Stromal Tumor-infiltrating Lymphocytes Density Research Articles

HER2-low and tumor infiltrating lymphocytes in triple-negative breast cancer: Are they connected?

Most patients with triple-negative breast cancer (TNBC) are not candidates for targeted therapy, leaving chemotherapy as the primary treatment option. Recently, immunotherapy has demonstrated promising results in TNBC, due to its immunogenicity. In addition, a novel antibody–drug conjugate, namely, trastuzumab-deruxtecan, has shown effectiveness in TNBC patients with low-HER2 expression (HER2-low). These novel treatment options raise the question about the potential association between the density of stromal tumor-infiltrating lymphocytes (sTILs) and the level of HER2 expression. We aimed to evaluate the association between the level of HER2 expression (HER2-low versus HER2-0) and density of sTILs in TNBC patients, and how they impact the response to neoadjuvant chemotherapy (NAC). This was a retrospective multicenter study including all TNBC patients diagnosed between 2018 and 2022. Central pathology review included sTILs percentages and level of HER2 expression. Tumors were reclassified as either HER2-0 (HER2 IHC 0) or HER2-low (IHC 1 + or 2 + with negative reflex test). Various clinicopathologic characteristics, including sTILs density, and response to NAC were compared between HER2-0 and HER2-low cases. In total, 753 TNBC patients were included in this study, of which 292 patients received NAC. Interobserver agreement between the original pathology report and central review was moderate (77% had the same IHC status after reclassification in either HER2-0 or HER2-low; k = 0.45). HER2-low TNBC represented about one third (36%) of the tumors. No significant difference in sTILs density or complete pathologic response rate was found between HER2-0 and HER2-low cases (p = 0.476 and p = 0.339, respectively). The density of sTILs (≥ 10% sTILs vs. < 10%) was independently associated with achieving a pCR (p = 0.011). In conclusion, no significant association was found between HER2-low status and density of sTILs nor response to NAC. Nonetheless, sTILs could be an independent biomarker for predicting NAC response in TNBC patients.

Initial interactions with the FDA on developing a validation dataset as a medical device development tool.

Quantifying tumor-infiltrating lymphocytes (TILs) in breast cancer tumors is a challenging task for pathologists. With the advent of whole slide imaging that digitizes glass slides, it is possible to apply computational models to quantify TILs for pathologists. Development of computational models requires significant time, expertise, consensus, and investment. To reduce this burden, we are preparing a dataset for developers to validate their models and a proposal to the Medical Device Development Tool (MDDT) program in the Center for Devices and Radiological Health of the U.S. Food and Drug Administration (FDA). If the FDA qualifies the dataset for its submitted context of use, model developers can use it in a regulatory submission within the qualified context of use without additional documentation. Our dataset aims at reducing the regulatory burden placed on developers of models that estimate the density of TILs and will allow head-to-head comparison of multiple computational models on the same data. In this paper, we discuss the MDDT preparation and submission process, including the feedback we received from our initial interactions with the FDA and propose how a qualified MDDT validation dataset could be a mechanism for open, fair, and consistent measures of computational model performance. Our experiences will help the community understand what the FDA considers relevant and appropriate (from the perspective of the submitter), at the early stages of the MDDT submission process, for validating stromal TIL density estimation models and other potential computational models. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Programmed death-ligand 1 and tumor-infiltrating lymphocytes (TILs) - low TIL density may predict poorer long-term prognosis in T1 laryngeal cancer.

We evaluated the prognostic role of programmed death-ligand 1 (PD-L1) and tumor-infiltrating lymphocytes (TILs) in T1 glottic laryngeal squamous cell carcinoma (LSCC). T1 glottic LSCC patients (n = 174) treated at five Finnish university hospitals between 2003 and 2013 were included. Tissue microarray (TMA) blocks were used for PD-L1 immunohistochemistry. TILs were scored from intratumoral and stromal regions in whole tissue sections. Of 174 patients, 92 (53%) had negative, 66 (38%) intermediate, and 16 (9%) high PD-L1 levels. Of 80 patients whose TILs were analyzed, 50 (63%) had low and 30 (38%) high stromal TIL density. Patients with a local recurrence or a new primary tumor of the larynx had lower TIL density than had other patients (p = 0.047). High PD-L1 expression with low stromal TIL density was associated with inferior 5-year disease-specific survival (85% vs. 100%, p = 0.02). In conclusion, in patients treated for T1 glottic LSCC, low stromal TIL density was associated with local recurrences and new primary tumors of the larynx. High PD-L1 expression with low stromal TIL density may be associated with worse survival in T1 glottic LSCC.

Tumor-infiltrating lymphocyte enrichment predicted by CT radiomics analysis is associated with clinical outcomes of non-small cell lung cancer patients receiving immune checkpoint inhibitors.

Enrichment of tumor-infiltrating lymphocytes (TIL) in the tumor microenvironment (TME) is a reliable biomarker of immune checkpoint inhibitors (ICI) in non-small cell lung cancer (NSCLC). Phenotyping through computed tomography (CT) radiomics has the overcome the limitations of tissue-based assessment, including for TIL analysis. Here, we assess TIL enrichment objectively using an artificial intelligence-powered TIL analysis in hematoxylin and eosin (H&E) image and analyze its association with quantitative radiomic features (RFs). Clinical significance of the selected RFs is then validated in the independent NSCLC patients who received ICI. In the training cohort containing both tumor tissue samples and corresponding CT images obtained within 1 month, we extracted 86 RFs from the CT images. The TIL enrichment score (TILes) was defined as the fraction of tissue area with high intra-tumoral or stromal TIL density divided by the whole TME area, as measured on an H&E slide. From the corresponding CT images, the least absolute shrinkage and selection operator model was then developed using features that were significantly associated with TIL enrichment. The CT model was applied to CT images from the validation cohort, which included NSCLC patients who received ICI monotherapy. A total of 220 NSCLC samples were included in the training cohort. After filtering the RFs, two features, gray level variance (coefficient 1.71 x 10-3) and large area low gray level emphasis (coefficient -2.48 x 10-5), were included in the model. The two features were both computed from the size-zone matrix, which has strength in reflecting intralesional texture heterogeneity. In the validation cohort, the patients with high predicted TILes (≥ median) had significantly prolonged progression-free survival compared to those with low predicted TILes (median 4.0 months [95% CI 2.2-5.7] versus 2.1 months [95% CI 1.6-3.1], p = 0.002). Patients who experienced a response to ICI or stable disease with ICI had higher predicted TILes compared with the patients who experienced progressive disease as the best response (p = 0.001, p = 0.036, respectively). Predicted TILes was significantly associated with progression-free survival independent of PD-L1 status. In this CT radiomics model, predicted TILes was significantly associated with ICI outcomes in NSCLC patients. Analyzing TME through radiomics may overcome the limitations of tissue-based analysis and assist clinical decisions regarding ICI.

Prognostic values of stromal tumor-infiltrating lymphocytes and lymphocyte-activation gene-3 in advanced gastric adenocarcinoma

Objective: To analyze density of stromal tumor-infiltrating lymphocytes (sTIL) and expression of lymphocyte-activation gene-3 (LAG-3) protein in advanced gastric adenocarcinomas, and to investigate the correlation of sTIL and LAG-3 with the prognosis in patients with advanced gastric adenocarcinoma. Methods: The clinicopathological characteristics and follow-up data of 260 patients with advanced gastric adenocarcinoma were collected at Fujian Cancer Hospital, from January 2011 to December 2014. The percentage of sTILs was reported semi-quantitatively using histological section evaluation, the LAG-3 protein was detected using immunohistochemistry, and the expression was correlated with the clinicopathological features and patient outcomes. Results: Among the 260 cases, high density of sTIL was detected in 173 cases (66.5%) while LAG-3 high expression was observed in 160 cases (61.5%). These cases were divided into four groups. Group Ⅰ: 48 cases (18.5%) were sTIL low/LAG-3 low; group Ⅱ: 52 cases (20.0%) were sTIL high/LAG-3 low; group Ⅲ: 39 cases (15.0%) were sTIL low/LAG-3 high; group Ⅳ: 121 cases (46.5%) were sTIL high/LAG-3 high. Kaplan-Meier survival analyses showed that patient prognoses were related to age, tumor size, tumor location, Lauren classification, perineural invasion, vascular invasion, TNM staging, postoperative adjuvant chemotherapy and molecular classification (P<0.05). Meanwhile, higher densities of sTIL and higher expression of LAG-3 were associated with better prognosis. Multivariate survival analysis showed age, tumor size, Lauren classification and postoperative adjuvant chemotherapy were independent prognostic factors for patient survival. The results showed a poor prognosis in low-sTIL/low-LAG-3 patients. Conclusions: Compared with low density of sTIL and low expression of LAG-3, high density of sTIL and high expression of LAG-3 are associated with better outcomes in patients with advanced gastric adenocarcinoma, respectively. Combined detecton of sTIL and LAG-3 may be more useful in gastric cancer than using either alone. Age, tumor size, Lauren classification and postoperative adjuvant chemotherapy are independent prognostic factors for patients with advanced gastric adenocarcinoma.

Artificial intelligence-powered whole-slide image analyzer reveals a distinctive distribution of tumor-infiltrating lymphocytes in neuroendocrine tumors and carcinomas.

e16214 Background: Immune checkpoint inhibitors (ICIs) have shown promising treatment outcomes for various types of tumors. However, in neuroendocrine tumors and carcinomas (NET/NEC), ICI has proven to be applicable for only limited cases. In addition, little is known about the immunoprofile of NET/NEC. Here we investigate the landscape of tumor-infiltrating lymphocytes (TIL) using artificial intelligence (AI)-powered H&amp;E whole-slide image (WSI) analyzer to elucidate the tumor microenvironment of NET/NEC. Methods: A total of 240 H&amp;E stained pathologic slides diagnosed with NET/NEC were obtained from Ajou University Medical Center in Korea (from January 2020 to December 2021). For spatial TIL analysis, we used Lunit SCOPE IO, an AI-powered H&amp;E WSI analyzer, which identifies and quantifies TIL within the cancer or stroma area. The AI was developed with a 13.5 x 109 μm2 area and 6.2 x 106 TIL from 17,849 H&amp;E WSI of multiple cancer types, annotated by 104 board-certified pathologists. Intra-tumoral TIL, stromal TIL, and combined (cancer + stroma) TIL density were defined as the TIL count divided by the area of interest respectively. NET with histological grade 1 and 2 were labeled as low grade and NET with histological grade 3 and together with NEC were labeled as high grade. Primary origins of the NET/NEC were grouped by colorectum, stomach, small intestine, hepatopancreatobiliary, lung, and other organs (including anus, appendix, breast, cervix, and larynx). Results: Total slides classified as low grade and high grade were 211 and 29, respectively; 175 samples were from colorectal, 19 from stomach, 16 from small intestine, 16 from hepatopancreaticobiliary, seven from lung, and seven from other organs. The median intra-tumoral TIL, stromal TIL, and combined TIL density were 4.2/mm2 (IQR 1.718 - 11.478), 139.1/mm2 (IQR 75.4 - 313.9), and 62.4/mm2 (IQR 36.3 - 162.6), respectively. The median intra-tumoral TIL density was significantly higher in patients with high grade NET/NEC compared with low grade (11.9/mm2 [IQR 4.51 - 30.9] vs 3.45/mm2 [IQR 1.63 - 9.81], p &lt; 0.001). However, statistical differences in stromal TIL density and combined TIL density were not observed between low grade and high grade NET/NEC. The highest intra-tumoral TIL density in the group classified according to primary origins was lung (n = 7, median: 16.5/mm2, IQR 5.01 - 34.1) and was followed by stomach (n = 19, median: 11.8/mm2, IQR 8.64 - 20.8), and small intestine (n = 16, median: 7.23/mm2, IQR 4.12 - 25.2). Conclusions: AI-powered TIL analysis reveals that the intra-tumoral TIL density is significantly higher in high grade NET/NEC than low grade NET. Our findings align with recent evidence that ICIs are effective against large cell NEC and small cell carcinoma.Therefore, AI-powered TIL analysis should be investigated as a predictive biomarker for ICI response in NET/NEC.

Tumor-infiltrating lymphocyte enrichment predicted by CT radiomic analysis is associated with clinical outcomes of immune checkpoint inhibitor in non–small cell lung cancer.

2663 Background: Enrichment of tumor-infiltrating lymphocytes (TIL) in the tumor microenvironment (TME) is a reliable biomarker of immune checkpoint inhibitors (ICI) in non-small cell lung cancer (NSCLC). Phenotyping through CT radiomics has the overcome the limitations of tissue-based assessment, including for TIL analysis. Here, we objectively assess TIL enrichment using an artificial intelligence-powered H&amp;E analyzer, Lunit SCOPE IO, and analyze its association with advanced quantitative imaging features extracted via radiomic analysis. Clinical significance of the selected radiomic features (RFs) is then validated in independent NSCLC patients who received ICI. Methods: In the training cohort, which included 235 NSCLC patients with both tumor tissue and corresponding CT images obtained within 1 month, we extracted 86 RFs from the CT images. From tissue, a patient’s TIL enrichment score (TILes) was defined as the fraction of tissue area with high intra-tumoral or stromal TIL density, divided by the whole TME area, as measured on an H&amp;E slide. From the corresponding CT images, the least absolute shrinkage and selection operator model was then developed using features that were significantly associated with TIL enrichment. The CT model was then applied to CT images from the validation cohort, which included 242 NSCLC patients who received ICI as ≥ second line. Results: Among the extracted RFs, 22 features were significantly associated with TILes (p &lt; 0.005). After excluding features of multicollinearity and/or zero-coefficient, two features, gray level variance (coefficient 1.71 x 10-3) and low gray level emphasis (coefficient -2.48 x 10-5), were finally included in the model. The two features were both computed from the size-zone matrix (SZM), the idea of which is to break down a given tumor volume into smaller spatially contiguous compartments of different sizes. In the validation cohort, the patients with high predicted TILes (≥ median) had significantly prolonged progression-free survival compared with those with low predicted TILes (median 3.81 months [95% CI 2.14 – 5.69] versus 1.94 months [95% CI 1.58 – 2.93], hazard ratio 0.69 [95% CI 0.53 – 0.90], p = 0.007). Conclusions: This CT radiomics model is able to assess TIL enrichment in TME, which is significantly associated with favorable ICI outcomes in NSCLC. Analyzing the TME through radiomics may overcome limitations of tissue-based analysis and inform clinical decisions, particularly related to use of ICI.

The prognostic value of stromal tumor-infiltrating lymphocytes in intrahepatic cholangiocarcinoma: a population-based study

Objective We assess the predictive value impacted by tumor-infiltrating lymphocytes (TILs) for overall survival (OS) and progression-free survival (PFS) in patients with intrahepatic cholangiocarcinoma (ICC) undergoing complete resection. Methods Sixty-eight patients with resectable ICC were included in this study. We studied stromal TIL density and scored it by staining sections from surgically resected ICC patients with hematoxylin and eosin (HE). The clinical data and prognosis of patients with ICC were obtained by searching clinical and follow-up records. Results A stromal TIL negative status was a predictor of poor OS (HR = 0.41, 95% CI 0.20–0.83, p = .01) and poor PFS (HR = 0.47, 95% CI 0.23–0.97, p = .04) independently. Low stromal TIL density was associated with high levels of CA125 (p = .03) and CA19-9 (p < .01). The high level of CA19-9 (p = .05), high differentiation (p = .02), a large diameter (p = .05), a positive bile duct/vascular cancer embolus (p = .03) and positive satellite nodules (p = .02) were tendencies to develop tumors for patients with a negative status of stromal TIL. Conclusion Our data prompt for the prediction of the PFS and OS of patients with ICC after complete resection, stromal TILs play an important role.

Impact of stromal tumor-infiltrating lymphocytes (sTILs) on response to neoadjuvant chemotherapy in triple-negative early breast cancer in the WSG-ADAPT TN trial

BackgroundHigher density of stromal tumor-infiltrating lymphocytes (sTILs) at baseline has been associated with increased rates of pathological complete response (pCR) after neoadjuvant chemotherapy (NACT) in triple-negative breast cancer (TNBC). While evidence supports favorable association of pCR with survival in TNBC, an independent impact of sTILs (after adjustment for pCR) on survival is not yet established. Moreover, the impact of sTIL dynamics during NACT on pCR and survival in TNBC is unknown.MethodsThe randomized WSG-ADAPT TN phase II trial compared efficacy of 12-week nab-paclitaxel with gemcitabine versus carboplatin. This preplanned translational analysis assessed impacts of sTIL measurements at baseline (sTIL-0) and after 3 weeks of chemotherapy (sTIL-3) on pCR and invasive disease-free survival (iDFS). Predictive performance of sTIL-0 and sTIL-3 for pCR was quantified by ROC analysis and logistic regression; Kaplan–Meier estimation and Cox regression (with mediation analysis) were used to determine their impact on iDFS.ResultsFor prediction of pCR, the AUC statistics for sTIL-0 and sTIL-3 were 0.60 and 0.63, respectively, in all patients; AUC for sTIL-3 was higher in NP/G. The positive predictive value (PPV) of “lymphocyte-predominant” status (sTIL-0 ≥ 60%) at baseline was 59.3%, though only 13.0% of patients had this status. To predict non-pCR, the cut point sTIL-0 ≤ 10% yielded PPV = 69.5% while addressing 33.8% of patients.Higher sTIL levels (particularly at 3 weeks) were independently and favorably associated with better iDFS, even after adjusting for pCR. For example, the adjusted hazard ratio for 3-week sTILs ≥ 60% (vs. < 60%) was 0.48 [0.23–0.99]. Low cellularity in 3-week biopsies was the strongest individual predictor for pCR (in both therapy arms), but not for iDFS.ConclusionThe independent impact of sTILs on iDFS suggests that favorable immune response can influence key tumor biological processes for long-term survival. The results suggest that the reliability of pCR following neoadjuvant therapy as a surrogate for survival could vary among subgroups in TNBC defined by immune response or other factors. Dynamic measurements of sTILs under NACT could support immune response-guided patient selection for individualized therapy approaches for both very low levels (more effective therapies) and very high levels (de-escalation concepts).Trial registration: Clinical trials No: NCT01815242, retrospectively registered January 25, 2013.

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

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

A Pathologist-Annotated Dataset for Validating Artificial Intelligence: A Project Description and Pilot Study

Purpose: Validating artificial intelligence algorithms for clinical use in medical images is a challenging endeavor due to a lack of standard reference data (ground truth). This topic typically occupies a small portion of the discussion in research papers since most of the efforts are focused on developing novel algorithms. In this work, we present a collaboration to create a validation dataset of pathologist annotations for algorithms that process whole slide images. We focus on data collection and evaluation of algorithm performance in the context of estimating the density of stromal tumor-infiltrating lymphocytes (sTILs) in breast cancer. Methods: We digitized 64 glass slides of hematoxylin- and eosin-stained invasive ductal carcinoma core biopsies prepared at a single clinical site. A collaborating pathologist selected 10 regions of interest (ROIs) per slide for evaluation. We created training materials and workflows to crowdsource pathologist image annotations on two modes: an optical microscope and two digital platforms. The microscope platform allows the same ROIs to be evaluated in both modes. The workflows collect the ROI type, a decision on whether the ROI is appropriate for estimating the density of sTILs, and if appropriate, the sTIL density value for that ROI. Results: In total, 19 pathologists made 1645 ROI evaluations during a data collection event and the following 2 weeks. The pilot study yielded an abundant number of cases with nominal sTIL infiltration. Furthermore, we found that the sTIL densities are correlated within a case, and there is notable pathologist variability. Consequently, we outline plans to improve our ROI and case sampling methods. We also outline statistical methods to account for ROI correlations within a case and pathologist variability when validating an algorithm. Conclusion: We have built workflows for efficient data collection and tested them in a pilot study. As we prepare for pivotal studies, we will investigate methods to use the dataset as an external validation tool for algorithms. We will also consider what it will take for the dataset to be fit for a regulatory purpose: study size, patient population, and pathologist training and qualifications. To this end, we will elicit feedback from the Food and Drug Administration via the Medical Device Development Tool program and from the broader digital pathology and AI community. Ultimately, we intend to share the dataset, statistical methods, and lessons learned.

The Prognostic Value of Stromal Tumor-Infiltrating Lymphocytes in Intrahepatic Cholangiocarcinoma

Objective: We conducted this study to assess the predictive value of tumor-infiltrating lymphocytes (TILs) for progression-free survival (PFS) and overall survival (OS) in intrahepatic cholangiocarcinoma (ICC) patients after complete resection. Methods: Sixty-eight patients with resectable ICC were included in this study. We studied stromal TIL density and scored it by staining sections from surgically resected ICC patients with hematoxylin and eosin (HE). The clinical data and prognosis of ICC patients were obtained by searching clinical and follow-up records. Results: A stromal TIL negative status was an independent predictor of poor OS (HR=2.84, 95% CI 1.48- 5.46, P&lt;0.01) and poor PFS (HR=2.88, 95% CI 1.44-5.76, P&lt;0.01). Low stromal TIL density was associated with high CA125 (P=0.03) and CA19-9 (P&lt;0.01) levels. Patients with a stromal TIL negative status tended to develop tumors with a high level of CA19-9 (P=0.05), high differentiation (P=0.02), a large diameter (P=0.05), a positive bile duct/vascular cancer embolus (P=0.03) and positive satellite nodules (P=0.02). Conclusion: Our data suggest that stromal TILs play an important role in predicting the PFS and OS of ICC patients after complete resection.

B7-H3 and B7-H4 Expression in Breast Cancer and Their Association with Clinicopathological Variables and T Cell Infiltration

Objectives: B7-H3 and B7-H4 proteins are expressed in breast cancer tissues, but their relationships with respect to tumor immune surveillance and outcomes in breast cancer are not conclusive. Methods: We first examined B7-H3 and B7-H4 mRNA expression in the Genotype-Tissue Expression (GTEx) and The Cancer Genome Atlas (TCGA) datasets. Next, mRNA and protein expression were assessed by RNAscope in situ hybridization (ISH) and immunohistochemistry in 10 pairs of breast cancer and matched normal tissues. Immunohistochemical staining of B7-H3, B7-H4, CD3, and CD8 was performed in tissue microarray slides containing 198 breast cancer samples. Association of B7-H3 and B7-H4 expression with survival was verified using the publicly accessible BreastMark tool. Results: B7-H3 and B7-H4 mRNA expression were significantly higher in breast cancer samples in the TCGA dataset than in normal breast tissues in the GTEx dataset. RNAscope ISH and immunohistochemistry showed that B7-H3 and B7-H4 mRNA and protein appeared to be mainly expressed in cancer cells. Expression of B7-H3 and B7-H4 tended to be associated with low-density scores of stromal tumor-infiltrating lymphocytes (TILs) as well as molecular subtypes. Expressions of B7-H3 and B7-H4 were negatively correlated with stromal CD3+ and CD8+ T cell infiltration density. B7-H3 and B7-H4 expression was not associated with survival, which was verified by BreastMark analysis. Conclusion: Expression levels of B7-H3 and B7-H4 were independent of clinical outcomes of breast cancer. There was an inverse relationship between the expression of B7-H3 and B7-H4 in breast cancer and the density of stromal TILs and CD8+ T lymphocytes. This inverse relationship may represent a promising target in the field of breast cancer immunotherapy.

Assessment of Tumor-infiltrating Lymphocytes Using International TILs Working Group (ITWG) System Is a Strong Predictor of Overall Survival in Colorectal Carcinoma: A Study of 1034 Patients.

The presence of increased tumor-infiltrating lymphocytes (TILs) is established as a positive prognostic factor in many malignancies including colorectal carcinoma (CRC). However, multiple different approaches have been used to assess TILs. In 2014, the International TILs Working Group (ITWG) proposed a standardized methodology for evaluating TILs, initially in the context of breast cancer, but subsequently expanded to other malignancies. To date, the efficacy of the ITWG system has not been investigated in a large cohort of all-stage CRC. We, therefore, sought to validate this system in CRC. We used the ITWG system to assess the density of stromal TILs in an unselected cohort of 1034 CRC patients undergoing primary tumor resection at our institution. The percentage TILs' score was categorized into 3 groups: low (0% to 10%), intermediate (15% to 50%), and high (55% to 100%). The mean survival was 53, 67, and 75 months, respectively (P=0.0001). This survival benefit remained statistically significant in multivariate analyses (P=0.0001) and subgroup analyses of mismatch repair-proficient CRCs (P=0.0001), mismatch repair-deficient CRCs (P=0.031), BRAFV600E-mutant CRCs (P=0.0001), and BRAF wild-type CRCs (P=0.001). The predictive value of TILs assessed using the ITWG system was superior to the assessment of intraepithelial lymphocyte performed prospectively using a standard system requiring ≥5 lymphocytes per high-powered field in direct contact with tumor cells or between tumor clusters. We conclude that the ITWG system for assessing TILs is a powerful predictor of all-cause survival in CRC independent of many prognostic factors and superior to the assessment of intraepithelial lymphocytes using a traditional system.

Prognostic Significance of Stromal and Intraepithelial Tumor-Infiltrating Lymphocytes in Small Intestinal Adenocarcinoma.

Assessment of tumor-infiltrating lymphocytes (TILs) may predict the prognosis and therapeutic benefit of immunotherapy in small intestinal adenocarcinoma (SIAC) patients. TILs were evaluated in 231 surgically resected SIACs and compared with microsatellite instability (MSI) and clinicopathologic variables. The average number of intraepithelial TILs (iTILs) and the average density of stromal TILs (sTILs) were calculated separately. High iTIL count (≥2 per high-power field) was associated with MSI-high, whereas high sTIL density (≥20% on ×200 magnification) was not. High iTIL count and high sTIL density were related to distal tumor location, medullary carcinoma, high Crohn-like lymphoid reaction counts, and fewer pancreatic invasions. SIAC patients with high iTIL count or high sTIL density had better survival than those with low values. On multivariate analysis, MSI, high sTIL density, proximal locations, lower N category, and absence of lymphovascular invasions and retroperitoneal seeding were the best independent prognostic predictors. High sTIL density can be used as a prognostic indicator and high iTIL count may provide a basis for the clinical use of targeted immunotherapy in SIAC patients.

Diffuse distribution of tumor-infiltrating lymphocytes is a marker for better prognosis and chemotherapeutic effect in triple-negative breast cancer.

High-density tumor-infiltrating lymphocytes (TILs) are a prognostic marker for triple-negative breast cancer (TNBC). However, lymphocytic infiltration is heterogeneous in its pattern. We aimed to explore the utility of TIL distribution patterns against TIL density for predicting TNBC prognosis and chemotherapeutic effects. Primary invasive TNBC cases were retrieved from a single institutional cohort, and archived samples were reviewed by two board-certificated pathologists. We used 154 consecutive surgical specimens from patients with standard adjuvant therapy, and 80 biopsies taken before primary systemic chemotherapy. The average density of stromal TILs was scored at 10% intervals, while the distribution pattern of TILs was evaluated as diffuse or non-diffuse. The association between TILs and prognosis or pathological complete response (pCR) was statistically analyzed. A diffuse pattern of TILs at primary surgery correlated with better prognosis (relapse-free survival [RFS], hazard ratio [HR] 3.71, 95% confidence interval [CI] 1.60-8.57; overall survival [OS], HR 3.87, 95% CI 1.46-10.27), as well as high TIL density (≥ 50%; RFS, HR 4.51, 95% CI 2.06-9.90; OS, HR 3.28, 95% CI 1.32-8.14). Diffuse TIL pattern and nodal status were independent prognostic factors in multivariate analysis. Diffuse TIL pattern upon biopsy was associated with higher pCR rate (diffuse, 46%; non-diffuse, 21%; P = 0.032). All high TIL cases had diffuse patterns and the best outcome. Interobserver concordance was moderate (k = 0.53-0.55; distribution pattern) to good (weighted k = 0.67-0.69; density), and it was faster to assess the distribution pattern than to assess the density of TIL. Showing similar clinical impacts to the TIL density, diffuse TILs could be a predictive marker for better prognosis and higher pCR. The assessment of TIL distribution pattern is simple, faster, and practical. Heterogeneous tumor immunity may contribute to further stratification of TNBC treatment.

Abstract P4-08-13: Prognostic significance of CD8+ tumor-infiltrating lymphocytes (TILs) in patients with early breast cancer (EBC) treated with dose-dense sequential adjuvant chemotherapy (dds-CT). An observational study (ACTRN12616001043426)

Abstract Background - aim: Information on the prognostic role of cytotoxic CD8+ T cells in the era of modern adjuvant CT is limited. The primary objective of the present report is to assess the prognostic impact of CD8+ cells in patients with intermediate or high-risk EBC (T1-3N1-2M0) treated with dds-CT. Secondary endpoints are safety, disease-free survival (DFS) and overall survival (OS). Patients and Methods: Patients (N=1,000) were treated with 4 cycles of Epirubicin, 75mg/m2, and Cyclophophamide, 600mg/m2 every 2 weeks followed by 4 cycles of Docetaxel (D), 100mg/m2 every 3 weeks with G-CSF support in all cycles. Trastuzumab was initiated concurrently with D and continued for a total of 1 year. Hormonal and radiation therapy were given post CT, as indicated. Formalin-fixed paraffin-embedded tumors were available for 642 patients (64.2%) and were centrally assessed for immunohistochemical subtypes (IHC4; N=526), stromal TILs density by morphology (N=636), as well as stromal and intratumoral cytotoxic CD8+ T cell numbers (N=554). TILs and CD8+ were assessed as continuous variables for associations and as 10% increments for outcome. Results: In total, 901/1,000 pts (90.1%) completed 8 cycles of CT. Severe (gradeIII-IV) toxicitiesincludedneutropenia (5.6%), leucopenia (3.6%), lymphopenia (2.1%), hand-footsyndrome (2.1%), and hepatotoxicity (1.8%). Febrileneutropenia occurred in 1.6% of the patients. The 5-year DFS and OS rates were 89.5% and 93.1%, respectively. Luminal A tumors were classified in 26.2%, Luminal B in 35.2%, luminal HER2 in 9.5%; HER2-enriched in 7.2%; and, triple-negative (TNBC) in 21.9% of informative patients. Among subtypes, stromal TILs density was higher in HER2-enriched and TNBC (p&amp;lt;0.001); intratumoral CD8+ values were higher in TNBC (p&amp;lt;0.001); and, stromal CD8+ were higher in HER2-enriched (p=0.034). In all patients, TILs density and intratumoral CD8+ cell numbers were not associated with DFS and OS, while increased stromal CD8+ were marginally associated with prolonged DFS (HR=0.98, 95%CI 0.96-1.00, p=0.066).Adjusted for histological grade, menopausal, ER/PgR and nodal status, higher stromal CD8+ were associated with prolonged DFS (HR=0.98, 95% CI 0.96-1.00, p=0.043). In TNBC, higher stromal TILs density conferred prolonged DFS (HR=0.97, 95%CI 0.94-0.99, p=0.029), which retained its prognostic significance in multivariate analysis (HR=0.97, 95% CI 0.94-1.00, p=0.049). Conclusions: In this study, dds-CT was well tolerated and active in patients with EBC. We confirm the presence of morphologically assessed higher TILs density, and of higher cytotoxic CD8+ T cell numbers in hormone receptor negative EBC, as well as the favorable prognostic impact of higher stromal TILs density in TNBC. In comparison to stromal TILs density, higher stromal CD8+ may confer favorable prognosis irrespectively of EBC subtype. Stromal CD8+ seems to be a marker worth further standardizing for reporting on immune cell infiltrates in EBC. Citation Format: Kourea HP, Koletsa T, Kotoula V, Koliou G-A, Batistatou A, Pentheroudakis G, Arapantoni-Dadioti P, Zagouri F, Bobos M, Sotiropoulou M, Papoudou-Bai A, Chrisafi S, Efstratiou I, Aravantinos G, Nicolaou I, Gogas H, Visvikis A, Christodoulou C, Petraki C, Koutras A, Psyrri A, Pectasides D, Fountzilas G. Prognostic significance of CD8+ tumor-infiltrating lymphocytes (TILs) in patients with early breast cancer (EBC) treated with dose-dense sequential adjuvant chemotherapy (dds-CT). An observational study (ACTRN12616001043426) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-08-13.

Abstract P4-06-25: B7-H3 and B7-H4 expression in ductal carcinoma in situ of the breast: Associations with clinicopathologic features and T-cell infiltration

Abstract Background: With the clinical success of immune check point blockades in treating malignant tumors, there are intense investigations in identifying new pathways to activate immune system by targeting immunoregulatory molecules. B7-H3 and B7-H4 play an inhibitory role in T cell function by limiting proliferation and cytokine production. Although several studies have investigated the expression of B7-H3 and B7-H4 in invasive breast cancers, the information of the B7-H3 and B7-H4 molecules in ductal carcinoma in situ (DCIS) remains uncertain. The present work was undertaken to evaluate the expression of B7-H3 and B7-H4 in DCIS and its association with clinicopathological features in patients with DCIS. In addition, the association of B7-H3 and B7-H4 expression with the T-cell infiltration was also assessed to investigate its roles in the regulation of tumor immune surveillance. Materials and methods: B7-H3 and B7-H4 expression was examined in 8 pairs of DCIS tissues and matched normal adjacent tissues at mRNA and protein levels by RNAscope in situ hybridization (ISH) and immunohistochemistry. Immunohistochemical staining of B7-H3 and B7-H4 was done in 79 DCIS samples with known hormone receptor (HR) and human epidermal growth factor 2 (HER2) expression using tissue microarray. In addition, immunohistochemical staining was also performed for the T cell lineage markers CD3 and CD8 in DCIS. Results: RNAscope ISH and immunohistochemistry of B7-H3 and B7-H4 confirmed their increases in DCIS tissues compared with their corresponding normal tissues. B7-H3 and B7-H4 mRNA and protein expression appeared to be concentrated mainly in the DCIS carcinoma cells. High B7-H3 and B7-H4 expression was observed in 58 (73.4%) and 62 (78.5%) cases with DCIS, respectively. High B7-H3 expression was significantly associated with high nuclear grade and presence of comedo-type necrosis (P &amp;lt; 0.05 and P &amp;lt; 0.05, respectively). B7-H3 expression was higher in the HR–/HER2+ subtypes than in the HR+/HER2– subtype (P &amp;lt; 0.05). B7-H3 and B7-H4 expression tended to be associated with low stromal tumor infiltrating lymphocytes density and was negatively related to the CD3+ and CD8+ T cell infiltrates density. Conclusions: These results suggest that B7-H3 and B7-H4 may play an important role in immune surveillance mechanisms of DCIS and may be useful targets for immune-based therapy to alter or prevent DCIS progression. Citation Format: Lee JS, Kim G-E, Park MH, Yoon JH. B7-H3 and B7-H4 expression in ductal carcinoma in situ of the breast: Associations with clinicopathologic features and T-cell infiltration [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-06-25.

Feasibility of Classification of Triple Negative Breast Cancer by Immunohistochemical Surrogate Markers

IntroductionRecently, Burstein et al identified 4 stable molecular subtypes of triple negative breast cancer (TNBC) by mRNA profiling: luminal androgen receptor (LAR), mesenchymal (MES), basal-like immune-activated (BLIA), and basal-like immune-suppressive (BLIS) types. The purpose of this study was to assess the feasibility of immunohistochemistry (IHC) surrogate panel in classifying the TNBC molecular subtypes using a large cohort of TNBC retrieved from a single institution. Materials and MethodsIHC for androgen receptor [AR], claudin-3, E-cadherin, cytokeratin 5/6 [CK5/6], epidermal growth factor receptor [EGFR], indoleamine 2,3-dioxygenase 1 [IDO1], and Forkhead box C1 [FOXC1] were performed using the tissue microarray constructed from 200 TNBC samples. ResultsThe 200 TNBCs were classified as LAR (AR+, n = 22; 11.0%), MES (claudin 3– and/or E-cadherin–, n = 23; 11.5%), basal-like (CK5/6+ and/or EGFR+, n = 85; 42.5%), mixed (n = 60; 30%), and unclassifiable type (n = 10; 5%). LAR type was associated with older patient age, apocrine histologic features, low density of stromal tumor-infiltrating lymphocytes (TIL), and low Ki-67 labeling index. MES type was associated with tumor cell discohesiveness and metaplastic features. Basal-like type was associated with younger patient age, high histologic grade, high stromal TIL density, and high Ki-67 labeling index. Basal-like TNBCs were further classified as BLIA (IDO1+ and FOXC1−, n = 27) or BLIS type (IDO1- and FOXC1+, n = 11). BLIS type was associated with large tumor size and low stromal TIL density, which had the worst prognostic outcome among 4 subtypes. ConclusionThe IHC surrogate panel may define TNBC subtypes with distinct clinicopathologic characteristics and prognostic significance.

The predictive value and role of stromal tumor-infiltrating lymphocytes in pancreatic ductal adenocarcinoma (PDAC)

ABSTRACTRecently, increasing evidence has indicated that the presence of tumor infiltrating immune cells has shown predictive significance for many solid tumors. Present study was performed to evaluate the predictive value of stromal tumor-infiltrating lymphocytes (TILs) for the presence of liver metastasis and overall survival in PDAC (pancreatic ductal adenocarcinoma) patients after complete resection and to explore the potential role of lymphocytes in PDAC. A total of 155 resectable patients with PDAC were enrolled in our study. Stromal TIL density was investigated in hematoxylin and eosin-stained sections of surgical specimens and scored. The effect and possible mechanism of lymphocytes on cancer cells was evaluated using co-culture techniques and ELISA test. Stromal TIL negative status (HR = 2.80, 95% CI 1.75-4.48, P < 0.01) was not only an independent predictor of worse OS (HR = 2.7, 95% CI 1.80-4.06, P = <0.01) but also a significant independent predictor of liver metastasis. Higher CEA (P = 0.01) or CA19-9 (P = 0.01) levels were associated with low stromal TIL density. Stromal TIL negative patients appeared to develop tumors with a higher CEA (P = 0.01), larger diameter (P = 0.05) and advanced stage (P = 0.02). The co-culture experiment suggests that lymphocytes can inhibit pancreatic cancer cell proliferation. Further ELISA and cell culture test indicate that lymphocytes may cause pancreatic cancer cells apoptosis through TNF-alpha secretion. Our data suggest a potential favorable role of stromal TILs in predicting liver metastasis and overall survival of patients with PDAC after complete resection. Lymphocytes may inhibit the growth of PDAC through TNF-alpha secretion, which suggest a potential therapeutic approach against PDAC.