.5%, <i>P</i> = 0.047) compared to sFOXP3 ≤3/HPF. However, no survival benefits were found from patients with iFOXP3 > 14/HPF. Presence of PD-1-positive immune cells in TNBC were associated with a significantly better DFS (93.4% vs 83.5%, <i>P</i> = 0.044), while PD-L1 expression had no impact on patient outcomes. Multivariate analysis revealed that sTIL ≥60% (HR 0.23, 95% CI 0.05-0.99, <i>P</i> = 0.048) or PD-1 positivity (HR 0.40, 95% CI: 0.16-0.96, <i>P</i> = 0.041) were independent prognostic factors for DFS, in addition to stage (<i>P</i> < 0.001). No other subsets of TILs nor PD-L1 was independently associated with relapse or survival (all <i>P</i> > 0.05). <h3>Conclusion</h3> High density of stromal TIL and presence of PD-1-positive immune cells may have a positive prognostic value in TNBC. These findings need further validation.

Ultrasonography Combined With Contrast-enhanced Ultrasonography Can Predict Lymphocyte-predominant Breast Cancer.

We investigated whether contrast-enhanced ultrasonography (CEUS) scores can predict lymphocyte-predominant breast cancer (LPBC). We evaluated 75 patients who underwent US and CEUS. LPBC was defined as tissues with ≥50% stromal tumour-infiltrating lymphocytes (TILs) preoperatively. Characteristic US images predicting LPBC were evaluated using TIL-US scores via three ultrasonic tissue characteristics: Shape, internal echo level, and posterior echoes. TIL-CEUS was evaluated based on TIL-US plus CEUS. TIL-US and TIL-CEUS cut-offs for predicting LPBC were 4 and 6 (area under the curve=0.93 and 0.96, respectively) points based on receiver operating characteristics curves. Sensitivity, specificity, and accuracy values (95% confidence intervaI) were 0.94 (0.77-0.99), 0.75 (0.70-0.77), and 0.80 (0.72-0.82); and 0.94 (0.78-0.99), 0.86 (0.81-0.87), and 0.88 (0.80-0.90) for TIL-US and TIL-CEUS, respectively. TIL-CEUS score was a significant single predictor for LPBC in multivariate logistic regression (p=0.001). TIL-CEUS can be used for preoperative LPBC prediction and detection.

Prognostic value of tumor-infiltrating lymphocytes (TILS) and their association with clinicopathological features in breast cancer: A retrospective study involving 53 cases

ObjectiveQuantitative analysis of tumor-infiltrating lymphocytes (TILs) is currently considered as a prognostic factor in several malignant tumors. The aim of our study was to assess the prognostic value of TILs in breast cancers and its association with other clinicopathological prognostic factors in non-specific type (NST) breast carcinoma in Tunisian population. MethodsRetrospective study included 53 women with NST breast carcinoma. The data were collected over a period of 13 months with a follow-up of 40 months for all the patients. The TILs were evaluated according to the 2014 recommendations of the international working group on TILs. ResultsTILs level was between 3% and 60% with mean of 21%. Ten patients had lymphocyte predominant breast cancer (LPBC). Statistical analysis had shown that the TILs level ≤50% was associated with the presence of vascular emboli and the absence of HER2 amplification. Inflammatory-type carcinoma and HER2 amplification significantly worsened OS. Presence of vascular emboli, lymph node metastases, inflammatory type of carcinoma, TILs ≤50%, and absence of estrogen receptors (ER) were associated with reduced RFS. In multivariate analysis, the presence of vascular emboli was an independent factor for OS. TILs ≤50%, inflammatory type of carcinoma and presence of vascular emboli were independent risk factors for RFS. ConclusionThis Tunisian pilot study showed higher level of TILs in NST breast carcinomas is associated with improved RFS. The therapeutic implications will benefit from multiple research studies including ours on the predictive value of TILs for neoadjuvant or adjuvant treatment.

A Study on Tumor-Infiltrating Lymphocytes in Different Subtypes of Breast Cancer

Summary The study aimed to investigate immune cell infiltration in different subtypes of breast cancer (BC). Retrospectively were selected 100 patients with primary BC, grouped into four molecular surrogate subtypes (Luminal A and Luminal B-like, HER2-positive and triple-negative - TN), determined by immunohistochemistry (IHC). In each patient, a percentage of stromal tumor-infiltrating lymphocytes (TILs) was determined by hematoxylin-eosin staining. IHC was performed using primary antibodies CD3, CD4, CD8, CD20, and FOXP3. Immunophenotyped lymphocytes were counted (separately intratumoral and stromal) and semi-quantitatively graded. In the studied tumors, 10% were defined as lymphocyte-predominant BC. A high count of intratumoral and stromal TILs subsets was found mainly in TN and HER2-positive BC. The stroma is the preferred localization for immune cells in all four BC subtypes. CD3+ T predominates over CD20+ B lymphocytes, with CD8+ T cytotoxic and FoxP3+ T regulatory cells dominating T subtypes. HER2 and TN are more immunogenic than Luminal A and Luminal B – like subtypes of BC. The T-cells’ immune response was predominant in the studied cases of BC, with a predominance of CD8+ Tc and Foxp3+ Treg cells located mainly in the stroma.

Molecular Characteristics of Lymphocyte-predominant Triple-negative Breast Cancer.

Tumor-infiltrating lymphocytes (TILs) are considered a prognostic marker for triple-negative breast cancer (TNBC). Immune checkpoint inhibitor (ICI)-based treatments are more effective for tumors with PD-L1-positive TILs, suggesting crucial roles of TILs in the local tumor immunity. However, factors attracting TILs are still largely unknown. Focusing on tumor antigenicity, we examined TNBC samples to identify the characteristics of TIL-high tumors. Nine treatment-naïve TNBCs (TIL-high: five, TIL-low: four) were subjected to next-generation sequencing (NGS). Loss of heterozygosity (LOH) of PTEN was also analyzed. A variety of copy number variations were observed, and no genes differed significantly between TIL-high and -low groups. However, PTEN loss was more frequently observed in the TIL-high group: 60% compared to 25% in TIL-low tumors. NGS correlated well with LOH analysis in identifying PTEN loss. All three tumors with PTEN loss in the TIL-high group showed high PD-L1. All nine samples were microsatellite-stable. Frequent PTEN loss and high expression of PD-L1 in TIL-high TNBC suggest that PTEN mutation may be a biomarker for ICIs.

Abstract PS6-46: Correlation of Tumor-infiltrating lymphocytes with clinicopathological features and treatment outcomes in non-metastatic breast cancer patients

Abstract Background Breast cancer is the second most common cancer in the world and most frequent among women. Tumor-infiltrating lymphocytes (TILs) can impact tumor progression and response to therapies. Here we aimed to study TILS using the International Immunooncology Biomarkers Working Group guidelines and correlating the TILS with clinicopathological characteristics, disease free and overall survival in non-metastatic breast cancer patients. Methods Non metastatic breast cancer patients (N = 86) who presented to our department between (2013 - 2015) were retrospectively evaluated. The assessment of TILS distribution was based on the TILS International Working Group 2014 recommendations. The eligible patients were reviewed retrospectively regarding the demographic status and clinical parameters and primary treatment information were extracted from the medical records. For all of the available patients, disease-free survival (DFS) and overall survival (OS) were calculated. Results In the study the majority of the patients were 45 years of age or less. The TILS percentage was measured using image analysis. All samples had a range of 1-100%, with a mean 22%. There was 9.3% of the patients having lymphocyte predominant breast cancer (≥50%), while 90.3% had TILS percentage of &amp;lt;50%. Dividing the patients into low (0-20%), intermediate (20-&amp;lt;50%) and high (50-100%). TILS percentage gave us 62.7%, 27.9% and 9.3% of the patients respectively. The distribution of TILS according to age showed a trend towards decreasing TILS with increasing age, and this was not statistically significant. The majority of tumors in the study were T2 (60.5%) followed by T1 (15.1%).. T1 and T2 tumors had significantly higher TILS percentages compared to T3 and T4, mean 24.94% versus mean 13.37% respectively. Using the t-test this showed a p&amp;lt;0.006. The majority of patients in the study were N1 at 62.8% followed by an equal percentage of 17.4% as both N0 and N2. Patients who were N0 had a significantly higher TILS percentage when compared with N1 and N2, mean 33.07% versus mean 20.0% respectively. This was statistically significant using the t-test with a p&amp;lt;0.032 (table 1). Also in this study, there were 74.4% tumors of the luminal subtype, 11.6% of the Her-2 enriched subtype and 12.8% of the triple negative subtype. Although there was no statistically significant correlation between any molecular subtype and TILS, there was a tendency for Her-2 enriched tumors and triple negative tumors to have a higher percentage of TILS in comparison with luminal subtypes as seen in table 1. We noticed that there is a subgroup of patients with exceptionally high TILS, (called LPBC with TILS ≥50%) had a beneficial response to treatment and confers additional survival benefit; every incremental increase of 10% also resulted in benefit to treatment and delayed relapse. High level of TILS associated with delayed relapse and a survival benefit. Conclusions We demonstrated that increasing TILS confer a relapse free benefit of breast cancer patient, regardless of the tumor subtype, indicating that TILS represent an breast cancer the patients. Keywords Tumor-infiltrating lymphocyte, Breast cancer, outcome of management Table 1:FinalNTumor infiltrating lymphocytes (TILS %)Min. - Max.Mean ± SD.MedianERNegative (0)212.0 - 100.027.33 ± 28.9318.0Weak (1+)63.0 - 40.019.67 ± 16.2917.0Moderate (2++)135.0 - 62.024.38± 20.6317.0Strong (3+++)451.0 - 87.019.78 ± 17.9217.0N/A15.0PRNegative (0)282.0 - 100.024.86 ± 26.1916.50Weak (1+)36.0 - 12.08.33 ± 3.217.0Moderate (2++)171.0 - 87.022.12 ± 22.1017.0Strong (3+++)371.0 - 62.021.68 ± 17.5518.0N/A15.0HER-2Negative (0)441.0 - 100.022.84 ± 22.9717.50Weak (1+)16.0Moderate (2++)114.0 - 87.025.09 ± 23.7625.0Strong (3+++)232.0 - 65.018.70 ± 17.1312.0N/A74.0 - 53.026.71 ± 22.1225.0BC subtypesLuminal A401.0 - 62.021.28 ± 17.9217.50Luminal B242.0 - 87.019.75 ± 18.9013.50Her2 Enriched104.0 - 65.023.20 ± 21.3616.50Triple -ve112.0 - 100.031.09 ± 35.0819.0N/A15.0 Citation Format: Neamat Elsayed Hegazy, Haitham Mohsen Abdel Motal, Hanan Yehia Tayel, Abdelsalam A Ismail. Correlation of Tumor-infiltrating lymphocytes with clinicopathological features and treatment outcomes in non-metastatic breast cancer patients [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS6-46.

113 Poster - PD-L1 and HSP-70 molecules are part of immunosupressive environment in the deep layer of the lymphocyte predominant breast cancer (LPBC)

Background: Tumor infiltrating lymphocytes (TILs) are involved in host imunity against tumor cells. However, in later phases of the disease high TIL infiltration is related to disease progression. Tumor immunogenicity is strongly correlated with the higher tumor mutation burden. Triple negative (TN) and HER-2 enriched breast cancers have the highest immunogenic potential so the aim of our study was to investigate the TIL infiltration and expression of PD-L1, HSP-70 in such tumors.

Microsatellite instability and mismatch repair protein expressions in lymphocyte-predominant breast cancer.

The frequency of microsatellite instability (MSI) is reportedly extremely low in breast cancer, despite widespread clinical expectations that many patients would be responsive to immune‐checkpoint inhibitors (ICI). Considering that some triple‐negative breast cancers (TNBC) responded well to ICI in a clinical trial and that a high density of tumor‐infiltrating lymphocytes (TILs) is frequently observed in other cancers with high levels of microsatellite instability (MSI‐H), we hypothesized that some TNBC with a high density of TILs would be MSI‐H. Medullary carcinoma (MedCa) of the breast, a rare histological type, is characterized by a high density of TILs. Considering that MedCa of the colon is often MSI‐H, we suspected that MedCa in breast cancer might also include MSI‐H tumors. Therefore, we conducted MSI tests on such breast cancers with a high density of TILs. The MSI status of 63 TIL‐high TNBC and 38 MedCa tumors, all from Asian women who had undergone curative surgery, were determined retrospectively. DNA mismatch repair (MMR) proteins and PD‐L1 expression were also investigated immunohistochemically. All samples were microsatellite stable, being negative for all microsatellite markers. TIL‐high TNBC with low MLH1 protein had higher levels of PD‐L1 in stromal immune cells (P = .041). MedCa tumors showed significantly higher PD‐L1 expression in immune cells than in TIL‐high TNBC (<.001). We found that MSI‐H tumors were absent in TIL‐high breast cancers. Examination of MMR proteins, not a purpose of Lynch syndrome screening, may merit further studies to yield predictive information for identifying patients who are likely to benefit from ICI.

Predicting therapeutic efficacy of endocrine therapy for stage IV breast cancer by tumor-infiltrating lymphocytes.

The tumor immune environment not only modulates the effects of immunotherapy, but also the effects of other anticancer drugs and treatment outcomes. These immune responses may be evaluated by measuring tumor-infiltrating lymphocytes (TILs), which has been frequently verified clinically. In the present study, the prediction of the therapeutic effect of endocrine therapy by TILs on stage IV breast cancer was clinically analyzed. Data from 40 patients who underwent endocrine therapy as the initial drug therapy for stage IV breast cancer were used. The correlation between TILs, evaluated according to standard methods, and prognosis, including the efficacy of endocrine therapy, was investigated retrospectively. Patients with ≥50% lymphocytic infiltration were considered to have lymphocyte-predominant breast cancer (LPBC). An analysis of outcomes revealed no difference in progression-free survival (PFS; P=0.171), time to treatment failure (TTF; P=0.054), or overall survival (OS; P=0.641) between the high TIL (>10%) and low TIL (≤10%) groups. Patients with LPBC (≥50%) exhibited a significant prolongation of PFS (P=0.005, log-rank), TTF (P=0.001) and OS (P=0.027) compared with non-LPBC patients. On receiver operating characteristics (ROC) curve analysis, better results were obtained with LPBCs [area under the curve (AUC)=0.700] than with TILs (AUC=0.606). The present findings suggest that a high level of lymphocytic infiltration in the tumor stroma may serve as a predictor of the therapeutic efficacy of endocrine therapy in patients with stage IV estrogen receptor-positive breast cancer.

ATM kinase regulates tumor immunoreactions in lymphocyte-predominant breast cancer through modulation of NKG2D ligand and TNF cytokines on tumor cells.

To explore impact of Ataxia telangiectasia mutated (ATM) kinase on immunoreactions in lymphocyte-predominant breast cancer (LPBC), particularly its role in triple negative breast cancer (TNBC), 194 cases of LPBC were identified with pertinent clinical information retrieved. The expressions of ATM, activated ATM (P-ATM), Fas ligand (FASL), tumor necrosis factor-related apoptosis-induced ligand (TRAIL), major histocompatibility complex class I chain-related protein A (MICA), CD8, and Forkhead box P3 (FOXP3) were assessed by immunohistochemically. We found that ATM expressed on tumor cells was correlated with upregulated expression of P-ATM and MICA (P < 0.05), down-regulated expression of FASL and TRAIL (P < 0.01), and decreased Ki-67 tumor labeling (P < 0.05). However, within the TNBC group, only a negative correlation with FASL expression was found (P = 0.001). ATM and MICA expressions were significantly down -regulated in TNBC (P < 0.01) compared to non-TNBC, while TRAIL was significantly upregulated (P < 0.01). Tregs were increased in TNBC (P < 0.05), with CD8 + TILs decreased (P < 0.01). Ki-67 index was higher in TNBC than in non-TNBC (P < 0.01). ATM may play an important role in immunoreaction of LPBC, probably through upregulation of MICA and down-regulation of FASL and TRAIL. The down-regulated ATM expression in TNBC might be responsible for impaired tumor immunoactivity, rapid tumor growth, and aggressive clinical course.

Abstract GS3-01: Investigating denosumab as an add-on treatment to neoadjuvant chemotherapy and two different nab-paclitaxel schedules in a 2x2 design in primary breast cancer - First results of the GeparX study

Abstract Background Pharmacologic inhibition of the receptor RANK with denosumab can prevent or delay bone metastasis in patients with breast cancer (BC) and may improve disease-free survival in postmenopausal woman with hormone receptor (HR) positive BC. The GeparSepto study demonstrated an increased pCR rate after weekly nab-paclitaxel compared to weekly standard solvent-based paclitaxel, whereas the ETNA did not. It remains still unclear which administration regimen should be preferred for nab-paclitaxel. GeparX investigates whether the addition of denosumab to anthracycline/taxan-containing neoadjuvant chemotherapy (NACT) increases the pCR rate and which nab-paclitaxel schedule should be preferred for primary BC. Methods GeparX, a phase II study, was planned to randomize 778 patients to NACT+/-denosumab (120mg s.c. q4w for 6 cycles), stratified by lymphocyte predominant BC (≤50% vs &amp;gt;50% stromal tumor infiltrating lymphocytes [sTILs]), subtype (HER2-/HR+ vs triple negative (TNBC) vs HER2+), and epirubicin/cyclophosphamide (EC, q2w vs q3w). Secondarily, patients are randomized to different taxane backbones: nab-paclitaxel 125mg/m² weekly+EC or nab-paclitaxel 125mg/m² day 1,8 q22 (2/3)+EC. Carboplatin was given in TNBC and ABP 980 + pertuzumab (biosimilar substudy) in HER2+ BC. Patients with primary cT1c-cT4a-d BC and centrally assessed HR, HER2, Ki-67, and sTILs on core biopsy could be enrolled. Co-primary objectives compare the pCR (ypT0 ypN0) rates of NACT+/-denosumab and the pCR rates between the two backbone nab-paclitaxel treatments. Secondary objectives are interaction of denosumab treatment with RANK expression; pCR per arm for both randomizations in TNBC and HER2+ BC; pCR in RANK high vs low; other pCR definitions for both randomizations; response rates; breast conservation rates; toxicity and compliance; survival. The biosimilar substudy evaluates safety and efficacy of the biosimilar trastuzumab ABP 980 in combination with pertuzumab for HER2+ positive BC. Sample size (primary endpoint) planning assumed a pCR improvement by denosumab from 35% to 46% (OR=1.58; 2-sided α=0.10; 92% power) and by different nab-paclitaxel schedule from 36% to 45% (OR=1.45; 2-sided α=0.10; 80% power; overall α=0.20), requiring 778 patients (2-sided α=0.10; 80% power). Results A total of 780 patients were randomized between 2/2017 and 3/2019. Baseline characteristics were well balanced. Median age was 49.0 years [range 22-80]; 37.7% of patients had cT1, 56.3% cT2, 6.0% cT3/ T4, and 38.1% were cN+; G3 was reported in 66.5% and Ki-67 &amp;gt;20% in 83.1%; 7.9% of tumors showed &amp;gt;50% sTILs. 40.6% of patients had TNBC, 39.7% HER2/HR+ BC and 19.6% HER2+ BC. 343 serious adverse events (SAEs) in a total of 214 patients were reported. The rate of patients with at least one SAE was higher with nab-paclitaxel 125mg/m² weekly than in 2/3 weeks (30.8% vs 24.1%) irrespective of the addition of denosumab. The last patient will be operated at the beginning of October 2019. Conclusions The results of the primary endpoints and selected secondary endpoints will be presented at the meeting. Funding: The trial was financially supported by Amgen and Celgene Citation Format: Jens-Uwe Blohmer, Theresa Link, Sherko Kümmel, Michael Untch, Marianne Just, Peter A Fasching, Andreas Schneeweiss, Pauline Wimberger, Oliver Stötzer, Jens Huober, Marc Thill, Christian Jackisch, Kerstin Rhiem, Claus Hanusch, Carsten Denkert, Knut Engels, Valentina Nekljudova, Sibylle Loibl. Investigating denosumab as an add-on treatment to neoadjuvant chemotherapy and two different nab-paclitaxel schedules in a 2x2 design in primary breast cancer - First results of the GeparX study [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr GS3-01.

Associations between absolute neutrophil count and lymphocyte-predominant breast cancer

IntroductionTumor-infiltrating lymphocytes (TILs) might be associated with host-cell mediated immunity, which could be partly reflected by peripheral blood cell counts. In addition, lymphocyte-predominant breast cancer (LPBC), which was defined as tumors having high TIL levels, showed a favorable prognosis among triple-negative breast cancer or HER2-positive breast cancer. We aimed to investigate whether peripheral blood cell counts are associated with LPBC. MethodsWe evaluated the percentage of stromal TILs in breast cancer patients who underwent primary surgery, using the standardized methodology proposed by the international TIL Working Group. Lymphocyte-predominant breast cancer (LPBC) was defined as tumors having high TIL levels (≥50%). Peripheral blood cell counts including absolute neutrophil counts (ANC), absolute lymphocyte counts (ALC) and neutrophil-to-lymphocyte ratio (NLR) was obtained from pretreatment laboratory data. ResultOf the 810 patients, 132 (16.3%) had LPBC, and 678 (83.7%) had non-LBPC. In a comparison of 3 markers of peripheral blood counts, LPBC had a significantly lower mean ANC than non-LPBC (3,304 vs. 3,564; P = 0.023), but the other means were not different. In multivariable analysis, each 1K increment in ANC corresponded to an odds ratio of 0.790 (95% CI, 0.642 to 0.971) for LPBC. In the ER-negative and high-Ki67 subgroups identified by interaction tests, significant inverse correlations between continuous ANC and TILs were noted. ConclusionLow peripheral ANC could be linked with LPBC, supporting the hypothesis that systemic immune cell counts might be associated with the tumor-immune microenvironment.

72P - Clinico-pathological relationship between androgen receptor (AR) and tumor infiltrating lymphocytes (TILs) in triple negative breast cancer (TNBC)

Background: Triple negative breast cancer (TNBC) is an aggressive subtype of with ill-defined therapeutic targets. Androgen receptor (AR) expression ranged from 32-53% in different reports presenting emerging biomarker in TNBC. Tumor-infiltrating lymphocytes (TIL) play an important prognostic and predictive value in TNBC. The relationship between AR, TIL and clinical behavior is still not fully understood. Methods: Thirty-six TNBC patients were retrospectively selected from a cohort of 800 patients diagnosed in 2012. Clinico-demographic data were collected. Formalin-fixed, paraffin embedded tissue specimens were evaluated for AR (+ ve if ≥ 1% expression), CD3, CD20, CD4, CD8 with IHC. Stromal TIL were quantified following the TIL Working Group recommendations. Lymphocyte-predominant breast cancer (LPBC) are defined as stromal TILs of ≥ 50%. Results: The mean age was 52.5 years and 27.8% were ≥60 years. The majority of patients (58.3%) were invasive duct carcinoma (IDC), while IDC with medullary features and invasive lobular carcinoma were 22% and 14% respectively. Grade III tumors were 52.8%. Stage III, IV were 54.3%, 8.6% respectively and N+ve were 77%. Seven patients of the total (19.4%) were AR+ve. TIL were assessed in 31 patients where LPBC was 32.3%. Median TIL was 15% and 33% (p = 0.07) and CD20 was 7% and 18% (p = 0.01) in AR+ve and AR-ve respectively. Mean CD3 was 93.3% and 80.6% (p = 0.007) and CD8 was 80.8% and 75% (p = 0.41) in AR+ve and AR-ve respectively. Majority of patients ≥60 years (66.7%) were lymphocyte deficient (TIL˂50%). CD8 was significantly lower in stage III/IV vs I/II (p = 0.04). LPBC type was significantly lower in N+ve vs N-ve patients (p = 0.03). Significant positive correlation presented between CD8 and CD3 (p˂0.001) while significant negative correlation presented between CD20 and CD3 (p = 0.03) and CD4 and CD8 (p = 0.04). Non statistically significant increased median OS in AR+ve vs AR-ve (44 vs 40 months, p = 0.84). Conclusions: The present study show that T cell marker (CD3) was more predominant in AR+ve TNBC. Older patients and N+ve were lymphocyte deficient (TIL˂50%). More studies are needed to focus on the clinical impact of the relation between AR and TIL in TNBC. Legal entity responsible for the study: Hagar Elghazawy. Funding: Has not received any funding. Disclosure: All authors have declared no conflicts of interest.