High Density Of Tumor-infiltrating Lymphocytes Research Articles

The combination of CDX2 expression status and tumor-infiltrating lymphocyte density as a prognostic factor in adjuvant FOLFOX-treated patients with stage III colorectal cancers.

Colorectal carcinomas (CRCs) with caudal-type homeobox 2 (CDX2) loss are recognized to pursue an aggressive behavior but tend to be accompanied by a high density of tumor-infiltrating lymphocytes (TILs). However, little is known about whether there is an interplay between CDX2 loss and TIL density in the survival of patients with CRC. Stage III CRC tissues were assessed for CDX2 loss using immunohistochemistry and analyzed for their densities of CD8 TILs in both intraepithelial (iTILs) and stromal areas using a machine learning-based analytic method. CDX2 loss was significantly associated with a higher density of CD8 TILs in both intraepithelial and stromal areas. Both CDX2 loss and a high CD8 iTIL density were found to be prognostic parameters and showed hazard ratios of 2.314 (1.050-5.100) and 0.378 (0.175-0.817), respectively, for cancer-specific survival. A subset of CRCs with retained CDX2 expression and a high density of CD8 iTILs showed the best clinical outcome (hazard ratio of 0.138 [0.023-0.826]), whereas a subset with CDX2 loss and a high density of CD8 iTILs exhibited the worst clinical outcome (15.781 [3.939-63.230]). Altogether, a high density of CD8 iTILs did not make a difference in the survival of patients with CRC with CDX2 loss. The combination of CDX2 expression and intraepithelial CD8 TIL density was an independent prognostic marker in adjuvant chemotherapy-treated patients with stage III CRC.

Phase 2 trial of avelumab in combination with gemcitabine in advanced leiomyosarcoma as a second-line treatment (EAGLES, Korean Cancer Study Group UN18-09).

In this single-arm, multicenter, phase 2 trial, the authors evaluated the efficacy and safety of avelumab plus gemcitabine in patients with leiomyosarcoma (LMS) who failed on first-line chemotherapy. Patients with advanced LMS received avelumab 10 mg/kg on days 1 and 15 (for up to 24 months) plus gemcitabine 1000 mg/m2 on days 1, 8, and 15 of a 28-day cycle until they developed disease progression or intolerable toxicity. The primary end point was the objective response rate (ORR). In total, 38 patients were enrolled. Of these, 35 patients were evaluable, and the ORR was 20% (95% confidence interval; [CI], 8%-37%). The disease control rate was 71%, and the median duration of response was 21.8 months (range, 7.6 to ≥43.3 months). The median progression free-survival was 5.6 months (95% CI, 4.5-6.8 months), and the median overall survival was 27.5 months (95% CI, 20.4-34.6 months). Grade 3-4 adverse events occurred in 70% of patients, of which neutropenia was the most common (54%). Immune-mediated adverse events occurred in five patients (14%; hypothyroidism, n=3; hepatitis, n=2). Patients who had a higher density of tumor-infiltrating lymphocytes (greater than the median) exhibited better ORR (35% vs. 8%; p=.104), progression-free survival (median, 7.3 vs. 3.3 months; p=.024), and overall survival (median, not reached vs. 21.5 months; p=.027). The combination of avelumab and gemcitabine demonstrated promising efficacy and manageable safety in patients with advanced LMS who progressed on first-line therapy. Tumor-infiltrating lymphocyte density may be an important factor in predicting the response to combining immunotherapy with chemotherapy.

Clinical impact of heterogeneously distributed tumor-infiltrating lymphocytes on the prognosis of colorectal cancer.

Tumor-infiltrating lymphocytes (TILs) exist in various malignancies, and have been viewed as a promising biomarker to predict the efficacy and outcome of treatment. However, the marked inter- and intra-tumor heterogeneity of TILs has resulted in some confusion regarding their impact on the prognosis of colorectal cancer (CRC). In this study, 78 CRC patients were enrolled and the CD3+ and CD8+ TILs densities at the tumor center (TC), the invasive margin (IM) and the tumor stroma (TS) were assessed by immunohistochemical staining. Their associations with clinicopathological features and progression free survival (PFS) were analyzed to evaluate the predictive and prognostic values of TILs. TILs were mainly distributed along the invasive margin. High density of TILs in tumor center and invasive margin was associated with smaller tumor size (CD3+TILsIM), reduced tumor invasion (CD3+TILsIM), absence of lymph node metastasis (CD3+TILsIM and CD8+TILsTC), earlier stage (CD3+TILsIM and CD8+TILsIM), and lower tumor grade (CD3+TILsIM and CD8+TILsTC). However, stromal TILs were not associated with any clinicopathological features. Kaplan-Meier survival analysis revealed that high densities of TILs always correlated with prolonged patient survival. The pathological N stage, CD3+ TILsIM and CD8+ TILsTC were found to be independent prognostic indicators. Additionally, early-stage CRC patients who developed recurrence after surgery, showed a higher CD3+/CD8+ TILs ratio in invasive margin. In the present study, it was clarified that CD3+ and CD8+ TILs were heterogeneously distributed in tumor tissues of CRC. The increase in intratumoral and peritumoral TILs had been shown to be strongly predictive of improved clinical outcome. More importantly, the immune signatures enabled to stratify early-stage CRC patients with high risk of recurrence, highlighting the prognostic power of TILs.

Homologous Recombination Deficiency Across Subtypes of Primary Breast Cancer.

Homologous recombination deficiency (HRD) is highly prevalent in triple-negative breast cancer (TNBC) and associated with response to PARP inhibition (PARPi). Here, we studied the prevalence of HRD in non-TNBC to assess the potential for PARPi in a wider group of patients with breast cancer. HRD status was established using targeted gene panel sequencing (360 genes) and BRCA1 methylation analysis of pretreatment biopsies from 201 patients with primary breast cancer in the phase II PETREMAC trial (ClinicalTrials.gov identifier: NCT02624973). HRD was defined as mutations in BRCA1, BRCA2, BRIP1, BARD1, or PALB2 and/or promoter methylation of BRCA1 (strict definition; HRD-S). In secondary analyses, a wider definition (HRD-W) was used, examining mutations in 20 additional genes. Furthermore, tumor BRCAness (multiplex ligation-dependent probe amplification), PAM50 subtyping, RAD51 nuclear foci to test functional HRD, tumor-infiltrating lymphocyte (TIL), and PD-L1 analyses were performed. HRD-S was present in 5% of non-TNBC cases (n = 9 of 169), contrasting 47% of the TNBC tumors (n = 15 of 32). HRD-W was observed in 23% of non-TNBC (n = 39 of 169) and 59% of TNBC cases (n = 19 of 32). Of 58 non-TNBC and 30 TNBC biopsies examined for RAD51 foci, 4 of 4 (100%) non-TNBC and 13 of 14 (93%) TNBC cases classified as HRD-S had RAD51 low scores. In contrast, 4 of 17 (24%) non-TNBC and 15 of 19 (79%) TNBC biopsies classified as HRD-W exhibited RAD51 low scores. Of nine non-TNBC tumors with HRD-S status, only one had a basal-like PAM50 signature. There was a high concordance between HRD-S and either BRCAness, high TIL density, or high PD-L1 expression (each P < .001). The prevalence of HRD in non-TNBC suggests that therapy targeting HRD should be evaluated in a wider breast cancer patient population. Strict HRD criteria should be implemented to increase diagnostic precision with respect to functional HRD.

Spatial Distribution and Densities of CD103+ and FoxP3+ Tumor Infiltrating Lymphocytes by Digital Analysis for Outcome Prediction in Breast Cancer.

The evaluation of tumor-infiltrating lymphocytes (TILs) for breast cancer prognosis is now established. However, the clinical value for their spatial distributions of specific immune subsets, namely CD103+ tissue-resident memory T cells FoxP3+ regulatory T ells, have not been thoroughly examined. Representative whole sections of breast cancers were subjected to CD103 and FoxP3 double staining. Their density, ratio, and spatial features were analyzed in tumor area and tumor-stromal interface. Their associations with clinicopathological parameters and patient's prognosis were analyzed. CD103 TILs were closer to tumor nests than FoxP3 TILs in the tumor-stromal interface. Their densities were associated with high-grade disease, TNBC, and stromal TILs. High stromal FoxP3 (sFoxP3) TILs and close proximity of sCD103 TILs to tumor were independently associated with better survival at multivariate analysis. Subgroup analysis showed the high FoxP3 TILs density associated better survival was seen in HER2-OE and TNBC subtypes while the proximity of CD103 TILs to tumor nests associated better survival was seen in luminal cancers. The prognostic impact of CD103 and FoxP3 TILs in breast cancer depends on their spatial localization. High sFoxP3 TIL density and the lower distance of CD103 TILs from the tumor nests had independent favorable prognostic values.

Identification of cytokine-predominant immunosuppressive class and prognostic risk signatures in glioma.

The advent of immune checkpoint blockade (ICB) therapies this year has changed the way glioblastoma (GBM) is treated. Meanwhile, some patients with strong PD-L1 expression remain immune checkpoint resistant. To better understand the molecular processes that influence the immune environment, there is an urgent need to characterize the immunosuppressive tumor microenvironment and identify biomarkers to predict patient survival outcomes. Our study analyzed RNA-sequencing data from 178 GBM samples. Their unique gene expression patterns in the tumor microenvironment were analyzed by an unsupervised clustering algorithm. Through these expression patterns, a panel of T-cell exhaustion signatures, immunosuppressive cells, and clinical features correlates with immunotherapy response. The presence or absence of immune status and prognostic signatures was then validated with the test dataset. 38.2% of GBM patients showed increased expression of anti-inflammatory cytokines, significant enrichment of T cell exhaustion signals, higher proportion of immunosuppressive cells (macrophages and CD4 regulatory T cells) and nine inhibitory checkpoints (CTLA4, PDCD1, LAG3, BTLA, TIGIT, HAVCR2, IDO1, SIGLEC7, and VISTA). The immunodepleted class (IDC) was used to classify these immunocompromised individuals. Despite the high density of tumor-infiltrating lymphocytes shown by IDC, such patients have a poor prognosis. Although PD-L1 was highly expressed in IDC, it suggested that there might be ICB resistance. There are many IDC predictive signatures to discover. PD-1 is strongly expressed in a novel immunosuppressive class of GBM, but this cluster may be resistant to ICB therapy. A comprehensive description of this drug-resistant tumor microenvironment could provide new insights into drug resistance mechanisms and improved immunotherapy techniques.

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 lymphocytes and tumor-associated macrophages as potential predictors of lymph node metastases in major salivary gland cancers.

The purpose of this study is to define if tumor-infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs) could represent potential predictors of lymph node metastases (LNM) in salivary gland cancers (SGC). A selected number of immunohistochemical markers related to TILs (CD3, CD4, CD68, and FOXP3) and TAMs (CD68 and CD163) were investigated on major salivary gland cancers. TIL and TAM densities were measured on digital images using the open-source QuPath both in the tumor interior (TI) and invasive margin (IM). Correlation with pathologic N classification and follow-up clinical data was investigated. A total of 25 consecutive patients (men: 11; median age: 62.0) were included. Densities of CD3+ IM (OR = 7.7, 95% CI 1.2-51.2), CD8+ TI (OR = 7.7, 95% CI 1.2-51.2), CD8+ IM (OR = 7.7, 95% CI 1.2-51.2), FOXP3+ TI (OR = 24.0, 95% CI 2.2-255.9), CD68+ TI (OR = 7.7, 95% CI 1.2-51.2), and CD163+ IM (OR = 7.7, 95% CI 1.2 - 51.2), and the Immunoscore CD8/CD3 (OR = 1.9, 95% CI 1.1-3.4) were significantly associated with LNM (p < 0.05). CD3+ TI density was significantly associated with tumor recurrence and death (HR = 5.8, 95% CI 1.5-22.6; p < 0.05). A high density of specific TIL and TAM subpopulations might be correlated with a higher probability of LNM in SGC.

Prognostic Impact of the Immune-Cell Infiltrate in N1-Positive Non–Small-Cell Lung Cancer

IntroductionThe tumoral immune milieu plays a crucial role for the development of non–small-cell lung cancer (NSCLC) and may influence individual prognosis. We analyzed the predictive role of immune cell infiltrates after curative lung cancer surgery. Materials and MethodsThe tumoral immune-cell infiltrate from 174 patients with pN1 NSCLC and adjuvant chemotherapy was characterized using immunofluorescence staining. The density and distribution of specific immune cells in tumor center (TU), invasive front (IF) and normal tissue (NORM) were correlated with clinical parameters and survival data. ResultsTumor specific survival (TSS) of all patients was 69.9% at 5 years. The density of tumor infiltrating lymphocytes (TIL) was higher in TU and IF than in NORM. High TIL density in TU (low vs. high: 62.0% vs. 86.7%; p = .011) and the presence of cytotoxic T-Lymphocytes (CTLs) in TU and IF were associated with improved TSS (positive vs. negative: 90.6% vs. 64.7% p = .024). High TIL-density correlated with programmed death-ligand 1 expression levels ≥50% (p < .001). Multivariate analysis identified accumulation of TIL (p = .016) and low Treg density (p = .003) in TU as negative prognostic predictors in squamous cell carcinoma (p = .025), whereas M1-like tumor- associated macrophages (p = .019) and high programmed death-ligand 1 status (p = .038) were associated with better survival in adenocarcinoma. ConclusionThe assessment of specific intratumoral immune cells may serve as a prognostic predictor in pN1 NSCLC. However differences were observed related to adenocarcinoma or squamous cell carcinoma histology. Prospective assessment of the immune-cell infiltrate and further clarification of its prognostic relevance could assist patient selection for upcoming perioperative immunotherapies.

Artificial intelligence–powered tumor-infiltrating lymphocytes analyzer to reveal distinct immune landscapes in breast cancer by molecular subtype and HER2 score.

1049 Background: Tumor-infiltrating lymphocytes (TIL) reflect an ongoing anti-tumor immune response in the tumor microenvironment (TME) and have been studied as a predictive as well as prognostic biomarker in breast cancer (BC). The immune profile differs depending on BC molecular subtypes, with triple-negative BC (TNBC) and HER2-positive BC more frequently infiltrated by higher numbers of TIL than hormone receptor (HR)-positive tumors. In this study, we analyze TIL and immune phenotype by molecular subtype in BC and explore differences based on HER2 scores using an AI-powered TIL analyzer. Methods: A total of 1,973 cases of BC with molecular subtype, HER2 immunohistochemistry (IHC) or in situ hybridization (ISH) results, and TIL analysis from H&amp;E slides from TCGA (n=1021), Samsung Medical Center (SMC, n=663), and KyungHee Medical Center (KHMC, n=289) were included in the analysis. The density of TIL were spatially analyzed from H&amp;E WSI, and the immune phenotype (IP) was analyzed for each 1 mm2-sized patch. Tumor microenvironment (TME) was categorized into three types: inflamed (TIL distributed intratumorally), Immune-excluded (TIL excluded, out of cancer stroma), immune-desert (scant TIL in TME), and the score for each IP was calculated as the percentage of the total patches in WSI. Results: TNBC showed the highest TIL density in both cancer epithelium (214.5 ± 299.7/mm2) and stroma (1133.3 ± 1245.9/mm2), and a high percentage of inflamed IP (35.7%), IS (28.4 ± 28.1) than other subtypes (inflamed IP: 17.5%, IS 16.2±20.2, both p&lt;0.001). HER2-positive BCs showed higher sTIL density (904.0 ± 901.7/mm2) compared to HR- positive BC (616.7 ± 794.1/mm2) but were associated with a higher percentage of immune-excluded IP (71.2%) and IES (57.6 ± 24.6). HR-positive BC had a high percentage of immune-desert IP (30.4%). In both HR-positive and negative BCs, higher IES was observed as the HER2 score increased. HER2-positive BC significantly showed a higher proportion of cancer stroma in the cancer region from WSI (Stromal ratio, 0.682 ± 0.149 vs. 0.578 ± 0.176, p&lt;0.001). Conclusions: Different TIL densities and IP were seen in BC for each molecular subtype, pointing to a distinct immune landscape. HER2 score was associated with immune-excluded IP despite having higher TIL density, which should be considered for immunotherapy in BC.

Abstract 2266: Tumor associated neutrophils (TAN) may produce neutrophil extracellular traps (NETs) which can suppress the infiltration of activated T cells in tumor tissue

Abstract Objective: High density of tumor-infiltrating lymphocytes (TIL) in the tumor is associated with favorable prognosis of cancer patients. It has been reported that many neutrophils also infiltrate in various tumor tissues as tumor-associated neutrophils (TAN) which produce considerable amounts of neutrophil extracellular traps (NETs). However, the crosstalk between TAN and TIL and its impact on tumor behavior have not been fully examined. Methods: Neutrophils and mononuclear cells (PBMC) were collected from the blood of healthy donor. Neutrophils were stimulated with PMA or LPS for 15 min, washed extensively, and incubated for another 4 hours to produce NETs. PBMC were cultured on anti-CD3 mAb-coated plate with recombinant-IL-2 for 7-14days. The migration of activated T cells through 3μm pore to CXCL-11 was examined in vitro. The localization and densities of TAN and TIL were immunohistochemically evaluated in 38 human serous ovarian cancer tissue. NETs were quantified with the density of CD66b(+) and Citrullinated Histon H3(+) cells. Results: Chemotactic migration of T cells to CXCL-11 was inhibited by 99%±1.6% (p&amp;lt;0.001) in the presence of PMA-activated neutrophils in lower chamber. The inhibition was similarly detected when NET components were removed by ultracentrifugation. Timelapse analysis also showed the NETs-depleted supernatant inhibited random migration, which was totally canceled by the pretreatment with catalase. When neutrophils were activated by LPS, T cell migration was also inhibited by 49%±19.9% (p&amp;lt;0.01), but not by NETs-depleted supernatant. In western blotting, CXCL11 was totally degraded by LPS-stimulated neutrophils, which was greatly reduced by the depletion of NET component and restored by DNase I as well as phenylmethylsulfonyl fluoride (PMSF) or Neutrophil elastase inhibitor (NEI). The reduced migration induced by LPS-stimulated neutrophils was also restored with these reagents. In ovarian cancer tissues, density of TIL was greater in tumors with diameter ≥10cm than that of tumors &amp;lt;10cm (p=0.04). However, the densities of TIL and NETs were irrelevant with age, tumor stage and neutrophil-to-lymphocyte ratio (NLR) in peripheral blood. Multicolor immunohistochemistry showed that TAN and TIL tended to be reciprocally distributed in tumor stroma in most of the ovarian cancer tissues. Cit-H3 was positively detected in 2.1%~75% of CD66b(+) TANs, and the proportion of NET in TAN greatly varied among tumors. In all cases, the densities of CD4(+) and CD8(+) T cells inversely correlated with that of NETs (r = -0.5182, p = 0.0012; r = -0.5684, p = 0.0003, respectively). Conclusion: Activated neutrophils negatively regulate T cell migration partially through NET formation. TAN may suppress T cell traffic into tumor tissue, which may attenuate cell-mediated immunity in tumor microenvironment and promote tumor progression. Citation Format: Kohei Tamura, Hideyo Miyato, Misaki Matsumiya, Rei Takahashi, Yuki Kaneko, Yurie Futoh, Kazuya Takahashi, Yuki Kimura, Akira Saito, Hideyuki Ohzawa, Yasushi Saga, Yuji Takei, Hiroyuki Fujiwara, Joji Kitayama. Tumor associated neutrophils (TAN) may produce neutrophil extracellular traps (NETs) which can suppress the infiltration of activated T cells in tumor tissue [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 2266.

VISTA+/CD8+ status correlates with favorable prognosis in Epithelial ovarian cancer.

Immunotherapy by blocking immune checkpoint regulators has emerged as a new targeted therapy for some cancers. Among them V-domain Ig suppressor of Tcell activation (VISTA) which is identified as a novel checkpoint regulator in ovarian cancer. This study aimed to investigate the VISTA role in Epithelial ovarian cancer (EOC), and its relationship with tumor-infiltrating lymphocytes (TILs) markers and its prognostic value. The expression of VISTA, CD3, CD8, CD4, FOXP3, and CD56 was assessed in 168 EOC tissue microarrays (TMA) by immunohistochemistry (IHC). In addition, associations between VISTA, TILs, clinicopathological variables, and overall survival (OS) were analyzed. VISTA expression in IGRov1 cells, as well as in PBMC of EOC patient, was evaluated by western blot. VISTA expression was detected in 64,28% of tissues, among which 42.3% were positive for tumor cells (TCs), and 47,9% were positive for immune cells (ICs). In univariate analysis, VISTA expression was significantly associated with a high density of TILs:CD3+ (p = 0,001), CD4+ (p = 0,002) and CD8+ (p≤0,001), in ICs but not in TCs. In terms of OS, multivariate analysis showed a significant association between the high density of CD8+ TILs and VISTA positive staining in ICs (p = 0,044), but not in TCs (p = 0,108). Kaplan-Meier curves demonstrated no correlation between VISTA expression and prolonged OS in both ICs (p = 0,841) and TCs (p = 0,090). Classification of EOC tumor microenvironment based on VISTA and CD8+TILs expression, demonstrated four immune subtypes: VISTA+/CD8+, VISTA+/CD8-, VISTA-/CD8+ and VISTA-/CD8-. The dual positive VISTA+/CD8+ subtype was significantly associated with prolonged OS in both TCs and ICs (p = 0,012 and p≤0,01, respectively), whereas patients with VISTA+/CD8- had the worst OS. Our results showed that VISTA is highly expressed in the IGRov1 cell line and LT-CD8 from a patient with EOC. Our results highlighted the association of VISTA expression and CD8+ TILs in EOC, with prolonged OS in patients with VISTA+/CD8+ and proposed VISTA as a potential immunotherapeutic target in EOC.

Discordance of PD-L1 expression in primary and metastatic ovarian high-grade serous carcinoma and its correlation with CD8 + tumor-infiltrating lymphocytes and patient prognosis.

Differential expression of programmed death-1 ligand (PD-L1) and its clinical significance in primary and metastatic ovarian high-grade serous carcinoma (HGSC) have not been defined. Thus, we investigated the PD-L1 expression of paired ovarian primary and omental metastatic HGSC and its correlation with CD8 + tumor-infiltrating lymphocyte (TILs) and patient survival. A total of 212 cases of ovarian HGSCs with matched primary ovarian and metastatic omental tumors accessioned between 2003 and 2018 were selected for further analysis. Using immunohistochemistry, we evaluated the density of CD8 + TILs and expression of PD-L1 on whole tissue sections. Applying tumor proportion score (TPS, cutoff 1%) and combined positive score (CPS, cutoff 1), the prevalence of PD-L1 expression was similar but with significant discordance in ovarian and omental tumor. Using TPS, patients with PD-L1-positive tumors demonstrated significantly worse recurrence free survival (RFS) and overall survival (OS) than patients with PD-L1-negative tumors. Using CPS, patients with PD-L1-positive ovarian tumors demonstrated significantly worse OS while no significant difference in RFS was found. Patients with PD-L1-positive omental tumors demonstrated significantly worse RFS and OS. Patients with omental PD-L1-positive tumors (TPS) were associated with poorer RFS and OS, while patients with ovarian PD-L1-positive tumors (TPS) were associated with OS not RFS, in COX multivariant analysis. Nonetheless, ovarian and omental high CD8 TILs density was not associated with worse OS in univariant and COX multivariant analysis. PD-L1 expression in ovarian and omental tumor associated with an increased CD8 + TILs density. PD-L1 expression by TPS was better correlated with survival than by CPS, and PD-L1 expression in omental tumors was a stronger prognostic indicator than that in ovarian tumors.

Artificial intelligence (AI)-powered immune phenotyping to predict outcomes of immuno-oncology (IO)-based regimens in hepatocellular carcinoma (HCC).

601 Background: Anti-PD-(L)1 monotherapy or combination with bevacizumab or ipilimumab are approved treatment options in HCC. Nevertheless, not all patients benefit from IO treatment, and discovery of predictive biomarkers for IO outcome has not been fully investigated. Immune phenotype analysis by AI-powered spatial tumor-infiltrating lymphocyte (TIL) analyzer in H&amp;E whole-slide images (WSI), Lunit SCOPE IO, has shown predictive effect across multiple cancer types. Here, we investigated the correlation between immune phenotype (IP) and the real-world outcomes in HCC. Methods: Real-world dataset of 177 patients with HCC were retrospectively collected from CHA Bundang Medical Center. Inflamed score was defined by the proportion of area with high intra-tumoral TIL density per 0.5 mm2-sized grid. IP status was sub-analyzed by PD-L1 combined positive score (CPS) and prognostic factors including Child-Pugh class and Barcelona Clinic Liver Cancer (BCLC) stage. We evaluated the correlation between IP and IO outcome by regimen (atezolizumab+bevacizumab [Atez+Bev], n=100; nivolumab+/-ipilimumab [Niv+/-Ipi], n=77). Results: The majority of patients (75.3%) were Child-Pugh class A (75.3%) and BCLC stage C (86.5%), respectively. Atez+Bev was mostly received as first-line therapy (92.0%), whereas Niv+/-Ipi as second-line or beyond (98.7%). The inflamed score was significantly higher in patients who had lymph node metastasis ( p=0.008), but it was not correlated with other metastatic sites, or any other prognostic factors. Of 177 patients, 105 (59.3%) patients were classified as having a high inflamed score (≥10%) and 72 (40.7%) patients as having lower than 10%. PD-L1 CPS was significantly higher in the sample with high score compared to those with low inflamed score ( p=0.01387). PFS of Niv+/-Ipi were significantly favorable in the patients with high inflamed score (3-m PFS rate; 44.0% vs 3.61%, 12-m PFS rate; 17.8% vs 0%, Hazard ratio [HR] 0.37, 95% confidence interval [CI] 0.22-0.63, p&lt;0.001), while PFS of Atez+Bev were not different according to inflamed score (3-m PFS rate; 67.5% vs 57.3%, 12-m PFS rate; 36.6% vs 31.0%, HR 0.80, 95% CI 0.48-1.32, p=0.400). Conclusions: Inflamed score, the proportion of area with high intra-tumoral TIL infiltration, can be a clinically significant predictor of PFS of Niv+/-Ipi, but it was not relevant for Atez+Bev treatment outcomes.

Analysis of PD-L1 and CD3 Expression in Glioblastoma Patients and Correlation with Outcome: A Single Center Report.

With the advent of immunotherapies, the field of cancer therapy has been revived with new hope, especially for cancers with dismal prognoses, such as the glioblastoma multiforme (GBM). Currently, immunotherapies should potentiate the host's own antitumor immune response against cancer cells, but it has been documented that they are effective only in small subsets of patients. Therefore, accurate predictors of response are urgently needed to identify who will benefit from immune-modulatory therapies. Brain tumors are challenging in terms of treatments. The immune response in the brain is highly regulated, and the immune microenvironment in brain metastases is active with a high density of tumor-infiltrating lymphocytes (TILs, CD3+ T cells) in certain patients and, therefore, may serve as a potential treatment target. In our study, we performed immunohistochemistry for CD3 and PD-L1 along the routine assessment of the O6-methylguanine-methyltransferase (MGMT) promoter methylation status and the IDH1 and 2 status in a single center cohort of 69 patients with GBM (58 primary tumors and 11 recurrences) who underwent standard multimodal therapies (surgery/radiotherapy/adjuvant temozolamide). We analyzed the association of PD-L1 tumor expression and TILs with overall survival (OS). The PD-L1 expression was observed in 25 of 58 (43%) newly diagnosed primary glioblastoma specimens. The sparse-to-moderate density of TILs, identified with CD3+ expression, was found in 48 of 58 (83%) specimens. Neither PD-L1 expression nor TILs were associated with overall survival. In conclusion, TILs and/or PD-L1 expression are detectable in the majority of glioblastoma samples, and even if they slightly relate to the outcome, they do not show a statistically significant correlation.

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.

High co-expression of immune checkpoint receptors PD-1, CTLA-4, LAG-3, TIM-3, and TIGIT on tumor-infiltrating lymphocytes in early-stage breast cancer

High expression of immune checkpoint receptors (ICRs) in the tumor microenvironment regulates the anti-tumor response. In this study, the differential expressions of ICRs on tumor-infiltrating lymphocytes (TILs) in patients with early-stage breast cancer were investigated.The study included 32 patients who underwent surgery with a diagnosis of early-stage breast cancer between September 2018 and March 2020. TIL isolation was performed using a MACS tumor separation device and tumor separation kit. PD-1, CTLA-4, LAG-3, TIM-3, and TIGIT expression of cytotoxic T and natural killer (NK) cells on TILs and peripheral blood lymphocytes (PBLs) were determined by flow cytometry.Patients with a high Ki-67 index, high TIL density, and HER-2 positivity were more likely to have increased CD16+CD56dim NK cells on TILs. Patients with T2 tumors were more likely to have increased expression of PD-1, LAG-3, and TIGIT on tumor-infiltrating CD8+ cytotoxic T cells than those with T1 tumors. PD-1, CTLA-4, TIGIT, LAG-3, and TIM-3 expression of CD8+ T and CD16-CD56bright NK cells in TILs showed significant positive correlations with each other. PD1+CD8+, TIGIT+CD16+, and CTLA-4+CD56+ cells in PBLs and TILs were found to be negatively correlated, whereas only TIM-3+ expression of CD8+ T and CD16+CD56dim cells in PBLs and TILs showed positive correlations.Our results suggest that CD16+CD56dim NK cells on TILs may play a major role in the immune response against HER2-positive or highly proliferating breast tumors in patients with early-stage breast cancer. Furthermore, various ICRs were found to be highly co-expressed with each other on TILs, including PD-1, CTLA-4, LAG-3, TIM-3, and TIGIT. These receptors may synergistically suppress the response to the tumor, which may trigger immune escape mechanisms in the early stage of carcinogenesis. However, ICR expressions other than TIM3 on PBLs were not found to accompany their counterparts on TILs.

Abstract 6309: Activated neutrophils inhibit chemotactic migration of activated T lymphocytes to CXCL11 by multiple mechanisms

Abstract Objective High density of tumor-infiltrating lymphocytes (TILs) in the tumor is associated with favorable prognosis of cancer patients, while that of tumor-associated neutrophils (TAN) shows inverse correlation with outcome. We examined the effects of activated neutrophils on chemotactic migration of activated T cells in vitro. Methods Neutrophils and mononuclear cells (PBMC) were collected from the blood of healthy donor. Neutrophils were stimulated with PMA (1uM) or LPS (10ug/ml) for 15 min, washed extensively, and incubated for another 4 hours. PBMC were cultured on anti-CD3 mAb-coated plate with recomnibant-IL-2 (10ng/ml) for 7-14days. The activated T cells were placed in culture inserts with 3μm pore and migration to CXCL-11 (1000ng/ml) placed in lower chamber with or without neutrophils was examined after 2 hours. Random migration of activated T cells on fibronectin coated plate was evaluated with timelapsed analysis using Biostudio system. Degradation of CXCL-11 was examined with western blotting. Results Neutrophils stimulated with PMA or LPS produced neutrophil extracellular traps (NETs). Migration of T cells to CXCL-11 was drastically inhibited in the presence of PMA-activated neutrophils. The inhibition was similarly detected when NETs was removed from PMA-activated neutrophils by ultracentrifugation. Timelapse analysis also showed the NET-depleted supernatant greatly inhibited random migration, which was totally canceled by the pretreatment with 800U/ml catalase. When neutrophils were activated by LPS, T cell migration was also significantly inhibited (p=0.02) but not by NET-depleted supernatant (p=0.99). In western blotting, CXCL11 was totally degraded by LPS-stimulated neutrophils, which was greatly reduced by the depletion of NET component. CXCL11 degraded by NETs was restored by protease inhibitor, 5mM phenylmethylsulfonyl fluoride (PMSF). The reduced migration induced by LPS-stimulated neutrophils was mostly restored with 2mM PMSF. Conclusion Activated neutrophils inhibit chemotactic migration of activated T cells through multiple mechanisms including ROS production and chemokine degradation. TAN may have suppressive effects on the accumulation of TILs. Citation Format: Kohei Tamura, Yuki Kaneko, Yurie Futoh, Kazuya Takahashi, Yuki Kimura, Akira Saito, Mineyuki Tojo, Hideyo Miyato, Hideyuki Ohzawa, Yasushi Saga, Yuji Takei, Hiroyuki Fujiwara, Joji Kitayama. Activated neutrophils inhibit chemotactic migration of activated T lymphocytes to CXCL11 by multiple mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6309.

Prognostic and Predictive Relevance of Tumor-Infiltrating Lymphocytes in Squamous Cell Head-Neck Cancer Patients Treated with Radical Radiotherapy/Chemo-Radiotherapy.

Microenvironmental conditions control the entrance and thriving of cytotoxic lymphocytes in tumors, allowing or preventing immune-mediated cancer cell death. We investigated the role of tumor-infiltrating lymphocyte (TIL) density in the outcome of radiotherapy in a series of squamous cell head–neck tumors (HNSCC). Moreover, we assessed the link between markers of hypoxia and TIL density. One-hundred twenty-one patients with HNSCC treated prospectively with radical radiotherapy/chemo-radiotherapy were analyzed. The assessment of TIL density was performed on hematoxylin and eosin biopsy sections before radiotherapy. TIL density ranged from 0.8 to 150 lymphocytes per ×40 optical field (median 27.5). Using the median value, patients were grouped into two categories of low and high TIL density. Early T-stage tumors had a significantly higher TIL density (p < 0.003), but we found no association with N-stage. Overexpression of HIF1α, HIF2α, and CA9 was significantly linked with poor infiltration by TILs (p < 0.03). A significant association of high TIL density with better disease-specific overall survival and improved locoregional relapse-free survival was noted (p = 0.008 and 0.02, respectively), which was also confirmed in multivariate analysis. It is concluded that HNSCC phenotypes that allow for the intratumoral accumulation of lymphocytes have a better outcome following radical radiotherapy/chemo-radiotherapy. Intratumoral-activated HIF- and CA9-related pathways characterize immunologically cold tumors and may be used as targets for therapeutic interventions.

Artificial intelligence (AI)–powered spatial analysis of tumor-infiltrating lymphocytes (TIL) for prediction of response to neoadjuvant chemotherapy (NAC) in triple-negative breast cancer (TNBC).

595 Background: Stromal TIL are a well-recognized prognostic and predictive biomarker in breast cancer. There is a need for tools assisting visual assessment of TIL, to improve reproducibility as well as for convenience. This study aims to assess the clinical significance of AI-powered spatial TIL analysis in the prediction of pathologic complete response (pCR) after NAC in TNBC patients. Methods: H&amp;E stained slides and clinical outcomes data were obtained from stage I – III TNBC patients treated with NAC in two centers in Korea. For spatial TIL analysis, we used Lunit SCOPE IO, an AI-powered H&amp;E Whole-Slide Image (WSI) analyzer, which identifies and quantifies TIL within the cancer or stroma area. Lunit SCOPE IO was developed with a 13.5 x 109 micrometer2 area and 6.2 x 106 TIL from 17,849 H&amp;E WSI of multiple cancer types, annotated by 104 board-certified pathologists. iTIL score and sTIL score were defined as area occupied by TIL in the intratumoral area (%) and the surrounding stroma (%), respectively. Immune phenotype (IP) of each slide was defined from spatial TIL calculation, as inflamed (high TIL density in tumor area), immune-excluded (high TIL density in stroma), or desert (low TIL density overall). Results: A total of 954 TNBC patients treated from 2006 to 2019 were included in this analysis. pCR (ypT0N0) was confirmed in 261 (27.4%) patients. The neoadjuvant regimens used were mostly anthracycline (97.8%) and taxane (75.1%) -based, with 116 (12.1%) patients receiving additional platinum and 41 (4.3%) patients treated as part of immune checkpoint inhibitor or PARP inhibitor clinical trials. The median iTIL score and sTIL score were 4.3% (IQR 3.2 – 5.8) and 8.1% (IQR 6.3 – 13.4), respectively. The mean iTIL score was significantly higher in patients who achieved pCR after NAC (5.8% vs. 4.5%, p &lt; 0.001), and a similar difference was observed with sTIL score (12.1%.1 vs. 9.4%, p &lt; 0.001). iTIL score was found to remain as an independent predictor of pCR along with cT stage and Ki-67 in the multivariable analysis (adjusted odds ratio 1.211 (95% CI 1.125 – 1.304) per 1 point (%) change in the score, p &lt;0.001). By IP groups, 291 (30.5%) patients were classified as inflamed, 502 (52.6%) as excluded, and 161 (16.9%) as desert phenotype. The patients with inflamed phenotype were more likely to achieve pCR (44.7%) than other phenotypes (19.8%, p &lt; 0.001). Conclusions: AI-powered spatial TIL analysis could assess TIL densities in the cancer area and surrounding stroma of TNBC, and TIL density scores and IP classification could predict pCR after NAC.