Density Of Tumor-infiltrating Lymphocytes Research Articles

Systemic Inflammatory Markers Combined with Tumor-Infiltrating Lymphocyte Density for the Improved Prediction of Response to Neoadjuvant Chemoradiotherapy in Rectal Cancer.

Previous studies have reported the utility of systemic inflammatory markers and CD8+ tumor-infiltrating lymphocyte (TIL) separately in predicting response to chemoradiotherapy (CRT) in rectal cancer; however, the efficacy of combining these markers remains unclear. This study aimed to elucidate the predictive efficacy of systemic inflammatory markers combined with CD8+ TIL density on response to neoadjuvant CRT in locally advanced rectal cancer. Ten systemic inflammatory markers and CD8+ TIL density were assessed in 267 patients with rectal cancer using pretreatment clinical data and biopsy samples. Response to CRT was determined using the Dworak tumor regression grade (TRG), with good responders classified as TRG3-4. Receiver operating characteristic curve analysis showed high areas under the curve for the lymphocyte-to-C-reactive protein ratio (LCR) and neutrophil×monocyte (N×M) value (0.58 and 0.62, respectively). In the multivariate analysis, LCR, N×M value, and CD8+ TIL density were independently associated with good responders (p=0.016, 0.005, and 0.002, respectively). Stratified analysis with these three markers showed a positive correlation between TRG3-4 ratio and the number of positive predictive factors (8.2%, 20.0%, 34.2%, and 59.1% in patients with 0, 1, 2, and 3 predictors, respectively). Overall and disease-free survival were significantly worse in patients with zero factors present compared with those with one to three factors present. LCR, N×M value, and CD8+ TIL density are independently associated with response to CRT. Assessing local TIL density along with systemic inflammatory markers may be useful for selecting a multimodal neoadjuvant approach in rectal cancer therapy.

ASO Author Reflections: Combinations of Systemic Inflammatory Markers and CD8+ Tumor-infiltrating Lymphocyte Density: Predictive Utility for Response to Neoadjuvant Chemoradiotherapy in Rectal Cancer.

ASO Author Reflections: Combinations of Systemic Inflammatory Markers and CD8+ Tumor-infiltrating Lymphocyte Density: Predictive Utility for Response to Neoadjuvant Chemoradiotherapy in Rectal Cancer.

Clinical Significance of Tumor Microenvironment in Acral Melanoma: A Large Single-Institution Study of Caucasians.

Background: The presence of tumor-infiltrating lymphocytes (TILs) in many studies is associated with a better prognosis in melanoma patients. Programmed death-ligand 1 (PD-L1) expression has a significant value in predicting several cancers, but its role in melanoma remains ambiguous. The study aims to report a comprehensive analysis of TILs characteristics and their impact on survival in primary acral melanoma (AM). Methods: Clinical and pathological features and survival outcomes were investigated in 70 patients with AM. Immunohistochemical quantitative analysis of TILs, including expression of CD4, CD8, FOXP3, PD-1, and PD-L1, on melanoma cells was performed. Results: Kaplan-Meier analysis showed significant differences in overall survival (OS) for CD4+ (p = 0.021), CD8+ (p = 0.037), FOXP3+ (p = 0.007), and TILs density (p = 0.043). In univariate analysis of immunohistochemical features, FOXP3, CD4, CD8, PD-1, and Melanoma Institute of Australia (MIA) grading TILs (grade, density, and distribution) were correlated with survival. The higher density of FOXP3-positive cells was an independent factor associated with better survival. Conclusions: High TILs content (classed as brisk Clark scale and marked/diffuse TILs MIA grade) regardless of its immunophenotype was associated with better survival outcomes in AM. PD-L1 expression on tumor cells did not influence OS and was independent of clinical and pathological characteristics. We demonstrated that TILs are significant biomarkers in sentinel lymph node status prediction.

POLE and Mismatch Repair Status, Checkpoint Proteins and Tumor-Infiltrating Lymphocytes in Combination, and Tumor Differentiation: Identify Endometrial Cancers for Immunotherapy.

ObjectiveAlthough Polymerase-epsilon (POLE)-mutated and mismatch repair (MMR)-deficient endometrial cancers (ECs) are considered as promising candidates for anti-PD-1/PD-L1 therapy, selecting only these patients may exclude other patients who could potentially respond to this treatment strategy, highlighting the need of additional biomarkers for better patient selection. This study aims to evaluate potential predictive biomarkers for anti-PD-1/PD-L1 therapy in addition to POLE mutation (POLEm) and MMR deficiency (MMRd).MethodsWe performed next generation sequencing for POLE from 202 ECs, and immunohistochemistry for MLH1, MSH2, MSH6, PMS2, CD3, CD8, PD-1 and PD-L1 on full-section slides from these ECs. We assessed the association of POLEm and MMRd with clinicopathologic features, expression of check point proteins, and density of tumor-infiltrating lymphocytes (TILs). Prognostic impact of these immune markers was also evaluated.ResultsPOLEm, MMRd and high-grade tumors exhibited elevated level of TILs. Increased expression of PD-1 and PD-L1 was observed in MMRd and high-grade ECs. A subgroup of MMR proficient ECs also harbored increased density of TILs, and positive expression of PD-1 and PD-L1. In addition, negative expression of checkpoint proteins and high density of TILs in combination was associated with good prognosis.ConclusionsCandidates for PD-1 blockade may extend beyond POLEm and MMRd ECs, additional factors such as tumor grade, and combination of TILs levels and expression of checkpoint proteins may need to be considered for better patient selection.

The density of tumor-infiltrating lymphocytes and prognosis in resectable hepatocellular carcinoma: a two-phase study.

Aim: Previous studies have focused on the subpopulations of tumor-infiltrating lymphocytes (TILs) in tumors. This study focuses only on the concentration of TILs in the tumor irrespective of type and elucidates its prognostic value.Methods: We used 315 HCC patients as the discovery phase and another 343 HCC patients as the validation phase. By following the standardized guideline, density of TILs were categorized into low (TILs < 10%), intermediate (10% ≦ TILs < 50%), and high (TILs ≧ 50%) levels. Associations of TILs with prognostic, immune-related, and genetic variables were examined.Results: We observed a dose-response relation of TILs with overall survival (intermediate: HR, 0.58; 95% confidence interval (CI), 0.36-0.93; high: HR, 0.37; 95% CI, 0.15-0.93) and disease-free survival (intermediate: HR, 0.35; 95% CI, 0.22-0.58; high: HR, 0.23; 95% CI, 0.09-0.58). The prognostic value of TILs was validated in the TCGA set. Mutation burden or the number of neoantigens were not associated with TILs intensity. However, hepatitis B or C virus infection patients had higher TILs intensity in the para-tumor tissue.Conclusions: The TILs intensity was associated with patients' survival. If confirmed, this would suggest that clinical routine assessment of TILs could provide prognostic information in HCC.

From the Editor’s Desk...

From the Editor’s Desk...

LAG-3 expression in the inflammatory microenvironment of glioma

PurposeImmune modulatory therapies including immune checkpoint inhibitors have so far failed to result in clinically meaningful efficacy in glioma. We aimed to investigate lymphocyte activation gene 3 (LAG-3), an inhibitory receptor on immune cells and target of second-generation immune checkpoint inhibitors, in glioma.Methods97 patients with diffuse glioma (68 with glioblastoma, 29 with WHO grade II-III glioma) were identified from the Neuro-Biobank of the Medical University of Vienna. LAG-3 expression in the inflammatory microenvironment was assessed by immunohistochemistry (monoclonal anti-LAG-3 antibody, clone 17B4) and correlated to CD3+ , CD8+ , CD20+ and PD-1+ tumor-infiltrating lymphocytes (TILs) and PD-L1 expression on tumor cells.ResultsLAG-3+ TILs could be observed in 10/97 (10.3%) IDH-wildtype samples and in none of the included IDH-mutant glioma samples (p = 0.057). Further, LAG-3+ TILs were only observed in WHO grade IV glioblastoma, while none of the investigated WHO grade II–III glioma presented with LAG-3+ TILs (p = 0.03). No association of O6-methylguanine-DNA-methyltransferase (MGMT) promoter methylation and presence of LAG-3+ TILs was observed (p = 0.726). LAG-3 expression was associated with the presence of CD3+ (p = 0.029), CD8+ (p = 0.001), PD-1+ (p < 0.001) TILs and PD-L1+ tumor cells (p = 0.021), respectively. No association of overall survival with LAG-3+ TIL infiltration was evident (median OS 9.9 vs. 14.2 months, p = 0.95).ConclusionsLAG-3 is only rarely expressed on TILs in IDH-wildtype glioma and associated with active inflammatory milieu as defined by higher TIL density. Immune microenvironment diversity should be considered in the design of future immunotherapy trials in glioma.

Nomograms to Predict the Density of Tumor-Infiltrating Lymphocytes in Patients With High-Grade Serous Ovarian Cancer.

BackgroundTumor-infiltrating lymphocytes (TILs) have important roles in predicting tumor therapeutic responses and progression, however, the method of evaluating TILs is complicated. We attempted to explore the association of TILs with clinicopathological characteristics and blood indicators, and to develop nomograms to predict the density of TILs in patients with high-grade serous ovarian cancer (HGSOC).MethodsThe clinical profiles of 197 consecutive postoperative HGSOC patients were retrospectively analyzed. Tumor tissues and matched normal fallopian tubes were immunostained for CD3+, CD8+, and CD4+ T cells on corresponding tissue microarrays and the numbers of TILs were counted using the NIH ImageJ software. The patients were classified into low- or high-density groups for each marker (CD3, CD4, CD8). The associations of the investigated TILs to clinicopathological characteristics and blood indicators were assessed and the related predictors for densities of TILs were used to develop nomograms; which were then further evaluated using the C-index, receiver operating characteristic (ROC) curves and calibration plots.ResultsMenopausal status, estrogen receptor (ER), Ki-67 index, white blood cell (WBC), platelets (PLT), lactate dehydrogenase (LDH), and carbohydrate antigen 153 (CA153) had significant association with densities of tumor-infiltrating CD3+, CD8+, or CD4+ T cells. The calibration curves of the CD3+ (C-index = 0.748), CD8+ (C-index = 0.683) and CD4+ TILs nomogram (C-index = 0.759) demonstrated excellent agreement between predictions and actual observations. ROC curves of internal validation indicated good discrimination for the CD8+ TILs nomogram [area under the curve (AUC) = 0.659, 95% CI 0.582–0.736] and encouraging performance for the CD3+ (AUC= 0.708, 95% CI 0.636–0.781) and CD4+ TILs nomogram (AUC = 0.730, 95% CI 0.659–0.801).ConclusionMenopausal status, ER, Ki-67 index, WBC, PLT, LDH, and CA153 could reflect the densities of T cells in the tumor microenvironment. Novel nomograms are conducive to monitor the immune status of patients with HGSOC and help doctors to formulate the appropriate treatment strategies.

Tumor mutation burden and immune microenvironment analysis of urothelial carcinoma.

494 Background: Tumor mutation burden (TMB) has been established as a biomarker for response to immune therapy and prognosis in various cancers. However, the correlation between TMB and immune microenvironment remains unwell studied, especially in urothelial carcinoma. This study was aimed to investigate the relationship between TMB and other immunotherapy related biomarkers, including genetic alterations, APOBEC signature, microsatellite instability (MSI), PD-L1 expression and immune cell infiltration in urothelial carcinoma. Methods: 131 patients with urothelial carcinoma admitted from October 2018 to May 2020 were included. Total DNA was isolated from FFPE or fresh tissues. Mutation profiles, APOBEC signature and MSI scores were obtained by next-generation sequencing based a 642 cancer genes panel assay. PD-L1 expression, CD8+ T-cells and tumor-infiltrating lymphocytes density were evaluated by immunohistochemistry. The correlation was analyzed by Wilcoxon signed-rank test. Results: The mutation landscape showed that TP53 mutation is the most common alterations (n = 64/131, 48.9%), followed by KMT2D alterations (n = 49/131, 37.4%), KDM6A mutations (n = 42/131, 32.1%), MUC17 mutations (n = 42/131, 32.1%). The median TMB was 5.06 Muts/Mb (0-118 Muts/Mb). 2 of 131 patients showed MSI-H, who exhibited a much higher TMB (41, 118 Muts/Mb). Further analysis showed that TMB in the patients with certain gene mutations (such as TP53, KMT2D, KDM6A and MUC17) was significantly higher than those wild type ones (p &lt; 0.05). Meanwhile, the high APOBEC-enrichment group has a higher TMB than the low APOBEC-enrichment group (p = 0.045). Furthermore,we observed that the patients with a higher PD-L1 expression (n = 28/131, 21.4%, at a combined positive score cut-off value of 10) also showed a significantly higher TMB (p = 0.016), and TMB in the patients with higher density of CD8+ T-cells (n = 42/131, 32.1%, at a cut-off value of 5%) was also significantly higher than that of the group with lower density of CD8+ T-cells (p = 0.039). Conclusions: This study provides new insights into the correlation between the TMB and the immune microenvironment in urothelial carcinoma. The result may be a reference to immunotherapy.

Abstract PS4-38: Association of tumor infiltrating lymphocytes (TILs) density and PD-L1 expression with pembrolizumab (P) plus gemcitabine (Gem) efficacy in patients with HER2-negative advanced breast cancer (ABC) from the GEICAM/2015-04 (PANGEA-Breast) study

Abstract Background Immune cells (ICs) infiltration and immune checkpoints have been shown to be important for BC patients’ (pts) prognosis and response to immunotherapy. We aimed to analyze the relation between TILs prevalence and PD-L1 expression with efficacy to an immunostimulatory combination with P and Gem in ABC HER2-negative pts previously treated with ≤4 chemotherapy and/or ≥2 hormone therapy lines from the PANGEA-Breast trial (NCT03025880). Methods Pre-treatment (ttm) metastatic BC samples were assessed for TILs density [% of occupied stromal area upon H&amp;E staining] and for PD-L1 immunohistochemistry expression using monoclonal anti-PD-L1 antibody clone 22C3 (Merck) by calculating ICs score (% of positive infiltrated ICs) and combined positive score [CPS; PD-L1 stained cells (tumor cells, lymphocytes, macrophages) divided by total viable tumor cells, multiplied by 100]. Cut-offs ≥5%, ≥10%, ≥30% were explored for TILs. PD-L1 scores were considered positive if ≥1%. Cut-offs (≥5%, ≥20%, ≥50%) were additionally assessed for PD-L1 as CPS. Logistic regression models were used to evaluate association between TILs density and PD-L1 expression with ttm efficacy in terms of Objective Response Rate [ORR; Complete + Partial Response (CR + PR)], Clinical Benefit Rate [CBR; CR + PR + Stable Disease ≥24 weeks] and Progression Free Survival (PFS), according to RECIST v1.1. Results Thirty-six pts were included, 58% had triple negative BC and 98% ECOG score ≤1. Median number of prior ttm lines was 4. ORR and CBR were 15.2% and 17%, respectively; median PFS was 3.1 months. TILs and PD-L1 were evaluated in 30 and 29 pts, respectively. No association was found between TILs density and ttm efficacy in terms of ORR, CBR and PFS. Analysis of PD-L1 ICs score did not reveal any significant association with ORR, CBR or PFS. However, pts with negative PD-L1 expression by CPS (&amp;lt;1%) had a significantly prolonged PFS [p-value=0.031; HR 0.39 (95%CI 0.16; 0.95)], not maintained at CPS &amp;lt;20% cut-off [p-value=0.062; HR 0.42 (95%CI 0.17; 1.08)]. Conclusions Our findings support that: 1) P plus Gem ttm in heavily pre-treated HER2-negative ABC pts obtains a modest ORR of 15.2%; 2) TILs density and PD-L1 expression in ICs does not predict its benefit; 3) PD-L1 in tumor cells scored as CPS impacts in worse outcome (PFS) but not in ORR, suggesting an eventual prognostic role in this population; 4) no long-term responders were observed with P plus Gem in this trial. Citation Format: Luis De la Cruz, María Gion, Josefina Cruz, Jose Luis Alonso, Vanesa Quiroga, Fernando Moreno, Marta Santisteban, Raquel Andrés, Esther Holgado, Natalia Palazón, Luz Milva Rodríguez, Asunción Soto, Javier Cortés, Alfonso Cortés, Manuel Ramos, Maribel Casas, Massimo Chiesa, Susana Bezares, Rosalía Caballero, Federico Rojo. Association of tumor infiltrating lymphocytes (TILs) density and PD-L1 expression with pembrolizumab (P) plus gemcitabine (Gem) efficacy in patients with HER2-negative advanced breast cancer (ABC) from the GEICAM/2015-04 (PANGEA-Breast) study [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS4-38.

Abstract PS4-37: A pro-tumorigenic mechanism of M2 tumor-associated macrophages (TAM) in triple-negative breast cancer

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. The success of immunotherapy in solid tumors has raised the hope for their utility in TNBCs as well. But only a subset of patients have a clinical response to check-point inhibitors. The cellular and molecular mechanisms that mediate the immunological response or tolerance are just beginning to be understood. Compared to ER/PR+ breast cancer, TNBC features a unique tumor microenvironment (TME) characterized by a large number of tumor-infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs). The density of TILs in and of themselves do not accurately predict response to neoadjuvant chemotherapy or survival. M2 tumor-associated macrophages (M2-TAMs) have been reported to associate with solid tumors to facilitate epithelial to mesenchymal transition (EMT), tumor invasiveness, metastasis, and resistance to therapy. In this study, we have characterized presence of M2-TAM in the TNBC immune environment by examining expression of several biomarkers. Methods:Surgically excised tumor specimens from 88 Indian women with TNBC were accessed from the longitudinal observational series of SJNAHS tissue bank under IERB approved protocols and assayed on nCounter® PanCancer Immune Profiling Panel which comprised of 740 genes and characterises 14 different immune cell types. Transcriptome analysis using Non-negative Matrix factorization (NMF) based unsupervised clustering was done to arrive at stable subtypes characterized by different tumour microenvironments within TNBC. CIEBERSORT tool was used to analyse for distribution of cell types. Identification of macrophages was done by Immunohistochemistry (IHC) for CD68 and CD163 markers in TNBCs and a control group of ER+HER2-.Results:NMF analysis with an enriched immune gene signature of 111 genes, yielded 3 subtypes (ST) (37%, 27% and 36% with relative proportions of ST1, ST2 and ST3) within the TNBC. The three subtypes had distinct survival patterns with ST1 having the best prognosis with enriched TH1 gene signature and ST3 had poorest (log rank p=0.5). On application of this gene signature to TNBC groups in METABRIC and TCGA data, similar pattern emerged with a significant survival pattern of ST 3 having the poorest outcome (p=0.005). A closer examination of ST 3 revealed a signature enriched for M2 macrophages also known as TAM. Immunohistochemistry for Pan macrophage marker CD68 and an M2 specific marker CD163 between TNBC and ER/PR+ revealed difference in the two groups was significantly different (Mann Whitney p=0.03), with TNBC exhibiting 2.4 fold higher expression of CD163. CD 68, was enriched in ST2 and ST3. CD163 which is an M2 specific marker was highest in ST3 as compared to ST 1&amp; 2 (p=0.04). This group also correlated with signal molecules secreted by macrophages containing growth factors, cytokines and chemokines, such as TGF-β, VEGF, IL-10 and CXCL and interleukins like IL4 and IL6 suggestive of TAM recruitment and polarization. It is likely that the TAM enriched subgroup is likely to be unresponsive to PD1/PDL1 inhibitors but could be targeted by novel therapeutic strategies to directly target M2-TAM. In vitro and In vivo analysis to target M2-TAM to evaluate the response to these therapeutic compounds in cell lines as well as a mouse syngeneic model is underway.Conclusions and future directions: As a cell type within the tumor microenvironment, that promotes invasion, M2-TAMs makes an ideal therapeutic target in TNBC. In addition, this sub-type lends itself to easy identification by a simple IHC assay. Citation Format: Aruna Korlimarla, Jyothi Prabhu, Chantirika Ragulan, Gnanapriya Shivakumar, Krisha Desai, Maggie Cheang, Srinath BS, Ravi Diwakar, Sandhya Apachu, Rekha Kumar, TS Sridhar, Savitha RajaRajan, Rohini Kaluve, Annie Alexander, Anguraj Sadanandam. A pro-tumorigenic mechanism of M2 tumor-associated macrophages (TAM) in triple-negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS4-37.

Tumor mutational burden and immune infiltrates in renal cell carcinoma and matched brain metastases

BackgroundTumor mutational burden (TMB) and density of tumor-infiltrating lymphocytes (TIL) have been postulated as predictive biomarkers for immunotherapy. Therefore, we investigated the concordance of TMB and TIL of primary/extracranial renal cell carcinoma (RCC) specimens and matched brain metastases (BM).Patients and methodsTwenty specimens from 10 patients were retrieved from the Vienna Brain Metastasis Registry (6/10 primary tumor, 4/10 lung metastasis, 10/10 matched BM). TMB was assessed using the TruSight Oncology 500 gene panel with libraries sequenced on a NextSeq instrument. TIL subsets (CD3+, CD8+, CD45RO+, FOXP3+, PD-L1+) were investigated using immunohistochemistry (Ventana Benchmark Ultra system) and automated tissue analysis (Definiens software).ResultsNo significant difference in TMB, CD3+, CD8+, CD45RO+, FOXP3+ or PD-L1+ expression was observed between extracranial and matched intracranial specimens (P > 0.05). Higher CD8+ TIL (P = 0.053) and CD45RO+ TIL (P = 0.030) densities in the primary tumor compared with the intracranial samples were observed in specimens collected after exposure to systemic treatment. Neither extracranial sample origin (lung metastasis versus primary RCC) nor extracranial disease status at BM diagnosis (progressive versus stable disease) were significantly associated with TMB or TIL densities in extracranial and intracranial samples (P > 0.05). No significant correlation was found between the median differences of TMB or TIL densities from extracranial to intracranial samples and BM-free survival.ConclusionThe comparable immunological microenvironment of extra- and intracranial tumor samples in our study underscores the immunological activation also in BM from RCC, and therefore, supports the development of immune modulatory treatments also in patients with brain metastatic RCC.

Clinicopathological Correlates of γδ T Cell Infiltration in Triple-Negative Breast Cancer.

Simple SummaryThe prognostic impact of the different tumor-infiltrating lymphocyte (TIL) subpopulations remains debated in solid cancers. We investigated the clinicopathological correlates and prognostic impact of TILs, particularly of γδ T cells, in 162 triple-negative breast cancer (TNBC) patients. A high γδ T cell density was significantly associated with younger age, higher tumor histological grade, adjuvant chemotherapy, BRCA1 promoter methylation, TIL density, and PD-L1 and PD-1 expression. In multivariate analyses, γδ T cell infiltration was an independent prognostic factor. However, this prognostic impact varied according to the tumor PIK3CA mutational status. High γδ T cell infiltration was associated with better survival in patients with PIK3CA wild-type tumors, without significant difference in the PIK3CA-mutated tumor subgroup. Altogether, these data suggest that high γδ T cell infiltrate is correlated with immune infiltration and might represent a prognostic tool in TNBC patients.The prognostic impact of the different tumor-infiltrating lymphocyte (TIL) subpopulations in solid cancers is still debated. Here, we investigated the clinicopathological correlates and prognostic impact of TILs, particularly of γδ T cells, in 162 patients with triple-negative breast cancer (TNBC). A high γδ T cell density (>6.625 γδ T cells/mm2) was associated with younger age (p = 0.008), higher tumor histological grade (p = 0.002), adjuvant chemotherapy (p = 0.010), BRCA1 promoter methylation (p = 0.010), TIL density (p < 0.001), and PD-L1 (p < 0.001) and PD-1 expression (p = 0.040). In multivariate analyses, γδ T cell infiltration (cutoff = 6.625 γδ T cells/mm2) was an independent prognostic factor (5-year relapse-free survival: 63.3% vs. 89.8%, p = 0.027; 5-year overall survival: 73.8% vs. 89.9%, p = 0.031, for low vs. high infiltration). This prognostic impact varied according to the tumor PIK3CA mutational status. High γδ T cell infiltration was associated with better survival in patients with PIK3CA wild-type tumors, but the difference was not significant in the subgroup with PIK3CA-mutated tumors. Altogether, these data suggest that high γδ T cell infiltrate is correlated with immune infiltration and might represent a candidate prognostic tool in patients with TNBC.

Activity and Immune Correlates of Programmed Death-1 Blockade Therapy in Patients With Advanced Large Cell Neuroendocrine Carcinoma

BackgroundThe efficacy of anti–programmed death receptor 1 (PD-1) therapy in patients with large cell neuroendocrine carcinoma (LCNEC) remains unclear. We investigated the outcome of anti–PD-1 therapy and its predictive markers by evaluating the immune-related tumor microenvironment. PatientsWe retrospectively reviewed patients with advanced LCNEC treated with systemic chemotherapy. We also evaluated PD ligand 1 (PD-L1) expression (clone: 22C3), CD8-positive tumor-infiltrating lymphocytes (TILs), and the mutational profiles. ResultsSeventy patients were enrolled, and 13 of 70 patients received anti–PD-1 therapy. The progression-free survival (PFS) and objective response rate (ORR) of the anti–PD-1 therapy were 4.2 months and 39%, respectively. The overall survival of patients treated with anti–PD-1 therapy (n = 13) was significantly better than those treated without anti–PD-1 therapy (n = 57) (25.2 months vs 10.9 months; P = .02). Among the 13 patients treated with anti–PD-1 therapy, 10 patients (90%) had PD-L1–negative tumors. Patients with a high density of tumoral CD8-positive TILs (≥38/mm2) had a significantly better ORR and PFS than those with a low density of tumoral CD8-positive TILs (ORR: P = .02; PFS: P = .003). Additionally, all 3 patients with TP53 mutation co-occurring with PIK3CA mutation (2 of 8 patients) or RB1 mutation (1 of 8 patients) responded to anti–PD-1 therapy. ConclusionsAnti–PD-1 therapy was effective regardless of PD-L1 positivity in patients with advanced LCNEC. Our investigation might suggest that the density of tumoral CD8-positive TILs and the presence of co-occurring mutations are predictors of the efficacy of anti–PD-1 therapy in patients with advanced LCNEC.

Case Report: Therapeutic Response to Chemo-Immunotherapy in an Advanced Large Cell Lung Carcinoma Patient With Low Values of Multiple Predictive Biomarkers

Immune checkpoint inhibitors have revolutionized the treatments of lung cancers, and multiple predictive biomarkers alone or in combination help clinicians with the appropriate therapeutic selections. Recently, chemo-immunotherapy has been recommended for treating advanced non-small cell lung cancers in patients without driver mutations. However, the clinical relevance of predictive biomarkers and the treatment efficacy of chemo-immunotherapy in large cell lung carcinoma (LCLC) remain unclear. Here, we reported a rare case of LCLC with none driver gene mutations and low values of multiple predictive biomarkers. These biomarkers included a low PD-L1 expression of 5–10%, a low tumor mutational burden (TMB) of 2.5 muts/mb, a low CD8(+) tumor-infiltrating lymphocyte density of 147.91 psc/mm². After one-cycle chemotherapy, the patient progressed rapidly and then was switched to pembrolizumab combining paclitaxel plus cisplatin. Interestingly, he achieved a partial response after two cycles of chemo-immunotherapy, showing multiple lymph nodes obviously shrunk on CT scan, and other clinical symptoms were relieved when compared with the baseline findings. After five cycles of chemo-immunotherapy, this advanced patient still benefited and was changed to maintenance immunotherapy monotherapy. This case suggests that chemo-immunotherapy may provide an effective therapeutic option for those LCLC patients with low values of multiple predictive biomarkers, particularly for those who progressed from first-line classical treatments.

Correlation of HPV16 Gene Status and Gene Expression With Antibody Seropositivity and TIL Status in OPSCC.

IntroductionHuman papillomavirus 16 (HPV16) is the main cause of oropharyngeal squamous cell carcinoma (OPSCC). To date, the links between HPV16 gene expression and adaptive immune responses have not been investigated. We evaluated the correlation of HPV16 DNA, RNA transcripts and features of adaptive immune response by evaluating antibody isotypes against E2, E7 antigens and density of tumor-infiltrating lymphocytes (TIL).Material and MethodsFFPE-tissue from 27/77 p16-positive OPSCC patients was available. DNA and RNA were extracted and quantified using qPCR for all HPV16 genes. The TIL status was assessed. Immune responses against E2 and E7 were quantified by ELISA (IgG, IgA, and IgM; 77 serum samples pre-treatment, 36 matched post-treatment).ResultsAmounts of HPV16 genes were highly correlated at DNA and RNA levels. RNA co-expression of all genes was detected in 37% (7/19). E7 qPCR results were correlated with higher anti-E7 antibody (IgG, IgA) level in the blood. Patients with high anti-E2 IgG antibody (>median) had better overall survival (p=0.0311); anti-E2 and anti-E7 IgA levels had no detectable effect. During the first 6 months after treatment, IgA but not IgG increased significantly, and >6 months both antibody classes declined over time. Patients with immune cell-rich tumors had higher levels of circulating antibodies against HPV antigens.ConclusionWe describe an HPV16 qPCR assay to quantify genomic and transcriptomic expression and correlate this with serum antibody levels against HPV16 oncoproteins. Understanding DNA/RNA expression, relationship to the antibody response in patients regarding treatment and outcome offers an attractive tool to improve patient care.

Brain Tumor Microenvironment and Angiogenesis in Melanoma Brain Metastases.

BackgroundHigh tumor-infiltrating lymphocytes (TILs) and hemorrhage are important prognostic factors in patients who have undergone craniotomy for melanoma brain metastases (MBM) before 2011 at the University of Pittsburgh Medical Center (UPMC). We have investigated the prognostic or predictive role of these histopathologic factors in a more contemporary craniotomy cohort from the University of North Carolina at Chapel Hill (UNC-CH). We have also sought to understand better how various immune cell subsets, angiogenic factors, and blood vessels may be associated with clinical and radiographic features in MBM.MethodsBrain tumors from the UPMC and UNC-CH patient cohorts were (re)analyzed by standard histopathology, tumor tissue imaging, and gene expression profiling. Variables were associated with overall survival (OS) and radiographic features.ResultsThe patient subgroup with high TILs in craniotomy specimens and subsequent treatment with immune checkpoint inhibitors (ICIs, n=7) trended to have longer OS compared to the subgroup with high TILs and no treatment with ICIs (n=11, p=0.059). Bleeding was significantly associated with tumor volume before craniotomy, high melanoma-specific expression of basic fibroblast growth factor (bFGF), and high density of CD31+αSMA- blood vessels. Brain tumors with high versus low peritumoral edema before craniotomy had low (17%) versus high (41%) incidence of brisk TILs. Melanoma-specific expression of the vascular endothelial growth factor (VEGF) was comparable to VEGF expression by TILs and was not associated with any particular prognostic, radiographic, or histopathologic features. A gene signature associated with gamma delta (gd) T cells was significantly higher in intracranial than same-patient extracranial metastases and primary melanoma. However, gdT cell density in MBM was not prognostic.ConclusionsICIs may provide greater clinical benefit in patients with brisk TILs in MBM. Intratumoral hemorrhage in brain metastases, a significant clinical problem, is not merely associated with tumor volume but also with underlying biology. bFGF may be an essential pathway to target. VEGF, a factor principally associated with peritumoral edema, is not only produced by melanoma cells but also by TILs. Therefore, suppressing low-grade peritumoral edema using corticosteroids may harm TIL function in 41% of cases. Ongoing clinical trials targeting VEGF in MBM may predict a lack of unfavorable impacts on TIL density and/or intratumoral hemorrhage.

Tumor-Infiltrating CD20+ B Lymphocytes: Significance and Prognostic Implications in Oral Cancer Microenvironment.

Simple SummaryThe complex interplay between the different cellular components in the tumor microenvironment (TME) dynamically modulates the antitumor immune response. This study investigates the prognostic relevance of CD20+ tumor-infiltrating B lymphocytes in oral squamous cell carcinoma (OSCC), and also possible relationships with other immune subtypes and key players within the oral TME.Immunohistochemical analysis of stromal/tumoral CD20+ B lymphocytes was performed in 125 OSCC patients. Correlations with immune profiles CD4+, CD8+, and FOXP3+ tumor-infiltrating lymphocytes (TILs), tumoral PD-L1, and stem-related factors NANOG and SOX2 were assessed, and also associations with clinical data and patient survival. There was a strong positive correlation between the infiltration of CD20+ B lymphocytes and other immune profiles (i.e., CD4+, CD8+, and FOXP3+ TILs, and CD68+ and CD163+ macrophages) both in stroma and tumor nests. Strikingly, CD20+ TILs were inversely correlated with NANOG/SOX2 expression. Stromal CD20+ TILs were significantly associated with T classification and second primary tumors. A stratified survival analysis showed that tumoral CD20+ TILs were significantly associated with prognosis in male and younger patients, with tobacco or alcohol consumption, high tumoral CD8+ TILs, low tumoral infiltration by CD68+ macrophages, positive PD-L1 expression, and negative NANOG/SOX2. Multivariate Cox analysis further revealed clinical stage and tumoral CD20+ TILs independently associated with disease-specific survival (HR = 2.42, p = 0.003; and HR = 0.57, p = 0.04, respectively). In conclusion, high CD20+ TIL density emerges as an independent good prognostic factor in OSCC, suggesting a role in antitumor immunity. This study also uncovered an inverse correlation between CD20+ TILs and CSC marker expression.

CD8+ tumor-infiltrating lymphocyte infiltration prediction with radiomic signature in locally advanced rectal cancer.

123 Background: Despite increasing use of cancer immunotherapies, no biomarker relevant to anti-tumor immunity has been developed in rectal cancer. The present study aims to develop a radiomic signature to predict the infiltration of CD8+ tumor-infiltrating lymphocytes (TILs). Methods: In total, 131 patients undergoing neoadjuvant chemoradiotherapy (CRT) from 2005 to 2012 were analyzed. The study included pre-CRT dataset A ( n = 113; n = 75 and 38 for training and validation) and post-CRT datasets B ( n = 32; predominance of tumor) and C ( n = 20; pure fibrosis). To build the radiomic signature, 38 features selected from pre-CRT T2-weighted MRI scans were incorporated into the least absolute shrinkage and selection operator method. Results: Higher CD8+ TIL infiltration was associated with better overall and progression-free survival ( P = 0.01 and 0.03, respectively). In pre-CRT dataset A, the area under the receiver operating characteristic curve values of radiomic score for predicting CD8+ TILs were 0.760 and 0.729 for training and validation subsets, respectively. A significant correlation was observed between the radiomic signature and CD8+ TIL density in the post-CRT dataset B (Pearson’s R = ‒0.372, P = 0.036), whereas no association was found within the dataset C accounting for pure fibrosis after CRT (Pearson’s R = ‒0.069, P = 0.77). Conclusions: We established the MRI-derived radiomic signature for predicting CD8+ TIL infiltration in the contemporary treatment era of rectal cancer. The noninvasive characterization of tumor immune status would provide insights into the role of radiomic-pathology modeling to predict the local immune phenotypes.

A template to quantify the location and density of CD3\u2009+\u2009and CD8\u2009+\u2009tumor-infiltrating lymphocytes in colon cancer by digital pathology on whole slides for an objective, standardized immune score assessment

BackgroundIn colon cancer, the location and density of tumor-infiltrating lymphocytes (TILs) can classify patients into low and high-risk groups for prognostication. While a commercially available ‘Immunoscore®’ exists, the incurred expenses and copyrights may prevent universal use. The aim of this study was to develop a robust and objective quantification method of TILs in colon cancer.MethodsA consecutive, unselected series of specimens from patients with colon cancer were available for immunohistochemistry and assessment of TILs by automated digital pathology. CD3 + and CD8 + cells at the invasive margin and in tumor center were assessed on consecutive sections using automated digital pathology and image analysis software (Visiopharm®). An algorithm template for whole slide assessment, generated cell counts per square millimeters (cells/mm2), from which the immune score was calculated using distribution volumes. Furthermore, immune score was compared with clinical and histopathological characteristics to confirm its relevance.ResultsBased on the quantified TILs numbers by digital image analyses, patients were classified into low (n = 83, 69.7%), intermediate (n = 14, 11.8%) and high (n = 22, 18.5%) immune score groups. High immune score was associated with stage I–II tumors (p = 0.017) and a higher prevalence of microsatellite instable (MSI) tumors (p = 0.030). MSI tumors had a significantly higher numbers of CD3 + TILs in the invasive margin and CD8 + TILs in both tumor center and invasive margin, compared to microsatellite stable (MSS) tumors.ConclusionA digital template to quantify an easy-to-use immune score corresponds with clinicopathological features and MSI in colon cancer.