Stromal Tumor-infiltrating Lymphocytes Research Articles

Neoadjuvant Chemotherapy Response in Triple-Negative Apocrine Carcinoma: Comparing Apocrine Morphology, Androgen Receptor, and Immune Phenotypes.

Apocrine differentiation and androgen receptor (AR) positivity represent a specific subset of triple-negative breast cancer (TNBC) and are often considered potential prognostic or predictive factors. To evaluate the response of TNBC to neoadjuvant chemotherapy (NAC) and to assess the impact of apocrine morphology, AR status, Ki-67 labeling index (Ki-67LI), and tumor-infiltrating lymphocytes (TILs). A total of 232 TNBC patients who underwent NAC followed by surgical resection in a single institute were analyzed. The study evaluated apocrine morphology and AR and Ki-67LI expression via immunohistochemistry from pre-NAC biopsy samples. Additionally, pre-NAC intratumoral TILs and stromal TILs (sTILs) were quantified from biopsies using a deep learning model. The response to NAC after surgery was assessed based on residual cancer burden. Both apocrine morphology and high AR expression correlated with lower Ki-67LI (P < .001 for both). Apocrine morphology was associated with lower postoperative pathologic complete response (pCR) rates after NAC (P = .02), but the difference in TILs between TNBC cases with and without apocrine morphology was not statistically significant (P = .09 for sTILs). In contrast, AR expression did not significantly affect pCR (P = .13). Pre-NAC TILs strongly correlated with postoperative pCR in TNBCs without apocrine morphology (P < .001 for sTILs), whereas TNBC with apocrine morphology demonstrated an indeterminate trend (P = .82 for sTILs). Although TIL counts did not vary significantly based on apocrine morphology, apocrine morphology itself was a more reliable predictor of NAC response than AR expression. Consequently, although apocrine morphology is a rare subtype of TNBC, its identification is clinically important.

The Impact of Neoadjuvant Chemotherapy on Ovarian Cancer Tumor Microenvironment: A Systematic Review of the Literature.

Immunotherapy, particularly the use of immune checkpoint inhibitors (ICIs), has shown limited efficacy in treating ovarian cancer (OC), possibly due to diverse T cell infiltration patterns in the tumor microenvironment. This review explores how neoadjuvant chemotherapy (NACT) impacts the immune landscape of OC, focusing on tumor-infiltrating lymphocytes (TILs), PD-1/PD-L1 expression, and their clinical implications. A comprehensive literature search across four databases yielded nine relevant studies. These studies evaluated stromal (sTILs) and intra-epithelial (ieTILs) TILs before and after NACT. sTIL responses varied, impacting prognostic outcomes, and ieTILs increased in some patients without clear survival associations. PD-L1 expression after NACT correlated with improved overall survival (OS), and increases in granzyme B+ and PD-1 correlated with longer progression-free survival (PFS). Remarkably, reduced FoxP3+ TILs post-NACT correlated with better prognosis. NACT often increases sTIL/ieTIL and CD8+ subpopulations, but their correlation with improved PFS and OS varies. Upregulation of co-inhibitory molecules, notably PD-L1, suggests an immunosuppressive response to chemotherapy. Ongoing trials exploring neoadjuvant ICIs and chemotherapy offer promise for advancing OC treatment. Standardized measurements assessing TIL density, location, and heterogeneity are crucial for addressing genetic complexity and immunological heterogeneity in OC.

The Impact of Neoadjuvant Chemotherapy on Ovarian Cancer Tumor Microenvironment: A Systematic Review of the Literature

Immunotherapy, particularly the use of immune checkpoint inhibitors (ICIs), has shown limited efficacy in treating ovarian cancer (OC), possibly due to diverse T cell infiltration patterns in the tumor microenvironment. This review explores how neoadjuvant chemotherapy (NACT) impacts the immune landscape of OC, focusing on tumor-infiltrating lymphocytes (TILs), PD-1/PD-L1 expression, and their clinical implications. A comprehensive literature search across four databases yielded nine relevant studies. These studies evaluated stromal (sTILs) and intra-epithelial (ieTILs) TILs before and after NACT. sTIL responses varied, impacting prognostic outcomes, and ieTILs increased in some patients without clear survival associations. PD-L1 expression after NACT correlated with improved overall survival (OS), and increases in granzyme B+ and PD-1 correlated with longer progression-free survival (PFS). Remarkably, reduced FoxP3+ TILs post-NACT correlated with better prognosis. NACT often increases sTIL/ieTIL and CD8+ subpopulations, but their correlation with improved PFS and OS varies. Upregulation of co-inhibitory molecules, notably PD-L1, suggests an immunosuppressive response to chemotherapy. Ongoing trials exploring neoadjuvant ICIs and chemotherapy offer promise for advancing OC treatment. Standardized measurements assessing TIL density, location, and heterogeneity are crucial for addressing genetic complexity and immunological heterogeneity in OC.

Tumor-Infiltrating Lymphocytes in Patients With Stage I Triple-Negative Breast Cancer Untreated With Chemotherapy

The absolute benefit of chemotherapy for all patients with stage I triple-negative breast cancer (TNBC) is unclear, and biomarkers are not currently available for selecting patients with an excellent outcome for whom neoadjuvant or adjuvant chemotherapy may have negligible benefit. High levels of stromal tumor-infiltrating lymphocytes (sTILs) are associated with favorable survival in TNBC, but data solely in stage I TNBC are lacking. To examine the outcomes of patients of all ages with stage I TNBC solely and who received neither neoadjuvant nor adjuvant chemotherapy, according to centrally reviewed sTIL levels at prespecified cutoffs. This cohort study used the Netherlands Cancer Registry to identify patients diagnosed with stage I TNBC between January 1, 2005, and December 31, 2015, who were not treated with chemotherapy. Only patients who did not receive neoadjuvant and/or adjuvant chemotherapy were selected. The clinical data were matched with their corresponding pathology data provided by the Dutch Pathology Registry. Data analysis was performed between February and October 2023. The primary end point was breast cancer-specific survival (BCSS) at 5, 10, and 15 years for the prespecified sTIL level cutoffs of 30%, 50%, and 75%. Hematoxylin and eosin-stained slides were used for central review of histologic subtype, grade, and lymphovascular invasion. The International Immuno-Oncology Biomarker Working Group guidelines were used to score the sTIL levels; these levels were determined for 1041 patients. A total of 4511 females with stage I TNBC (mean [SD] age at diagnosis, 64.4 [11.1] years; median follow-up, 11.4 [95% CI, 10.9-11.9] years) were included. Most tumors (952 [91.5%]) were invasive carcinomas of nonspecial histologic subtype. Most patients (548 [52.6%]) had pT1cN0 tumors. Median (range) sTIL level was 5% (1%-99%). A total of 775 patients (74.4%) had sTIL levels below 30%, 266 (25.6%) had 30% or greater, 203 (19.5%) had 50% or greater, and 141 (13.5%) had 75% or greater. Patients with pT1abN0 tumors had a more favorable outcome vs patients with pT1cN0 tumors, with a 10-year BCSS of 92% (95% CI, 89%-94%) vs 86% (95% CI, 82%-89%). In the overall cohort, sTIL levels of at least 30% were associated with better BCSS compared with sTIL levels less than 30% (96% and 87%, respectively; hazard ratio [HR], 0.45; 95% CI, 0.26-0.77). High sTIL levels of 50% or greater were associated with a better outcome than low sTIL levels of less than 50% (HR, 0.27; 95% CI, 0.10-0.74) in patients with pT1C tumors, with a 10-year BCSS of 95% increasing to 98% with sTIL levels of 75% or greater. Results of this study showed that patients with stage I TNBC and high level of sTILs who did not receive neoadjuvant or adjuvant chemotherapy had excellent 10-year BCSS. The findings further support the role of sTILs as integral biomarkers in prospective clinical trials of therapy optimization for this patient population.

Clinical, histopathological, and molecular biomarkers in a multicentric cohort of Mexican women with triple-negative breast cancer.

e13152 Background: Triple negative breast cancer (TNBC) is a heterogeneous entity with a marked variation in biological behavior, treatment responses, and patient outcomes. Thus, the investigation of clinical, histopathological, and molecular biomarkers is essential to understand this variability and potentially inform more effective management approaches across different patient profiles. Methods: We conducted a retrospective multicenter study of women &gt;18 years diagnosed with TNBC between 2006 and 2021 in two Mexican centers. We systematically gathered clinical and histopathological data, including an assessment of H&amp;E-stained slides for histopathological features and tumor-infiltrating lymphocytes (TILs), alongside immunohistochemistry analyses for androgen receptors (AR), CD8, and doublecortin-like kinase 1 (DCLK1). Statistical analyses were conducted using R version 4.3.2. Results: In our cohort, comprising 289 patients, the average age at diagnosis was 49.13 years, with a median overall survival (OS) of 63 months and a 5-year OS rate of 55%. The distribution of clinical stages at diagnosis was as follows: I (n=12, 4.2%), II (n=94, 32.5%), III (n=130, 45.0%), and IV (n=53, 18.3%). CD8 expression was higher in patients with no recurrence compared to patients with stage IV at diagnosis (p&lt;0.001), and it decreased with advancing clinical stages. We also found statistically significant differences in OS between negative and positive CD8 expression, favoring the latter (5-year OS 51.8% vs. 72.2%, p=0.032). Disease-free survival (DFS) and OS also differed significantly between low (≤10%) and high (&gt;10%) stromal TILs, favoring the high expression group (5-year DFS 51.9% vs. 70.8%, p=0.0066; 5-year OS 50.1% vs. 70.3%, p=0.0018). CD8 levels positively correlated with stromal and intratumoral TILs. Positive AR expression (&gt;10%) was seen in 8.9% of cases, with no observed differences in DFS or OS according to negative or positive status (5-year DFS 56.0% vs. 72.2%, p=0.42; 5-year OS 55.0% vs. 60.3%, p=0.68). Finally, DCLK1 positivity (≥10%) was seen in 23% of cases, with no influence on survival (negative vs. positive status, 5-year DFS 58.5% vs. 53.8%, p=0.63; 5-year OS 54.6% vs. 58.1%, p=0.60). Conclusions: Our study underscores the prognostic significance of CD8 and TILs in predicting DFS and OS among TNBC patients. Notably, the expressions of AR and DCLK1 were not independently associated with survival, although their prognostic impact in combination with other biomarkers is still an area of active research. Overall, our findings advocate for a more detailed examination of the molecular characteristics of TNBC within our demographic, encompassing an expanded array of biomarkers and a thorough investigation of the tumor microenvironment.

Association between stromal tumor-infiltrating lymphocytes (TILs) and pathologic complete response (pCR) in patients with early breast cancer (BC) treated with neoadjuvant chemotherapy and HER2-directed therapies in NSABP B-41.

3017 Background: In the phase 3 NSABP B-41 trial, there was an association between pCR and survival in 519 women with early HER2-positive BC treated with neoadjuvant chemotherapy in combination with trastuzumab (T), lapatinib (L), or both (TL). There is a need to identify prognostic biomarkers to tailor patients’ treatment, and the quantification of stromal TILs represents a promising, accessible, and reproducible option. In this analysis, we studied the association between TILs and pCR. Methods: Eligible patients had a baseline core biopsy sample, known pCR status, and no consent withdrawal for the use of specimens. Slides were scanned using an Aperio GT 450 automated scanner to create whole slide images. TIL analysis was then completed based on the international TIL working group guidelines (RS). Patients were grouped into three TIL score categories based on tumor TIL %: low (1-5%), intermediate (6-30%), and high (31-95%). The dose-response relationship between TIL percentage and pCR was explored via generalized linear models with splines. Results: 257 patients were included. 89 (35%) received T, 92 (36%) L, and 76 (29%) TL. 149 (58%) had ER-positive BC. TIL categories in this cohort: 49% low, 22% intermediate, and 29% high. TIL expression was similar across BC intrinsic subtypes and by ER. There was no association between the TIL group and pCR, irrespective of the treatment group (p=0.98). Among tumors with low TILs, T or TL had similar pCR compared to L alone (51.3 v 49%; p=0.95). Among tumors with high TILs, T or TL had higher pCR than L (61.2 v 40.4%; p=0.04). Among patients with ER-positive BC, high TILs were associated with a numerical increase in pCR rate (56% vs 39.6%; p=0.11). Among patients with ER-negative tumors, high TILs were associated with a numerical decreased rate of pCR (50% v. 66.7%; p=0.15). The p-value of the Breslow test of homogeneity of the odds ratios across ER status was 0.016, suggesting that the association between TILs (0-30 v. &gt;30) and pCR varies across ER status.No interaction between the TIL percentage and age, nodal status, menopausal status, or histologic grade was noted. Conclusions: This analysis revealed that patients with a high TIL percentage treated with T or TL compared to L were more likely to achieve a pCR. These findings can be partially explained by the mechanism of action of the HER2-directed agents. T leads to antibody-dependent cell-mediated cytotoxicity, and a higher percentage of TILs may enhance this mechanism, whereas L has intracellular tyrosine kinase disruption. The association between TILs and pCR varied across ER status. Our next steps include additional studies assessing the interaction between TILs and long-term outcomes, as well as gene expression and TILs to validate this biomarker for future clinical trials in HER2+ early BC. Clinical trial information: NCT00486668 .

Tumor Infiltrating Lymphocytes as an Independent Prognostic Factor in Undifferentiated Nasopharyngeal Carcinoma.

Nasopharyngeal carcinoma (NPC) is a common type of cancer in Southeast Asia. This cancer usually spreads locally and to nearby lymph nodes. One unique feature of NPC is its many immune cells called tumor-infiltrating lymphocytes (TILs). Recent studies have suggested that TILs in many types of cancer can indicate a better prognosis. However, the role of TILs in NPC is still a matter of debate. Further research is necessary to determine whether TILs can be used as a prognostic factor of NPC's outcome. A retrospective cohort study was conducted at Sardjito Hospital to examine the records and pathological sections of patients treated for the undifferentiated subtype of NPC. Two pathologists analyzed the presence of TILs using HE-stained slides. TILs were evaluated in stromal compartments, and their association with clinicopathological variables was analyzed using the Chi-square and Fisher exact tests. The study compared overall survival in tumor patients with varying TIL levels using Kaplan-Meier survival curves and the log-rank test. A Cox regression model was used for univariate and multivariate analyses to test the significance of different factors. Out of the total 61 subjects, 16 (26.2%) had high stromal TILs (≥ 70%), and 45 (73.8%) had low stromal TILs (<70%). The subjects' sex, age, and tumor stage did not affect the OS. However, high stromal TILs (≥ 70%) showed a significant association with a longer OS (log-rank test p = 0.006, HR 0.37, 95% CI 0.17-0.79, log-rank p = 0.006). Moreover, multivariate analysis confirmed that TILs were an independent prognostic indicator for OS (aHR 0.015). TILs correlate positively with overall survival in the undifferentiated NPC subtype and are an independent prognostic indicator.

Are we ready to use PSMA as a theranostic biomarker in clinical practice for patients with breast cancer?

e13114 Background: Subtype classification for breast cancer (BC) patients is important for risk-stratification. Unfortunately, this parameter is not able to discriminate perfectly between high- and low-risk disease and the identification of an accurate biomarker to determine aggressiveness is still a clinical need. Prostate-specific membrane antigen (PSMA) could be important given that it has been reported to be expressed in BC tumor cells and even more in endothelial cells of tumor vessels. Methods: We analyzed 101 BCs of whom 22 luminal A, 34 Luminal B, 22 HER2 positive and 23 triple negative (TN) BC to assess whether PSMA expression by immunohistochemistry was different in tumor subtypes and was related to ki67 expression and stromal (tumour-infiltrating lymphocytes) TILs. PSMA positivity was calculated as the number of positive vessels on 10 fields at 40X magnifications. Kruskal Wallis’s test was used to assess difference in PSMA positivity among BC subgroups and Dunn’s test was performed for post-hoc comparisons; Spearman’s rho coefficient was calculated to analyze the correlation among PSMA, KI67 and TILs. Results: The median age at diagnosis of BC patients was 66 (min-max: 25-87 years). Seventy (69.3%) patients had ER&gt;0 and 64 (63.4%) had PgR&gt;0. The ductal histotype was present in 92 patients (91%). The majority of the patients had grade 3 tumors (55.4%). Considering the number of positive vessels, our results showed that median PSMA was higher in TNBC compared to Luminal A, Luminal B and HER2 positive tumors (p&lt;0.001, p=0.001 and p=0.041, respectively). Ki67 was higher in TNBC compared to Luminal A, Luminal B and HER2 positive tumors (p&lt;0.001, p&lt;0.001 and p=0.001, respectively). We saw a correlation between PSMA and ki67, especially in HER2 positive tumors (p&lt;0.001), while a correlation between PSMA and TILs was observed in Luminal A (p=0.028). The analysis of PSMA expression on the lymph nodes showed the same trend observed for the primary tumors given that it was higher in TNBC compared to HER2 positive and luminal cancers. Conclusions: Our results suggest that PSMA could be used as a theranostic biomarker in BC considering that it is highly expressed in more aggressive tumors and the possibility to treat PSMA expressing patients for anti-angiogenesis and/or radionuclide treatment.

Deep learning-based quantitative assessment of tumor-infiltrating lymphocytes from hematoxylin and eosin-stained slides in triple-negative breast cancer: A prognostic study.

553 Background: Tumor-infiltrating lymphocytes (TILs) are pivotal in predicting responses to preoperative chemotherapy in triple-negative breast cancer. Yet, their clinical adoption as a diagnostic marker is limited by the challenge of achieving high accuracy with objective measures. This study introduces a novel AI-based method to quantitatively assess stromal TILs within the tumor bed using hematoxylin and eosin (H&amp;E)-stained slides, examining their correlation with disease-free survival (DFS) and overall survival (OS). Methods: In this study, 68 cases of breast cancer with axillary lymph node metastasis at the time of resection were selected from a total of 3,902 resections conducted between 2002 and 2016. We then developed an AI-based model to detect the epithelium and lymphocytes (CD3/CD20) from H&amp;E-stained slides. This model was trained using annotated datasets, which included 26,509 images of the epithelium and 12,273 images of lymphocytes. The tumor bed areas were precisely defined by pathologists. Subsequently, the AI model identified epithelial and lymphocyte regions within these predefined areas. The TILs score was then calculated as the ratio of the lymphocyte area to the total stromal area within the tumor bed, excluding the epithelial areas identified by the AI model. Finally, we assessed the prognostic significance of the TILs score for DFS and OS through ROC analysis and log-rank tests. Results: ROC analysis identified an optimal TILs score threshold of 0.0042 for both DFS and OS, with an AUC of 0.743 (95% CI, 0.622-0.864) for DFS and 0.686 (95% CI, 0.559-0.812) for OS. This threshold significantly stratified patients into groups with distinct DFS and OS outcomes (P=0.003 and P=0.008, respectively). Conclusions: AI quantification of TILs offers a promising prognostic tool in triple-negative breast cancer, enhancing the objectivity and clinical utility of pathological analyses. Accurate TILs measurement could revolutionize prognostication and treatment planning.

Immunohistochemical Profile of Triple-Negative Breast Cancers: SOX10 and AR Dual Negative Tumors Have Worse Outcomes

Triple-negative breast cancer (TNBC) refers to an estrogen receptor–negative, progesterone receptor–negative, and HER2-negative breast cancer. Although accepted as a clinically valid category, TNBCs are heterogeneous at the histologic, immunohistochemical, and molecular levels. Gene expression profiling studies have molecularly classified TNBCs into multiple groups, but the prognostic significance is unclear except for a relatively good prognosis for the luminal androgen receptor subtype. Immunohistochemistry (IHC) has been used as a surrogate for basal and luminal subtypes within TNBC, but prognostication of TNBC using IHC is not routinely performed. We aimed to study immunophenotypic correlations in a well-annotated cohort of consecutive TNBCs, excluding postneoadjuvant chemotherapy cases. Tissue microarrays were constructed from a total of 245 TNBC cases. IHC stains were performed and consisted of luminal (AR and INPP4B), basal (SOX10, nestin, CK5, and EGFR), and diagnostic (GCDFP15, mammaglobin, GATA3, and TRPS1) markers. Survival analysis was performed to assess the significance of clinical-pathologic variables including age, histology, grade, lymphovascular invasion, Nottingham prognostic index category, American Joint Committee on Cancer (AJCC) stage, stromal tumor-infiltrating lymphocytes at 10% increment, CD8+ T-cell count, Ki-67 index, PD-L1 status, and chemotherapy along with the results of IHC markers. Apocrine tumors show prominent reactivity for luminal markers and GCDFP15, whereas no special-type carcinomas are often positive for basal markers. TRPS1 is a sensitive marker of breast carcinoma but shows low or no expression in apocrine tumors. High AJCC stage, lack of chemotherapy, and dual SOX10/AR negativity are associated with worse outcomes on both univariable and multivariable analyses. Lymphovascular invasion and higher Nottingham prognostic index category were associated with worse outcomes on univariable but not multivariable analysis. The staining for IHC markers varies based on tumor histology, which may be considered in determining breast origin. Notably, we report that SOX10/AR dual negative status in TNBC is associated with a worse prognosis along with AJCC stage and chemotherapy status.

Luminal androgen receptor subtype and tumor-infiltrating lymphocytes groups based on triple-negative breast cancer molecular subclassification.

In our previous study, we developed a triple-negative breast cancer (TNBC) subtype classification that correlated with the TNBC molecular subclassification. In this study, we aimed to evaluate the predictor variables of this subtype classification on the whole slide and to validate the model's performance by using an external test set. We explored the characteristics of this subtype classification and investigated genomic alterations, including genomic scar signature scores. First, TNBC was classified into the luminal androgen receptor (LAR) and non-luminal androgen receptor (non-LAR) subtypes based on the AR Allred score (≥ 6 and < 6, respectively). Then, the non-LAR subtype was further classified into the lymphocyte-predominant (LP), lymphocyte-intermediate (LI), and lymphocyte-depleted (LD) groups based on stromal tumor-infiltrating lymphocytes (TILs) (< 20%, > 20% but < 60%, and ≥ 60%, respectively). This classification showed fair agreement with the molecular classification in the test set. The LAR subtype was characterized by a high rate of PIK3CA mutation, CD274 (encodes PD-L1) and PDCD1LG2 (encodes PD-L2) deletion, and a low homologous recombination deficiency (HRD) score. The non-LAR LD TIL group was characterized by a high frequency of NOTCH2 and MYC amplification and a high HRD score.

Abstract PO3-15-05: B cell immunology predict response and survival in PD-1/PD-L1 targeting immune checkpoint inhibition of metastatic breast cancer

Abstract Critically understudied in the era of T cell immunotherapies, aggregation of B cells within tertiary lymphoid structures (TLS) has recently been linked to improved response and survival in immune checkpoint inhibition (ICI). While this has been reported for several cancers, including melanoma, lung cancer, bladder and renal cell cancer, there is an unmet need to understand molecular patterns in tumors with limited mutational burden (TMB) and low overall abundance of tumor infiltrating lymphocytes (TILs). Here, we report on the landscape of B cells in metastatic breast cancer, inferred by bulk sequencing (WGS/RNAseq) and multiplexed ion beam imaging (MIBI-TOF) in 203 patients across the breast cancer biology (ER/PR/HER2), collected from real world data (CATCH diagnostic and register study, NCT05652569). Median age was 53 years (range, 23-79), with a median of 3 systemic treatment lines for advanced or metastatic disease. The majority of biopsies were taken from the liver (49%), followed by breast (15%), lymph node (13%), skin (6%) and lung (6%). Immunohistochemistry based subtyping revealed 61% of the HR+/HER2- (or Luminal HER2-), followed by 26% of HR-/HER2- (TNBC), and 15% of HER2+ subtype. Immunological subtyping by stromal TILs and CD8+ infiltrates defined 3 subgroups with 57% being desert, 18% inflamed and 19% of no special type (6% NA). The majority (52%) was scored CD20+ with minimal colocalization in 45% of samples. MIBI-TOF demonstrated a continuum of coordinated B cell and T cell aggregation in the tumor microenvironment with lymphoid aggregates in 29% and mature TLS structures in 26% of patients. No correlation with conventional biomarkers for immunogenicity (PD-L1, TMB) has been observed. Chart review identified 21 patients treated by immune checkpoint blockade (ICI) targeting PD-1 (Pembrolizumab) or PD-L1 (Atezolizumab), either as monotherapy or in combination with chemotherapy/targeted therapy. Overall response rate (ORR) at week 12 was 57%, disease control rate (DCR) was 71%. RNA signature levels for germinal center signalling, B cell activation and antibody secretion correlated with quality of B cell aggregation and, importantly, significantly with response to ICI (Kruskal Wallis p=0,028). B cell derived prediction of response was independent of PD-L1 protein expression, breast cancer subtype, BRCA mutation status or TMB. Downstream single cell analysis, computational immunogenomics as well as spatial neighbourhoods will be reported at the meeting. These results extent B cell centered molecular insights to metastatic breast cancer and may pave the way for a composite biomarker, selecting patients for ICI who benefit meaningful and would not have been identified by the current standard of care. Citation Format: Carlo Fremd, Markus Schulze, Miray Cetin, Hannah Boehm, Bénédicte Lenoire, Oezlem Akilli-Oeztuerk, Inka Zörnig, Pornpimol Charoentong, Yanhong Lyu, Catch Collaborators, Daniel Hübschmann, Felix Kommoss, Verena Thewes, Andreas Schneeweiss, Marc Zapatka, Felix Hartmann, Peter Lichter. B cell immunology predict response and survival in PD-1/PD-L1 targeting immune checkpoint inhibition of metastatic breast cancer [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 PO3-15-05.

Abstract PO4-25-04: MHC Class I and II Expression, Functional Tertiary Lymphoid Structure (TLS), and Outcomes in Early-Stage HER2-Positive (HER2+) Breast Cancer in NCCTG N9831 (Alliance)

Abstract Background: Tertiary lymphoid structure (TLS) is an organized form of ectopic lymphoid aggregates (LA) with secondary lymphoid organ structure. Several studies showed that the presence of TLS associates with improved outcomes in multiple cancers when treated with immune checkpoint inhibitors. However, data is currently limited regarding TLS and outcomes in HER2+ breast cancer patients (pts) treated with adjuvant trastuzumab. Furthermore, distinguishing TLS and simple LA is challenging in standard hematoxylin and eosin (H&amp;E) staining, particularly when the germinal center is absent. Emerging studies also showed the prognostic value of MHC expression in breast cancer but mainly in triple-negative breast cancer. In this study, we evaluated integrated pathological quantification and genomic data to assess functional TLS in association with MHC expression and outcomes in the N9831 trial. Methods: Pathological evaluation of LA in H&amp;E slides from pts treated in Arm A (chemotherapy alone) and Arm C (chemotherapy with concurrent trastuzumab) in N9831 was performed. NanoString was used to quantify mRNA of MHC class I and II expression as well as TLS-related immune genes, including IFNG, ICOSLG, CXCL13, CXCR3, BCL6, IL21R, ICOS, PDCD1, CXCR5, CXCL9, TBX21, CD38, CXCL10, CLXCL11, IL21, CD200, CD19, MS4S1. Wilcoxon rank sum test, Chi-squared test, Kaplan-Meier method, and Cox regression model were used to evaluate the association between LA and baseline characteristics, MHC expression, and outcomes. Results: LA was evaluated in 526 pts in Arm A and 485 in Arm C. Greater number of LA was significantly associated with stromal tumor-infiltrating lymphocytes (sTILs), hormone receptor negative, and higher tumor grade, but not stage and age. Using multivariable Cox regression analysis, increasing numbers of LA were associated with improved recurrence-free survival in both arms combined (RFS, p 0.028). However, when evaluating each arm separately, LA was associated with improved RFS only in Arm A (HR 0.6, 95%CI 0.43-0.84, p 0.003) but not in Arm C (HR 0.72, 95%CI 0.47-1.1, p 0.134). We further evaluated TLS-related immune genes in 252 pts in Arm C with LA ≥ 1. As a continuous variable, higher expression of BCL6 (HR 0.61, 95%CI 0.41-0.92, p 0.019) and IL21R (HR 0.78, 95%CI 0.62-0.98, p 0.03) were associated with improved RFS in Arm C pts with LA ≥ 1. However, these genes were not significantly associated with outcomes in Arm C pts without LA with BCL6 (HR1.07, 95%CI 0.67-1.71, p 0.776) and IL21R (HR 0.96, 95%CI 0.71-1.29, p 0.775). Moreover, we evaluated differential gene expression between tumors with LA ≥ 1 with high BCL6 vs. LA 0. All MHC class I and II (HLA A, B, C, E, DQ, DM, DO, DP, and DQ) were significantly higher in tumors with LA ≥ 1 with high BCL6 (p &amp;lt; 0.001). Conclusion: A greater number of LA was associated with improved outcomes in pts with early-stage HER2+ breast cancer, particularly when treated with chemotherapy alone. Using histologic and genomic integration with the combination of pathological LA and TLS-related immune genes, we identified that pts with functional TLS with LA ≥ 1 and higher expression of BCL6 or IL21R had significantly improved outcomes when treated with trastuzumab. High MHC class I and II expressions are associated with the presence of functional TLS, underscoring the crucial role of antigen presentation in the generation of an effective adaptive immune response. Support: U10 CA180821, U10 CA180882, U24 CA196171, https://acknowledgments.alliancefound.org; Genentech; Clinicaltrials.gov Id: NCT00005970 Citation Format: Saranya Chumsri, Tracy Shachner, Zhuo Li, Nadine Norton, Alvaro Moreno-Aspitia, Gerardo Colon-Otero, Edith Perez, Aziza Nassar, E. Thompson, Keith Knutson. MHC Class I and II Expression, Functional Tertiary Lymphoid Structure (TLS), and Outcomes in Early-Stage HER2-Positive (HER2+) Breast Cancer in NCCTG N9831 (Alliance) [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 PO4-25-04.

Abstract PO4-03-04: Characterization of Gut Microbiome of patients with early-stage breast cancer treated with neoadjuvant chemotherapy

Abstract Objective: The gut microbiome (GM) has been identified as one of the major environmental factors that can regulate the development and maintenance of the immune system. In oncology, while the importance of GM has been evaluated in more immunogenic tumors such as melanoma, lung carcinoma and clear cell carcinoma, its importance in early-stage breast cancer (eBC) is underexplored. The aim of this study was to characterize the GM of patients with eBC who underwent neoadjuvant chemotherapy (NACT) and to evaluate its association with clinicopathologic prognostic factors and outcomes. Methods: This was a prospective study conducted at two Brazilian institutions. Fecal samples were collected at baseline (time 1) and prior to surgery (time 2) from 55 patients who received NACT with anthracycline/taxane. Gut microbiome was analyzed by 16S rRNA amplicon sequencing to characterize the α (InvSimpson index) and β (weighted UniFrac distance metric) diversity, as well as taxonomic composition. Clinicopathologic prognostic factors were retrieved from medical records. Pathological complete response (pCR) was defined as the absence of invasive carcinoma in breast and axillary nodes (ypT0/Tis ypN0). Stromal tumor-infiltrating lymphocytes (sTIL) were reported according to the TIL working group criteria. Information about prior use (&amp;lt; 60 days before stating on NACT) of antibiotic (ATB) was obtained directly from patients. Statistical significance was set at P ≤ 0.05 and analysis of composition of microbiomes (ANCOM-BC2) was used to identify enriched genera with p-values adjusted using FDR correction ≤ 0.25. Results: Median age was 49 years (range 31-73). Eleven patients (20%) reported prior antibiotic use at baseline. About 34.5% of patients had HR+/HER2- tumors, 45.4% had triple negative breast cancer (TNBC), and 20% had HER2-positive breast cancer. Fourteen (25%) patients achieved a pCR. Out of 55, 47 (85.4%) had sTIL information. There was no significant difference in alpha diversity (p = 0.6) or beta-diversity (p = 0.8) between the pCR and residual disease group nor within clinical pathologic factors (age, staging, histological tumor grade, subtype of breast cancer based on IHC, sTIL). The most abundant taxon in both time 1 and time 2 were the same (Blautia at genus level, Lachnospiraceae at family level, and Lachnospirales at order level), and longitudinal samples collected during NACT showed no significant changes in GM composition. At baseline, we found an enrichment for Clostridia sp. among patients who did not use ATB (p &amp;lt; 0.05, pFDR ≤ 0.25). Additionally, we found enrichment for Bifidobacterium (p &amp;lt; 0.05, pFDR ≤ 0.25) among patients with HER2 subtype who presented residual disease. Conclusions: Gut microbiome analysis in early breast cancer is feasible and to our knowledge, this is the first study to evaluate the intestinal microbiota of Brazilian patients with breast cancer. We did not observe any significant association between GM diversity and taxonomic composition with clinical pathologic factors and pCR in this study, nor did we observe changes in the ecological balance of the GM following NACT in breast cancer. Further studies are needed to explore the potential influence of the gut microbial ecosystem on tumor biology and response to neoadjuvant chemo-immunotherapy. Citation Format: Ludmila Thommen, Vitor Heidrich, Rosângela Vieira de Andrade, Luiza Nardin Weis, Maria Sueli Soares Felipe, Melissa Lole Da Cas Vita, Thiago David Alves Pinto, Anamaria Camargo, Romualdo Barroso-Sousa. Characterization of Gut Microbiome of patients with early-stage breast cancer treated with neoadjuvant chemotherapy [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 PO4-03-04.

Abstract PS16-07: Tumor infiltrating lymphocyte stratification refines AJCC TNM staging based outcomes of early-stage triple negative breast cancer treated with neoadjuvant anthracycline-free, docetaxel and carboplatin chemotherapy

Abstract Background: Stromal tumor-infiltrating lymphocytes (sTILs) quantification is associated with pathological response to neoadjuvant chemotherapy (NAC) and long term outcomes in setting of adjuvant anthracycline based chemotherapy. A previous study in TNBC patients treated with adjuvant anthracycline chemotherapy shows that at a cut point of 30%, sTILs can up and downstage traditional AJCC stage groups. Additive impact of sTILs on refining outcomes beyond pathological response and TNM stage for patients treated with anthracycline-free chemotherapy is not known. This study aimed to investigate impact of sTILs on outcomes in a large cohort of TNBC patients treated with Docetaxel plus carboplatin NAC. Methods: Patients with stage I (T size &amp;gt; 1cm) to III TNBC scheduled to receive 6 cycles of NAC docetaxel (75 mg/m2) plus carboplatin (AUC 6) (TCb) every 3 weeks from two studies (NCT01560663 and NCT02302742) were combined for this analysis. sTILs were evaluated on pre-treatment H&amp;E slide using standard criteria centrally by one of the investigators (RS). Pathological complete response (pCR) was defined as ypT0/is ypN0. Logistic regression analysis was used to examine the effect of multiple variables on Event free survival (EFS) and overall survival (OS). Results: For 474 patients included in this analysis, median age was 52 years, 8% were Black, 13% had germline BRCA1/2 mutation, 44% had clinical Lymph node (LN) positive disease and 13%, 62% and 25% respectively had TNM stage I, II and III disease. Median sTILs were 5% (range 1-95%) and 25% had &amp;gt;30% sTILs. pCR and RCB 0+1 rates were 50.2% and 60.4% respectively. On multivariable analysis, T stage (OR=0.51, p=0.030), nodal status (OR=0.56, p=0.028), Ki67 (OR=2.74, p&amp;lt; 0.001) and sTILs (OR=2.01, p=0.014) were associated with pCR. pCR rate in those with sTILs &amp;lt; 30 vs sTILs &amp;gt;30 was 45.9% and 63.9% respectively (p=0.0014). At median follow-up of 58 months, 5 years EFS was 81.05% in all patients, 93.94% in those with pCR and 68.67% in those without pCR, 5 year OS was 84.83% in all patients, 96.53% in those with pCR and 74.13% in those with residual disease. On multivariate analysis lower T stage, negative LN status and increasing sTILS were associated with better EFS (T stage: HR =1.98, p=0.018; LN status: HR=2.92, p&amp;lt; 0.001; sTILs: HR:0.46, p=0.04) and OS (T stage: HR=1.89, p=0.040; LN status: HR=3.13, p=0.001; sTILs: HR=0.30, p=0.008). At a cut-point of 30%, sTILs up and downstaged anatomic AJCC TNM stage groups (table 1). Conclusions and Relevance: In patients treated with anthracycline-free TCb chemotherapy, sTILs were independent predictors of EFS and OS beyond clinicopathological features. Notably, 30% sTILs cut-point stratified outcomes beyond anatomical TNM staging. These, findings can aid in patient stratification for chemotherapy backbone de-escalation in future trials and have the potential to inform patient selection for adjuvant treatment escalation and de-escalation trials. Tumor infiltrating lymphocyte stratification refines AJCC TNM staging based outcomes of early-stage triple negative breast cancer treated with neoadjuvant anthracycline-free, docetaxel and carboplatin chemotherapy. Table. Citation Format: Miguel Martín, Rachel Yoder, Roberto Salgado, María del Monte-Millán, Enrique Álvarez, Isabel Echavarria, Joshua Staley, Coralia Bueno-Muiño, Yolanda Jerez Gilarranz, Andrew Godwin, María Cebollero, Oscar Bueno, José Ángel García-Sáenz, Fernando Moreno Antón, Uriel Bohn, Henry L Gómez, Tatiana Massarrah, Sara López-Tarruella, Priyanka Sharma. Tumor infiltrating lymphocyte stratification refines AJCC TNM staging based outcomes of early-stage triple negative breast cancer treated with neoadjuvant anthracycline-free, docetaxel and carboplatin chemotherapy [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 PS16-07.

Abstract PS03-03: The spatially resolved single-cell atlas of the tumor immune architecture revealed the central role of IFN-alpha and plasmacytoid dendritic cells in triple-negative breast cancer in the Mayo Clinic cohort and FinXX trial

Abstract Background: Multiple studies have confirmed the central role of preexisting immune response measured by stromal tumor-infiltrating lymphocytes (sTILs) in triple-negative breast cancer (TNBC). Emerging studies showed that not only the number of TILs but also the location of TILs is important. There are 3 distinct immune architectures described based on the amount and locations of TILs, namely immune enriched (IN), immune excluded (IE), and immune desert (ID). Here we evaluated outcomes and characteristics associated with each immune landscape. Methods: NanoString Digital Spatial Profiling (DSP) and CosMx, a spatial multi-omics single-cell imaging platform, were performed in 75 samples from the Mayo Clinic (MC) TNBC cohort (Leon-Ferre BCRT 2018). NanoString IO360 was performed in 114 samples from the FinXX trial ( NCT00114816) . Firstly, tumors with sTIL quantified by H&amp;E ≤ 30% were classified as ID. The rest of the tumors with high sTIL &amp;gt; 30% were categorized according to the intratumoral CD8 protein expression by DSP, with IE having intratumoral CD8 in the lower median and IN having intratumoral CD8 in the upper median. Chi-square test, gene set enrichment, Cox regression, and Kaplan-Meier analysis were used. Differential expression listed as log 2-fold change (FC) was estimated from the linear mixed model with significance defined as two-sided p&amp;lt; 0.05. Results: ID is associated with low Ki67 &amp;lt; 5% (23.3% vs. 9.6% ID, p 0.02) as well as apocrine (11/13, 84.6%) and metaplastic histology (10/12, 83%). In both univariate and multivariate analysis, patients with IN had significantly improved recurrence-free survival (RFS) compared to those with ID (HR 0.37, 95%CI 0.18-0.74, p 0.005). Despite having high sTILs, IE had poor outcomes similar to ID (HR 0.84, 95%CI 0.36-1.98). Strikingly, we identified that IE patients had significantly lower plasmacytoid dendritic cells (pDCs) compared to IN (mean 0 vs. 0.26/100 tumor cells, 95%CI 0.08-0.43 , p 0.01). Using Gene Set Enrichment Analysis to evaluate differential hallmarks between IN and IE, we identified IF Nɑ and IFNγ (FDR &amp;lt; 0.001) responses as significantly enriched in IN group, consistent wi th the function of pDCs, which are a subset of dendritic cells specialized in secreting high levels of type I interferon. To validate this finding, we further evaluated the 11 leading edge gene IFNɑ signature in the FinXX trial. A high IFNɑ signature score was associated with significantly improved outcomes in the FinXX trial (HR 0.21, 95%CI 0.09-0.51 , p &amp;lt; 0.001). Similar findings were observed using Kaplan-Meier analysis in the FinXX trial with significantly improved RFS (p 0.0006) and overall survival (p 0.0001) in patients with high IFNɑ signature scores. Furthermore, we evaluated the differential gene expression unique to IN tumors in the Mayo cohort. Expressions of MHC class I and class II in tumor cells, including HLA-A, HLA-B, HLA-C, HLA-DRA, HLA-DRB1, HLA-DPA1, and HLA-E, were associated with IN and significantly improved outcomes (p &amp;lt; 0.05) compared to ID and IE. Conclusions: Highlighting the importance of spatial context, we identified that patients with IE tumors had poor outcomes despite having high TILs. Moreover, u sing an in-depth analysis with spatially defined context, we identified the central role of pDC and the significance of IF Nɑ in TNBC. Support: Breast Cancer Research Foundation, Mayo Clinic Breast Cancer SPORE (P50CA116201-17) W81XWH-15-1-0292, P50CA015083, R35CA253187 Citation Format: Saranya Chumsri, Yi Liu, Yaohua Ma, Jodi Carter, Mark Gregory, Sarah Church, Jason Reeves, Heather Ann Brauer, Sarah Warren, Heikki Joensuu, Edith Perez, Roberto Leon-Ferre, David Hillman, Judy Boughey, James Ingle, Krishna Kalari, Fergus Couch, Matthew Goetz, Keith Knutson, E. Thompson. The spatially resolved single-cell atlas of the tumor immune architecture revealed the central role of IFN-alpha and plasmacytoid dendritic cells in triple-negative breast cancer in the Mayo Clinic cohort and FinXX trial [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 PS03-03.

Abstract GS01-01: Biomarker Results in High-risk Estrogen Receptor Positive, Human Epidermal Growth Factor Receptor 2 Negative Primary Breast Cancer Following Neoadjuvant Chemotherapy ± Nivolumab: an Exploratory Analysis of CheckMate 7FL

Abstract Background: CheckMate 7FL (CM 7FL; NCT04109066) is a prospective, phase 3, randomized, multicenter, double-blind trial investigating nivolumab (NIVO) in combination with neoadjuvant chemotherapy (NACT) and adjuvant endocrine therapy (ET) in patients (pts) with high-risk, estrogen receptor-positive (ER+), human epidermal growth factor receptor 2-negative (HER2−) primary breast cancer (BC). The primary endpoint (pathological complete response, pCR) was met, resulting in a statistically significant improvement with added NIVO to NACT; residual cancer burden (RCB) 0–1 rate was meaningfully improved. NIVO benefit was larger in pts with PD-L1+ tumors (SP142 assay, % immune cells ≥ 1%). This analysis focuses on additional biomarkers to explore associations with NIVO treatment effect. Methods: The study enrolled newly diagnosed pts, stages T1c–2 N1–2 or T3–4 N0–2, grade 2 (ER 1–‍10%) or 3 (ER ≥ 1%). Pts were randomized 1:1 to NACT + NIVO 360 mg Q3W/NIVO 240 mg Q2W (arm A) or NACT + placebo (PBO; arm B). Clinical efficacy data included pCR (ypT0/is ypN0) and RCB 0–1 rates in the modified ITT (mITT) population and the SP142 PD-L1+ subgroup. Baseline PD-L1 expression from core biopsies was evaluated with the Dako 28-8 assay using the combined positive score (CPS) algorithm. Various cutoffs (CPS ≥ 1, 10, and 20) were used for this exploration. Concordance between PD-L1 SP142 and 28-8 CPS was evaluated, with assays performed on different slides from the same tumor biopsy blocks. Results of efficacy by ER and/or progesterone receptor (PR) expression, as well as Ki67 index and stromal tumor-infiltrating lymphocytes, will be presented at the meeting. Results: Overall, 830 pts were screened, 521 randomized, and 517 treated, with the primary efficacy population (mITT; n = 510) including all randomized pts but excluding 11 pts from Russian sites with insufficient follow-up. A total of 510 and 349 pts (68.4% of mITT population) were evaluated by PD-L1 SP142 and 28-8 CPS assays, respectively. The prevalence of PD-L1+ by CPS was balanced in arms A and B (52% vs 50% had CPS ≥ 1; 19% vs 16% had CPS ≥ 10, and 11% vs 9% had CPS ≥ 20, respectively). NIVO effect was larger in pts with tumors with increasing PD-L1 expression, with a ∆pCR rate (unweighted rate difference between arms A and B) of 16.6% in CPS ≥ 1 (40.4% vs 23.8% in arms A/B; 95% CI, 2.8 to 29.4), 32.4% in CPS ≥ 10 (65.7% vs 33.3% in arms A/B; 95% CI, 7.3 to 52.3), and 52.3% in CPS ≥ 20 (78.9% vs 26.7% in arms A/B; 95% CI, 18.6 to 72.4). In comparison, the ∆pCR rate was 10.7% in the mITT population (24.5% vs 13.8% in arms A/B; 95% CI, 3.9 to 17.4) and 5.7% in pts with CPS &amp;lt; 1 (14.0% vs 8.2% in arms A/B; 95% CI, -4.0 to 15.5; refer to the Table for additional details). Unweighted rate differences between arms A and B for RCB 0–1 (∆RCB 0–1 rate) followed a similar trend and were observed to be 17.0% in CPS ≥ 1, 34.4% in CPS ≥ 10, and 52.3% in CPS ≥ 20. In comparison, the ∆RCB 0–1 rate was 9.4% in the mITT population and 3.3% in pts with CPS &amp;lt; 1 (refer to the Table for additional details). Further biomarker data and cutoffs will be presented at the meeting. Conclusions: Greater PD-L1 expression was associated with higher pCR and RCB 0–1 rates, suggesting that pts with PD-L1+, high-risk, ER+/HER2− primary BC can achieve substantial pCR rates with the addition of NIVO to NACT. Table. Summary pCR and RCB 0–1 rates by PD-L1 SP142 and 28-8 CPS at various cutoffs. Note: CPS data with the cutoff at 5 will be presented at the meeting. Citation Format: Sherene Loi, Giuseppe Curigliano, Roberto Salgado, Roberto Iván Romero Díaz, Suzette Delaloge, Carlos Ignacio Rojas García, Marleen Kok, Cristina Saura, Nadia Harbeck, Elizabeth Mittendorf, Denise Yardley, Lajos Pusztai, Alberto Suárez Zaizar, Andrei Ungureanu, Felipe Ades, Rajalakshmi Chandra, Raheel Nathani, Misena Pacius, Thomas Spires, Jenny Wu, Heather McArthur. Biomarker Results in High-risk Estrogen Receptor Positive, Human Epidermal Growth Factor Receptor 2 Negative Primary Breast Cancer Following Neoadjuvant Chemotherapy ± Nivolumab: an Exploratory Analysis of CheckMate 7FL [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 GS01-01.

Abstract PO1-24-11: Distinct patterns of MHC class I, PD-L1 expression, and T-cell infiltration in different subtypes of ductal carcinoma in situ of the breast

Abstract Background: Ductal carcinoma in situ (DCIS) of the breast encompasses various subtypes with distinct behaviors. Immune cell infiltration within the tumor microenvironment is thought to influence DCIS progression. Tumor-infiltrating lymphocytes (TILs) serve as a surrogate marker for the adaptive immune response. The interaction between programmed death 1 (PD-1) and its ligand PD-L1 suppresses effector T-cell function. Major histocompatibility complex (MHC) class I molecules play a critical role in presenting tumor antigens to cytotoxic T cells. Despite the significance of PD-L1 and MHC class I in immune evasion, limited data exist on their correlation with T-cell infiltration in DCIS subtypes. Methods: Using immunohistochemistry, we examined MHC class I and PD-L1 expression, along with CD3+ and CD8+ T lymphocytes, in 131 DCIS cases through tissue microarrays. DCIS subtypes included hormone receptor-positive (HR+)/human epidermal growth factor 2-negative (HER2-), HR+/HER2+, HR-/HER2+, and triple-negative (TN) subtypes. Results: Among the 128 interpretable cases, the distribution was as follows: HR+/HER2- (50.8%), HR+/HER2+ (14.1%), HR-/HER2+ (26.5%), and TN (8.6%). HER2+ subtypes (HR+/HER2+ and HR-/HER2+) exhibited higher stromal TILs, CD3+ T cells, and CD8+ T cells compared to HR+/HER2- subtype. MHC class I loss was observed in 16.4% (21/128) of cases, with the highest frequency in the HR+/HER2- subtype (29.2%), followed by HR-/HER2+ (5.9%), HR+/HER2+ (0%), and TN (0%) subtypes. DCIS cases lacking MHC class I expression displayed lower stromal CD8+ T cell infiltration than those with intact MHC class I. PD-L1 expression in immune cells (≥1%) was detected in 18.8% (24/128) of cases and correlated with increased stromal TILs, CD3+ T cells, and CD8+ T cells. PD-L1 expression was more prevalent in HER2+ subtypes than in HR+/HER2- subtype. PD-L1-positive DCIS cases mostly retained MHC class I expression (95.8%), except for one case (4.2%) in the HR-/HER2+ subtype. The highest numbers of stromal CD3+ and CD8+ T cells were observed in DCIS cases with intact MHC class I and positive PD-L1 expression. The correlation between MHC class I and PD-L1 expression and infiltration of CD3+ and CD8+ T cells was maintained in HR+/HER2- and HR-/HER2+ subtypes. Notably, tumoral CD3+ and CD8+ T cells showed no significant association with MHC class I or PD-L1 expression. Furthermore, MHC class I and PD-L1 expression did not predict tumor recurrence. Conclusion: We observed distinct patterns of MHC class I and PD-L1 expression and T-cell infiltration among DCIS subtypes. Enhancing our understanding of MHC class I, PD-L1, and immune subsets, including CD8+ T cells, may contribute to the development of immune modulating therapies for DCIS, particularly in the HER2+ subtype. Citation Format: Nah Ihm Kim, Min Ho Park, Ji Shin Lee. Distinct patterns of MHC class I, PD-L1 expression, and T-cell infiltration in different subtypes of ductal carcinoma in situ of the breast [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 PO1-24-11.

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 (&amp;lt; 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&amp;lt; 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 PO1-18-04: Neoadjuvant pembrolizumab + decitabine followed by standard neoadjuvant chemotherapy for locally advanced HER2- breast cancer (NCT02957968)

Abstract Background: Higher levels of stromal tumor-infiltrating lymphocytes (sTILs) in breast cancers are associated with increased likelihood of pathologic complete response (pCR) to chemotherapy and improved outcomes. DNA methyltransferase inhibitors (DNMTi) can augment immune responses to cancers by upregulating tumor antigen and MHC expression, decreasing numbers and activity of myeloid derived suppressor cells (MDSC), and increasing responsiveness of T lymphocytes. We have shown that the DNMTi decitabine augments the effectiveness of immunotherapy against murine triple negative breast cancer (TNBC) using murine 4T1 and E0771 mammary carcinoma models. Methods: In a single-arm phase 2 study, patients with HER2-negative breast cancer who were candidates for neoadjuvant chemotherapy (NCT) received decitabine (15 mg/m2 × 4 doses over 5 days) followed by 2 doses of pembrolizumab (pembro) (200 mg, 2 weeks apart) – collectively, window immunotherapy – prior to starting NCT. Two research biopsies were obtained: 1 prior to the window immunotherapy and 1 afterwards, prior to starting NCT. Biopsies were analyzed using established procedures to quantify sTILs and PD-L1 expression. Patients proceeded to NCT and tumor resection per standard of care and were followed up for immune-related adverse events (irAEs) and response. The primary endpoint was change in sTILs. Key secondary endpoints were occurrence of irAEs and pCR following neoadjuvant treatment. After the study was opened, reported results of a phase 3 trial (KEYNOTE 522) led to allowing patients with TNBC to receive additional pembro (200 mg q3w) concurrently with standard NCT and adjuvantly following resection (Cohort A2). Results: 46 patients (median age 54.5 yrs, range 28-72; 71.7% White, 28.3% Black; 100% female) were enrolled and treated on study. 21 patients had TNBC and did not receive neoadjuvant pembro concurrently with NCT nor adjuvant pembro (Cohort A), 7 patients had TNBC and did receive neoadjuvant and/or adjuvant pembro (Cohort A2), and 18 patients were ER+ or PR+ and received neither concurrent nor adjuvant pembro (Cohort B). Blood samples collected after decitabine administration before the first pembro dose showed a 59% decrease (P&amp;lt; 0.01) in monocytic MDSCs compared to baseline; decrease in granulocytic MDSCs was not statistically significant. 37 patients had paired biopsies adequate for sTIL evaluation with a mean change from 23.4% to 30.3% (absolute change 6.9%, P&amp;lt; 0.001). Cohorts A/A2 experienced an absolute sTIL increase of 7.4% (P&amp;lt; 0.01); Cohort B experienced absolute sTIL increase of 6.1% (P=0.01). PD-L1 expression in tumors (H-score using MoAb 22C3 clone) increased by 43% (P&amp;lt; 0.01) across all cohorts. 16 of the 39 patients (41.0%) who proceeded to resection achieved pCR (n=12 of 27 [44.4%] in Cohorts A/A2 and n=4 of 13 [30.8%] in Cohort B). The most frequently reported irAEs were adrenal insufficiency (n=5, 10.9%), maculopapular rash (n=3, 6.5%), and hypothyroidism (n=3, 6.5%). Conclusions: Treatment in the pre-neoadjuvant window with decitabine and pembro could potentially sensitize breast cancers to standard NCT by recruitment of TILs to the tumor tissue. The treatment was well -tolerated at the tested doses. Funding: financial support and drug (pembrolizumab) were provided by Merck Citation Format: Harry Bear, Xiaoyan Deng, Dipankar Bandyopadhyay, Michael Idowu, Maciej Kmieciak, Monique Williams, Giovanni Archer, Lindsey Gwaltney, Patrick Dillon, Daniel Flora, Daniel Stover, Andrew Poklepovic, Mary Hackney, Masey Ross, Hetal Vachhani, Raphael Louie, Kandace McGuire, Amelia Grover, Tasnim Rahman, Amber Hendrix. Neoadjuvant pembrolizumab + decitabine followed by standard neoadjuvant chemotherapy for locally advanced HER2- breast cancer (NCT02957968) [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 PO1-18-04.