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Purpose: ATM is the most commonly mutated DNA damage and repair gene in non–small cell lung cancer (NSCLC); however, limited characterization has been pursued. Experimental Design: Clinicopathologic, genomic, and treatment data were collected for 5,172 patients with NSCLC tumors which underwent genomic profiling. ATM IHC was performed on 182 NSCLCs with ATM mutations. Multiplexed immunofluorescence was performed on a subset of 535 samples to examine tumor-infiltrating immune cell subsets. Results: A total of 562 deleterious ATM mutations were identified in 9.7% of NSCLC samples. ATM-mutant (ATMMUT) NSCLC was significantly associated with female sex (P = 0.02), ever smoking status (P < 0.001), non-squamous histology (P = 0.004), and higher tumor mutational burden (DFCI, P < 0.0001; MSK, P < 0.0001) compared with ATM–wild-type (ATMWT) cases. Among 3,687 NSCLCs with comprehensive genomic profiling, co-occurring KRAS, STK11, and ARID2 oncogenic mutations were significantly enriched among ATMMUT NSCLCs (Q < 0.05), while TP53 and EGFR mutations were enriched in ATMWT NSCLCs. Among 182 ATMMUT samples with ATM IHC, tumors with nonsense, insertions/deletions, or splice site mutations were significantly more likely to display ATM loss by IHC (71.4% vs. 28.6%; P < 0.0001) compared with tumors with only predicted pathogenic missense mutations. Clinical outcomes to PD-(L)1 monotherapy (N = 1,522) and chemo-immunotherapy (N = 951) were similar between ATMMUT and ATMWT NSCLCs. Patients with concurrent ATM/TP53 mutations had significantly improved response rate and progression-free survival with PD-(L)1 monotherapy. Conclusions: Deleterious ATM mutations defined a subset of NSCLC with unique clinicopathologic, genomic, and immunophenotypic features. Our data may serve as resource to guide interpretation of specific ATM mutations in NSCLC.

Supplementary Figure 8. Oncoprint plot showing the distribution of the most commonly mutated genes in NSCLCs with complete ATM loss by IHC (ATMLOST), intact ATM expression (ATMINTACT), and ATM heterogeneous loss (ATMHET LOST) by IHC.

Supplementary Figure 25. (A) Intratumoral, (B) tumor-stroma interface, and (C) total CD8+ PD1+ T cells according to ATM/TP53 co-mutation status. (D) Intratumoral, (E) tumor-stroma interface, and (F) total FOXP3+ immune cells according to ATM/TP53 co-mutation status.

Supplementary Table 6. Baseline clinicopathologic features of patients with ATM mutated NSCLC according ATM protein expression by immunohistochemistry.

Supplementary Figure 15. (A) Objective response rate, (B) progression-free survival, and (C) overall survival to chemo-immunotherapy among patients with advanced/metastatic PD-L1 negative (

Purpose:ATM is the most commonly mutated DNA damage and repair gene in non–small cell lung cancer (NSCLC); however, limited characterization has been pursued.Experimental Design:Clinicopathologic, genomic, and treatment data were collected for 5,172 patients with NSCLC tumors which underwent genomic profiling. ATM IHC was performed on 182 NSCLCs with ATM mutations. Multiplexed immunofluorescence was performed on a subset of 535 samples to examine tumor-infiltrating immune cell subsets.Results:A total of 562 deleterious ATM mutations were identified in 9.7% of NSCLC samples. ATM-mutant (ATMMUT) NSCLC was significantly associated with female sex (P = 0.02), ever smoking status (P < 0.001), non-squamous histology (P = 0.004), and higher tumor mutational burden (DFCI, P < 0.0001; MSK, P < 0.0001) compared with ATM–wild-type (ATMWT) cases. Among 3,687 NSCLCs with comprehensive genomic profiling, co-occurring KRAS, STK11, and ARID2 oncogenic mutations were significantly enriched among ATMMUT NSCLCs (Q < 0.05), while TP53 and EGFR mutations were enriched in ATMWT NSCLCs. Among 182 ATMMUT samples with ATM IHC, tumors with nonsense, insertions/deletions, or splice site mutations were significantly more likely to display ATM loss by IHC (71.4% vs. 28.6%; P < 0.0001) compared with tumors with only predicted pathogenic missense mutations. Clinical outcomes to PD-(L)1 monotherapy (N = 1,522) and chemo-immunotherapy (N = 951) were similar between ATMMUT and ATMWT NSCLCs. Patients with concurrent ATM/TP53 mutations had significantly improved response rate and progression-free survival with PD-(L)1 monotherapy.Conclusions:Deleterious ATM mutations defined a subset of NSCLC with unique clinicopathologic, genomic, and immunophenotypic features. Our data may serve as resource to guide interpretation of specific ATM mutations in NSCLC.

Supplementary Table 1. Baseline clinicopathologic features of the 5172 patients with NSCLC which underwent comprehensive tumor genomic profiling at the Dana-Farber Cancer Institute and Memorial Sloan Kettering Cancer Center.

Supplementary Figure 6. (A) Tumor mutational burden and (B) PD-L1 tumor proportion score distributions according to ATM protein expression by IHC (lost versus intact).

Supplementary Figure 9. (A) Disease free- and overall survival among patients with (A) stage I, and (B) stage II NSCLC according to ATM mutation status.

Supplementary Figure 3. Co-mutation plot showing co-occurrent and mutual exclusive mutations among (A) ATMMUT and (B) ATMWT NSCLCs.

Supplementary Figure 4. (A) Volcano plot showing gene mutations significantly enriched among NSCLCs harboring ATM missense mutations that were predicted to benign in silico versus NSCLCs without any ATM mutation. (B) Volcano plot showing gene mutations significantly enriched among ATMWT and ATMMUT samples in the TCGA-NSCLC cohort.

Supplementary Figure 7. (A) Histograms showing the distribution of deleterious class I and class II ATM mutations with lost or intact ATM protein expression by IHC. (B) Histograms showing the distribution of deleterious missense, nonsense, splice site, and insertion/deletion mutations with lost or intact ATM protein expression by IHC. (C) proportion of NSCLCs with predicted benign and predicted pathogenic ATM missense mutations with lost and intact ATM expression by IHC.

Supplementary Table 4. Baseline clinicopathologic features of patients with ATM mutated NSCLC according ATM protein expression by immunohistochemistry.

Supplementary Table 2. List of potential ATM germline variants identified among 3800 patients with NSCLC at the DFCI

Supplementary Table 5. List of ATM mutations with matched IHC

Supplementary Table 3. Baseline clinicopathologic features of patients with ATMMUT and ATMWT NSCLC at the Dana-Farber Cancer Institute and Memorial Sloan Kettering Cancer Center.

Supplementary Table 7. Baseline clinicopathologic features of patients with ATM mutated and ATM wild-type NSCLCs which underwent multiplexed immunofluorescence at the DFCI.

ImportanceCurrently, predictive biomarkers for response to immune checkpoint inhibitor (ICI) therapy in lung cancer are limited. Identifying such biomarkers would be useful to refine patient selection and guide precision therapy.ObjectiveTo develop a machine-learning (ML)-based tumor-infiltrating lymphocytes (TILs) scoring approach, and to evaluate TIL association with clinical outcomes in patients with advanced non–small cell lung cancer (NSCLC).Design, Setting, and ParticipantsThis multicenter retrospective discovery-validation cohort study included 685 ICI-treated patients with NSCLC with median follow-up of 38.1 and 43.3 months for the discovery (n = 446) and validation (n = 239) cohorts, respectively. Patients were treated between February 2014 and September 2021. We developed an ML automated method to count tumor, stroma, and TIL cells in whole-slide hematoxylin-eosin–stained images of NSCLC tumors. Tumor mutational burden (TMB) and programmed death ligand-1 (PD-L1) expression were assessed separately, and clinical response to ICI therapy was determined by medical record review. Data analysis was performed from June 2021 to April 2022.ExposuresAll patients received anti–PD-(L)1 monotherapy.Main Outcomes and MeasuresObjective response rate (ORR), progression-free survival (PFS), and overall survival (OS) were determined by blinded medical record review. The area under curve (AUC) of TIL levels, TMB, and PD-L1 in predicting ICI response were calculated using ORR.ResultsOverall, there were 248 (56%) women in the discovery cohort and 97 (41%) in the validation cohort. In a multivariable analysis, high TIL level (≥250 cells/mm2) was independently associated with ICI response in both the discovery (PFS: HR, 0.71; P = .006; OS: HR, 0.74; P = .03) and validation (PFS: HR = 0.80; P = .01; OS: HR = 0.75; P = .001) cohorts. Survival benefit was seen in both first- and subsequent-line ICI treatments in patients with NSCLC. In the discovery cohort, the combined models of TILs/PD-L1 or TMB/PD-L1 had additional specificity in differentiating ICI responders compared with PD-L1 alone. In the PD-L1 negative (Conclusions and RelevanceIn these cohorts, TIL levels were robustly and independently associated with response to ICI treatment. Patient TIL assessment is relatively easily incorporated into the workflow of pathology laboratories at minimal additional cost, and may enhance precision therapy.

MicroRNAs (miRs) are small non-coding RNA molecules, which are involved in the development of various malignancies, including prostate cancer (PCa). miR-17-5p is considered the most prominent member of the miR-17-92 cluster, with an essential regulatory function of fundamental cellular processes. In many malignancies, up-regulation of miR-17-5p is associated with worse outcome. In PCa, miR-17-5p has been reported to increase cell proliferation and the risk of metastasis. In this study, prostatectomy specimens from 535 patients were collected. Tissue microarrays were constructed and in situ hybridization was performed, followed by scoring of miR-17-5p expression on different tumor compartments. High expression of miR-17-5p in tumor epithelium was associated with biochemical failure (BF, p p = 0.019). In multivariate analyses, high miR-17-5p expression in tumor epithelial cells was an independent negative prognostic factor for BF (HR 1.87, 95% CI 1.32–2.67, p

9066 Background: Current biomarker(s) for immuno-oncology (IO) therapy response prediction in lung cancer are limited. Additional predictive biomarkers are useful to help refine patient selection and guide precision therapy. Methods: Biopsy and surgical specimens stained with hematoxylin-eosin (H&E) were subjected to whole-slide scanning for 446 advanced stage non-small cell lung cancer (NSCLC) treated with single agent immune check point inhibitors (ICI). A machine learning model was trained on H&E images for classification of tumor infiltrating lymphocytes (TILs), tumor cells, and stromal cells in specific tissue types. Results: TIL levels were found to be highly variable, with a range of 12 to 4270 cells/mm2, and median of 319 (Q1 = 159, Q3 = 681). TIL levels were assessed on tissue samples from multiple organs which had shown primary or metastatic NSCLC, and were similar across all specimen sites except the liver, for which median TIL levels were significantly lower, at 90 cells/mm2. There was no correlation between tumor mutational burden (TMB) and TIL levels, while high TIL levels were correlated with high PD-L1 (≥ 50%) expression. Patients who experienced a partial/complete response to ICI therapy had a trend to higher median TILs compared to those who had progressive/stable disease (350 versus 310 cells/mm2, P = 0.09). In a multivariable analysis after controlling for covariates (incl. sex, age, cigarette smoking, ECOG, PD-L1, TMB & treatment line), a higher TIL level (≥ 250 cells/mm2) was an independent predictor of IO response for both progression-free survival (PFS; HRadj 0.70; 95% CI, 0.55 - 0.89; P = 0.003) and overall survival (HRadj 0.73; 95% CI, 0.56 - 0.95; P = 0.02). In a ROC analysis considering single biomarkers, PD-L1 had the highest AUC (0.68, P < 0.001), while TIL (AUC = 0.53, P = 0.08) and TMB (AUC = 0.55, P = 0.05) had similar AUC values for classifying responders from non-responders based on objective response rate. Using weighted linear regression approach to combine the biomarkers, paired PD-L1/TMB had the greatest AUC (0.70, P < 0.001) compared to PD-L1 single assay. In the PD-L1 negative (< 1%, N = 50) subgroup, TIL levels had superior predictive performance for classification of IO responders (AUC = 0.77, P = 0.02) compared to TMB (AUC = 0.57, P = 0.3), such that patients with a high TIL level (≥ 250 cells/mm2) had an improved PFS (median PFS: 2.7 vs 2.2 months; HR = 0.48; 95% CI, 0.26 - 0.87; P = 0.02). Conclusions: Digital TIL quantification with use of machine learning is feasible. TIL levels appear to be a robust and independent biomarker of likelihood of response to IO treatment in NSCLC, especially in the PD-L1 negative subgroup. The findings of this study are under validation in additional lung cancer cohorts.

9065 Background: The presence of Tertiary Lymphoid Structures (TLS) in multiple cancer types has been recognized as a potential predictive biomarker for response to immune-checkpoint blockade. However, there is no standardized method to quantify their presence. In this context, Artificial Intelligence (AI)-based assessment of histology images may well contribute to improve reproducibility, accuracy and speed of TLS quantification. Methods: We developed an automated workflow for quantification of TLS on digitized H&E slides through A) pixel-level classification of tissue using supervised artificial neural networks model, B) object-level cell classification of candidate TLS regions, C) merging the two approaches for curation and validation of TLS versus non-TLS regions. 433 advanced stage non-small cell lung cancer (NSCLC) patients treated with first or subsequent line of anti-PD-(L)1 single agent at DFCI were included in this study. Results: TLS were detected in 37% (n = 161) of the patients H&E slides, with the highest score of 4.7 TLS per mm2 (interquartile range: Q1 = 0, Q2 = 0, Q3 = 0.03 TLS/mm2). TLS density (per mm2) was significantly higher in surgically resected (n = 246; TLSPOS= 49%) compared to bioptic samples (n = 187; TLSPOS= 21%). No association was observed between TLS and tumor mutational burden (TMB) or PD-L1 protein expression as continuous variables. Among clinically actionable mutations, EGFR (all subtypes) mutated patients (n = 38) had a significantly lower number of TLS compared to patients without EGFR mutations. Patients with ≥ 0.01 TLS/mm2 had a significantly higher objective response rate (32% vs 22%, p = 0.03), a significantly longer median progression-free survival (PFS, 4.8 vs 2.7 months, HR: 0.73, 95% CI: 0.59-0.90, p = 0.004), and a significantly improved median overall survival (OS, 16.5 vs 12.5 months, HR: 0.72, 95% CI: 0.57-0.92, p = 0.008). In multivariable analysis, after adjusting for PD-L1 (≥ vs < 50%), TMB (≥ vs < 10 mu/Mb), sex, age, ECOG score, smoking and line of treatment, TLS/mm2 (≥ vs < 0.01) levels were found to be an independent positive predictive factor for both PFS (HR:0.69, 95% CI: 0.54-0.88, p = 0.003) and OS (HR: 0.70, 95% CI: 0.52-0.93, p = 0.01). Conclusions: These findings suggest that TLS status is an independent predictor of immunotherapy effectiveness in NSCLC, with predictive value similar to that of PD-L1 expression and TMB. This novel AI system has potential for automated identification and quantification of the TLS on digital histopathological slides, and could be utilized in a standard pathology workflow with relative ease. These findings are currently being validated in other solid tumors and cohorts.

Background We previously proposed an immune cell score (tumour node metastasis (TNM)-Immune cell score) classifier as an add-on to the existing TNM staging system for non-small cell lung cancer (NSCLC). Herein, we examined how to reliably assess a tertiary lymphoid structure (TLS) score to refine the TNM staging system. Methods Using immunohistochemistry (CD8/cytokeratin), we quantified TLS in resected NSCLC whole-tumour tissue sections with three different scoring models on two independent collections (total of 553 patients). In a pilot setting, NanoString gene expression signatures were analysed for associations with TLS. Results The number of TLSs significantly decreased in stage III patients as compared to stage II. The TLS score was an independent positive prognostic factor, regardless of the type of (semi)-quantification strategy used (four-scale semi-quantitative; absolute count of total TLS; subpopulation of mature TLS) or the endpoint (disease-specific survival; overall survival; time to recurrence). Subgroup analyses revealed a significant prognostic impact of TLS score within each pathological stage, patient cohort and main histological subtype. Targeted gene expression analysis showed that high TLS levels were associated with the expression of B cell and adaptive immunity genes/metagenes including tumour inflammation signature. Conclusions The TLS score increases the prognostic power in each pathological stage and hence has the potential to refine TNM staging in resected NSCLC.

Kidney involvement confers significant morbidity and mortality in patients with systemic lupus erythematosus (SLE). The pathogenesis of lupus nephritis (LN) involves diverse mechanisms instigated by elements of the autoimmune response which alter the biology of kidney resident cells. Processes in the glomeruli and in the interstitium may proceed independently albeit crosstalk between the two is inevitable. Podocytes, mesangial cells, tubular epithelial cells, kidney resident macrophages and stromal cells with input from cytokines and autoantibodies present in the circulation alter the expression of enzymes, produce cytokines and chemokines which lead to their injury and damage of the kidney. Several of these molecules can be targeted independently to prevent and reverse kidney failure. Tertiary lymphoid structures with true germinal centers are present in the kidneys of patients with lupus nephritis and have been increasingly recognized to associate with poorer renal outcomes. Stromal cells, tubular epithelial cells, high endothelial vessel and lymphatic venule cells produce chemokines which enable the formation of structures composed of a T-cell-rich zone with mature dendritic cells next to a B-cell follicle with the characteristics of a germinal center surrounded by plasma cells. Following an overview on the interaction of the immune cells with kidney resident cells, we discuss the cellular and molecular events which lead to the formation of tertiary lymphoid structures in the interstitium of the kidneys of mice and patients with lupus nephritis. In parallel, molecules and processes that can be targeted therapeutically are presented.

Source at https://doi.org/10.1038/s41598-019-46510-3. Preclinical evidence suggests that stromal expression of platelet-derived growth factor receptors (PDGFRs) stimulates tumor development and diminishes intratumoral drug uptake. In non-small cell lung cancer (NSCLC), the clinical relevance of stromal PDGFR expression remains uncertain. Tumor specimens from 553 patients with primary operable stage I-IIIB NSCLC was obtained and tissue micro-arrays (TMA) were constructed (Norwegian cohort). Immunohistochemistry (IHC) was used to evaluate the expression of PDGFRα and -β in stromal cells and to explore their impact on patient survival. Results were validated in a non-related cohort consisting of TMAs of 367 stage I (A and B) NSCLC patients (Swedish cohort). High stromal PDGFRα expression was an independent predictor of increased survival in the overall populations and SCC (squamous cell carcinoma) subgroups of both investigated cohorts. PDGFRβ was an independent predictor of poor survival in the overall Norwegian cohort and an independent predictor of increased survival in the ADC (adenocarcinoma) subgroup of the Swedish cohort. Tumors displaying the combination PDGFRα-low/PDGFRβ-high exhibited inferior survival according to increasing stage in the Norwegian cohort. This study confirms that high stromal expression of PDGFRα is a predictor of increased survival in NSCLC. Further exploration of the prognostic impact of PDGFRβ and the relationship between PDGFRα and -β is warranted.

Source at https://doi.org/10.1016/j.neo.2019.01.005. Macrophages are important inflammatory cells that regulate innate and adaptive immunity in cancer. Tumor-associated macrophages (TAMs) are thought to differentiate into two main phenotypes: proinflammatory M1 and protumorigenic M2. Currently, the prognostic impact of TAMs and their M1 and M2 phenotypes is unclear in non–small cell cancer (NSCLC). The present study was set up to evaluate an approach for identifying common M1 and M2 macrophage markers and explore their clinical significance in NSCLC. Using multiplex chromogenic immunohistochemistry, tissue microarrays of 553 primary tumors and 143 paired metastatic lymph nodes of NSCLC specimens were stained to detect various putative macrophage phenotypes: M1 (HLA-DR/CD68), M2 (CD163/CD68), M2 (CD204/CD68), and pan-macrophage (CD68/CK). Correlation analyses were performed to examine the relationship between TAMs and adaptive/innate immune infiltrates. HLA-DR+/CD68+M1 TAM level significantly decreased from pathological stage I to III. In a compartment-specific correlation analysis, moderate to strong correlations were observed between both TAM subsets (M1 and M2) with CD3-, CD8-, CD4-, and CD45RO-positive immune cells. Survival analyses, in both stromal and intratumoral compartments, revealed that high levels of HLA-DR+/CD68+M1 (stroma, hazard ratio [HR] = 0.73, P = .03; intratumor, HR = 0.7, P = .04), CD204+M2 (stroma, HR = 0.7, P = .02; intratumor, HR = 0.6, P = .004), and CD68 (stroma, HR = 0.69, P = .02; intratumor, HR = 0.73, P = .04) infiltration were independently associated with improved NSCLC-specific survival. In lymph nodes, the intratumoral level of HLA-DR+/CD68+M1 was an independent positive prognostic indicator (Cox model, HR = 0.38, P = .001). In conclusion, high levels of M1, CD204+M2, and CD68 macrophages are independent prognosticators of prolonged survival in NSCLC.

Source at https://doi.org/10.1038/s41598-018-36854-7. © The Author(s) 2019 A large number of miRNAs influence key cellular processes involved in prostate tumorigenesis. Previous studies have demonstrated high expression of miRNAs in human prostate cancer (PC) tissues and cell lines. In previous microarray data, we found miR-141 to be upregulated and miR-145 to be downregulated in PC. In this large PC cohort (n = 535), we explored the prognostic role of miR-141 and miR-145 in PC. Tumor epithelial (TE) and tumor stromal (TS) areas were evaluated separately and combined (TE + TS). In situ hybridization was used to evaluate the expression of the miRNAs. We found that miR-141 (TE) correlated significantly to Gleason score ≥8 (p = 0.040) and large tumor size (≥20 mm, p = 0.025) and miR-141 (TE + TS) to Gleason grade (p = 0.001). MiR-145 correlated to pT-stage (p = 0.038), tumor size (p = 0.025), Gleason grade (p = 0.051) and PSA (p = 0.032). In univariate analysis miR-141 (TE + TS) was significantly associated with biochemical failure-free survival (BFFS, p = 0.007) and clinical failure-free survival (CFFS, p = 0.021). For miR-145, there were no differences between patients with high versus low expression. In multivariate analysis overexpression of miR-141 in tumor epithelium and tumor stroma was significantly associated with BFFS (HR = 1.07 CI95% 1.00–1.14, p = 0.007). To conclude, high expression of miR-141 appears associated with increased risk of biochemical PC recurrence.

2622 Background: We previously showed that CDKN2A genomic alterations (GAs) are associated with resistance to ICI (Adib E, Clinical Cancer Research, 2021). The majority of such GAs are homozygous deletions, which commonly (̃50-80%) include MTAP, located 100kb telomeric of CDKN2A. MTAP loss leads to 5′-deoxy-5′-methylthioadenosine (MTA) accumulation and immunosuppressive effects in tumors. We examined combined CDKN2A/MTAP deletion vs. CDKN2A deletion/mutation alone as predictors of poor ICI response. Methods: We curated clinical data for cancer patients (pts) treated with ICI at Dana-Farber Cancer Institute through 6/2021, who had targeted panel sequencing. Inclusion criteria were: ICI in metastatic setting, ≥2 cycles, no concurrent systemic therapy, cancer type with > 50 pts treated. CDKN2A/ MTAP GAs were defined as a deep deletion affecting both genes; CDKN2A only GAs included both homozygous deletions and truncating mutations. Hazard ratios (HR) for overall survival (OS) and time-to-treatment failure (TTF) were derived using multivariable Cox regression, adjusted for prior lines of therapy, treatment type (single vs. combination ICI), tumor mutational burden and ECOG PS. We also used a machine learning approach to quantify the density of tumor-infiltrating lymphocytes (TILs) in digital whole-slide H&E images of 144 melanoma pts with available genomic data. Results: 921 pts with 6 cancer types were studied: non-small cell lung cancer (NSCLC, n = 366), melanoma (mel, n = 228), urothelial carcinoma (UC, n = 120), esophagogastric carcinoma (EGC,n = 90), head and neck squamous cell carcinoma (HNSCC, n = 58), and renal cell carcinoma (RCC, n = 59). UC pts with MTAP/ CDKN2A GAs had shorter OS and TTF than pts without GA in either gene (OS HR = 1.9[1.1-3.4], p = 0.005; TTF HR = 1.8[1.0-3.1], p = 0.0016) after adjusting for covariates. Similar results were seen for melanoma (OS HR = 2.5[1.4-2.6],p = 0.00065; TTF HR = 1.9[1.1-3.2],p = 0.018). There was no significant difference between pts with CDKN2A GA only and those without GA in either gene for OS or TTF in either UC or melanoma. CDKN2A/MTAP status was not associated with significantly shorter survival for NSCLC and EGC; while the analysis was confounded by HPV events for HNSCC, and underpowered for RCC. ML-based analysis of digital slides for melanoma, showed that tumors with CDKN2A GAs only (n = 42) had similar median density of TILs compared to tumors without GAs in either gene (n = 84; 920 vs. 943 TILs/mm2; p = 0.42). In contrast, tumors with co-occurring CDKN2A/ MTAP GAs had lower TIL density (529 TIL/mm2, n = 17 vs. 925 TIL/mm2, n = 126 (pooled); p = 0.018, Wilcoxon rank sum). Conclusions: In this study, we showed that co-occurrence of MTAP/CDKN2A GAs, but not CDKN2A GA only, was associated with worse outcomes in pts with UC and melanoma treated with ICI. Lower TIL density was also seen in melanoma tissue samples with combined MTAP/CDKN2A GA.

The demographic shift toward an older population will increase the number of prostate cancer cases. A challenge in the treatment of prostate cancer is to avoid undertreatment of patients at high risk of progression following curative treatment. These men can benefit from early salvage treatment. An explorative cohort consisting of tissue from 16 patients who underwent radical prostatectomy, and were either alive or had died from prostate cancer within 10 years postsurgery, was analyzed by mass spectrometry analysis. Following proteomic and bioinformatic analyses, major vault protein (MVP) was identified as a putative prognostic biomarker. A publicly available tissue proteomics dataset and a retrospective cohort of 368 prostate cancer patients were used for validation. The prognostic value of the MVP was verified by scoring immunohistochemical staining of a tissue microarray. High level of MVP was associated with more than 4-fold higher risk for death from prostate cancer (hazard ratio = 4.41, 95% confidence interval: 1.45–13.38; P = 0.009) in a Cox proportional hazard models, adjusted for Cancer of the Prostate Risk Assessments Post-surgical (CAPRA-S) score and perineural invasion. Decision curve analyses suggested an improved standardized net benefit, ranging from 0.06 to 0.18, of adding MVP onto CAPRA-S score. This observation was confirmed by receiver operator characteristics curve analyses for the CAPRA-S score versus CAPRA-S and MVP score (area under the curve: 0.58 versus 0.73). From these analyses, one can infer that MVP levels in combination with CAPRA-S score might add onto established risk parameters to identify patients with lethal prostate cancer., Mass spectrometry of an explorative cohort identified a promising biomarker, major vault protein. Using two independent validation cohorts, we verified an association between high MVP and adverse outcome. MVP was associated with prostate cancer-specific death independently of a composite risk model.

The TNM classification is well established as a state-of-the-art prognostic and treatment-decision-making tool for non-small cell lung cancer (NSCLC) patients. However, incorporation of biological data may hone the TNM system. This article focuses on choosing and incorporating subsets of tissue-infiltrating lymphocyte (TIL), detected by specific immunohistochemistry and automatically quantified by open source software, into a TNM-Immune cell score (TNM-I) for NSCLC. We use common markers (CD3, CD4, CD8, CD20 and CD45RO) of TILs to identify TIL subsets in tissue micro-arrays comprising tumor tissue from 553 patients resected for primary NSCLC. The number of TILs is automatically quantified using open source software (QuPath). Their prognostic efficacy, alone and within a TNM-I model, is evaluated in all patients and histological subgroups. Compared with previous manual semi-quantitative scoring of TILs in the same cohort, the present digital quantification proved superior. As a proof-of-concept, we construct a TNM-I, using TNM categories and the CD8+ TIL density. The TNM-I is an independent prognosticator of favorable diagnosis in both the overall cohort and in the main histological subgroups. In conclusion, CD8+ TIL density is the most promising candidate marker for a TNM-I in NSCLC. The prognostic efficacy of the CD8+ TIL density is strongest in lung squamous cell carcinomas, whereas both CD8+ TILs and CD20+ TILs, or a combination of these, may be candidates for a TNM-I in lung adenocarcinoma. Furthermore, based on the presented results, digital quantification is the preferred method for scoring TILs in the future., Digitally quantified CD8+ TIL density is a positive prognosticator for NSCLC, which combines with pStage into a TNM-Immune cell score (TNM-I). The combined score provides superior prognostication versus TNM alone. Moreover, histological adaptation of the TNM-I is likely further to increase its prognostic efficacy.

Published version. Source at http://doi.org/10.1038/bjc.2016.92. License CC BY-NC-SA 4.0. background: Operable non-small cell lung cancer (NSCLC) patients whose tumours have spread to regional or central lymph nodes at the time of diagnosis have dismal prognoses compared with those who have limited disease. The current TNM staging system for NSCLC poorly distinguishes patients with lymph-node metastases who will succumb to, and those who will eventually be cured from, their disease. This novel study: (1) evaluates the presence of different subsets of intraepithelial tumour-infiltrating lymphocytes (TILs) in lymph nodes with metastases from NSCLC patients; (2) explores the impact of intraepithelial TILs in lymph nodes on survival; (3) correlates their presence with both intraepithelial and stromal TILs in their corresponding primary tumours. methods: Metastatic lymph-node tissue from 143N+ NSCLC patients was collected and tissue microarrays were constructed. Immunohistochemistry was used to evaluate the presence of intraepithelial CD3+, CD4+, CD8+, CD20+ and CD45RO+ TILs and their impact on survival. results: A high level of intraepithelial CD45RO+ TILs in lymph-node metastases from N+ NSCLC patients was an independent positive prognostic factor for disease-specific survival in all patients (HR=0.58, P=0.029) and in squamous cell carcinoma (HR=0.31, P=0.006), but not in adenocarcinoma patients. conclusions: The presence of intraepithelial CD45RO+ cells in lymph-node metastases from N+ NSCLC patients predicts favourable disease-specific survival and outperforms the established TNM staging system in the SCC subgroup.

Published version also available at http://dx.doi.org/10.1016/j.neo.2015.11.004 Tumor-infiltrating lymphocytes (TILs) are vital in limiting cancer progression and may supplement the TNM classification. CD45RO+ memory TILs show major prognostic impact in various malignancies but have not been extensively explored in non–small cell lung cancer (NSCLC). In this study, we aimed to evaluate their potential in a NSCLC TNM-Immunoscore. Tissue microarrays were constructed from tumor tissue samples from two cohorts including in total 536 patients (University Hospital of North Norway, n = 285; Nordland Hospital, n = 251) with primary resected stage I to IIIA NSCLC. The density of CD45RO+ and CD8+ TILs in tumor epithelial and stromal compartments of the tumors was evaluated by immunohistochemistry. In univariate analyses, intraepithelial CD45RO+ TIL density (T-CD45RO) was a significant prognostic factor for disease-specific survival (P = .007), limited to the squamous cell carcinoma (SCC) histology subgroup (P b .001), where it was significant in both cohorts (University Hospital of North Norway, P = .003; Nordland Hospital, P = .022). Combining T-CD45RO and stromal CD8+ TIL density (S-CD8) increased the prognostic impact in SCC (P b .001) and showed a significant impact within all pathological stages (I, P = .025; II, P b .001; III, P = .001). In the multivariate analysis, T-CD45RO was an independent positive prognostic factor for SCC (hazard ratio 2.65, 95% confidence interval 1.64-4.28, P b .001), and in combination with S-CD8, the prognostic impact increased vastly (high + high versus low + low: hazard ratio 6.50, 95% confidence interval 3.54-11.91, P b .001). In conclusion, T-CD45RO was an independent prognostic factor for SCC NSCLC. When combined with S-CD8, the prognostic impact increased and was significant within each pathological stage. We propose CD45RO as a candidate marker for TNM-Immunoscore in SCC NSCLC.

The presence of tumor-infiltrating lymphocytes (TILs) positively impacts the outcome of non-small cell lung cancer (NSCLC) patients. Most previous studies have assessed TILs using different immunohistochemical assays. The purpose of this study was to develop and validate a histopathological scoring model for the assessment of TILs in whole-tissue hematoxylin and eosin (HE)-stained section slides of NSCLC patients and to evaluate the model in an immunoscore setting. Therefore, TIL was evaluated manually on HE slides from 537 surgical specimens of primary resected stage I-III NSCLC patients. Using stromal TIL score as a stepwise discrete variable, increasing survival was seen with rising TIL level: disease-specific survival (DSS; P = .008), overall survival (P = .036) and disease-free survival (P = .006). Subgroup analysis revealed that high stromal TILs level was associated with superior DSS (P = .047) in patients with squamous cell carcinoma, but not in patients with adenocarcinoma. Multivariable analysis confirmed that high TIL levels independently predict improved prognosis for all endpoints in the overall cohort. In conclusion, high stromal TIL level is an independent favorable prognostic factor in stage I-III NSCLC patients. The comprehensive histological evaluation conducted in this study may be helpful in streamlining TIL quantification for routine clinical use in a future NSCLC immunoscore setting.

Source at: http://doi.org/10.1371/journal.pone.0192157 Objectives: Selective targeting of cancer-associated fibroblasts (CAFs) has been proposed to synergize with immune-checkpoint inhibitors. While the roles of CAFs in cancer development are well described, their immune-regulatory properties remain incompletely understood. This study investigates correlations between CAF and immune-markers in tumor stroma from nonsmall cell lung cancer (NSCLC) patients, and examines whether a combination of CAF and immune cell scores impact patient prognosis. Methods: Tumor specimens from 536 primary operable stage I-III NSCLC patients were organized in tissue microarrays. Expression of protein-markers was evaluated by immunohistochemistry. Results: Fibroblast and stromal markers PDGFRα, PDGFRβ, FAP-1 and vimentin showed weak correlations while αSMA, and Masson’s trichrome did not correlate with any of the investigated markers. Hierarchical clustering indicated the existence of different CAF-subsets. No relevant correlations were found between any CAF-marker and the immune-markers CD3, CD4, CD8, CD20, CD68, CD1a, CD56, FoxP3 and CD45RO. High density of fibroblast-activation protein positive mesenchymal cells (CAFFAP) was associated with better prognosis in tumors with high infiltration of CD8 and CD3 T-lymphocytes. Conclusions: The presented data suggest that CAFs, irrespective of identity, have low influence on the degree of tumor infiltration by inflammatory- and/or immune-cells. However, CAFFAP may exert immuno-adjuvant roles in NSCLC, and targeting CAFs should be cautiously considered.

Source at https://doi.org/10.1038/s41598-018-29520-5. The role of steroid hormones in carcinogenesis of the prostate is to some extent unraveled thorough the effect of androgen deprivation therapy on prostate cancer (PCa) progression. Other members of the steroid hormone family, such as progesterone, are also implicated in PCa, but progesterone’s role remains undefined. This study aimed to examine the distribution of progesterone receptor isoforms (PGRA, PGRB) in PCa tissue and their association with clinical endpoints. This was conducted retrospectively by collecting radical prostatectomy specimens from 535 patients. Tissue was analyzed using tissue microarray, where representative tumor areas were carefully selected. Protein expression was evaluated through immunohistochemistry, in stromal and epithelial tissue. Associations between receptor expression and clinical data were considered using statistical survival analyses. Herein, we discovered a solely stromal PGRA- and a stromal and epithelial PGRB expression. Further, a high PGRB expression in tumor tissue was associated with an unfavorable prognosis in both univariate and multivariate analyses: Biochemical failure (HR: 2.0, 95% CI: 1.45–2.76, p

Source at https://doi.org/10.1371/journal.pone.0189000 Background: Prostate cancer (PC) stratification needs new prognostic tools to reduce overtreatment. Phosphatase of regenerating liver (PRL-3) is a phosphatase found at high levels in several cancer types, where its expression is associated with survival. A recent PC cell line study has shown it to be involved in PC growth and migration. Methods: We used a monoclonal antibody to evaluate the expression of PRL-3 in PC tissue of patients in an unselected cohort of 535 prostatectomy patients. We analyzed associations between PRL-3 expression and biochemical failure-free survival (BFFS), clinical failure-free survival (CFFS) and PC death-free survival (PCDFS). Results: Cytoplasmic PRL-3 staining in tumor cells was significantly correlated to expression of molecules in the VEGFR-axis, but not to the clinicopathological variables. High PRL-3 was not significantly associated with survival in the univariate analysis for BFFS (p = 0.131), but significantly associated with CFFS (p = 0.044) and PCDFS (p = 0.041). In multivariate analysis for the various end points, PRL-3 came out as an independent and significant indicator of poor survival for BFFS (HR = 1.53, CI95% 1.10–2.13, p = 0.012), CFFS (HR = 2.41, CI95% 1.17–4.98, p = 0.017) and PCDFS (HR = 3.99, CI95% 1.21–13.1, p = 0.023). Conclusions: PRL-3 is independently associated with all PC endpoints in this study. Since high PRL-3 expression also correlates with poor prognosis in other cancers and functional studies in PC support these findings, PRL-3 emerges as a potential treatment target in PC.

Background Lymphocyte activation gene-3 (LAG-3) is an immune checkpoint receptor and a putative therapeutic target in non–small-cell lung cancer (NSCLC). We explored the prognostic effect of LAG-3+ tumor-infiltrating lymphocytes (TILs) in primary tumors and metastatic lymph nodes in NSCLC and its potential for inclusion in an immunoscore, supplementing the TNM classification. Materials and Methods Primary tumor tissue from 553 stage I-IIIB NSCLC patients and 143 corresponding metastatic lymph nodes were collected. The expression of LAG-3 was evaluated by immunohistochemistry on tissue microarrays. Results On univariate analysis, LAG-3+ TILs in the intraepithelial and stromal compartments of primary tumors and in the intraepithelial and extraepithelial compartments of metastatic lymph nodes were associated with improved disease-specific survival (DSS). On multivariate analysis, stromal LAG-3+ TILs were a significant independent predictor of improved DSS (hazard ratio [HR], 0.59; 95% confidence interval [CI], 0.43-0.82; P = .002). Stromal LAG-3+ TILs did not have prognostic impact across all pathologic stages. In the metastatic lymph nodes, intraepithelial (HR, 0.61; 95% CI, 0.38-0.99; P = .049) and extraepithelial (HR, 0.54; 95% CI, 0.29-0.70; P Conclusion LAG-3+ TILs are an independent positive prognostic factor in stage I-IIIB NSCLC. LAG-3 in metastatic lymph nodes is a candidate marker for an immunoscore in NSCLC.