Tumor-infiltrating Lymphocytes Counts Research Articles

Utility of neutrophil-to-lymphocyte ratio as an indicator of tumor immune status in non-small cell lung cancer.

Neutrophil-to-lymphocyte ratio (NLR) has been reported as a prognostic biomarker in non-small cell lung cancer (NSCLC); however, the underlying biological rationale remains unclear. The present study aimed to explore the potential utility of NLR as a surrogate biomarker for immune response to cancer and to elucidate the underlying mechanism. This retrospective study included the medical records of 120 patients with NSCLC who underwent surgery at the study institution in 2012. NLR in peripheral blood was determined from blood test within 30days before surgery. Tumor immune status was evaluated using immunohistochemical staining to identify CD3+, CD8+ and FOXP3+ tumor-infiltrating lymphocytes (TILs), and the relationship of NLR, with clinicopathologic characteristics including 5-year overall survival (OS), and the tumor immune status was investigated. The median values of NLR and TIL count were used as cutoff points. The 5-year OS was significantly better in patients with low NLR (<2.2) than in those with high NLR (≥2.2) (70.1% vs. 56.8%, P=0.042) and in patients with high CD3+ TIL count (≥242) than in those with low CD3+ TIL count (<242) (70% vs. 56.8%, P=0.019). Additionally, the CD3+ TIL count was negatively correlated with preoperative NLR (P=0.005). NLR might potentially reflect the immune status of tumor microenvironment, explaining its impact on prognosis of patients with NSCLC.

GATA3 Positively Correlates with BCL2 Expression in Indolent and Aggressive Histological Types of Cutaneous Basal Cell Carcinoma.

Some histological basal cell carcinoma (BCC) types demonstrate more aggressive behavior than others. They are known as high-risk BCC and are more challenging in therapy, contrary to indolent (low-risk) BCC types. Identifying novel protein markers to predict aggressiveness and potential therapeutic targets in challenging cases is recommended. GATA3 is a transcription factor critical for epithelial and lymphocytic differentiation. This study investigated the immunohistochemical expression of GATA3 in indolent and aggressive BCC and its association with BCL2 expression. Retrospectively collected indolent and aggressive BCC groups (24 cases each) were immunohistochemically stained with anti-GATA3 and BCL2 antibodies. The mean expression score (by area percentage) and TIL counts were determined and compared using ImageJ analysis. Stromal tumor-infiltrating lymphocytes (TIL) were counted per high-power field (HPF) on hematoxylin and eosin (H&E) staining. GATA3 and BCL2 expressions were significantly higher in the indolent group than in the aggressive group. GATA3 expression significantly correlated with BCL2 score and TIL counts. Higher GATA3 expression was significantly associated with a more indolent BCC histological type, higher BCL2 expression, and higher TIL count. GATA3 is a possible target for immunomodulation experiments to improve BCC immunotherapy outcomes.

Biomarker-driven targeted therapy in patients with recurrent platinum-resistant epithelial ovarian cancer (BRIGHT): protocol for an open-label, multicenter, umbrella study.

Platinum-resistant, recurrent ovarian cancer has an abysmal prognosis with limited treatment options. Poly-(ADP-ribose)-polymerase (PARP), angiogenesis, and immune checkpoint inhibitors might improve the outcomes of platinum-resistant, recurrent ovarian cancer, but accurate patient selections for those therapies remain a significant clinical challenge. To evaluate the efficacy and safety of biomarker-driven combinatorial therapies of pamiparib, tislelizumab, bevacizumab, and nab-paclitaxel in platinum-resistant, recurrent ovarian cancer. A precision medicine combination of PARP inhibitors, anti-angiogenic therapy, immunotherapy, and chemotherapy will improve disease outcomes of platinum-resistant, recurrent ovarian cancer by accounting for genomic and immunologic features. The BRIGHT Trial is a prospective, open-label, multicenter, phase II, umbrella study planning to enroll 160 patients with serous, endometrioid, or clear cell platinum-resistant, recurrent ovarian cancer from 11 clinical centers in China. Patients are assigned to one of three experimental arms based on biomarkers. Patients with BRCA1/2 mutations will receive pamiparib plus bevacizumab (arm 1, n=40) regardless of CD8+ tumor-infiltrating lymphocytes count. Patients with wild-type BRCA1/2 (BRCAwt) and ≥3 CD8+ tumor-infiltrating lymphocytes count will receive the combination of tislelizumab, bevacizumab, and nab-paclitaxel (arm 2, n=50), while BRCAwt patients with <3 CD8+ tumor-infiltrating lymphocytes count will receive bevacizumab plus dose-dense nab-paclitaxel (arm 3, n=50). After completing patient enrollment in arm 2, another 20 BRCAwt patients with ≥3 CD8+ tumor-infiltrating lymphocytes count will be included as an arm 2 expansion. Treatment will continue until disease progression or intolerable toxicity, and all adverse events will be recorded. Eligible patients include those aged ≥18 with serous, endometrioid, or clear cell ovarian cancer, platinum-resistant recurrence, and Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. Objective response rate (ORR) assessed by the investigators by the RECIST 1.1 criteria. 160 patients. Recruitment is estimated to be completed by 2024 and results may be published by 2027. ClinicalTrials.gov: NCT05044871.

Pre-Operative Immunonutrition Enhances Postoperative Outcomes and Elevates Tumor-Infiltrating Lymphocyte Counts in Colorectal Cancer Patients: A Meta-Analysis of Randomized Controlled Trials

Objective This study (CRD42023464989) aimed to explore the effects of pre-operation immunonutrition on safety and immune related factors in colorectal cancer patients undergoing surgery. Methods We systematically searched PubMed, Embase, and Wanfang databases to collect all clinical randomized controlled trials of the application of pre-operation immunonutrition for patients with colorectal cancer, published until July 2023. The primary outcomes were safety and immune related factors. Results A total of 16 studies were finally included. Preoperative immunonutrition could reduce the postoperative infection rate (risk ratio (RR) = 0.56, 95% confidence interval (CI): 0.36, 0.88; p = .01), and wound infection rate (RR = 0.44, 95% CI: 0.27, 0.70; p < .001) in patients with colorectal cancer. For length of stay (mean difference (MD) = −1.10, 95% CI: −2.70, 0.49; p = .17), it was similar between groups. Meanwhile, patients in the pre-operation immune nutrition group also had significantly increased infiltrative lymphocytes CD16 + (MD = 0.04, 95% CI: 0.02, 0.06; p < .001), and CD56 + (MD = 0.05, 95% CI: 0.03, 0.06; p < .001) cells in the tumor tissues, compared to the control group. Conclusion Immunonutrition intervention has the potential to reduce postoperative infectious complications and improve tumor infiltrative lymphocytes in patients with colorectal cancer undergoing surgery.

High PD-L1 expression on immune cells along with increased density of tumor-infiltrating lymphocytes predicts a favorable survival outcome for patients with loco-regionally advanced head and neck cancer: early results from a prospective study.

In the era of personalized medicine and treatment optimization, use of immune biomarkers holds promise for estimating the prognosis of patients with head and neck squamous cell carcinoma (HNSCC) undergoing definitive treatment. To evaluate the prognostic potential of immune biomarkers, we conducted a prospective monocentric cohort study with loco-regionally advanced HNSCC patients indicated for definitive radiotherapy/radiochemotherapy at the Department of Oncology, Ostrava University Hospital, Czech Republic, between June 2020 and August 2023. We focused on the expression of programmed death ligand 1 (PD-L1) and tumor-infiltrating lymphocytes (TILs) relative to overall survival (OS) and specific survival rates. Associations between biomarkers and survival rates were assessed by crude and adjusted hazard ratios (cHR, aHR, respectively) obtained from Cox proportional hazards regression. Among a total of 55 patients within a median follow-up of 19.7 months, there were 21 (38.2%) all-cause deaths and 15 (27.3%) cancer-related deaths. An overall survival (OS) rate of 61.8% and a disease-specific survival (DSS) rate of 72.7% were recorded. A significant association between survival rates and a ≥10% difference in PD-L1 expression on immune versus tumor cells (high PD-L1IC expression) was documented regardless of the type of analysis (univariate or multivariate). In addition, a stronger association was confirmed for OS and the composite biomarker high PD-L1IC expression along with either median-higher CD8+ TIL count or increased TIL density ≥30%, as indicated by an aHR of 0.08 (95% CI, 0.01 to 0.52) and 0.07 (95% CI, 0.01 to 0.46), respectively. Similar results were demonstrated for other specific survival rates. The early outcomes of the present study suggest the utility of a strong prognostic factor involving a composite biomarker high PD-L1IC expression along with increased TIL density in HNSCC patients undergoing definitive radiotherapy and radiochemotherapy. The study is registered with Clinicaltrials.gov. - NCT05941676.

Abstract 4149: Real-time intravital trafficking of tumor infiltrating lymphocytes in breast cancer models

Abstract Cancer immunotherapy utilizing the immunoreactive efficiency of immune cells via adoptive cell therapy (ACT) has been a novel method of cancer treatment. In tumor microenvironment, immune cell reactivity of tumor-infiltrating lymphocytes (TILs) can facilitate cancer cell attack, which finally derives efficient suppression of tumor growth. However, the conventional evaluation for cancer immunotherapy has been generally performed by ex vivo methods, providing restricted information on lymphocyte motility and immune cell dynamics within the tumor microenvironment. In vivo data with the dynamic behavior of TILs in breast cancer can provide new insight to investigate novel targets of immune checkpoint inhibitors. In this research, longitudinal intravital imaging of motility trafficking of TILs in breast cancer animal models has been performed by utilizing intravital microscopy to unveil the immunoreactive dynamics of T cell activity in early stage. In vivo analysis of TIL motility within different breast cell lines, including 4T1 (mouse breast cancer cell), MDA-MB-231 (human triple-negative breast cancer cell), and MCF7 (human breast cancer cell), respectively, has been investigated for immune responses to cancer. We’ve monitored spatiotemporal dynamics of TIL over 11 days in breast cancer xenograft mouse model generated by tumor implantation into the dorsal skinfold imaging chamber and adoptive T cell transfer by blood circulation. In the results, the adoptive immunoreactivity of TIL has been differentiated by the timepoint of adoptive T cell transfer, distance proximity of T cells in tumors, and breast cancer types. Furthermore, anti-cancer effect coordinated with dendritic cells behaviors showed induced immune responses with accumulated dendritic cells with increased TIL count over time in breast cancer model treated with various cancer therapy such as immunotherapy, radiation therapy and anti-cancer drug (anti-PD-L1), which implied a relationship between programmed death-ligand 1 expression levels and TIL/dendritic cell motility. The result demonstrates that intravital TIL motility trafficking analysis in cancer model can be an invaluable assessment method to explain the detailed cellular/molecular mechanism of immune response to cancer involved with T lymphocytes, dendritic cells, and immune checkpoint inhibitors to accelerate the efficiency of cancer immunotherapy. Citation Format: Soyeon Ahn, Kubra Akyildiz, Hyunseok Kim, Pilhan Kim. Real-time intravital trafficking of tumor infiltrating lymphocytes in breast cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4149.

Prevalence and type of MMR expression heterogeneity in colorectal adenocarcinoma: therapeutic implications and reporting.

Mismatch repair (MMR) immunohistochemical (IHC) evaluation has entered pathology routine practice as the first-line screening method to identify patients with MMR deficient (MMRd)/microsatellite instability (MSI) colorectal cancer (CRC), and its misdiagnosis may significantly impact the personalization of CRC patient care. To determine the prevalence of MMR protein intratumor heterogeneity in real-world practice, we collected a series of 8282 CRCs tested for MMR proteins in the setting of Lynch syndrome universal screening. Four heterogenous cases were also investigated for tumor infiltrating lymphocytes count, MSI status, and consensus molecular subtypes by Nanostring nCounter® Platform. Overall, 1056 (12.8%) CRCs showed a MMR altered status, with 46 cases showing a heterogeneous MMR profile (0.56% of the total, and 4.36% of all MMRd cases). To conclude, the authors make some critical remarks regarding the approach to MMR heterogeneity in clinical practice and routine diagnostics.

The prognostic value of the histological shape of tumor negative sentinel nodes in breast cancer.

Sentinel lymph node (SLN) metastasis is an important predictor of prognosis in breast cancer (BC) patients, guiding treatment decisions. However, patients with the same BC subtype and tumor negative SLN (SLNneg) can have different survival outcomes. We hypothesized that the host anti-tumor immune reaction in SLNneg is important and results in morphometrically measurable changes in SLN size or shape which are related to patient prognosis. Surface area, circumference, long axis and short axis were histologically measured in 694 SLNneg from 356 cases of invasive BC and 67 ductal carcinoma in situ cases. The area occupied by fat was categorized as less or more than 50%. The long to short axis (L/S) ratio was calculated. The relationship between SLNneg morphometries and clinicopathological variables like tumor-infiltrating lymphocytes (TILs) within the primary tumor, as well as prognosis at 10 years follow up were analyzed. The mean SLNneg surface area was 78.7mm2, circumference 40.3mm, long axis 13.1mm, short axis 8.2mm and L/S ratio 1.7. Larger surface area, long axis and short axis, including age >55 years were associated with higher body mass index (BMI) and SLN fat over 50% (p<0.003). In invasive BC, a high SLNneg L/S ratio (≥1.9) was related to poorer disease-free (HR=1.805, 95%CI 1.182-2.755, p=0.006) and overall (HR=2.389, 95%CI 1.481-3.851, p<0.001) survival. A low SLNneg L/S ratio (<1.9) was associated with high TILs in the primary BC (≥10%) (p=0.005). However a high TIL count was not of prognostic relevance. This is the first study to suggest that morphometric characteristics of axillary SLNneg, like L/S ratio, could be used to predict prognosis in patients with SLNneg invasive BC of all subtypes. The association between low L/S ratio and high TILs suggest that SLN shape is related to immunological functioning of the SLN and could be used in addition to TIL evaluation. Regarding the dubious role of TILs in hormone receptor positive breast cancer, SLNneg morphometry to gain information about host immune status could especially be of benefit in this subtype. Further studies are warranted to better understand the underlying biological mechanisms.

High Tumor-Infiltrating Lymphocyte Count Is Associated with Distinct Gene Expression Profile and Longer Patient Survival in Advanced Ovarian Cancer.

Cancer-related immunity plays a significant role in the outcome of ovarian cancer, but the exact mechanisms are not fully explored. A retrospective, real-life observational study was conducted including 57 advanced ovarian cancer patients. Immunohistochemistry for CD4+, CD8+, and CD45+ was used for assessing tumor-infiltrating immune cells. Furthermore, an immune-related gene expression assay was performed on 12-10 samples from patients with less than and more than 1-year overall survival (OS), respectively. A higher number of CD4+ (p = 0.0028) and CD45+ (p = 0.0221) immune cells within the tumor microenvironment were associated with longer OS of patients. In a multivariate setting, higher CD4+ T cell infiltration predicted longer OS (p = 0.0392). Twenty-three differentially expressed genes-involved in antigen presentation, costimulatory signaling, matrix remodeling, metastasis formation, and myeloid cell activity-were found when comparing the prognostic groups. It was found that tumor-infiltrating immune cell counts are associated with peculiar gene expression patterns and bear prognostic information in ovarian cancer. SOX11 expression emerged and was validated as a predictive marker for OS.

Granzyme B (GZMB)-Positive Tumor-Infiltrating Lymphocytes in Lung Adenocarcinoma: Significance as a Prognostic Factor and Association with Immunosuppressive Proteins.

Granzyme B (GZMB) is a serine protease produced by cytotoxic lymphocytes that reflects the activity of anti-tumor immune responses in tumor-infiltrating lymphocytes (TILs); however, the prognostic significance of GZMB+ TILs in lung adenocarcinoma is poorly understood. We analyzed 273 patients with pathological stage (pStage) I-IIIA lung adenocarcinoma who underwent surgery at Kyushu University from 2003 to 2012. We evaluated GZMB+ TIL counts by immunohistochemistry. We set the cut-off values at 12cells/0.04mm2 for GZMB+ TILs and divided the patients into GZMB-High (n=171) and GZMB-Low (n=102) groups. Then, we compared the clinicopathological characteristics of the two groups and clinical outcomes. Programmed cell death ligand-1 (PD-L1) and indoleamine 2,3-dioxygenase 1 (IDO1) expression in tumor cells was also evaluated, and combined prognostic analyses of GZMB+ TILs with PD-L1 or IDO1 were performed. GZMB-Low was significantly associated with pStage II-III, PD-L1 positivity, and IDO1 positivity. Disease-free survival (DFS) and overall survival (OS) in the GZMB-Low group were significantly worse than in the GZMB-High group. In multivariable analysis, GZMB-Low was an independent prognostic factor for both DFS and OS. Furthermore, combined prognostic analyses of GZMB+ TILs with PD-L1 or IDO1 showed that GZMB-Low with high expression of these immunosuppressive proteins had the worst prognosis. We analyzed GZMB+ TIL counts in lung adenocarcinoma and elucidated its prognostic significance and association with PD-L1 and IDO1. GZMB+ TIL counts might reflect the patient's immunity against cancer cells and could be a useful prognostic marker of lung adenocarcinoma.

The prognostic and predictive role of tumor-infiltrating lymphocytes (FoxP3 + and CD8 +) and tumor-associated macrophages in early HER2 + breast cancer

PurposeIn HER2-positive (HER2 +) breast cancer, tumor-infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs) may influence the efficacy of the HER2-antibody trastuzumab and the patient’s outcome. In this HER2 + patient cohort, our aim was to study the numbers of FoxP3 + regulatory TILs and CD8 + cytotoxic TILs, their correlations with CD68 + and CD163 + TAMs, and the prognostic and predictive value of the studied factors.MethodsWe evaluated 139 non-metastatic HER2 + breast cancer patients operated between 2001 and 2008. The FoxP3+TIL count (FoxP3+TILs) was assessed using the hotspot method, and the CD8 + TIL count (CD8+mTILs) utilizing a digital image analysis from invasive margin areas. The ratios between CD8+mTILs and FoxP3+TILs as well as CD8+mTILs and TAMs were calculated.ResultsFoxP3 + TILs and CD8 + mTILs correlated positively with each other (p<0.001). FoxP3+TILs had a positive correlation with CD68+and CD163+TAMs (p≤0.038), while CD8 + mTILs correlated only with CD68+TAMs (p<0.001). In the HER2 + and hormone receptor-positive Luminal B subgroup, high numbers of FoxP3+TILs were associated with shorter disease-free survival (DFS) (54% vs. 79%, p = 0.040). The benefit from adjuvant trastuzumab was extremely significant among patients with a high CD8 + mTILs/CD68 + TAMs ratio, with overall survival (OS) 84% vs. 33% (p = 0.003) and breast cancer-specific survival (BCSS) 88% vs. 48% (p = 0.009) among patients treated with or without trastuzumab, respectively.ConclusionIn the HER2 + Luminal B subgroup, high FoxP3 + TILs were associated with shorter DFS. A high CD8 + mTILs/CD68 + TAMs ratio seems to associate with impressive efficacy of trastuzumab.

The epigenetic modifier lysine methyltransferase 2C is frequently mutated in gastric remnant carcinoma.

Gastric remnant carcinoma (GRC), which occurs in the stomach after partial gastrectomy, is a rare and aggressive form of gastric adenocarcinoma (GAC). Comprehensive profiling of genomic mutations in GRC could provide the basis for elucidating the origin and characteristics of this cancer. Herein, whole-exome sequencing (WES) was performed on 36 matched tumor-normal samples from patients with GRC and identified recurrent mutations in epigenetic modifiers, notably KMT2C, ARID1A, NSD1, and KMT2D, in 61.11% of cases. Mutational signature analysis revealed a low frequency of microsatellite instability (MSI) in GRC, which was further identified by MSIsensor, MSI-polymerase chain reaction, and immunohistochemistry analysis. Comparative analysis demonstrated that GRC had a distinct mutation spectrum compared to that of GAC in The Cancer Genome Atlas samples, with a significantly higher mutation rate of KMT2C. Targeted deep sequencing (Target-seq) of an additional 25 paired tumor-normal samples verified the high mutation frequency (48%) of KMT2C in GRC. KMT2C mutations correlated with poor overall survival in both WES and Target-seq cohorts and were independent prognosticators in GRC. In addition, KMT2C mutations were positively correlated with favorable outcomes in immune checkpoint inhibitor-treated pan-cancer patients and associated with higher intratumoral CD3+ , CD8+ tumor-infiltrating lymphocyte counts, and PD-L1expression in GRC samples (p = 0.018, 0.092, 0.047, 0.010, and 0.034, respectively). Our dataset provides a platform for information and knowledge mining of the genomic characteristics of GRC and helps to frame new therapeutic approaches for this disease.

Do tumor infiltrating lymphocytes (TILs) predict benefits from trastuzumab therapy for HER2 positive breast cancer? Meta-analysis of individual patient data from 4097 women in 5 trials.

508 Background: High TIL counts are associated with a lower risk of breast cancer recurrence, especially in women with ER negative, HER2 negative tumors and, possibly, greater benefit from trastuzumab in women with HER2 positive cancer: the FinHER trial reported a differential effect of trastuzumab based upon TIL status. We performed a meta-analysis of randomized trials of trastuzumab in early breast cancer to attempt to validate this finding. Methods: TILs were quantified in 4097 women in 5 randomized controlled trials (NSABP B-31, FinHER, HERA, Intergroup N9831, PACS-04). All trials contributed to the Early Breast Cancer Trialists' Collaborative Group individual patient data meta-analysis of trastuzumab for women with HER2 positive tumors which found a significant benefit for trastuzumab therapy. TILs were assessed using established International Guidelines, with HERA using digital TIL-scores. The primary outcome was time to first recurrence. Cox regression analyses, adjusted for trial, treatment allocation, and nodal status, were used to quantify the prognostic value of TILs; and standard stratified logrank tests were used to assess the differential effect of trastuzumab therapy. Results: The median percentage TILs was 13% (Interquartile Range 5-30), with fewer than 10% of patients exhibiting TILs &gt;50%. The prognostic value of TILs was confirmed, with patients with higher TILs being at lower risk for recurrence (adjusted hazard ratio per 10% increase in TILs 0.87 (95% CI 0.84-0.90), p&lt;.0001) with similar effects in both treatment groups. Outcomes improved steadily with increasing TILs, and unadjusted 10-year recurrence rates fell from 30% in women with TILs &lt;10% to 15% in those with TILs 70% or greater. Consequently, analyses of the predictive effect of TILs were stratified into 5 groups (0-9, 10-19, 20-39, 40-59, 60+). Overall, there was a highly significant benefit of trastuzumab on recurrence (HR 0.62 (0.54-0.70) p&lt;.0001), but there was no evidence of any interaction between TILs and the proportional reduction in recurrence (p=0.8 for heterogeneity and trend). Conclusions: While higher TILs are associated with lower recurrence rates, there was no indication that the proportional reduction in recurrence with trastuzumab varied by TILs, although the number of patients with high levels was limited. Owing to a lower underlying recurrence rate, absolute benefits from trastuzumab were lower, but still substantial, in women with high TIL tumors. Clinical trial information: NCT00045032 , ISRCTN76560285 , NCT00005970 , NCT00004067 , NCT00054587 . [Table: see text]

Association between Tumor-Infiltrating Lymphocytes and Histopathological Types of Thyroid Carcinoma in Department of Anatomical Pathology, University of Indonesia/Dr. Cipto Mangunkusumo National Central General Hospital

Background&#x0D; The presence of tumor-infiltrating lymphocytes (TILs) has been widely explored as one of the factors that can affect the success of immunotherapy in various cancers. This study aims to investigate the TILs in various types of thyroid carcinoma (TC) and its association with the aggressive histopathological type compared to the less aggressive histopathological type of TC.&#x0D; Methods&#x0D; The population covered by this retrospective study was TCs diagnosed at the Department of Anatomic Pathology, Faculty of Medicine, University of Indonesia/Dr. Cipto Mangunkusumo National Central General Hospital, period 2015-2019. The research sample was divided into group of TCs with aggressive histopathological type and less aggressive histopathological type with a total of 25 samples for each group. TILs was calculated in 10 high power fields (HPF) with 400 kali magnification. The analyses were conducted using Mann-Whitney and Kruskal Wallis test.&#x0D; Results&#x0D; TILs count was found with a median value of 43 (3-660) in the aggressive histopathological group and 36 (9-387) in the less aggressive histopathological group (p:0.96, CI:95%). The highest TILs was found in anaplastic thyroid carcinoma (ATC) and papillary thyroid carcinoma (PTC) with anaplastic components. TILs count was also found with a median value of 17.5 (3-75) in male and 48 (9-660) in female (p:0.02, CI:95%).&#x0D; Conclusion&#x0D; There was no significant association between TILs and histopathological types of TC (aggressive and less aggressive). However, there is a tendency for high TILs in ATC and PTC with anaplastic components. There is also a significant difference in TILs between male and female gender.

TIM-3 Qualifies as a Potential Immunotherapeutic Target in Specific Subsets of Patients with High-Risk Soft Tissue Sarcomas (HR-STS).

(1) Background: The expression of T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3), an immune checkpoint receptor on T cells, has been associated with dismal outcomes and advanced tumor stages in various solid tumors. The blockade of TIM-3 is currently under examination in several clinical trials. This study examines TIM-3 expression in high-risk soft tissue sarcomas (HR-STS). (2) Methods: Tumor cell expression of TIM-3 on protein level was analyzed in pre-treatment biopsies of patients with HR-STS. TIM-3 expression was correlated with clinicopathological parameters including tumor-infiltrating lymphocyte (TIL) counts, programmed cell death 1 (PD-1) and programmed cell death ligand 1 (PDL-1) expression in patients with HR-STS. Survival dependent on the expression of TIM-3 was analyzed. (3) Results: TIM-3 expression was observed in 101 (56%) out of 179 pre-treatment biopsies of patients with HR-STS. TIM-3 expression was significantly more often observed in undifferentiated pleomorphic sarcomas (UPS) compared to other histological subtypes (p < 0.001), high TIL counts (p < 0.001), and high PD-1 (p < 0.001) and PD-L1 expression (p < 0.001). TIM-3 expression did not have a prognostic impact on survival in patients with HR-STS. (4) Conclusions: This is the first study to demonstrate a significant tumor cell expression of TIM-3 in specific subsets of patients with HR-STS. TIM-3 qualifies as a potential immunotherapeutic target in HR-STS.

Abstract P1-04-14: Predicting the likelihood of response in PDL-1 positive metastatic triple negative breast cancer treated with an immune checkpoint inhibitor

Abstract Background: Patients with metastatic triple negative breast cancer (mTNBC) have a poor prognosis with median survival of 18 months or less. While the combination of immune checkpoint inhibitors (ICIs) and chemotherapy has shown promise in mTNBC, biomarkers beyond PDL-1 are needed to better predict which individuals will benefit from this treatment approach. In this study, we assessed the ability of spatial characteristics in predicting clinical best response in patients with PDL-1 positive mTNBC treated with an ICI and chemotherapy. Methods: Women with advanced unresectable or mTNBC treated with an ICI plus chemotherapy at Emory University between 2019 and 2021 with available biopsy specimens were retrospectively evaluated. Different cell types (tumor, stroma, immune cells) were identified by morphology on H&amp;E staining. A cellular network was created by connecting each cell centroid to its adjacent centroids within a 30-μm distance. The resulting spatial neighborhood network was used to assess tumor density and quantify immune infiltration. The immune infiltration score was defined as the number of immune to tumor cell neighbors divided by the total number of immune cell neighbors in a region of 75-μm. A final immunoscore was calculated for each patient by averaging the immune infiltration scores in regions with high tumor cell density. Tumor infiltrating lymphocytes (TILs) were manually quantified. Responders were defined as those with a complete response (CR), partial response (PR), or stable disease (SD), while those with progressive disease (PD) were categorized as non-responders. A continuous response score was developed from tumor measurements of a target lesion on serial imaging. Pearson’s correlation coefficients were used to assess the relationship between continuous response scores and tumor characteristics. Responders and non-responders were compared using Mann-Whitney U tests. Results: Fifteen women with PDL-1 positive mTNBC treated with ICI plus chemotherapy and available tissue were included. All patients had relapsed disease, and 10 patients (67%) received an ICI and chemotherapy as first line treatment for mTNBC. Eight patients (53%) received atezolizumab and nab-paclitaxel while the remaining 7 (47%) patients received pembrolizumab with an approved chemotherapeutic agent. Seven patients (47%) experienced a PR, 3 (20%) with a CR, and 1 (7%) had SD. Four patients (27%) had PD with no clinical benefit. Higher immunoscores (-0.17, p=0.6) and TILs (-0.21, p=0.5) were numerically associated with continuous response scores. However, there were no significant differences in immunoscores (0.28 vs 0.26, p=0.6) or TIL counts (2 vs 5, p=0.6) between responders (N=11) and non-responders (N=4). Interestingly, patients with response to treatment had lower tumor densities compared to non-responders (7.5 vs 17.3, p=0.02). Conclusions: Spatial analysis of tumor density and immune infiltration, including immunoscore shows evidence of correlation with response. Tumor density was the only parameter significantly associated with response. The study identified novel tumor characteristics that need to be considered in the prediction of response to ICIs plus chemotherapy in mTNBC. The current findings are hypothesis generating and need validation in additional tissue samples to determine the role of tumor density as a predictor of response to ICI. Citation Format: Jeffrey Aldrich, Thomas Hu, Jeffrey Switchenko, Ahmet Coskun, Yesim Gokmen-Polar, Sunil Badve, Manali Bhave. Predicting the likelihood of response in PDL-1 positive metastatic triple negative breast cancer treated with an immune checkpoint inhibitor [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 P1-04-14.

Abstract P2-11-23: Evaluation of T-cell infiltrating lymphocytes vs. Th2 in triple negative breast cancers (TNBC)

Abstract Purpose. Triple-negative breast cancers (TNBC) are defined as negative for hormonal receptors and human epidermal growth factor receptors 2 and account for 15% of breast cancers. TNBC carry the worst prognosis mainly because of their high proliferation index and the absence of efficient targeted therapies due to their molecular heterogeneity, even though some promising options are emerging. Besides, they do share a very high and distinct tumor-infiltrating lymphocytes (TILs) infiltration. Amongst TILs, CD8+ cytotoxic and CD4+ helper T-cells (TC) proved to be markers of a good outcome. However, pathologists routinely quantify overall TILs density only. Anti-PD-1/PD-L1 immunotherapies recently became available for immunosuppressive TNBC. Unfortunately, neither PD-1/PD-L1 expression nor TILs quantification is able to predict patients’ responses accurately. Thus, we aimed at precisely characterizing TILs sub-populations and evaluating their prognostic significance before their predictive one. Methods. We selected 91 patients at the time of surgery before any adjuvant therapy from January 2013 to December 2018 in Rennes, France. TNBC tumors went through TILs quantification on hematoxylin and eosin slides by a trained pathologist and immune microenvironment characterization using flow cytometry. We then compared the prognostic value of immune microenvironment subpopulations vs total TILs count. Results. TNBCs contained a mean of 22.8±25.9% TILs, including CD4+ TC (14.1%) mainly made of Th2 (11.7%), CD8+ TC (11.1%), and myeloid cells (8.4%) such as antigen presenting cells (APC). TILs groups and percentages were correlated with the abundance of these cellular subpopulations (p≤0.004). TILs percentage was predictive of overall survival (OS), while high APC infiltration was of relapse-free survival (RFS) in univariate analyses (p=0.044 &amp; p≤0.030 respectively). Only Th2 infiltration was predictive of both RFS and OS in univariate (p=0.009 &amp; p=0.008 respectively) and multivariate analyses (p=0.002 &amp; p=0.010 respectively). When considering the different Th populations, only Th2 was a better predictive factor of survival than total leukocytes. Discussion. The development of immune therapies aiming at unlocking the anti-tumor immune response has revealed the desperate need to better characterize the tumor immune infiltrate. TILs quantification by pathologists is the only parameter routinely measured so far but it must be standardized since variability can affect their correlation with pCR. In our study, and contrary to most previous ones, stromal TILs were not significantly associated with RFS and OS. Thus, the characterization of TILs subtypes is critical since they can have either a pro- or an anti-tumorigenic function. Thus, we counteracted the limitations of TILs pathological quantification by finely characterizing the immune infiltrate using flow cytometry. Th2 infiltrate was the most frequent, as previously reported, and counterintuitively, was associated with a favorable prognostic value. This is rarely shown as Th2 infiltration is frequently reported to favor pro-tumorigenic immune tolerance. This proves that a more refined characterization of the intra-tumoral immune landscape is essential to derive interesting therapeutic perspectives, either by recapacitating Th2 response or by targeting their interaction with other immune subsets, like fibroblasts, macrophages, dendritic cells, or eosinophils and IgE. Finally, immunotherapies modulating Th2 response could also be used around the time of surgery that has been proved to induce immunomodulation. Conclusion. The characterization of TILs composition is essential to better understand the potential antitumoral functions of these cells and to substantially improve the associated prognostic and predictive values. Citation Format: Susie Brousse, Florence Godey, Elodie Laffont, Patrick Tas, Boris Campillo-Gimenez, Vincent Lavoué, Matthieu Le Gallo. Evaluation of T-cell infiltrating lymphocytes vs. Th2 in triple negative breast cancers (TNBC) [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-11-23.

Prognostic significance for recurrence of fibroblast growth factor receptor 2 in intrahepatic cholangiocarcinoma patients undergoing curative hepatic resection.

The fibroblast growth factor receptor 2 (FGFR2) fusion gene is frequently found as a genetic abnormality in the FGFR pathway in patients with intrahepatic cholangiocarcinoma (ICC). The FGFR fusion protein, produced from the FGFR fusion gene, is thought to cause tumor cell growth. To date, there have been few reports on the relationship between pathologic FGFR2 expression and prognosis in patients who have undergone hepatectomy for ICC, and on the relationship between FGFR2 and tumor-infiltrating lymphocytes (TILs). We enrolled 92 patients who underwent hepatectomy for ICC and performed immunohistochemical staining for FGFR2 and cluster of differentiation 8, and hematoxylin and eosin staining for evaluating TILSs. The relationships between the FGFR2 and clinicopathological characteristics and outcomes were analyzed, and patients were classified into positive (n=18) and negative (n=74) FGFR2 groups. The FGFR2-positive group contained more men (p<0.0001) and had lower serum albumin (p=0.0355) and higher carcinoembryonic antigen (p=0.0099). Furthermore, multivariable analyses revealed that the FGFR2-positive group had worse disease-free survival (DFS) (p=0.0002). Multivariate analysis showed that the independent prognostic factors for DFS were maximum tumor size (≥5cm) (p=0.0011), tumor localization (perihilar type) (p=0.0180), and FGFR2 positivity (p=0.0029). There was no significant difference in TILs count between the two groups. We showed that FGFR2 high expression was an independent prognostic factor for recurrence of resected ICC.

Markers associated with genomic instability, immunogenicity and immune therapy responsiveness in Metaplastic carcinoma of the breast: Expression of γH2AX, pRPA2, P53, PD-L1 and tumor infiltrating lymphocytes in 76 cases

BackgroundMetaplastic breast cancer (MpBC) is an aggressive subtype of breast carcinoma that is often resistant to conventional chemotherapy. Therefore, novel treatment strategies are urgently needed. Immune check point inhibitors have shown activity in programmed death-ligand 1 (PD-L1) – positive metastatic triple negative breast carcinoma (TNBC), which raises the possibility that immunotherapy may also be effective in MpBC as most of the MpBCs are triple negative. The aim of the present study was to assess genomic instability and immunogenicity in tumor specimens of patients with MpBC.MethodsA total of 76 patients diagnosed with MpBC over a 15-year period were included in the study. We performed immunohistochemical analyses for tumor cell PD-L1, immune cell PD-L1 and p53 on tissue microarrays (TMAs), analyzed stromal and intratumoral tumor infiltrating lymphocytes (TILs) from hematoxylin and eosin-stained (H&E) slides and scored gamma-H2AX (γH2AX) and phosphorylated-RPA2 (pRPA2) from whole tissue sections. We correlated marker expression with clinicopathologic features and clinical outcome.ResultsAll tumors expressed γH2AX and pRPA2 with median expressions of 43% and 44%. P53- (68%), tumor cell PD-L1- (59%) and immune cell PD-L1-positivity (62%) were common in MpBCs. Median stromal TIL and intratumoral TIL counts were 5% and 0. The spindle and squamous cell carcinomas expressed the highest levels of PD-L1 and TILs, and carcinoma with mesenchymal differentiation the lowest.ConclusionsMpBC appears to be an immunogenic cancer with high genomic instability and frequent PD-L1-positivity, implying that check point inhibitors might be effective in MpBC. Expression levels of PD-L1 and TILs varied across different histologic subtypes, suggesting that immunotherapy might be less effective in carcinoma with mesenchymal differentiation.

Tumor-Infiltrating Lymphocytes and Immune Response in HER2-Positive Breast Cancer.

Human epidermal growth factor receptor 2-positive (HER2-positive) breast cancer accounts for 15 to 25% of breast cancer cases. Although therapies based on the use of monoclonal anti-HER2 antibodies present clinical benefit for a subtype of patients with HER2-positive breast cancer, more than 50% of them are unresponsive to targeted therapies or they eventually relapse. In recent years, reactivation of the adaptive immune system in patients with solid tumors has emerged as a therapeutic option with great potential for clinical benefit. Since the approval of the first treatment directed against HER2 as a therapeutic target, the range of clinical options has expanded greatly, and, in this sense, cellular immunotherapy with T cells relies on the cytotoxicity generated by these cells, which ultimately leads to antitumor activity. Lymphocytic infiltration of tumors encompasses a heterogeneous population of immune cells within the tumor microenvironment that exhibits distinct patterns of immune activation and exhaustion. The prevalence and prognostic value of tumor-infiltrating lymphocyte (TIL) counts are associated with a favorable prognosis in HER2-positive breast cancers. This review discusses emerging findings that contribute to a better understanding of the role of immune infiltrates in HER2-positive breast cancer. In addition, it summarizes the most recent results in HER2-positive breast cancer immunotherapy and anticipates which therapeutic strategies could be applied in the immediate future.