CD8-positive Tumor-infiltrating Lymphocytes Research Articles

Understanding the impact of spatial immunophenotypes on the survival of endometrial cancer patients through the ProMisE classification

ObjectivesWe focused on how the immunophenotypes based on the distribution of CD8-positive tumor-infiltrating lymphocytes (TILs) relate to the endometrial cancer (EC) molecular subtypes and patients’ prognosis.Patients and methodsTwo cohorts of EC patients (total n = 145) were analyzed and categorized using the Molecular Risk Classifier for Endometrial cancer (ProMisE): POLEmut (POLE mutation), MMRd (mismatch repair deficiency), NSMP (no specific molecular profile), and p53abn (p53 abnormality). CD8-positive TILs, within the central tumor and the invasive margin, were examined by using immunohistochemical staining and advanced image-analysis software. It was investigated whether these immunophenotypes correlate with the molecular subtypes and patients' survival. RNA-sequencing (RNA-seq) was used to explore tumor-derived factors influencing these immunophenotypes.ResultsThree distinct immunophenotypes (inflamed, excluded, and desert) based on the CD8-positive TIL patterns were identified in EC patients. Notably, the inflamed phenotype was most frequently observed in the POLEmut and MMRd subtypes, while the desert phenotype was predominant in the NSMP subtype; however, other immunophenotypes were also observed. All p53abn subtype showed the non-inflamed (excluded or desert) phenotype. The prognosis was markedly poorer in the patients with the non-inflamed phenotype than in those with the inflamed phenotype. The RNA-seq analysis showed that the expression of MYC target genes and type-1 interferon response genes was enriched in the non-inflamed phenotype in MMRd and NSMP subtypes, respectively.ConclusionEvaluating not only the molecular classification but also the immunophenotype may lead to more personalized immunotherapy in EC and elucidating the mechanisms that underlie the formation of the three immunophenotypes could lead to the discovery of new immunotherapy targets.

Abstract 2519: Alteration of tumor immune microenvironment after recurrence and its impact on the efficacy of anti-PD-1 antibody in resected non-small cell lung cancer patients

Abstract Background: Tumor immune microenvironment (TIME), including programmed death-1 ligand-1 (PD-L1) and CD8-positive tumor-infiltrating lymphocytes (TIL), has been suggested as a potential biomarker to predict the efficacy of anti-PD-1 antibody. Postoperative recurrence in patients with non-small cell lung cancer (NSCLC) is treated with therapeutic strategies similar to those for metastatic disease. However, in postoperative recurrence cases, PD-L1 evaluation is generally based on archival resection specimens, because there is little opportunity for a new biopsy of the recurrent site. Therefore, it remains unclear whether TIME of recurrent lesion is similar to TIME of primary lesion at surgery, and the efficacy of anti-PD-1 antibody for postoperative recurrent NSCLC patients based on TIME status at surgery is unknown. Materials and Methods: Cohort A included 59 patients with advanced and 25 patients with postoperative recurrence NSCLC who treated with first-line pembrolizumab monotherapy. PD-L1 expression and CD8-TIL were evaluated immunohistochemically in tumor specimens obtained at diagnosis in the advanced cases and at surgery in the postoperative recurrence cases. Tumor proportion score for PD-L1 and the extent of CD8-TIL were evaluated and classified as high or low. TIME was categorized into 4 subclasses, according to the respective PD-L1 and CD8-TIL, as follows: (class I) high/high; (class II) low/low; (class III) high/low; and (class IV) low/high. Progression-free survival (PFS) of pembrolizumab was compared based on TIME. Cohort B consisted of 50 NSCLC patients who underwent radical resection, and whose tumor specimens were obtained from recurrent site. As in cohort A, TIME was assessed for paired resection and recurrent tumor samples. Results: In cohort A, the median PFS of pembrolizumab for the advanced cases were 25.7 months in class I (n=25), 2.3 months in class II (n=8), 8.0 months in class III (n=20), and 6.5 months in class IV (n=6) (p<0.001), respectively; while those for the postoperative recurrence cases were 9.0 months (n=7), 4.3 months (n=7), 7.6 months (n=6), and 2.5 months (n=5) (p=0.55), respectively. In cohort B, TIME for resected primary lesion were observed in 9 with class I, 20 with class II, 3 with class III and 18 patients with class IV, respectively. The concordance of PD-L1 category (high vs low) between resected primary lesion and recurrent lesion was 88.0%, while that of TIME classification was 54.0%. Conclusion: TIME classification using tumor specimens just before administration of anti-PD-1 antibody was useful predictive biomarker. However, TIME alterations between resection and postoperative recurrence were observed in many cases, suggesting that TIME based on tumor specimens at surgery may not be useful for predicting the efficacy of anti-PD-1 antibody for postoperative recurrent NSCLC. Citation Format: Yoshiya Matsumoto, Tetsuya Watanabe, Megumi Mizutani, Akira Sugimoto, Hiroaki Nagamine, Yoko Tani, Kenji Sawa, Takuma Tsukioka, Hiroyasu Kaneda, Shigeki Mitsuoka, Noritoshi Nishiyama, Tomoya Kawaguchi. Alteration of tumor immune microenvironment after recurrence and its impact on the efficacy of anti-PD-1 antibody in resected non-small cell lung cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2519.

Abstract 3806: Impact of tumor-infiltrating lymphocytes, tertiary lymphoid structures, and neutrophil-to-lymphocyte ratio on the prognosis of patients with lung metastases from uterine leiomyosarcoma

Abstract In the tumor microenvironment, tumor-infiltrating lymphocytes (TILs) and tertiary lymphoid structures (TLSs) are known to play a major role in terms of antitumor immunity. It has been reported that the presence of TILs and TLSs is related to the prognosis of various cancers. In addition, neutrophil-to-lymphocyte ratio (NLR) has also been associated with the prognosis in them. We investigated the significance of TILs, TLSs and NLR as a prognosis indicator for lung metastases from uterine leiomyosarcoma. We retrospectively recruited 102 patients undergoing metastasetomy for lung metastases from uterine leiomyosarcoma at Okayama University Hospital from January 2006 to December 2019. TILs were evaluated by immunohistochemical staining of surgically resected specimens of lung metastases using anti-CD3/CD8/CD103/Foxp3 antibodies. TLSs were also evaluated using anti-CD20 antibodies. We counted the number of CD3/CD8/CD103/Foxp3-positive cells and classified TILs into high and low groups. We measured the density of CD20-positive cells and classified TLSs into high- and low-density groups. NLR was calculated from the blood examination immediately before the most recent pulmonary metastasectomy. We determined the cut-off value for NLR using a time-dependent receiver operating characteristic (ROC) curve analysis and classified the patients into NLR-high and NLR-low groups according to the cut-off value. In terms of TILs, both CD3 and CD8 were correlated with the prognosis. The overall survival (OS) was significantly better in patients with CD3-high group (p = 0.018), CD8-high group (p = 0.017), TLSs-high group (p = 0.041) and NLR-low group (p = 0.018), respectively. The status of CD103 and Foxp3 did not affect the prognosis. We assessed the impact of the combination of CD8-positive TILs or TLSs and NLR on the prognosis. Regarding CD8-positive TILs and NLR, the OS of CD8-high/NLR-low group was significantly better than that of CD8-low/NLR-high group (p = 0.0078). In terms of TLSs and NLR, the OS of TLSs-high/NLR low group was significantly better than that of TLSs-low/NLR-high group (p = 0.010). In conclusion, CD3-positive TILs, CD8-positive TILs, TLSs, and NLR are correlated with the prognosis, respectively. The combination of CD8-positive TILs or TLSs and NLR may be indicators to predict the prognosis of the patients with lung metastases from uterine leiomyosarcoma. Citation Format: Fumiaki Mukohara, Naoki Matsuda, Hiromasa Yamamoto, Tomohiro Habu, Daisuke Mizuno, Ryo Yoshichika, Atsushi Matsuoka, Tomoaki Higashihara, Naohiro Hayashi, Mao Yoshikawa, Masayoshi Ohki, Kazuhiko Shien, Ken Suzawa, Katsuhito Takahashi, Shinichi Toyooka. Impact of tumor-infiltrating lymphocytes, tertiary lymphoid structures, and neutrophil-to-lymphocyte ratio on the prognosis of patients with lung metastases from uterine leiomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3806.

CD8-positive Tumor-infiltrating Lymphocytes and Prognosis in Radiotherapy for Uterine Cervical Squamous Cell Carcinoma.

Prognostic factors, including CD8-positive tumor-infiltrating lymphocytes (CD8+TILs), in definitive radiotherapy (RT) for squamous cell carcinoma (SqCC) of the uterine cervix need to be studied. This study aimed to explore these factors in a retrospective cohort. Patients with SqCC who underwent definitive RT comprising external beam RT and intracavitary brachytherapy at our facility between April 2006 and November 2013 were evaluated. CD8 immunohistochemistry was performed in pre-treatment biopsy samples to analyze the prognostic significance of CD8+TILs in the tumor nest. Positive staining was defined as at least one CD8+ lymphocyte infiltrating the tumor area in the specimen. In total, 150 consecutive patients were included. Among them, 66 (43.7%) patients had International Federation of Gynecology and Obstetrics (FIGO, 2008 edition) stage IIIA or higher progressive disease. The median follow-up period was 61 months. In the entire cohort, the 5-year cumulative rates of overall survival (OS), progression-free survival (PFS), and pelvic recurrence-free rate (PRFR) were 75.6%, 69.6%, and 84.8%, respectively. Of the 150 patients, 120 (80.0%) patients were CD8+TIL positive. The independent favorable prognostic factors were FIGO stage I or II disease, administration of concurrent chemotherapy, and CD8+TILs for OS (p=0.028, 0.005, and 0.038, respectively); FIGO stage I or II disease and CD8+TILs for PFS (p=0.015 and <0.001, respectively); and CD8+TILs for PRFR (p=0.017). The presence of CD8+TILs in the tumor nest may be a favorable prognostic factor of survival after definitive RT in patients with SqCC of the uterine cervix.

Abstract 1268: The effect of metformin for neutrophil extracellular traps (NETs) on tumor immune microenvironment of colorectal cancer with type 2 diabetes mellitus

Abstract Background: Although metformin (Met) is well known to suppress tumor growth, the detailed mechanisms of its anti-tumor effect remain unknown. In recent years, it has been pointed out that tumor-associated neutrophils (TAN) can produce neutrophil extracellular traps (NETs) in the tumor microenvironment (TME) which can tumor invasion and metastasis. Here, in this study, we investigated the effect of Met on tumor-associated NETs and its effect on the prognosis of cancer patients. Method: We retrospectively examined the outcome of colorectal cancer (CRC) patients with type 2 diabetes mellitus (T2DM) who received curative surgery in Jichi Medical University Hospital and asked the impact of Met intake on their outcome. In addition, we performed multicolor immunohistochemistry to examine the phenotypes of tumor-infiltrating lymphocytes (TIL), TAN and NETs in surgically removed tissues specimens in 40 (colorectal cancer) CRC patients who had taken Met before surgery and propensity score-matched 40 patients who had not. Results: A total of 1858 patients underwent curative colectomy from April 2010 to March 2021. Among them, 283 patients had T2DM at the time of surgery, and 62 patients had been treated with drugs including Met. The postoperative disease-free survival rate (DFS) was significantly improved in the Met intake group compared with non-intake group (HR=0.21, p&amp;lt;0.01). The number of CD66b(+) TANs significantly decreased in Met-intake group (Median(M)=169 (36-553)/mm2 vs 99 (34-322)/mm2, p&amp;lt;0.01). The number of tumor-associated NETs defined as CD66b(+) cit-H3(+) cells significantly decreased in Met-intake group (M=36 (8-272)/mm2 vs 20 (0-104)/mm2, p&amp;lt;0.01), and tumor-associated NETs/TANs ratio significantly decreased in Met-intake group (29.9% vs 19.8%, p&amp;lt;0.01). The number of CD3(+) TILs significantly increased in Met intake group (M=122 (28-257)/mm2 vs 142 (48-386)/mm2, p&amp;lt;0.05). The number of CD8(+) TILs significantly increased in Met-intake group (M=60 (14-180)/mm2 vs 99 (52-318)/mm2, p&amp;lt;0.01), and CD8/CD3 ratio significantly increased in Met-intake group (51.2% vs 72.1%, p&amp;lt;0.01). In addition, there was a negative correlation between TANs or tumor-associated NETs and CD8 positive TILs in all 80 patients (r=-0.26, p&amp;lt;0.05). Conclusion: Met can inhibit neutrophil infiltration and NETs formation while promote the infiltration of cytotoxic T cells in TME, which may lead to the improvement of the outcome of the patients with CRC. Citation Format: Akira Saito, Hideyuki Ohzawa, Yuki Kaneko, Kohei Tamura, Yurie Futoh, Kazuya Takahashi, Yuki Kimura, Rie Kawashima, Hideyo Miyato, Joji Kitayama, Naohiro Sata. The effect of metformin for neutrophil extracellular traps (NETs) on tumor immune microenvironment of colorectal cancer with type 2 diabetes mellitus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1268.

Identification of inflamed-phenotype of small cell lung cancer leading to the efficacy of anti-PD-L1 antibody and chemotherapy

BackgroundPlatinum etoposide plus anti-programmed cell death ligand-1 (PD-L1) antibody therapy is the standard of care for extensive-stage small cell lung cancer (ES-SCLC). However, patient characteristics associated with the efficacy of the combination therapy in SCLC are unclear. MethodsWe retrospectively reviewed post-surgical limited-stage (LS)-SCLC and ES-SCLC patients treated with atezolizumab plus carboplatin and etoposide (ACE). The association between SCLC subtypes based on transcriptomic data and pathological findings, including CD8-positive tumor-infiltrating lymphocyte (TIL) status, was investigated in the LS-SCLC cohort. The association between the efficacy of ACE therapy, pathological subtypes, and TIL status was evaluated in the ES-SCLC cohort. ResultsThe LS-SCLC cohort (N = 48) was classified into four SCLC subtypes (ASCL1 + NEUROD1 [SCLC-A + N, N = 17], POU2F3 [SCLC-P, N = 15], YAP1 [SCLC-Y, N = 10], and inflamed [SCLC-I, N = 6]) based on transcriptomic data. SCLC-I showed enriched immune-related pathways, the highest immune score (CD8A expression and T-cell–inflamed gene expression profiles), and epithelial–mesenchymal transition (EMT), in transcriptional subtypes. Immunohistochemical staining (IHC) showed that SCLC-I had the highest density of CD8-positive TILs in transcriptional subtypes. In the ES-SCLC cohort, the efficacy of ACE therapy did not differ according to pathological subtypes. The progression-free survival (PFS) of TILHigh patients was significantly longer than that of TILLow patients (PFS: 7.3 months vs. 4.0 months, p < 0.001). ConclusionTumors with a high density of TILs, which represent the most immunogenic SCLC subtype (SCLC-I), based on transcriptomic data could benefit from ACE therapy.

Association between Tumor Mutational Burden, Stromal CD8+ Tumor-Infiltrating Lymphocytes, and Clinical Factors in Cervical Cancers Treated with Radiotherapy.

Tumor mutational burden (TMB) and stromal CD8-positive tumor-infiltrating lymphocytes (CD8+TILs) serve important roles in antitumor immune responses to radiotherapy. This study aimed to elucidate the association between TMB, CD8+TILs, and clinical factors in patients with cervical cancer treated with radiotherapy. Patients with squamous cell carcinoma of the uterine cervix treated with definitive radiotherapy, and with available somatic mutation data and immunohistochemical staining data from identical tumor tissues, were enrolled retrospectively. The association between TMB and/or CD8+TIL density and patient characteristics, mutation profiles, and treatment outcome was analyzed. The study analyzed 44 patients (median follow-up period, 61 months). There was no significant correlation between TMB and CD8+TIL density, or between TMB or CD8+TIL density and patient characteristics. TMB-high or CD8+TIL density-low status was associated with worse overall survival and distant metastasis-free survival; the predictive value of these factors became greater when used in combination. TMB-high or CD8+TIL density-high status was associated with ARID1A mutations. These data indicate independence of TMB and CD8+TIL density and the involvement of ARID1A alterations in antitumor immune responses in patients with cervical cancers treated with radiotherapy, warranting further mechanistic research and prospective validation.

CD8/PD-L1 immunohistochemical reactivity and gene alterations in cutaneous squamous cell carcinoma.

In recent years, several immune checkpoint inhibitors targeting programmed death-ligand 1 (PD-L1) or PD-1 have been developed for cancer therapy. The genetic background of tumors and factors that influence PD-L1 expression in tumor tissues are not yet elucidated in cutaneous squamous cell carcinoma (cSCC). CD8-positive tumor-infiltrating lymphocytes (TILs) are known to be related to tumor immunity. Here, we aimed to study the relationship between CD8/PD-L1 immunohistochemical reactivity and gene alterations in cSCC. Tumorigenic genes were examined to identify gene alterations using next-generation sequencing (NGS). We collected 27 cSCC tissue samples (from 13 metastatic and 14 non-metastatic patients at primary diagnosis). We performed immunohistochemical staining for CD8 and PD-L1, and NGS using a commercially available sequencing panel (Illumina Cancer Hotspot Panel V2) that targets 50 cancer-associated genes. Immunohistochemically, CD8-positive TILs showed a high positive score in cSCC without metastasis; in these cases, cSCC occurred predominantly in sun-exposed areas, the tumor size was smaller, and the total gene variation numbers were notably low. The tumor depth, PD-L1 positivity, and gene variation number with or without tumor metastasis were not related, but the gene variation number tended to be higher in cSCCs arising in non-sun-exposed areas. Tumor metastasis was more common in cSCC arising in non-sun-exposed areas, which decreased the number of TILs or CD8-positive cells. From a genetic perspective, the total gene alterations were higher in cSCC with metastasis. Among them, ERBB4 and NPM1 are presumably involved in cSCC tumorigenesis; in addition, GNAQ, GNAS, JAK2, NRAS, IDH2, and CTNNB1 may be related to tumor metastasis. These results provide information on potential genes that can be targeted for cSCC therapy and on immune checkpoint inhibitors that may be used for cSCC therapy.

Implication of IL6-positive Cancer-associated Fibroblasts in Merkel Cell Carcinoma Pathogenesis: A Possible Modulator of Immune Microenvironment.

The role of cancer-associated fibroblasts (CAFs) in the pathogenesis of Merkel cell carcinoma (MCC) remains unknown. This study aimed to investigate the clinicopathological significance of CAF subpopulations and their association with tumor-infiltrating lymphocytes (TILs) in patients with MCC. Clinicopathological features and the status of microenvironment fibrosis (MF) around tumor masses were evaluated in 20 MCC patient and tissue sections. Alpha-smooth muscle actin (α-SMA)-positive CAFs (α-SMA+CAFs), interleukin-6-positive CAFs (IL6+CAFs), CD4-positive TILs (CD4+TILs), and CD8-positive TILs (CD8+TILs) in MCC tissue samples were investigated using immunohistochemistry. In a total of 20 MCC patients, high-MF was detected in 12 (60%) patients which was significantly associated with worse progression-free survival (p=0.048), but not with overall survival. CD4+/CD8+ TILs were frequently detected in MCC tissues. High-intra-tumoral CD8+TIL was significantly associated with better overall and progression-free survival (p=0.04 and p=0.015) in our cohort. High-αSMA+ CAFs were detected in 11 (55.0%) patients and high-IL6+CAFs in 10 (50.0%) patients. A negative association was found between high-IL6+CAF and high-intra-tumoral CD8+TILs (p=0.005). Patients with high IL6+CAFs showed worse overall/progression-free survival than patients with low-IL6+CAFs (p=0.022 and p=0.035). IL6+CAFs may largely influence the tumor immune microenvironment of MCC by modulating distinct T-cell populations and functions. This study provides a possible therapeutic target to overcome resistance to immune therapies in MCC.

Prognostic value of Dickkopf-1 and \xdf-catenin expression according to the antitumor immunity of CD8-positive tumor-infiltrating lymphocytes in biliary tract cancer

The role of β-catenin and Dickkopf-1 (DKK1) is dependent on the specific immunobiology of T cell inflammation in biliary tract cancer (BTC). We aimed to analyze the role of DKK1 or β-catenin as a prognostic factor in BTC, and determine the clinical associations of ß-catenin and DKK1 with CD8+ tumor-infiltrating lymphocytes (TIL). We used data from The Cancer Genome Atlas Research Network and the clinicopathological data of 145 patients with BTC who had undergone primary radical resection between 2006 and 2016. CD8+ TIL expression was a significant predictor of favorable overall survival (OS) and relapse-free survival (RFS) (median OS, 34.9 months in high-TIL, 16.7 months in low-TIL, P < 0.0001 respectively; median RFS, 27.1 months in high-TIL, 10.0 months in low-TIL, P < 0.0001 respectively). In the high-CD8+ TIL BTC group, the tumor expression of β-catenin and DKK1 had a significant negative impact on either OS or RFS. In the low-TIL BTC group, there were no differences according to ß-catenin and DKK1 expression. Cox regression multivariate analysis demonstrated that CD8+ TIL and β-catenin retained significant association with OS. Among patients with resected BTC, the β-catenin and DKK1 protein and high CD8+ TIL levels were associated with poor and good clinical outcomes, respectively.

IFN-Gamma Expression in the Tumor Microenvironment and CD8-Positive Tumor-Infiltrating Lymphocytes as Prognostic Markers in Urothelial Cancer Patients Receiving Pembrolizumab.

Simple SummaryPembrolizumab, an immune checkpoint inhibitor, has shown therapeutic benefit for advanced urothelial carcinoma (aUC) patients, but only a limited population achieves a long-term response. Prediction of treatment outcomes for aUC patients receiving immune checkpoint inhibitors is a clinical challenge. We assessed the associations between the expression of multiple immune markers in the tumor microenvironment using immunohistochemistry of tumor tissues obtained from 26 aUC patients who received second-line pembrolizumab treatment. We found that high infiltration of CD8-positive lymphocytes was significantly associated with a favorable objective response and overall and progression-free survival. Furthermore, expression of interferon-gamma (IFNγ) showed a significant positive correlation with post-progression survival. Finally, we demonstrated that the coincidence of low infiltration of CD8-positive lymphocytes and low IFNγ expression was an independent prognostic factor for an unfavorable response to pembrolizumab.Although immune checkpoint inhibitors have shown benefit for advanced urothelial carcinoma (aUC) patients, prognostication of treatment efficacy and response duration remains a clinical challenge. We evaluated the expression of immune markers in the tumor microenvironment and assessed their associations with response to and survival after pembrolizumab treatment in 26 aUC patients. High levels of CD8+ tumor-infiltrating lymphocytes (TILs) were associated with favorable objective responses (23.0% vs. 15.3%, p = 0.0425), progression-free survival (median, 8.8 vs 2.1 months; hazard ratio (HR), 0.24; 95% confidence interval (CI), 0.07–0.66, p = 0.0060), and overall survival (median, >24.0 vs. 5.3 months; HR, 0.17; 95% CI, 0.04–0.56, p = 0.0034) compared with low levels. High interferon-gamma (IFNγ) expression levels were associated with longer post-progression survival (median, 4.9 vs. 1.0 months; HR, 0.18; 95% CI, 0.04–0.59, p = 0.0027) compared with low expression. Multivariate analysis incorporating clinical prognosticators demonstrated that the coincidence of low CD8+ T cells/IFNγ was an independent factor for unfavorable overall survival after pembrolizumab treatment (HR, 4.07; 95% CI, 1.36–12.73; p = 0.0125). The combination of low CD8+ TILs and IFNγ expression was an independent prognostic factor with predictive ability equivalent to previously reported clinical prognosticators.

Immunohistochemical expression of programmed death-ligand 1 and CD8 in glioblastomas

BackgroundGlioblastoma is the most aggressive primary malignant brain tumor in adults and is characterized by poor prognosis. Immune evasion occurs via programmed death-ligand 1 (PD-L1)/programmed death receptor 1 (PD-1) interaction. Some malignant tumors have responded to PD-L1/PD-1 blockade treatment strategies, and PD-L1 has been described as a potential predictive biomarker. This study discussed the expression of PD-L1 and CD8 in glioblastomas.MethodsThirty cases of glioblastoma were stained immunohistochemically for PD-L1 and CD8, where PD-L1 expression in glioblastoma tumor tissue above 1% is considered positive and CD-8 is expressed in tumor infiltrating lymphocytes. The expression of each marker was correlated with clinicopathologic parameters. Survival analysis was conducted to correlate progression-free survival (PFS) and overall survival (OS) with PD-L1 and CD8 expression.ResultsDiffuse/fibrillary PD-L1 was expressed in all cases (mean expression, 57.6%), whereas membranous PD-L1 was expressed in six of 30 cases. CD8-positive tumor-infiltrating lymphocytes (CD8+ TILs) had a median expression of 10%. PD-L1 and CD8 were positively correlated (p = .001). High PD-L1 expression was associated with worse PFS and OS (p = .026 and p = .001, respectively). Correlation of CD8+ TILs percentage with age, sex, tumor site, laterality, and outcomes were statistically insignificant. Multivariate analysis revealed that PD-L1 was the only independent factor that affected prognosis.ConclusionsPD-L1 expression in patients with glioblastoma is robust; higher PD-L1 expression is associated with lower CD8+ TIL expression and worse prognosis.

MA09.05 PD1-Positive Tertiary Lymphoid Structure as a Predictive Factor of Durable Clinical Effect in Immunotherapy for NSCLC

There is urgent need for accurate biomarkers in immunotherapy (IO) for advanced or recurrent lung cancer. In 2020, tertiary lymphoid structure (TLS) of cancer tissue was reported to be related with IO response and is attracting attention as a new biomarker. In the current study, we investigated whether TLS and its lymphocyte distribution are associated with durable clinical effect (DCR) by IO in postoperative recurrent non-small cell lung cancer (NSCLC) patients. Between 2016 and 2019, 55 patients were treated with IO for recurrent NSCLCs. Tumor pathology and TLS were evaluated by H&E and immunohistochemical staining. DCR was defined as ‘tumor maintaining stable disease or better for more than one year after IO’, and the patients were divided to DCR and non-DCR group. Their clinicopathological factors, the number of TLS or tumor infiltrating lymphocytes (TIL) and lymphocytic subset in TLS were compared between the two groups. The median age of all 55 patients was 68 years and 43 (78%) of them were men. Histological types of their tumors were 39 (71%) adenocarcinomas, 8 (15%) squamous cell carcinomas, and 3 (6%) large cell neuroendocrine carcinomas. DCR was observed in 14 (26%) patients, and included significantly younger patients (70 years old or less), more PD-L1 expression scores (TPS), more CD8-positive TIL, more PD1-positive TLS (PD1+TLS) and more immune-related adverse event (irAE, Grade 3 and more) than non-DCR group. There were not significant differences in pathological findings including histological type, pathological stage, pleural invasion, vessel invasion, predominant subtypes in adenocarcinoma. Multivariate analysis revealed that age, PD1+TLS and irAE were independent predictive factors of DCR. In DCR group, 1- and 3-year overall survival (OS) rate after IO treatment was 100 and 81%, respectively, while those in non-DCR group were 37 and 7%, respectively. Their post-IO OS curves were significantly different (p <0.01). We explored predictive factors for DCR in IO-treated recurrent NSCLC patients. PD1+ TLS was an independent positive factor of DCR.

Prognostic value of dickkopf-1 and ß-catenin expression according to the antitumor immunity of CD8-positive tumor-infiltrating lymphocytes in biliary tract cancer.

e16172 Background: Despite recent advancements in the understanding of the molecular biology of biliary tract cancer (BTC), target therapy and immunotherapy have demonstrated only limited efficacy, with cytotoxic systemic therapy still being the most effective treatment in BTC, except for surgery. Thus, this study aimed to analyze the role of DKK1 or β-catenin as a prognostic factor in BTC and determine the clinical association of ß-catenin and DKK1 with CD8+ tumor-infiltrating lymphocyte (TIL). Methods: We used data in The Cancer Genome Atlas (TCGA) Research Network and the clinicopathological data of 145 patients with BTC who had undergone primary radical resection between 2006 and 2016. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded tissue sections, and whole tissue sections of representative tumor samples were used for antigen retrieval. Results: CD8+TIL expression was a significant predictor of favorable overall survival (OS) and recurrence-free survival (RFS) (median OS, 34.9months in TIL-high, 16.7months in TIL-low, P &lt; 0.0001 respectively; median RFS, 27.1months in TIL-high, 10months in TIL-low, P &lt; 0.0001 respectively). Positive ß-catenin expression and high DKK1 expression was also associated with a shorter OS (median OS, 23.95months in positive ß-catenin, 26.1months in negative ß-catenin, P = 0.1009 respectively; median OS, 19.4months in high DKK1, 31.65months in Low DKK1, P = 0.0093 respectively), but not RFS (p = 0.1466, at ß-catenin respectively; p = 0.2924, in DKK1 respectively). In the CD8+TIL-high BTC group, the tumor expression of β-catenin and DKK1 had a significant negative impact on either OS or RFS (p = 0.0146 and p = 0.0112, at ß-catenin respectively; p = 0.0950 and p = 0.3904, in DKK1 respectively). However, in the TIL-low BTC group, there were no differences in OS or RFS according to ß-catenin and DKK1 expression (p = 0.5108 and p = 0.8431, at ß-catenin respectively; p = 0.1127 and p = 0.1095, in DKK1 respectively). Cox regression multivariate analysis demonstrated that CD8+ TIL (hazard ratio [HR], 0.490; 95% confidence interval (CI), 0.303-0.791; p = 0.004) and β-catenin (HR, 1.652; 95% CI, 1.035-2.639; p = 0.036) retained significant association with OS after adjustment for all variables. Conclusions: Among patients with resected BTC, β-catenin and DKK1 protein levels are associated with poor clinical outcomes, whereas high CD8+ TIL levels are associated with good clinical outcomes. This confirms the differential clinical role of Wnt/β-catenin and DKK1 proteins according to TIL expression in BTC.

Tumor microenvironment disparity in multiple primary lung cancers: Impact of non-intrinsic factors, histological subtypes, and genetic aberrations

IntroductionMultiple primary lung cancers (MPLCs) occur in common carcinogenetic risks such as lifestyle, biological aging, immune responses, hormones, and metabolism. Although MPLCs harbor various genetic profiles within the same individuals, differences in the tumor microenvironment (TME) are unclear. We investigated the impact of genetic aberrations, non-intrinsic factors, and pathological subtypes on tumor immunity. Materials and MethodsIn total, 73 surgically resected specimens from 32 patients with MPLC were analyzed. PD-L1 expression in tumor cells (TCs) and immune cells (ICs), CD3-positive tumor-infiltrating lymphocytes (TILs), CD8/CD3 ratios, and FOXP3-positive TILs that compose TMEs were evaluated by immunohistochemistry and classified on a score of 0–2. 38 tumors were sequenced for somatic mutations in 409 cancer-associated genes. ResultsFemales and never or light smokers had a higher incidence of PD-L1-negative tumors and a higher concordance rate. PD-L1 positivity in TCs and ICs was significantly less frequent in EGFR-mutated than in wild-type tumors. Differences in the score of TMEs were observed between the KRAS-mutated-only tumor and the KRAS and TP53-co-mutated tumors, and between the KRAS-mutated-only tumor and the KRAS and STK11-co-mutated tumors. Significantly more FOXP3-high TILs were observed in invasive pathological subtypes than in non-invasive ones. ConclusionComparing TMEs among MPLCs revealed that non-smokers or light smokers and females were unlikely to express PD-L1 regardless of tumor site and confirmed that the EGFR mutations and co-occurring KRAS and STK11 or TP53 mutations were associated with TME. Pathological subtypes may impact the efficacy of immune therapy due to their potential correlations with regulatory T cells.

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.

Clinical Significance of PD-L1 Expression and CD8-Positive Tumor-Infiltrating Lymphocytes in Patients with Cavitary Lung Adenocarcinoma

Clinical Significance of PD-L1 Expression and CD8-Positive Tumor-Infiltrating Lymphocytes in Patients with Cavitary Lung Adenocarcinoma

An increase of CD8+\u2009T cell infiltration following recurrence is a good prognosticator in HNSCC

Programmed death-ligand 1 (PD-L1) expression and CD8-positive tumor-infiltrating lymphocyte (CD8+ TIL) infiltration are essential biomarkers for immune checkpoint inhibitor therapy. The objective of this study was to compare the expression of those biomarkers between initial and recurrent HNSCCs using paired analysis. Prognostic significance of those immunological changes was also investigated. Forty-two consecutive patients with locally recurrent HNSCCs were included. Immunohistochemical staining of CD8 and PD-L1 was done for both initial and recurrent tumor specimens. The IHC findings were verified with mRNA expression profiling. Also, the prognostic impact was analyzed based on overall survival (OS). Recurrent-to-initial (R/I) ratios of CD8+ TILs and PD-L1 were widely variable. CD8+ TIL density and PD-L1 expression decreased in 59.5% and 69% of patients, respectively (R/I ratio < 1). The R/I ratio of CD8A mRNA was significantly higher in patients with a CD8 R/I ratio > 1 (1.7 ± 1.5 vs. 0.6 ± 0.6, p = 0.042). CD8 R/I ratio (> 1) was a good prognosticator for OS (HR 0.293, 95% CI 0.091–0.945, p = 0.040). CD8+ TIL infiltration and PD-L1 expression changed variably following local recurrence of HNSCC. The increase of CD8+ TILs at recurrence was an excellent independent prognosticator.

Expression of PD-L1, PD-L2, and IDO1 on tumor cells and density of CD8-positive tumor-infiltrating lymphocytes in early-stage lung adenocarcinoma according to histological subtype.

This study examined the expression of programmed cell death-ligand 1 (PD-L1), programmed cell death-ligand 2 (PD-L2), and indoleamine 2,3-dioxygenase-1 (IDO1) in tumor cells and cluster of differentiation 8 (CD8)-positive tumor-infiltrating lymphocytes (TILs) in early-stage lung adenocarcinoma according to histological subtypes. We evaluated PD-L1, PD-L2, and IDO1 expression in tumor cells and CD8-positive TILs in surgically resected specimens from 196 stage 0 or I lung adenocarcinoma patients by immunohistochemical staining. We also examined the relationships between the expression of PD-L1, PD-L2, and IDO1 in tumor cells and the density of CD8-positive TILs and clinical factors. Patients were divided into three groups: A, adenocarcinoma in situ and minimally invasive adenocarcinoma (N = 32); B, lepidic predominant invasive adenocarcinoma (IAD; LPA; N = 66); and C, IAD except for LPA (N = 98). PD-L1 was expressed only in Group C, but not in Groups A or B. The positive ratio of PD-L2 was significantly higher in Group C (63.3%), and that of IDO1 was also significantly higher in Group C (65.3%). The density of CD8-positive TILs was significantly higher in Group C (45 ± 2.4). There was no significant difference between the positive ratios of PD-L2 and IDO1 and the density of CD8-positive TILs in Group A (50.0%, 21.9%, and 36 ± 4.1, respectively) or Group B (60.6%, 25.8%, and 44 ± 3.0, respectively). No cases in Groups A and B expressed PD-L1. The expression of immune-related factors, especially PD-L1 and IDO1, was significantly associated with Group C. This is the first report of the detailed examination of PD-L1, PD-L2, IDO1, and CD8 expression in lung adenocarcinoma subtypes with lepidic predominant components. Our results could help identify patients who would benefit from perioperative immunotherapy.

Treatment outcomes of patients with adenocarcinoma of the uterine cervix after definitive radiotherapy and the prognostic impact of tumor-infiltrating CD8+ lymphocytes in pre-treatment biopsy specimens: a multi-institutional retrospective study.

The current study aimed to evaluate the outcomes of patients with adenocarcinoma (AC) of the uterine cervix after definitive radiotherapy (RT) and to evaluate prognostic factors, including immunity-related molecules. A total of 71 patients with AC of the uterine cervix from multiple Japanese institutions were retrospectively analysed. Histological subtypes were diagnosed according to the 2014 World Health Organization classification. All patients underwent definitive RT comprising external beam RT and intracavitary brachytherapy with or without concurrent chemotherapy. Immunohistochemical studies were performed to detect the expression of programmed cell death-ligand 1(PD-L1) and CD8. The 5-year locoregional control (LC), overall survival (OS) and progression-free survival (PFS) rates for all patients were 61.8, 49.7 and 36.1%, respectively. The LC, OS and PFS rates were not significantly different among the histological subtypes. Membranous PD-L1 expression was not significantly associated with prognosis. Patients with CD8-positive tumor-infiltrating lymphocytes (CD8+TILs) in the tumor nests had significantly better OS than patients without CD8+TILs in the tumor nests (5-year OS: 53.8 vs 23.8%, P = 0.038). As expected, the International Federation of Gynecology and Obstetrics (FIGO) stage (2008) III–IVA and maximum tumor diameter > 40 mm were significantly associated with worse prognosis. In summary, the presence of CD8+TILs in the tumor nests has the potential to be an independent favorable prognostic factor for patients with AC of the uterine cervix after definitive RT.