Tumor-infiltrating Lymphocytes Group Research Articles

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 PS16-06: Tumor-infiltrating lymphocytes and pathologic response to neoadjuvant chemotherapy with the addition of platinum and pembrolizumab in TNBC: A single-center real-world study

Abstract Background Previous studies have presented compelling evidence suggesting that the inclusion of platinum agents alongside anthracyclines and taxanes could potentially enhance treatment outcomes in high-risk triple-negative breast cancer (TNBC). Moreover, pembrolizumab has been integrated into clinical practice as a part of standard-of-care for non-metastatic TNBC with high risk. In light of these findings, we undertook a real-world study to investigate the impact of incorporating platinum agents and pembrolizumab on achieving pathologic complete response (pCR) in TNBC patients undergoing neoadjuvant chemotherapy (NAC). Furthermore, we specifically examined the influence of tumor-infiltrating lymphocytes (TILs) on the treatment outcomes. Patients and Methods In this real-world study conducted at Gangnam Severance Hospital, Seoul, Republic of Korea, we analyzed a cohort of 398 patients with TNBC who underwent surgery following NAC between March 2007 and November 2022. Among them, 247 patients received an anthracycline-taxanes (A-T), 120 received a carboplatin regimen including A-T, and 31 received a pembrolizumab regimen including A-T-carboplatin as part of their neoadjuvant chemotherapy treatment. TIL was evaluated in biopsied samples prior to NAC according to the guideline of TIL international working group. The high TIL was defined with a cutoff of 50%. Results Among the 398 patients analyzed, 87 (21.9%) had high TIL tumors. The pCR rates were 32% in the anthracycline-taxane (A-T) regimen group, 57% in the A-T-carboplatin regimen group, and 68% in the pembrolizumab with A-T-carboplatin regimen group. Within the high TIL group, the pCR rate did not increase with the addition of carboplatin (51.8% in the A-T group and 41.7% in the A-T-carboplatin group), but reached 85.7% with the addition of pembrolizumab and carboplatin. Among the low TIL group, the pCR rate increased from 26.7% to 61.1% with the addition of carboplatin, but there was no difference in the pCR rate between the carboplatin and pembrolizumab groups (61.1% and 60.9%, respectively). In clinically node-positive patients, the pCR rate significantly increased with pembrolizumab in the high TIL group (40.9% versus 100%, p=0.035). However, in low TIL patients, the addition of carboplatin alone significantly increased the pCR rate to 62.3%, whereas the addition of pembrolizumab did not show the same effect. Conclusions Our real-world data consistently demonstrates an increased pCR rate with the addition of carboplatin and pembrolizumab. Among patients with high TIL, the addition of carboplatin did not result in an elevated pCR rate. However, the addition of pembrolizumab tended to maximize the pCR rate, surpassing 80%. On the other hand, among patients with low TIL, the addition of carboplatin significantly increased the pCR rate, while the addition of pembrolizumab did not have the same effect. Efforts should be made to improve the response to pembrolizumab-containing regimens for patients with low baseline TIL levels. Citation Format: Min Ji Kim, Yoonwon Kook, Seung Ho Baek, Junghyun Kim, Sohyun Moon, Seung Eun Lee, Jee Hung Kim, Soong June Bae, Sung Gwe Ahn, Joon Jeong. Tumor-infiltrating lymphocytes and pathologic response to neoadjuvant chemotherapy with the addition of platinum and pembrolizumab in TNBC: A single-center real-world study [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 PS16-06.

Clinicopathological characteristics and immune microenvironment of breast squamous cell carcinoma

Objective: To investigate the clinicopathological characteristics of breast squamous cell carcinoma and to analyze the relationship between its immune microenvironment tumor infiltrating lymphocytes (TILs) and prognosis. Methods: Forty-four cases of primary squamous cell carcinoma of the breast diagnosed and treated in the First Affiliated Hospital of Air Force Medical University, Xi'an, China from January 2006 to July 2022 were selected. Their clinicopathological characteristics were analyzed. The cell composition of TILs was evaluated using immunohistochemistry (Mainly markers of B lymphocytes, T lymphocytes and plasma cells). The relationship between TILs and prognosis was also analyzed. Results: The 44 patients of breast squamous cell carcinoma were all female and all were invasive carcinoma. Eight cases (8/44, 18.2%) were squamous cell carcinoma, while 36 cases (36/44, 81.8%) were mixed squamous cell carcinoma. The mixed components included non-specific carcinoma and spindle cell metaplastic carcinoma (17 cases each). One case contained ductal carcinoma in situ of the breast and 1 case contained tubular carcinoma. The proportion of squamous cell carcinoma was 10% to 90%. The cases with pure squamous cell carcinoma often had a large cystic cavity, which was lined by atypical squamous epithelium, while infiltrating squamous cell carcinoma nests were seen in the breast tissue around the cystic cavity. Immunohistochemical staining showed that p63 and CK5/6 were expressed in the squamous cell carcinoma component, but ER, PR and HER2 were not, except for one case of HER2 1+. The positive rates of TRPS1 and PDL-1 were 76% and less than 1%, respectively. Fifteen cases were in the high TILs group (TILs≥30%) and 29 cases were in the low TILs group (TILs<30%). Twenty-three patients were followed up for 5 to 118 months. Among them, 12 died within 3 years and 9 were alive at the end of the follow up. There was no significant difference in TNM stage, TILs and prognosis between simple squamous cell carcinoma and mixed squamous cell carcinoma. Conclusions: Breast squamous cell carcinoma can be divided into simple squamous cell carcinoma and mixed squamous cell carcinoma. There are differences in gross findings and histology between the simple and mixed squamous cell carcinoma of the breast. Sufficient samples should be taken to avoid missing the diagnosis of a minor squamous component. The prognosis of patients with high TILs is significantly better than that of patients with low TILs. The expression rate of TRPS1 in primary squamous cell carcinoma of breast is high and helpful to the differential diagnosis from metastatic squamous cell carcinoma.

Prognostic implications of tumor-infiltrating lymphocytes within the tumor microenvironment in gastric cancer

Tumor-infiltrating lymphocytes (TILs) play a regulatory role in the tumor-associated immune response and are important in the prognosis and treatment response of several cancers. However, because of its heterogeneity, the prognostic value of TILs in gastric cancer (GC) is still controversial. Thus, this study aimed to investigate the association between the density of TILs and patients' outcomes in GC. Patients with gastric adenocarcinoma who underwent curative intent gastrectomy were retrospectively investigated. The groups for analysis were determined on the basis of TIL intensity and percentage of CD3+T-cell infiltration by immunohistochemical. Furthermore, Epstein-Barr virus (EBV), microsatellite instability (MSI), T-cell ratio of CD4 to CD8, and programmed death protein ligand 1 (PD-L1) status were evaluated. A total of 345 patients were enrolled: 124 patients with GCs (35.9%) were classified as the low-CD3+TIL group, and 221 patients with GCs (64.1%) were classified as the high-CD3+TIL group. Poorly differentiated histology (P=.014), EBV-positive status (P<.001), PD-L1-positive status (P=.001), and CD4<CD8 (P<.001) were associated with high-CD3+GC. There was no difference regarding MSI status, the degree of tumor invasion (pT), the presence of lymph node metastasis, and pTNM stage between low- and high-CD3+groups. In survival analysis, the high-CD3+group had better disease-free survival and overall survival rates than had the low-CD3+group (P=.055 and P=.041, respectively). In the multivariate analysis, total gastrectomy, lymph node metastasis, advanced pT stage, and low CD3+levels were independent factors related to worse survival. High CD3+TILs levels were significantly associated with improved survival and could serve as prognostic biomarkers in GC. In addition, CD3+T-cell infiltration was related to both EBV-positive and PD-L1-positive GC and may assist in the investigation of targets in immunotherapy.

Role of stromal PD-L1 expression in colorectal liver metastasis

Background and AimThe outcomes of immune checkpoint blockade for colorectal cancer (CRC) treatment are unsatisfactory. Furthermore, the efficacy of immune checkpoint blockade for liver metastasis of various cancer is poor. Here, we investigated the relationship between stromal programmed death-ligand 1 (PD-L1) expression and the prognosis of patients with colorectal cancer liver metastasis (CRLM).MethodsThe present study enrolled 84 CRLM patients who underwent surgery (R0) for CRC. Immunohistochemistry was performed to analyze stromal PD-L1 expression in CRLM.ResultsStromal PD-L1 was expressed in 52.3% of CRLM samples, which was associated with fewer not optimally resectable metastases (p = 0.04). Stromal PD-L1 also tended to associate with a lower tumor grade (p = 0.08). Stromal PD-L1-positive patients had longer overall survival (p = 0.003). Multivariate analysis identified stromal PD-L1 expression (p = 0.008) and poorer differentiation (p < 0.001) as independent prognostic indicators. Furthermore, stromal PD-L1 expression was correlated to a high number of tumor-infiltrating lymphocytes (TILs). Stromal PD-L1– and low TIL groups had shorter OS than stromal PD-L1 + and high TIL groups (46.6% vs. 81.8%, p = 0.05) Stromal PD-L1-positive patients had longer disease-free survival (DFS) (p = 0.03) and time to surgical failure (p = 0.001). Interestingly, stromal PD-L1 expression was positively related to the desmoplastic subtype (p = 0.0002) and inversely related to the replacement subtype of the histological growth pattern (p = 0.008).ConclusionsStromal PD-L1 expression may be a significant prognostic marker for CRLM.

Safety, efficacy, and biomarker analysis of response to engineered tumor-infiltrating lymphocytes secreting anti-PD-1 antibody in recurrent glioblastoma: An open-label, two-arms, phase 1 study.

2042 Background: Adoptive cell therapy using tumor-infiltrating lymphocytes (TILs) is effective against solid tumors with convincing evidence. But T-cells infused were exhausted when they encountered PD-1-mediated immunosuppression in the microenvironment. Here, we generated a modified autologous TILs secreting antibody targeting PD-1 (PD1-TIL), and conducted a first-in-human, two arms, phase 1 study (NCT03347097) in recurrent glioblastoma who had a poor prognosis with estimated 6-9 months of median overall survival (OS). Methods: Patients were included to be treated with PD1-TIL or TIL cell products intravenously every one month following surgery. The primary endpoint was safety and feasibility. The secondary endpoints were disease control rate (DCR), OS, immunologic responses and biomarker analysis. The clinical response to cell therapy was assessed by MRI according to iRANO criteria. Here, patients with clinical benefit (CR, PR, and SD) were defined as responder. Results: A total of 21 consecutive patients (median age 45 years, range 20–64) received infusions of cell products (7 patients for PD1-TIL with 1 excluded, 14 patients for TIL with 2 excluded). 52% (11/21) and 29% (6/21) of 21 patients had methylated MGMT promoter and IDH1/2 mutant respectively. No significant differences in baseline characteristics between groups were found. Both of PD1-TIL and TIL infusions were well tolerated with no unexpected high-grade adverse events. Of 7 evaluable patients in PD1-TIL group, 1 had PR for more than 26 months, 2 had SD lasting for 7.7 months and 24 months, and 4 had PD after infusion. The DCR, 1-year and 2-years survival rates in PD1-TIL group were 43% (3/7), 86% (6/7) and 29% (2/7) respectively. In TIL group, 3 patients had SD and the others had PD. The DCR, 1-year and 2-years survival rates in TIL group were half of that in PD1-TIL group. Survival analysis showed that patients treated by PD1-TIL had extended OS than those received TIL (16.1 vs. 11.2 months, P = 0.047). Among 21 patients, clinical responders had significantly improved OS (30.9 vs. 10.7 months, P = 0.001) than non-responders. We also found a high capacity for engraftment and persistence of infused TILs in peripheral blood which could be detectable at 6 months after treatment. Existence of neoantigen-reactive T cell clonotypes in cell products may be important for responding because missing of missense mutations predicted to generate a neoantigen after cell therapy was a remarkable signature in responders. Responsive tumors were also associated with tumor microenvironment profiles. Conclusions: This first-in-human study establish the preliminary feasibility, safety and encouraging efficacy of PD1-antibody-secreting TILs, and may constitute a new treatment strategy by T-cell genetic engineering in glioblastoma. Clinical trial information: NCT03347097 .

Frequency of Her2-low in colorectal cancer and its relations with the tumor microenvironment

BackgroundTo date, little is known regarding human epithelial growth factor receptor (HER2) low-expressing colorectal cancer (CRC). Due to promising rising therapies with HER2-antibody-drug conjugates we aimed to analyze the frequency of HER2-low in patients with CRC. Additionally we characterized the clinicopathologic background of this group and its potential relationship with the tumor microenvironment represented by budding and tumor infiltrating lymphocytes (TILs). Methods319 patients with CRC, stages I–IV, were enrolled. HER2-immunohistochemistry (IHC) as well as fluorescence in situ hybridization (FISH) were performed on tissue microarrays. IHC was evaluated semiquantitatively and software-assisted using the HERACLES Diagnostic Criteria for CRC. HER2-low was defined as IHC 1 + or 2 +/FISH negative. HER2-IHC results were compared with budding, TILs and their combinations. ResultsThe HER2 low-expressing subset represented almost one half of all CRC (47.1 %). Assessment was highly reproducible with different methods. HER2-low cases were significantly more often lower T-, N-, and tumor stage and had less L1 compared with HER2-0. Additionally, they showed more often TILs > 5 % (p = 0.001). The difference between HER2-0 and HER2-low was highly significant between the four budding/TILs-groups (p < 0.001). Cases with low budding/high TILs were more often HER2-low. The highest difference was seen between the low budding/high TILs-group and the low budding/low TILs-group (p < 0.001). ConclusionsHER2-low expression in CRC is frequent and involves nearly one half of all patients. We could show a relationsship between HER2-low expression and the tumor microenvironment. Special attention should be paid to the low budding/high TILs group in future research.

Abstract P2-20-12: Abundance of tumor-infiltrating lymphocytes is associated with pathologic complete response to neoadjuvant chemotherapy but not with survival in ER-positive/HER2-negative breast cancer

Abstract INTRODUCTION: Abundance of tumor infiltrating lymphocytes (TILs) is well known to be associated with achievement of pathologic complete response (pCR) after neoadjuvant chemotherapy and is a surrogate of better survival in triple negative breast cancer (TNBC). However, the association remains unclear in ER-positive/HER2-negative, which is the most common subtype in breast cancer. Further, pathological quantification of TILs is known for its ambiguity. We hypothesized that abundance of TILs, quantified by transcriptomic signatures, in ER-positive/HER2-negative is associated with different biology and is not a surrogate of survival unlike in TNBC. METHODS: A total of 6035 primary breast cancer patients from three cohorts (TCGA, METABRIC, and SCAN-B) with full transcriptome and clinical data were analyzed. Lymphocyte fractionation in the tumor microenvironment of a bulk tumor was estimated from the transcriptome using two different deconvolution tools, CIBERSORTx and xCell. RESULTS: First, the correlation between TIL score measured histologically on TCGA patients and gene expression of CD3D, CD3E, CD3G, CD8A, CD4, and total lymphocytes, and total T-cells measured by xCell or CIBERSORTx were analyzed. Total sum of lymphocytes by xCell correlated the strongest, thus it was adopted as the TIL score. The TIL score strongly correlated with CD3G, CD3E, CD3D, CD8A, and CD4 gene expressions in both TCGA and GSE96058 cohorts (all r &amp;gt; 0.48). Among the infiltrated cells; M1 and M2 macrophages, dendritic cells, mast cells and monocytes were all higher, and microvascular endothelial cells and pericytes were lower in TIL score high tumors in ER-positive/HER2-negative (all p &amp;lt; 0.01) where M2 and mast cells were not in HER2+ and TNBC in both 2 cohorts. Enrichment of immune related gene sets IL2/Stat5 signaling, IL6/Jak/Stat3 signaling, inflammatory response, and Complement were less in high TIL group of ER-positive/HER2-negativethan HER2 and TNBC. In agreement, cytolytic activity was lower in TIL high of ER-positive/HER2-negative compared from TNBC. On the other hand, high TIL group in ER-positive/HER2-negative had significantly higher intratumor heterogeneity, homologous recombination deficiency, mutation burden, and neoantigens, which were not the case in the other subtypes. In agreement, all the cell proliferation-related gene sets; E2F targets, G2M checkpoint, mitotic spindle, Myc targets v1 and v2, were more strongly enriched in high TIL group of ER-positive/HER2-negative compared from HER2+ and TNBC. MKI67 gene expression and Nottingham histological Grade were uniformly elevated with high TIL regardless of subtypes. In three independent neoadjuvant cohorts, we found that pathologic complete response rates of high TIL group. On the other hand, high TIL group did not show any survival benefit compared with low TIL group in ER-positive/HER2-negative, unlike in HER2+ or TNBC. CONCLUSION: High tumor-infiltrating lymphocytes in ER-positive/HER2-negative breast cancer was associated with less immune response and higher genetic alteration and cell proliferation compared to the other subtypes, leading to better response of neoadjuvant chemotherapy, but no benefit in survival outcome. Citation Format: Rongrong Wu, Masayuki Nagahashi, Masanori Oshi, Yasuo Miyoshi, Takashi Ishikawa, Kazuaki Takabe. Abundance of tumor-infiltrating lymphocytes is associated with pathologic complete response to neoadjuvant chemotherapy but not with survival in ER-positive/HER2-negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-20-12.

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.

Tumor-Infiltrating Lymphocyte Therapy or Ipilimumab in Advanced Melanoma

BackgroundImmune checkpoint inhibitors and targeted therapies have dramatically improved outcomes in patients with advanced melanoma, but approximately half these patients will not have a durable benefit. Phase 1–2 trials of adoptive cell therapy with tumor-infiltrating lymphocytes (TILs) have shown promising responses, but data from phase 3 trials are lacking to determine the role of TILs in treating advanced melanoma.MethodsIn this phase 3, multicenter, open-label trial, we randomly assigned patients with unresectable stage IIIC or IV melanoma in a 1:1 ratio to receive TIL or anti–cytotoxic T-lymphocyte antigen 4 therapy (ipilimumab at 3 mg per kilogram of body weight). Infusion of at least 5×109 TILs was preceded by nonmyeloablative, lymphodepleting chemotherapy (cyclophosphamide plus fludarabine) and followed by high-dose interleukin-2. The primary end point was progression-free survival.ResultsA total of 168 patients (86% with disease refractory to anti–programmed death 1 treatment) were assigned to receive TILs (84 patients) or ipilimumab (84 patients). In the intention-to-treat population, median progression-free survival was 7.2 months (95% confidence interval [CI], 4.2 to 13.1) in the TIL group and 3.1 months (95% CI, 3.0 to 4.3) in the ipilimumab group (hazard ratio for progression or death, 0.50; 95% CI, 0.35 to 0.72; P<0.001); 49% (95% CI, 38 to 60) and 21% (95% CI, 13 to 32) of the patients, respectively, had an objective response. Median overall survival was 25.8 months (95% CI, 18.2 to not reached) in the TIL group and 18.9 months (95% CI, 13.8 to 32.6) in the ipilimumab group. Treatment-related adverse events of grade 3 or higher occurred in all patients who received TILs and in 57% of those who received ipilimumab; in the TIL group, these events were mainly chemotherapy-related myelosuppression.ConclusionsIn patients with advanced melanoma, progression-free survival was significantly longer among those who received TIL therapy than among those who received ipilimumab. (Funded by the Dutch Cancer Society and others; ClinicalTrials.gov number, NCT02278887.)

Evaluating Tumor-Infiltrating Lymphocytes in Breast Cancer: The Role of Conventional Ultrasound and Contrast-Enhanced Ultrasound.

Tumor-infiltrating lymphocytes (TILs) have emerged as an efficient biomarker predicting treatment response and prognosis of breast cancer (BC). This study aimed to evaluate the association between conventional ultrasound and contrast-enhanced ultrasound (CEUS) imaging features with TIL levels in invasive BC patients. We retrospectively included 267 women with invasive BC who had undergone conventional ultrasound and CEUS. Patients were divided into low (≤10%) and high (>10%) TIL groups. Conventional ultrasound and CEUS features were analyzed by two sonographers. The associations between the TIL levels and imaging features were evaluated. Of the 267 patients, 122 with high TILs and 145 with low TIL levels. High TIL tumors were more likely to have a circumscribed margin, oval or round shape, and enhanced posterior echoes on ultrasonography (p < 0.05). In contrast, low TIL tumors were more likely to have an irregular shape, un-circumscribed, indistinct and spiculated margin (p < 0.05). In CEUS, high TIL tumors showed a more regular shape, clearer margin, more homogeneous enhancement and higher peak intensity (PI) value (p < 0.05). Logistic analysis indicated that shape, posterior features, PI, and enhanced homogeneity were independent predictors for high TIL tumors. The model combined the four independent predictors have a moderate performance in predicting high TIL tumors with AUC 0.79, sensitivity 0.72, and specificity 0.78. Conventional ultrasound and CEUS features were associated with TIL levels in invasive BC. Consequently, the results suggested that preoperative conventional ultrasound and CEUS may be a useful noninvasive imaging biomarker for individualized treatment decisions.

CD8+ TILS Expression in Invasive Breast Carcinoma (No Special Type)

Invasive breast carcinoma (IBC) is the most cancer in women (24%) and the cause of cancer death in women worldwide. Based on data from globocan 2018 shows the incidence of breast cancer is around 2.08 million (11.6%) which is the second rank of all cancers after lung cancer with a mortality rate of 626.6 thousand (6.6%) which is also the most common cause of death in women. The basic role of the immune system in maintaining homeostasis by immunosurveillance and initiation of the inflammatory reactions that include coordination of innate and adaptive immune cells activation against tumor cells is one of the most important in the mechanism of tumor cell elimination. Prognosis of IBC were influenced by several factors, including tumor histology grade and Tumor Infiltrating Lymphocytes (TILs). Infiltration of lymphocytes in the tumor microenvironment/TILs through CD8+ T lymphocytes is known to be an important component of adaptive immunity that eliminates tumor cells. CD8+ T cells present tumor antigens on the surface of the major histocompatibility complex class I (MHC class I). Based on its importance in clinical application and it’s role in biological concepts, this study was conducted to determine the expression of CD8+ in Invasive Breast Carcinoma of No Special Type (IBC-NST) grades 1,2 and 3. Analytical study with a cross-sectional design on IBC-NST samples from 2017 until 2020 at the Laboratory of Anatomic Pathology, Faculty of Medicine, Hasanuddin University Makassar from May to August 2021. Immunoexpression data were analyzed to determine its relationship with grade. Eighty samples met the inclusion and exclusion criteria, there were 17 samples (21.3%) with grade 1, 32 samples (40%) with grade 2, and 31 samples (38.8%) with grade 3. In the high CD8+ TILs group, from a total of 27 samples, 10 samples with grade 1, 6 samples with grade 2, and as many as 11 samples with grade 3. In the low CD8+ TILs group, from a total of 53 samples, there were 7 samples with grade 1, 26 samples with grade 2, and 20 samples with grade 3. Based on the Chi-square test, p value = 0.017 (p <0.05). There is a significant difference in CD8+ TILS in Invasive Breast Carcinoma of No Special Type grade 1, grade 2 and grade 3.

Tumor Infiltrating Lymphocytes are Prognostic Factors and Can Be Markers of Sensitivity to Chemoradiotherapy in Head and Neck Squamous Cell Carcinoma.

Background:Tumor-infiltrating lymphocytes (TILs) are assessed by the ratio of the area of lymphocytes infiltrating the stroma. TILs are important in breast cancer and malignant melanoma and are being established as a marker of prognosis and sensitivity to chemotherapy. This has resulted in various therapies being developed in fields such as breast cancer. However, the evaluation of TILs in head and neck squamous cell carcinoma (HNSCC) is not progressing, and the prognosis is still poor. Thus, investigating whether or not the evaluation of TILs is also effective in HNSCC and prognoses can be predicted with just biopsy samples alone is required. Methods:This study included 153 patients who were diagnosed with HNSCC between January 2010 and December 2019, underwent treatment, and could be followed up thereafter at our institution. Results:TILs, overall survival (OS), and progression-free survival (PFS) were evaluated in all patients, the chemoradiotherapy arm, and the surgery arm. The cut-off value for TILs was 50%. In all patients, OS was 69.8% and 40.2% (P = 0.01) and PFS was 58.4% and 31.6% (P = 0.003) in the high and low TIL groups, respectively. Multivariate analyses revealed that TILs independently predicted prognosis. In the chemoradiotherapy arm, OS was 70.8% and 31.6% (P = 0.012) and PFS was 63.4% and 20.3% (P = 0.001) in the high and low TIL groups, respectively. No significant differences were noted in the surgery arm. Conclusions: In HNSCC, TILs can be used as a prognosis predictor and chemoradiotherapy biomarker. Assessments can be performed just with hematoxylin–eosin staining and is very simple. This will greatly contribute to report personalized therapy progress. Further evaluations and, thus, prospective clinical multicenter trials are needed to use TILs in clinical practice for HNSCC.

The prognostic significant of tumor budding, tumor stroma ratio and tumor-infiltrating lymphocytes in gallbladder adenocarcinoma

Aim: Tumor microenvironment plays an important role in onset and progression of the cancer. Tumor budding (TB), tumor stroma and tumor-infiltrating lymphocytes are component of the tumor microenvironment. It was aim to determine the relationship of TB, tumor stroma ratio (TSR) and tumor-infiltrating lymphocytes (TILs) with clinicopathological prognostic parameters in gallbladder adenocarcinoma. Materials and Methods: Thirty cholecystectomy cases that were diagnosed as adenocarcinoma between 2010 and 2020, that did not receive neoadjuvant therapy and of which archive slides could be accessed, were included in the study. TB, TSR and TILs were evaluated. A p&amp;lt;0.05 value was statistically significant. Results: High TB score was significantly associated with higher histological grade (p=0.008), higher pT stage, lymphovascular invasion (LVI) (p=0.038), lymph node metastasis (p=0.046) and distant metastasis (p=0.036) . Patients with high TB scores had a shorter overall survival (p&amp;lt;0.001). In the high TILs group, lower histological grade (p=0.004), less LVI (p=0.029), fewer distant metastases (p=0.021) and lower TSR (p=0.008) were detected. Increased TCR was associated with higher histological grade (p=0.015) and increased distant metastasis (p=0.013). There was no significant effect of TCR on overall survival (p=0.239). Conclusion: TB can be used as a novel prognostic histological characteristic in the gallbladder cancer patients. We have concluded that it is early to recommend TILs and TSR as the prognostic indicators in gallbladder cancers. In the future, further studies can be conducted on a larger number of GBC cases with a multicenter participation to clarify the prognostic value of TILs and TSR.

Abstract P3-03-24: Machine learning approach to predict the level of tumor-infiltrating lymphocytes of breast cancer via MRI-based radiomics

Abstract Background: The level of tumor-Infiltrating lymphocytes (TILs) has important guiding significance for the individual treatment of breast cancer. In clinical, it is necessary to obtain tumor samples via invasive biopsy for pathological assessment. Our study aimed to investigate the feasibility of using machine learning algorithms to predict the level of TILs based on Multi-parametric MRI-radiomics in breast cancer. Materials and Methods: 207 patients with histologically-proven breast cancer (including 81 patients with HR+/HER2- subtype, 95 patients with HER2+ subtype, and 31 patients with TN subtype) who underwent breast MRI from January 2017 to March 2021 were retrospectively retrieved and analyzed. A total of 4198 quantitative radiomics features were extracted from the tumor regions of interest (ROI) on multi-parametric MRI, including T1-weighted Dynamic Contrast-Enhancement (T1-DCE) images, Fat-suppressed T2-weighted images (FS-T2WI) and Apparent Diffusion Coefficient (ADC) map using the Pyradiomics software. Feature selection was then performed using the least absolute shrinkage and selection operator (LASSO) and Boruta algorithm to identify the radiomics features most relevant to the level of TILs. Six machine learning models, including Logistic Regression (LR), Support Vector Machine (SVM), Random Forests (RF), Linear Discriminant Analysis (LDA), Gaussian Bayes (GNB) and Multilayer Perceptron (MLP) were developed with leave-one-out cross-validation (LOOCV) for predicting the level of TILs in breast cancer. The predictive performance of those classifiers for the level of TILs was evaluated through the receiver operating characteristic (ROC) curves, the area under curve (AUC), accuracy, sensitivity and specificity in the testing set. Results: Among the 207 patients, 94 (45.4%) showed high TILs levels (≥30%), while 113 (54.6%) showed low TILs levels (&amp;lt;30%). There was no inter-group distribution difference of clinicopathologic characteristics (including age, clinical TNM stage of tumor, ER, PR, HER2 and Ki-67 index) between the low TILs group and the high TILs group. After the feature selection step, 11 top-class features (including 5 features from T1-DCE images, 3 features from FS-T2WI, and 3 features from ADC-map) were selected to develop the radiomics models. The MLP classifier achieved the most excellent predictive performance in the testing dataset [AUC: 0.865, 95% confidence interval (CI), 0.788-0.942, with an accuracy of 82.8%]. Moreover, the Rad-score in the high TILs group was higher than that in the low TILs group (p = 0.014). We also evaluate the model performances in different molecular subtypes, and the MLP classifier had the best performance in HER2+ subtype with an AUC of 0.916 and an accuracy of 86.9%. While for HR+/HER2- subtype and TN subtype, the AUCs were 0.848 and 0.781, and the accuracies were 81.3% and 73.7%, respectively. Conclusion: Multi-parametric MRI-based radiomics model could provide a non-invasive tool to predict the TILs levels of breast cancer, which might help to guide clinical decision-making in individual treatment for patients with breast cancer. Citation Format: Yuhong Huang, Ying Lin. Machine learning approach to predict the level of tumor-infiltrating lymphocytes of breast cancer via MRI-based radiomics [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-03-24.

Abstract P5-06-14: A novel combination of a 2 gene score &amp; TIL as a predictive Biomarker for responders to novel therapies in Indian TNBC - A population with greater proportion of TNBCs

Abstract Introduction: Triple-Negative Breast Cancer (TNBC) comprises approximately 30% of all breast cancers in Indian women. Given their aggressive nature, TNBCs have high rates of systemic metastasis and mortality with only chemotherapy available for treatment. Compelling evidence has demonstrated the prognostic value of tumor-infiltrating lymphocytes (TILs), in many cancers especially in Breast Cancer and play a critical role in tumour progression, response to therapeutics and prognosis. They are typically measured by H&amp;E staining and immunohistochemistry for research purpose and degree of infiltrate is also known to differ among subtypes. Nonetheless, the literature regarding the types of immune cells characterizing TILs and their prognostic utility in TNBC has been conflicting for lack of accurate “functional” TIL assessments. Herein we have used a combination signature of TILs and a 2 gene immune function expression based signature to develop an unique classifier, to identify a subgroup within TNBC that has better clinical outcome, such as survival and response to treatment. Our findings also suggest a possible use of the score as a predictive biomarker for response to immune checkpoint therapy Patients and Methods: Surgically excised TNBC tumor specimens from 44 women from a single treating hospital in Bangalore, India were accessed under IERB approved protocols and assayed on nCounter® PanCancer Immune Profiling Panel which comprised of 740 genes and characterises 14 different immune cell types. Analysis using Non-negative Matrix factorization (NMF) based unsupervised clustering was done to arrive at stable subtypes characterized by different tumor microenvironments within TNBC. SSGSEA analysis was done to identify immune cell types. TILs were characterised by a pathologist on H&amp;E sections according to guidelines from TIL working group. The Immune classifier was validated on Breast Cancer data sets from TCGA. Results: NMF followed by SAM and PAM analysis yielded 2 stable subtypes (ST1 and ST2) of proportions 59% and 41% respectively. TIL groups also were divided into Dense and Mild groups which were 52% and 48% respectively. Despite similar distributions of the groups, ST1 and ST2 had distinct survival with ST2 having poorer outcome (p=0.023) while the dense and mild TIL groups did not separate as distinct groups. On a closer examination using SSGSEA analysis with Rooney et al signature, ST1 was enriched with T-cells, Dendritic cells, MHC-class 1 and very significantly high cytolytic score (CYT), which was defined by Granzyme A and Perforin expressions, proteins secreted by cytotoxic T cells (p=0.0074). The CYT Score ranged from -35 to 170 and cutoff was based on 75th percentile. We next hypothesized that a combined score of TIL and CYT would represent a functional TIL group with high immune activity. We arrived at a CYT+TIL score based on 75th percentile cut off of CYT and 2 groups of TIL. Of the resultant four groups, TIL High-CYT high that constituted about 20% of all TNBCs indicated an elevated immune response because of their microenvironment constitution and this can be identified using our simple immune classifier. On the other hand, in the TIL-high and CYT-low group, despite lymphocyte migration, outcome was not favorable. The classifier was applied to TNBC from TCGA (n=96) and similar results seen, with ST1 and ST2 which had distinctly separate survival and had similar patterns on CYT high groups. Conclusion: We developed a simple immune classifier with 2 gene signature c and H&amp;E slide TIL assessment for identifying a group with TNBC that can be better targeted with therapies. Our signature is predictive for selecting TNBCs which are potential responders to Immune therapies and has the potential for quick clinical adoption though it requires validation on a larger set. Citation Format: Aruna Korlimarla, Hari PS, Jyothi S Prabhu, Chantirika Ragulan, Ravi B Diwakar, Sandhya Apachu, Maggie Cheang, Rekha V Kumar, BS Srinath, TS Sridhar, Savitha Rajarajan, Annie Alexander, Anguraj Sadanandam. A novel combination of a 2 gene score &amp; TIL as a predictive Biomarker for responders to novel therapies in Indian TNBC - A population with greater proportion of TNBCs [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-06-14.

Prognostic value of the density of tumor-infiltrating lymphocytes in colorectal cancer liver metastases.

Tumor-infiltrating lymphocytes (TILs) have been reported to reflect the anti-tumor immune status of patients and to be correlated with their prognosis and therapeutic outcomes. However, the characteristics of the local immune status in metastatic tumors is poorly understood, as primary tumors have been the focus in most previous studies. In addition, the local immune status may be influenced by preoperative chemotherapy. The present study aimed therefore to investigate the relationship between the degree of TIL infiltration and the prognosis in patients with curative resection of colorectal cancer liver metastases and to examine the effects of preoperative chemotherapy on the function of immune cells. A total of 108 patients who underwent curative resection of colorectal cancer liver metastases in our department between May 1996 and January 2017 were enrolled in the present study. Peripheral blood samples were obtained within two weeks before surgery. TIL infiltration was evaluated by immunohistochemical staining of surgically resected specimens of liver metastases using anti-CD8/CD3 antibodies. The mean number of TILs in five different fields was calculated, and patients were classified into a high-TIL group and a low-TIL group. Furthermore, patients were divided into three groups as follows: i) A group of patients who did not receive preoperative chemotherapy; ii) a group of patients who received short-term preoperative chemotherapy for <6 months; and iii) a group of patients who received long-term preoperative chemotherapy for ≥6 months. The results demonstrated that the density of TILs in colorectal liver metastases was not correlated with the absolute peripheral lymphocyte count in all patients. Furthermore, the degree of CD8+TIL infiltration in liver metastases was significantly lower in the recurrence group compared with the recurrence-free group following hepatectomy. In all patients with colorectal liver metastases, the degree of CD8+TIL infiltration was significantly associated with the relapse-free and overall survival. In patients without preoperative chemotherapy, the degree of CD8+TIL infiltration was significantly associated with the relapse-free survival, and a high CD8+TIL presence tended to have a better effect on the overall survival than a low CD8+TIL presence. In the short-term chemotherapy group, the degree of CD8+TIL infiltration was significantly associated with the relapse-free and overall survival. In the long-term chemotherapy group, there were no significant differences between the high- and low- CD8+TIL groups in the relapse-free and overall survival. In contrast to CD8+TILs, CD3+TILs showed a poor prognostic ability. In summary, the degree of CD8+TIL infiltration in colorectal cancer liver metastases may be correlated with patient prognosis. However, in patients who received long-term chemotherapy before surgery, the degree of TIL infiltration was not necessarily associated with prognosis as the anti-tumor effects of TILs may decrease. The degree of CD8+TIL infiltration may therefore be considered as a useful prognostic factor in patients with colorectal liver metastases, but the prognostic accuracy may decrease in patients who received long-term chemotherapy.

Stromal tumor-infiltrating lymphocytes level as a prognostic factor for resected intrahepatic cholangiocarcinoma and its prediction by apparent diffusion coefficient.

Tumor-infiltrating lymphocytes (TILs) are a prognostic factor or an indicator of chemotherapy response for various malignancies. The aim of this study was to investigate the prognostic impact of TILs in resected intrahepatic cholangiocarcinoma (IHCC). We also investigated the usefulness of the apparent diffusion coefficient (ADC) in diffusion-weighted magnetic resonance imaging (DW-MRI) to predict TILs. We enrolled 23 patients with IHCC who underwent initial hepatic resection in Tokushima University Hospital from 2006 to 2017. We evaluated stromal TILs in the tumor marginal area and central area in surgical specimens. Patients were divided into low vs high stromal TILs groups. We analyzed the patients' clinicopathological factors, including prognosis, according to the degree of stromal TILs. We also analyzed the correlation between stromal TILs and the minimum ADC value. Stromal TILs in the marginal area reflected overall survival more accurately than that in the central area. Additionally, marginal low TILs was significantly associated with lymph node metastasis and portal vein invasion. Both overall- and disease-free survival rates in the marginal low TILs group were significantly worse than those in the marginal high TILs group (P < 0.05). In the multivariate analysis, marginal low TILs were an independent prognostic factor for both overall- and disease-free survival (P < 0.05), and marginal low TILs were significantly associated with lower minimum ADC values (P < 0.02). Stromal TILs, especially in the marginal area, might demonstrate prognostic impact in patients with IHCC. Moreover, the ADC values from MRI may predict TILs in IHCC tumor tissue.

Abstract 2828: The prognostically significant immunological indices for patients who underwent curative resection of pulmonary metastases of colorectal cancer

Abstract Background: In recent years, it has been revealed that the immunological state of the host affects with the effect of cancer treatments. The neutrophil-to-lymphocyte ratio (NLR) as a systemic inflammatory marker and the density of tumor-infiltrating lymphocytes (TILs) at cancer microenvironment have been reported to be useful prognostic indices in various cancers, including colorectal cancer (CRC). The aim of this study is to evaluate the prognostic significance of immunological indices in patients who underwent curative resection of pulmonary metastases of CRC. Patients and Methods: The medical records of 17 patients who underwent potentially curative resection for pulmonary metastases of CRC were retrospectively reviewed. The correlation between the relapse-free survival (RFS) or the overall survival (OS) after curative resection of pulmonary metastases of CRC and the clinicopathological factors, NLR or TILs were evaluated. The NLR was calculated from the blood sample which was obtained within 2 weeks before pulmonary resection by dividing the absolute neutrophil count by the absolute lymphocyte count. According to the receiver operating characteristic (ROC) curve analysis, patients with an NLR of ≥ 2.5 were classified as high-NLR group. The density of TILs was examined by immunohistochemistry of pulmonary metastatic tumor sample, by staining anti-CD3 and anti-CD8 antibody. The number of CD3+TILs and CD8+TILs at the border of tumor was counted in a randomly selected field at a magnification of ×400. The mean of the values obtained in five different areas was used. According to the ROC curve analysis, the patients with the number of CD3+TILs ≥ 31 were classified as high- CD3+TILs group and patients with the number of CD8+TILs ≥ 21 were classified as high- CD8+TILs group. Results: The RFS and OS after resection of pulmonary metastases were significantly worse in the high-NLR group than in the low-NLR group (p=0.001, p=0.003, respectively). The RFS after resection of pulmonary metastases was significantly better in the high-CD3+TILs group than in the low-CD3+TILs group (p=0.041). Multivariate analysis revealed that the number of pulmonary metastases and the NLR before resection of pulmonary metastases were independent prognostic factors of the RFS (p=0.007, p=0.002, respectively) and the NLR before resection of pulmonary metastasis was the independent prognostic factor of the OS (p=0.009). Conclusion: The immunological indices such as the NLR and the density of TILs, may have prognostic significance in patients who underwent curative resection of pulmonary metastases of CRC. Citation Format: Yuki Okazaki, Masatsune Shibutani, En Wang, Hisashi Nagahara, Tatsunari Fukuoka, Yasuhito Iseki, Shinichiro Kashiwagi, Hiroaki Tanaka, Kiyoshi Maeda, Kosei Hirakawa, Masaichi Ohira. The prognostically significant immunological indices for patients who underwent curative resection of pulmonary metastases of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2828.

Programmed death ligand 1 (PD-L1) in colon cancer and its interaction with budding and tumor-infiltrating lymphocytes (TILs) as tumor-host antagonists

PurposeTo analyze the role of programmed death ligand 1 (PD-L1) immunohistochemisty in the context of tumor microenvironment in colon cancer (CC) with focus on the interaction between tumor budding and tumor-infiltrating lymphocytes (TILs) and to elucidate its potential value for immunooncologic treatment decisions.MethodsThree hundred forty seven patients with CC, stages I to IV, were enrolled. PD-L1 immunohistochemistry was performed using two different antibodies (clone 22C3 pharmDx, Agilent and clone QR1, Quartett). Tumor proportion score (TPS) as well as immune cell score (IC) was assessed. Budding and TILs were assessed according to the criteria of the International Tumor Budding Consensus Conference (ITBCC) and International TILs Working Group (ITWG). Correlation analyses as well as survival analyses were performed.ResultsPD-L1 positivity significantly correlated with TILs > 5% and MMR deficiency, and PD-L1-positive cases (overall and IC) showed significantly longer overall survival (OS) with both antibodies.The parameters “high grade,” “right-sidedness,” and “TILS > 5% regardless of MMR status” evolved as potential parameters for additional immunological treatment decisions. Additionally, TPS positivity correlated with low budding. More PD-L1-positive cases were seen in both high TIL groups. The low budding/high TIL group showed longer disease-free survival and longer OS in PD-L1-positive cases.ConclusionOverall, PD-L1 positivity correlated with markers of good prognosis. PD-L1 immunohistochemistry was able to identify parameters as additional potential candidates for immune therapy. Furthermore, it was able to stratify patients within the low budding/high TIL group with significant prognostic impact.