High Density Of Tumor-infiltrating Lymphocytes Research Articles

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

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

The inflamed immune phenotype (IIP): A clinically actionable artificial intelligence (AI)-based biomarker predictive of immune checkpoint inhibitor (ICI) outcomes across >16 primary tumor types.

2621 Background: The IIP, defined by enriched intratumoral tumor-infiltrating lymphocytes (TIL), is a potential tumor-agnostic biomarker of responsiveness to ICI therapy. Here, we validate the IIP, as assessed by Lunit SCOPE IO, an AI-powered spatial TIL analyzer that runs on routine H&E-stained whole-slide images (WSI), for clinical outcome prediction in a large, multi-center international cohort of ICI-treated patients, demonstrating its utility as a practical biomarker to guide ICI treatment planning. Methods: Lunit SCOPE IO was developed using 17,849 H&E WSI of multiple cancer types, annotated by 104 board-certified pathologists (13.5 x 109 µm2 area and 6.2 x 106 TIL). IIP+ tumors were defined as those with ≥ 20% of all 1 mm2 tumor tiles in a WSI classified as having a high intratumoral TIL density. We evaluated the correlation between IIP and ICI treatment outcomes (overall response rate (ORR) and progression-free survival (PFS), assessed by RECIST v1.1) in a real-world dataset of 1,806 patients ( > 16 primary tumor types) retrospectively collected from Stanford University Medical Center, Samsung Medical Center, Chonnam National University Hospital, Seoul National University Bundang Hospital, and Northwestern University. IIP status was sub-analyzed by PD-L1 22C3 tumor proportion score (TPS, n = 798), microsatellite status, and tumor mutational burden (TMB, n = 130). Results: The IIP+ phenotype (35.2%, 636 of 1,806) was highly enriched in nasopharyngeal carcinoma (68.0%), melanoma (56.3%), renal cell carcinoma (52.9%), and non-small cell lung cancer (NSCLC, 33.7%). The IIP+ proportion by PD-L1 TPS ( < 1% / ≥ 1%) was 21.6% and 40.7%, respectively. While 33.3% of microsatellite unstable (MSI-H) or TMB-high (≥ 10/Mb) tumors were IIP+, a substantial proportion (26.1%) of microsatellite stable (MSS), TMB-low tumors were IIP+. The ORR in IIP+ patients was significantly higher (26.0% vs. 15.8% in IIP-, p < 0.001). Median PFS for IIP+ was 5.3 months (95% CI 4.6-6.9 m), significantly longer than that for IIP- (3.1 m, 95% CI 2.8-3.6 m), with a hazard ratio (HR) of 0.68 (95% CI 0.61-0.76, p < 0.001). The association held after excluding NSCLC patients (n = 909) (HR 0.69, 95% CI 0.59-0.81, p < 0.001). On subgroup analysis, IIP+ correlated significantly with prolonged PFS, regardless of ICI regimen (mono / combo therapy) or PD-L1 TPS ( < 1% / ≥ 1%). Of note, IIP+ was predictive of favorable PFS only in the MSS, TMB-low group (n = 88, HR 0.56, 95% CI 0.33-0.96), but not in the MSI-H or TMB-high groups. Conclusions: The IIP, as evaluated by Lunit SCOPE IO, may represent a practical, clinically-actionable biomarker predictive of favorable ICI treatment outcomes across diverse cancer patient populations, including those with PD-L1 negative, MSS/TMB-low tumors, in whom predictive biomarkers are urgently needed.

Concordance, Correlation, and Clinical Impact of Standardized PD-L1 and TIL Scoring in SCCHN.

Simple SummaryIn patients with relapsed or metastasized squamous cell cancer of the head and neck (R/M SCCHN), the PD-L1 Combined Positive Score (CPS) is currently the only predictive biomarker for treatment with anti-PD-1 agents. However, ambiguous results have been determined regarding the overall response rates of immunotherapeutic agents based on PD-L1 status, which may be partially attributed to spatiotemporal heterogeneity. Furthermore, tumor-infiltrating lymphocytes (TILs) have proven to be of significant prognostic value, yet lack of a standardized method for quantification impedes their integration into the current armamentarium of biomarkers in SCCHN. In this paper, concordance of PD-L1 CPS and stromal TILs was investigated in different paired samples of SCCHN subtypes. The results were then linked to well-known clinicopathological variables and prognosis.Background: The clinical significance of tumor-infiltrating lymphocytes (TILs) and programmed cell death-ligand 1 (PD-L1) expression has been thoroughly researched in squamous cell carcinoma of the head and neck (SCCHN). To address the impact of intra- and intertumoral heterogeneity in these biomarkers, we explored the concordance of PD-L1 combined positive score (CPS) and stromal TILs in different paired tissue sample types, while evaluating their internal relationship and prognostic impact. Methods: A total of 165 tissue blocks from 80 SCCHN patients were reviewed for TILs and PD-L1 CPS. Concordance between paired tissue samples was evaluated, and their association with several clinicopathological variables, overall survival (OS), and disease-free survival (DFS) was determined. Results: Biopsies and paired resection material were severely discordant in 39% and 34% of samples for CPS and TIL count, respectively, of which CPS was underscored in 27% of biopsies. In paired primary tumor–metastatic lesions, the disagreement was lower for CPS (19%) but not for TIL count (44%). PD-L1 CPS was correlated with prolonged OS when calculated from tissue acquirement, while extended OS and DFS were observed for high TIL density. Conclusion: Intertumoral and, especially, intratumoral heterogeneity were confounding factors when determining PD-L1 CPS and TIL count on paired tissue samples, indicating the increasing necessity of assessing both biomarkers on representative tissue material. Although TILs hold valuable prognostic information in SCCHN, the robustness of PD-L1 as a biomarker in SCCHN remains ambiguous.

Prognostic value of immune factors in the tumor microenvironment of patients with pancreatic ductal adenocarcinoma

BackgroundBoth activated tumor-infiltrating lymphocytes (TILs) and immune-suppressive cells, such as regulatory T cells (Tregs), in the tumor microenvironment (TME) play an important role in the prognosis of patients with pancreatic ductal adenocarcinoma (PDAC).MethodsThe densities of TILs, programmed death receptor 1 (PD-1) + T cells, and forkhead box P3 (Foxp3) + T cells were analyzed by immunohistochemical staining. The associations of the immunological status of the PDAC microenvironment with overall survival (OS) time and disease-free survival (DFS) time were evaluated.ResultsPDAC patients with a high density of TILs in the TME or PD-1-positive T cells in tertiary lymphoid aggregates (TLAs) demonstrated a significantly better prognosis than those with a low density of TILs or PD-1-negativity, respectively. Moreover, PDAC patients with high levels of Foxp3-expressing T cells showed a worse prognosis than those with low levels of Foxp3-expressing T cells. Importantly, even with a high density of the TILs in TME or PD-1-positive T cells in TLAs, PDAC patients with high levels of Foxp3-expressing T cells showed a worse prognosis than patients with low levels of Foxp3-expressing T cells. A PDAC TME with a high density of TILs/high PD-1 positivity/low Foxp3 expression was an independent predictive marker associated with superior prognosis.ConclusionCombined assessment of TILs, PD-1+ cells, and Foxp3+ T cells in the TME may predict the prognosis of PDAC patients following surgical resection.

PD-L1 expression, tumor-infiltrating lymphocytes, mismatch repair deficiency, EGFR alteration and HPV infection in sinonasal squamous cell carcinoma

The antitumor efficacies of immune checkpoint inhibitors (ICIs) and the usefulness of potential predictive markers such as programmed death-ligand 1 (PD-L1) expression, density of tumor-infiltrating lymphocytes (TILs) and microsatellite instability (MSI) in sinonasal squamous cell carcinoma (SNSCC) have not been fully elucidated. We retrospectively analyzed 131 SNSCCs with immunohistochemistry for PD-L1 expression, TIL subpopulations and loss of mismatch repair (MMR) proteins as a surrogate for MSI-high. We also comprehensively evaluated the mutual relationships among these immuno-markers, high-risk human papillomavirus (HPV) infection, epidermal growth factor receptor (EGFR) gene status, and KRAS mutation. PD-L1 expression (tumor proportion score ≥ 1%) was detected in 60 (45.8%) SNSCC cases and was significantly associated with worse overall survival (OS) (p = 0.0240). High density of cluster of differentiation 8 (CD8)-positive TILs was significantly associated with better progression-free survival (PFS) (p = 0.0368), and high density of forkhead box protein P3-positive TILs was significantly associated with better PFS and OS (p = 0.0007 and 0.0143, respectively). With respect to the combination of CD8 + TIL and PD-L1 expression, the high-CD8/PD-L1-negative group showed the most favorable prognosis, whereas the low-CD8/PD-L1-positive group showed the worst prognosis. MMR loss was detected in 3 (2.3%) of the 131 cases. HPV infection (6.1%), EGFR mutation (14.5%), EGFR copy number gain (26%), and MMR loss were essentially mutually exclusive; patients in these molecular groups showed significant differences in prognosis but not in the degree of PD-L1 expression or TILs. Among the nine ICI-treated patients, three (33.3%) were responders, and the EGFR-wild type cases (n = 7) showed better clinical responses to an ICI compared to the EGFR-mutant cases (n = 2). Among the patients with residual/recurrent EGFR-wild type tumors (n = 43), ICI treatment significantly improved OS (p = 0.0281). The results suggest that the evaluation of immuno-markers and molecular subclassification may be helpful for prognostic prediction and selecting an individualized therapeutic strategy for patients with SNSCC.

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

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

194P Stromal tumor-infiltrating lymphocyte scores in core needle biopsy and its correlation to complete pathological response to neoadjuvant systemic therapy

Neoadjuvant systemic therapy (NAST) is increasingly being used to treat locally advanced breast cancer with the goal of less extensive surgery of the breast and axilla. The best possible outcome of NAST is complete pathological response (pCR) in both sites. A high density of tumor-infiltrating lymphocytes (TIL) is prognostic for pCR to NAST in some histologic subtypes. The aim of our study was to prospectively assess TIL and pCR correlation on core needle biopsy (CNB) samples in patients before NAST.

Molecular Characterization of Biliary Tract Cancer Predicts Chemotherapy and Programmed Death 1/Programmed Death-Ligand 1 Blockade Responses.

Biliary tract cancer (BTC) exhibits diverse molecular characteristics. However, reliable biomarkers that predict therapeutic responses are yet to be discovered. We aimed to identify the molecular features of treatment responses to chemotherapy and immunotherapy in BTCs. We enrolled 121 advanced BTC patients (68 cholangiocarcinomas [33 intrahepatic, 35 extrahepatic], 41 gallbladder cancers, and 12 Ampulla of Vater cancers) whose specimens were analyzed by clinical sequencing platforms. All patients received first-line palliative chemotherapy; 48 patients underwent programmed death 1 (PD-1)/programmed death-ligand 1 (PD-L1) blockade therapy after failed chemotherapy. Molecular and histopathological characterization was performed using targeted sequencing and immunohistochemical staining to investigate treatment response-associated biomarkers. Genomic analysis revealed a broad spectrum of mutational profiles according to anatomical location. Favorable responses to chemotherapy were observed in the small-duct type compared with the large-duct type intrahepatic cholangiocarcinoma, with frequent mutations in BRCA1-associated protein-1/isocitrate dehydrogenase 1/2 and KRAS proto-oncogene, GTPase/SMAD family member 4 genes, respectively. The molecular features were further analyzed in BTCs, and transforming growth factor beta and DNA damage response pathway-altered tumors exhibited poor and favorable chemotherapy responses, respectively. In PD-1/PD-L1 blockade-treated patients, KRAS alteration and chromosomal instability tumors were associated with resistance to immunotherapy. The majority of patients (95.0%) with these resistance factors show no clinical benefit to PD-1/PD-L1 blockade and low tumor mutational burdens. Low tumor-infiltrating lymphocyte (TIL) density in tumors with these resistance factors indicated immune-suppressive tumor microenvironments, whereas high intratumoral TIL density was associated with a favorable immunotherapy response. This study proposes predictive molecular features of chemotherapy and immunotherapy responses in advanced BTCs using clinical sequencing platforms. Our result provides an intuitive framework to guide the treatment of advanced BTCs benefiting from therapeutic agents based on the tumors' molecular features.

Artificial intelligence-powered spatial analysis of tumor infiltrating lymphocytes (TIL) to reflect target gene expressions of novel immuno-oncology agents.

e14534 Background: Novel immuno-oncology (IO) agents are promising but showing their efficacy in early phase clinical trials has been challenging due to limited enrichment strategies using practical biomarker platforms. We hypothesize that an artificial intelligence (AI)-powered spatial analysis of TIL using practically feasible H&amp;E slides, can reflect a specific target gene expression derived from RNA sequencing. This enhances its potential application in early development of novel IO agents. Methods: An AI-powered spatial TIL analyzer, namely Lunit SCOPE IO, was developed with data from 2.8 x 109 micrometer2 H&amp;E-stained tissue regions and 5.9 x 106 TILs from 3,166 whole slide images of multiple cancer types, annotated by board-certified pathologists. Inflamed Score and Immune-Excluded Score was defined as the proportion of all tumor-containing 1 mm2-size tiles within a WSI classified as being of inflamed immune phenotype (high TIL density within cancer epithelium) and immune-excluded phenotype (low TIL density within cancer epithelium, but high TIL density within stroma), respectively. We used RNA sequencing data and H&amp;E images from The Cancer Genome Atlas database, excluding those of mesenchymal origin (n = 7,467). Spearman's rank correlation between each gene expression and IS or IES, respectively, was calculated. Correlation coefficient &gt; 0.2 and false discovery rate (FDR) &lt; 1% was considered as a significant correlation. Results: In a total of 20,304 genes, 871 (4.3%) and 1,155 (5.7%) genes were significantly correlated with Inflamed Score (IS) and Immune-Excluded Score (IES), respectively. The IS was highly related to genes reflecting immune cytolytic activity and targets of approved immune checkpoint inhibitors (Table). Interestingly, it was also significantly correlated with target genes of novel IO such as TIGIT, LAG3, TIM3, IDO, Adenosine receptor A2A, OX40, ICOS, M-CSF, IL2, IL7, and IL12. Moreover, the IES was exclusively correlated with the target genes of CEACAM, TGFB, and IL1. Conclusions: Expression levels of novel I-O target genes are correlated with three scores derived from AI-powered TIL analysis using H&amp;E slides, which can be easily applied to clinical research.[Table: see text]

Artificial intelligence-powered spatial analysis of tumor-infiltrating lymphocytes predicts survival after immune checkpoint inhibitor therapy across multiple cancer types.

2607 Background: Tumor infiltrating lymphocytes (TIL) are a potential tumor-agnostic biomarker for immune checkpoint inhibitor (ICI) therapy. We previously reported the clinical application of an artificial intelligence-powered spatial TIL analyzer, Lunit SCOPE IO, for predicting ICI treatment outcomes in advanced non-small cell lung cancer (NSCLC). Here, we expand the clinical application of Lunit SCOPE IO as a tumor-agnostic ICI biomarker across multiple cancer types. Methods: Lunit SCOPE IO was trained and validated with a 2.8 x 109 micrometer2 area and 5.9 x 106 TILs from 3,166 H&amp;E Whole-Slide Images (WSI) of multiple cancer types, annotated by 52 board-certified pathologists. The Inflamed Score (IS) was defined as the proportion of all tumor-containing 1 mm2-size tiles within a WSI classified as being of the inflamed immune phenotype (high TIL density within cancer epithelium). We first evaluated the correlation between the IS and TMB, MSI-H, and immune cytolytic activity ( GZMA and PRF1) across 22 cancer types from The Cancer Genome Atlas (TCGA, n = 7,467). Subsequently, the correlation between the IS and overall survival after ICI treatment was evaluated in a real-world dataset of patients with 9 different tumor types (n = 1,013), retrospectively collected from Stanford University Medical Center, Chonnam National University Hospital, Samsung Medical Center, and Seoul National University Bundang Hospital. Results: Lunit SCOPE IO accurately detected CE, CS, and TILs with an area under the receiver-operating-characteristic curve of 0.970, 0.949, and 0.925, respectively. In the TCGA pan-cancer cohort, Lunit SCOPE IO’s IS correlated significantly with immune cytolytic activity (Spearman rho = 0.504, p &lt; 0.001), TMB-high (≥ 10 mutations/Mb, fold change 1.39, p &lt; 0.001) and MSI-H (fold change 1.45, p &lt; 0.001). The IS-positive proportions of microsatellite-stable (MSS) and TMB-low cases were 42.5% and 17.1%, using the thresholds of IS ≥ 20% and ≥ 50% as presumptive clinical cutoffs. In the real-world ICI clinical dataset (n = 1,013), an IS ≥ 20% correlated significantly with favorable overall survival after ICI treatment (cancer type-adjusted hazard ratio [HR] 0.70, 95% confidence interval [CI] 0.59-0.83, p &lt; 0.0001). Furthermore, this association remained significant after the exclusion of NSCLC patients (n = 519) (adjusted HR 0.68, 95% CI 0.53-0.86, p = 0.0016) indicating that the effect was not driven solely by one major tumor type. Conclusions: The Inflamed Score (IS), as evaluated by Lunit SCOPE IO, correlates with favorable overall survival after ICI treatment across multiple tumor types. AI-powered spatial TIL analysis of the tumor microenvironment may be able to detect a significant proportion of ICI responders, and offers promise as a new companion diagnostic, particularly in patients with MSS/TMB-low tumors.

Kynurenine/Tryptophan Ratio as a Potential Blood-Based Biomarker in Non-Small Cell Lung Cancer.

The enzyme indoleamine 2,3-dioxygenase 1 (IDO1) degrade tryptophan (Trp) into kynurenine (Kyn) at the initial step of an enzymatic pathway affecting T cell proliferation. IDO1 is highly expressed in various cancer types and associated with poor prognosis. Nevertheless, the serum Kyn/Trp concentration ratio has been suggested as a marker of cancer-associated immune suppression. We measured Kyn and Trp in blood samples of a wide cohort of non-small-cell lung cancer (NSCLC) patients, before they underwent surgery, and analyzed possible correlations of the Kyn/Trp ratio with either IDO1 expression or clinical–pathological parameters. Low Kyn/Trp significantly correlated with low IDO1 expression and never-smoker patients; while high Kyn/Trp was significantly associated with older (≥68 years) patients, advanced tumor stage, and squamous cell carcinoma (Sqcc), rather than the adenocarcinoma (Adc) histotype. Moreover, high Kyn/Trp was associated, among the Adc group, with higher tumor stages (II and III), and, among the Sqcc group, with a high density of tumor-infiltrating lymphocytes. A trend correlating the high Kyn/Trp ratio with the probability of recurrences from NSCLC was also found. In conclusion, high serum Kyn/Trp ratio, associated with clinical and histopathological parameters, may serve as a serum biomarker to optimize risk stratification and therapy of NSCLC patients.

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.

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.

High EZH2 expression in ductal carcinoma in situ diagnosed on breast core needle biopsy is an independent predictive factor for upgrade on surgical excision

PurposeApproximately 25 % of DCIS diagnosed on breast core needle biopsy (CNB) is upgraded to invasive carcinoma on surgical excision. Risk factors to predict the upgrade on excision are not well established, leading many patients to be over or under-treated. EZH2 was shown to be associated with aggressive behavior of cancer from many sites, including breast cancer. We aimed to analyze EZH2 expression and tumor infiltrating lymphocytes (TILs) in DCIS as predictive factors for an upgrade on excision. MethodsWe assessed EZH2 expression in 34 DCIS cases diagnosed on CNB and upgraded to invasive carcinoma on excision. Then, we compared these cases with 60 control cases that were not upgraded on excision. A staining score for DCIS (0–12) was obtained by multiplying the staining intensity (0–3) and the percentage of positive cells (1–4). The nuclear staining score ≥6 was considered as ‘high’ expression. Results46 of 94 (49 %) DCIS on CNB showed high EZH2 expression. EZH2 expression was directly correlated with TILs density, nuclear grade, HER2 expression, Ki-67 index and negative ER status. On univariate analysis, upgrade on excision was associated with high EZH2 expression, high TILs density, negative ER status and high Ki-67 index. Multivariate analysis revealed the high EZH2 expression as the only independent predictive factor for upgrade on excision. ConclusionsOur study revealed the high EZH2 expression as the only independent predictive factor for an upgrade on excision. Future studies should focus on the evaluation of EZH2 expression in tumor-microenvironment interaction in terms of diagnostic, treatment and prognostic purposes.

Prognostic Impact of Tumor-Infiltrating Lymphocytes in Laryngeal Squamous Cell Carcinoma Patients.

Tumor-infiltrating lymphocytes (TILs) has been shown to be associated with the prognosis of many tumors, yet few studies have investigated their roles in laryngeal squamous cell carcinoma (LSCC). We aim to investigate the prognostic values of tumor-infiltrating CD3+ /CD4+ /CD8+ /Foxp3+ T-cells and neutrophils in LSCC patients that received total or partial laryngectomy. Retrospective case series of LSCC patients who underwent total or partial laryngectomy from 2013 to 2014 at Eye, Ear, Nose, and Throat Hospital of Fudan University. In our study, 41 tumor tissues from patients with LSCC were retrospectively assessed using immunohistochemistry for CD3+ /CD4+ /CD8+ /Foxp3+ T-cells and CD66b+ neutrophils. Overall survival (OS) and disease-free survival (DFS) were recorded using Kaplan-Meier methods. Generally, patients with high density of TILs (CD3, CD4, CD8) showed improved OS or DFS. Specifically, high density of CD3+ TILs were associated with better OS, yet poorer OS and DFS for CD66b+ neutrophils. Patients with an Immunoscore of 0-1 experienced the worst OS and DFS, compared with Immunoscore 2-4 (P = .0111 for OS, P = .0391 for DFS). In Cox proportional hazards analysis adjusted for N stage and T stage, only stroma CD66b+ neutrophils densities were able to predict OS, with odds ratios of 4.819 (95% confidence interval [CI] 1.149-20.206; P = .032*), and DFS 2.888 (95% CI 1.043-7.997; P = .041*). The density of TILs and CD66b+ neutrophils may help predict the prognosis of patients with LSCC after surgery. 3 Laryngoscope, 131:E1249-E1255, 2021.

Microsatellite instability and mismatch repair protein expressions in lymphocyte-predominant breast cancer.

The frequency of microsatellite instability (MSI) is reportedly extremely low in breast cancer, despite widespread clinical expectations that many patients would be responsive to immune‐checkpoint inhibitors (ICI). Considering that some triple‐negative breast cancers (TNBC) responded well to ICI in a clinical trial and that a high density of tumor‐infiltrating lymphocytes (TILs) is frequently observed in other cancers with high levels of microsatellite instability (MSI‐H), we hypothesized that some TNBC with a high density of TILs would be MSI‐H. Medullary carcinoma (MedCa) of the breast, a rare histological type, is characterized by a high density of TILs. Considering that MedCa of the colon is often MSI‐H, we suspected that MedCa in breast cancer might also include MSI‐H tumors. Therefore, we conducted MSI tests on such breast cancers with a high density of TILs. The MSI status of 63 TIL‐high TNBC and 38 MedCa tumors, all from Asian women who had undergone curative surgery, were determined retrospectively. DNA mismatch repair (MMR) proteins and PD‐L1 expression were also investigated immunohistochemically. All samples were microsatellite stable, being negative for all microsatellite markers. TIL‐high TNBC with low MLH1 protein had higher levels of PD‐L1 in stromal immune cells (P = .041). MedCa tumors showed significantly higher PD‐L1 expression in immune cells than in TIL‐high TNBC (<.001). We found that MSI‐H tumors were absent in TIL‐high breast cancers. Examination of MMR proteins, not a purpose of Lynch syndrome screening, may merit further studies to yield predictive information for identifying patients who are likely to benefit from ICI.

Prognostic value of tumor-infiltrating lymphocyte subtypes in residual tumors of patients with triple-negative breast cancer after neoadjuvant chemotherapy.

Background:After neoadjuvant chemotherapy (NAC), non-pathological complete response of breast cancer patients can benefit from tailored adjuvant chemotherapy. However, it is difficult to select patients with poorer prognosis for additional adjuvant chemotherapy to maximize the benefits. Our study aimed to explore whether the subtypes of tumor-infiltrating lymphocytes (TILs) in residual tumors (RT) is related to the prognosis of triple-negative breast cancer (TNBC) after NAC.Methods:Data from patients with primary TNBC consecutively diagnosed at the Breast Disease Center of Peking University First Hospital from 2008 to 2014 were retrieved, and the cases with RT in the breast after NAC were enrolled. TILs subtypes in RT were observed by double-staining immunohistochemistry, and counted with the median TILs value per square millimeter as the cut-off to define high versus low TILs density in each subtype. The relationships between the TIL density of each subgroup and the clinicopathological characteristics of the RT after NAC patients were analyzed by Fisher exact test. Disease-free survival (DFS) and overall survival (OS) were analyzed by the Kaplan-Meier method and log-rank statistics.Results:A total of 37 eligible patients were included in this study, and the median follow-up period was 50 months (range 17–106 months). There was no significant correlation between the infiltrate density of CD4+, CD8+, CD20+, and CD68+ lymphocytes and clinic-pathological characteristics. Significantly better prognosis was observed in patients with high CD4+-TILs (DFS: P = 0.005, OS: P = 0.021) and high CD8+-TILs (DFS: P = 0.018) and low CD20+-TILs (OS: P = 0.042). Further analysis showed that patients with CD4+/CD20+ ratio greater than 1 (DFS: P = 0.001, OS: P = 0.002) or CD8+/CD20+ ratio greater than 1 (DFS: P = 0.009, OS: P = 0.022) had a better prognosis.Conclusions:Subtypes of TILs in RT is a potential predictive biomarker of survival in TNBC patients after NAC.

Association of cancer progression with elevated expression of programmed cell death protein 1 ligand 1 by upper tract urothelial carcinoma and increased tumor-infiltrating lymphocyte density

BackgroundIncreased expression of programmed cell death 1 ligand 1 (PD-L1) by tumor cells is thought to be a mechanism through which solid cancers promote immune tolerance. However, the association between PD-L1 expression and the prognosis of upper urinary tract urothelial carcinoma (UTUC) remains unknown.MethodsWe examined immunohistochemical PD-L1 expression and the tumor-infiltrating lymphocyte density (TILD) in 79 patients with UTUC who underwent nephroureterectomy. We classified the tumors into four types based on the combination of PD-L1 expression and TILD, and studied the clinicopathological characteristics of these four tumor types.ResultsElevated expression of PD-L1 by tumor cells and a higher TILD were associated with a worse histological grade, higher pT stage, and higher peripheral blood neutrophil-to-lymphocyte ratio. Elevated expression of PD-L1 by tumor cells, a higher TILD, and type I, III, or IV tumors with elevated expression of either PD-L1 or TILD showed a positive correlation with poorer differentiation and local invasion. These three variables were associated with shorter progression-free survival and overall survival in univariate analysis, but only the latter was an independent determinant according to multivariate analysis. The patients who had type II tumors with lower PD-L1 expression and a lower TILD showed more favorable survival than the other three groups.ConclusionsThese findings suggest that PD-L1 expression and TILs in the tumor microenvironment influence the progression of UTUC. Accordingly, it is important to understand the immunologic characteristics of the tumor microenvironment to develop more effective treatment strategies for this cancer.

Response to ipilimumab therapy in metastatic melanoma patients: potential relevance of CTLA-4+ tumor infiltrating lymphocytes and their in situ localization.

Immune checkpoint inhibitors, including ipilimumab (IPI), achieve a clinical benefit in a small proportion of melanoma patients highlighting the need to investigate predictive biomarkers. In this study, we characterized tumor infiltrating lymphocytes (TILs), focusing on the CTLA-4+ subset, and evaluated their possible predictive significance. We characterized TIL density, cell type, and localization in 40 melanoma lesions from 17 patients treated with IPI. Associations of TILs with IPI timing, tissue localization, and response to IPI were estimated using a linear mixed-effects modelling approach. We found that most of TIL subsets increased in situ upon IPI therapy, with particular reference to FoxP3+ cells. TILs and TIL subsets, such as CD3+, CD45RO+, CTLA-4+, CD4+, CD8+ T cells, CD20+ B cells, and NKp46+ NK cells, showed significantly different spatial distributions in the tumor microenvironment being higher at the invasive margin (IM) as compared to the tumor center (TC) (P value < 0.001 for TIL score and P value < 0.05 for all subsets). Remarkably, high TIL score and density of CD3+, CD8+ T cells, and CTLA-4+ immune cells were significantly associated with a better response to IPI (P values = 0.002, 0.023, 0.007, and 0.001, respectively, for responders vs non-responders). In conclusion, we provide a detailed analysis of CTLA-4+ TIL distribution in melanoma tissues taking into account localization, relationship with CD3+/CD8+ TILs, and changes in response to IPI treatment. We identified that CTLA-4+ TILs may represent a marker of IPI response, alone or with CD3+/CD8+ subsets, although this requires confirmation in larger studies.

Quantitative digital image analysis of tumor-infiltrating lymphocytes in HER2-positive breast cancer.

As visual quantification of the density of tumor-infiltrating lymphocytes (TILs) lacks in precision, digital image analysis (DIA) approach has been applied in order to improve. In several studies, TIL density has been examined on hematoxylin and eosin (HE)-stained sections using DIA. The aim of the present study was to quantify TIL density on HE sections of core needle biopsies using DIA and investigate its association with clinicopathological parameters and pathological response to neoadjuvant chemotherapy in human epidermal growth factor receptor 2 (HER2)-positive breast cancer. The study cohort comprised of patients with HER2-positive breast cancer, all treated with neoadjuvant anti-HER2 therapy. DIA software applying machine learning-based classification of epithelial and stromal elements was used to count TILs. TIL density was determined as the number of TILs per square millimeter of stromal tissue. Median TIL density was 1287/mm2 (range, 123-8101/mm2). A high TIL density was associated with higher histological grade (P = 0.02), estrogen receptor negativity (P = 0.036), and pathological complete response (pCR) (P < 0.0001). In analyses using receiver operating characteristic curves, a threshold TIL density of 2420/mm2 best discriminated pCR from non-pCR. In multivariate analysis, high TIL density (> 2420/mm2) was significantly associated with pCR (P < 0.0001). Our results indicate that DIA can assess TIL density quantitatively, machine learning-based classification algorithm allowing determination of TIL density as the number of TILs per unit area, and TIL density established by this method appears to be an independent predictor of pCR in HER2-positive breast cancer.