High-throughput transcriptome profiling indicates ribosomal RNAs to be associated with resistance to immunotherapy in non-small cell lung cancer (NSCLC).

BACKGROUND: Despite the impressive outcomes with immune checkpoint inhibitor (ICI) in non-small cell lung cancer (NSCLC), only a minority of the patients show long-term benefits from ICI. In this study, we used retrospective cohorts of ICI treated patients with NSCLC to discover and validate spatially resolved protein markers associated with resistance to programmed cell death protein-1 (PD-1) axis inhibition. METHODS: Pretreatment samples from 56 patients with NSCLC treated with ICI were collected and analyzed in a tissue microarray (TMA) format in including four different tumor regions per patient using the GeoMx platform for spatially informed transcriptomics. 34 patients had assessable tissue with tumor compartment in all 4 TMA spots, 22 with leukocyte compartment and 12 with CD68 compartment. The patients' tissue that was not assessable in fourfold redundancy in each compartment was designated as the validation cohort; cytokeratin (CK) (N=22), leukocytes CD45 (N=31), macrophages, CD68 (N=43). The human whole transcriptome, represented by~18,000 individual genes assessed by oligonucleotide-tagged in situ hybridization, was sequenced on the NovaSeq platform to quantify the RNAs present in each region of interest. RESULTS: 54,000 gene variables were generated per case, from them 25,740 were analyzed after removing targets with expression lower than a prespecified frequency. Cox proportional-hazards model analysis was performed for overall and progression-free survival (OS, PFS, respectively). After identifying genes significantly associated with limited survival benefit (HR>1)/progression per spot per patient, we used the intersection of them across the four TMA spots per patient. This resulted in a list of 12 genes in the tumor-cell compartment (RPL13A, GNL3, FAM83A, CYBA, ACSL4, SLC25A6, EPAS1, RPL5, APOL1, HSPD1, RPS4Y1, ADI1). RPL13A, GNL3 in tumor-cell compartment were also significantly associated with OS and PFS, respectively, in the validation cohort (CK: HR, 2.48; p=0.02 and HR, 5.33; p=0.04). In CD45 compartment, secreted frizzled-related protein 2, was associated with OS in the discovery cohort but not in the validation cohort. Similarly, in the CD68 compartment ARHGAP and PNN interacting serine and arginine rich protein were significantly associated with PFS and OS, respectively, in the majority but not all four spots per patient. CONCLUSION: This work highlights RPL13A and GNL3 as potential indicative biomarkers of resistance to PD-1 axis blockade that might help to improve precision immunotherapy strategies for lung cancer.

Spatially resolved tissue imaging to analyze the tumor immune microenvironment: beyond cell-type densities.

INTRODUCTION: The tissue immune microenvironment is associated with key aspects of tumor biology. The interaction between the immune system and cancer cells has predictive and prognostic potential across different tumor types. Spatially resolved tissue-based technologies allowed researchers to simultaneously quantify different immune populations in tumor samples. However, bare quantification fails to harness the spatial nature of tissue-based technologies. Tumor-immune interactions are associated with specific spatial patterns that can be measured. In recent years, several computational tools have been developed to increase our understanding of these spatial patterns. TOPICS COVERED: In this review, we cover standard techniques as well as new advances in the field of spatial analysis of the immune microenvironment. We focused on marker quantification, spatial intratumor heterogeneity analysis, cell‒cell spatial interaction studies and neighborhood analyses.

Distinct genomic and immunologic tumor evolution in germline TP53-driven breast cancers.

Pathogenic germline TP53 alterations cause Li-Fraumeni Syndrome (LFS), and breast cancer is the most common cancer in LFS females. We performed first of its kind multimodal analysis of LFS breast cancer (LFS-BC) compared to sporadic premenopausal BC. Nearly all LFS-BC underwent biallelic loss of TP53 with no recurrent oncogenic variants except ERBB2 (HER2) amplification. Compared to sporadic BC, in situ and invasive LFS-BC exhibited a high burden of short amplified aneuploid segments (SAAS). Pro-apoptotic p53 target genes BAX and TP53I3 failed to be up-regulated in LFS-BC as was seen in sporadic BC compared to normal breast tissue. LFS-BC had lower CD8+ T-cell infiltration compared to sporadic BC yet higher levels of proliferating cytotoxic T-cells. Within LFS-BC, progression from in situ to invasive BC was marked by an increase in chromosomal instability with a decrease in proliferating cytotoxic T-cells. Our study uncovers critical events in mutant p53-driven tumorigenesis in breast tissue.

Vascular mimicry as a facilitator of melanoma brain metastasis.

Melanoma has the highest propensity among solid tumors to metastasize to the brain. Melanoma brain metastases (MBM) are a leading cause of death in melanoma and affect 40-60% of patients with late-stage disease. Therefore, uncovering the molecular mechanisms behind MBM is necessary to enhance therapeutic interventions. Vascular mimicry (VM) is a form of neovascularization linked to invasion, increased risk of metastasis, and poor prognosis in many tumor types, but its significance in MBM remains poorly understood. We found that VM density is elevated in MBM compared to paired extracranial specimens and is associated with tumor volume and CNS edema. In addition, our studies indicate a relevant role of YAP and TAZ, two transcriptional co-factors scarcely studied in melanoma, in tumor cell-vasculogenesis and in brain metastasis. We recently demonstrated activation of the Hippo tumor suppressor pathway and increased degradation of its downstream targets YAP and TAZ in a metastasis impaired cell line model. In the current study we establish the utility of anti-YAP/TAZ therapy in mouse models of metastatic melanoma whereby treatment effectively inhibits VM and prolongs survival of mice with MBM. The data presented herein suggest that VM may be an important and targetable mechanism in melanoma and that VM inhibition might be useful for treating MBM, an area of high unmet clinical need, thus having important implications for future treatment regimens for these patients.

Up-regulated PLA2G10 in cancer impairs T cell infiltration to dampen immunity.

T cells are often absent from human cancer tissues during both spontaneously induced immunity and therapeutic immunotherapy, even in the presence of a functional T cell-recruiting chemokine system, suggesting the existence of T cell exclusion mechanisms that impair infiltration. Using a genome-wide in vitro screening platform, we identified a role for phospholipase A2 group 10 (PLA2G10) protein in T cell exclusion. PLA2G10 up-regulation is widespread in human cancers and is associated with poor T cell infiltration in tumor tissues. PLA2G10 overexpression in immunogenic mouse tumors excluded T cells from infiltration, resulting in resistance to anti-PD-1 immunotherapy. PLA2G10 can hydrolyze phospholipids into small lipid metabolites, thus inhibiting chemokine-mediated T cell mobility. Ablation of PLA2G10's enzymatic activity enhanced T cell infiltration and sensitized PLA2G10-overexpressing tumors to immunotherapies. Our study implicates a role for PLA2G10 in T cell exclusion from tumors and suggests a potential target for cancer immunotherapy.

Objective Analysis and Clinical Significance of the Spatial Tumor-Infiltrating Lymphocyte Patterns in Non-Small Cell Lung Cancer.

PURPOSE: The spatial arrangement of lymphocytes in the tumor bed (e.g., immune infiltrated, immune excluded, immune desert) is expected to reflect distinct immune evasion mechanisms and to associate with immunotherapy outcomes. However, data supporting these associations are scant and limited by the lack of a clear definition for lymphocyte infiltration patterns and the subjective nature of pathology-based approaches. EXPERIMENTAL DESIGN: We used multiplexed immunofluorescence to study major tumor-infiltrating lymphocyte (TIL) subsets with single-cell resolution in baseline whole-tissue section samples from NSCLC patients treated with immune checkpoint inhibitors (ICI). The spatial TIL patterns were analyzed using a qualitative pathologist-based approach, and an objective analysis of TIL density ratios in tumor/stromal tissues. The association of spatial patterns with outcomes was studied for different TIL markers. RESULTS: The analysis of CD8+ TIL patterns using qualitative assessment identified prominent limitations including the presence of a broad spectrum of phenotypes within most tumors and limited association with outcomes. The utilization of an objective method to classify NSCLCs showed the existence of at least three subgroups with partial overlap with those defined using visual patterns. Using this strategy, a subset of cases with "immune excluded-like" tumors showed prominently worse outcomes, suggesting reduced sensitivity to ICI; however, these results need to be validated. The analysis for other TIL subsets showed different results, underscoring the relevance of the marker selected for spatial TIL pattern evaluation and opportunities for market integration. CONCLUSIONS: Our results identified major challenges associated with the qualitative spatial TIL pattern evaluation. We devised a novel objective strategy to overcome some of these limitations that has strong biomarker potential.

Digital spatial profiling of melanoma shows CD95 expression in immune cells is associated with resistance to immunotherapy.

Although immune checkpoint inhibitor (ICI) therapy has dramatically improved outcome for metastatic melanoma patients, many patients do not benefit. Since adverse events may be severe, biomarkers for resistance would be valuable, especially in the adjuvant setting. We performed high-plex digital spatial profiling (DSP) using the NanoString GeoMx® on 53 pre-treatment specimens from ICI-treated metastatic melanoma cases. We interrogated 77 targets simultaneously in four molecular compartments defined by S100B for tumor, CD68 for macrophages, CD45 for leukocytes, and nonimmune stromal cells defined as regions negative for all three compartment markers but positive for SYTO 13. For DSP validation, we confirmed the results obtained for some immune markers, such as CD8, CD4, CD20, CD68, CD45, and PD-L1, by quantitative immunofluorescence (QIF). In the univariable analysis, 38 variables were associated with outcome, 14 of which remained significant after multivariable adjustment. Among them, CD95 was further validated using multiplex immunofluorescence in the Discovery immunotherapy (ITX) Cohort and an independent validation cohort with similar characteristics, showing an association between high levels of CD95 and shorter progression-free survival. We found that CD95 in stroma was associated with resistance to ICI. With further validation, this biomarker could have value to select patients that will not benefit from immunotherapy.

Atezolizumab plus stereotactic ablative radiotherapy for medically inoperable patients with early-stage non-small cell lung cancer: a multi-institutional phase I trial.

Stereotactic ablative radiotherapy (SABR) is a standard-of-care for medically-inoperable-early-stage non-small cell lung cancer (NSCLC). One third of patients progress and chemotherapy is rarely used in this population. We questioned if addition of the immune-checkpoint-inhibitor (ICI) atezolizumab to standard-of-care SABR can improve outcomes. We initiated a multi-institutional single-arm phase I study (NCT02599454) enrolling twenty patients with the primary endpoint of maximum tolerated dose (MTD); secondary endpoints of safety and efficacy; and exploratory mechanistic correlatives. Treatment is well tolerated and full dose atezolizumab (1200 mg) is the MTD. Efficacy signals include early responses (after 2 cycles of ICI, before initiation of SABR) in 17% of patients. Biomarkers of functional adaptive immunity, including T cell activation in the tumor and response to ex-vivo stimulation by circulating T cells, are highly predictive of benefit. These results require validation and are being tested in a phase III randomized trial.

International Association for the Study of Lung Cancer Study of Reproducibility in Assessment of Pathologic Response in Resected Lung Cancers After Neoadjuvant Therapy.

INTRODUCTION: Pathologic response has been proposed as an early clinical trial end point of survival after neoadjuvant treatment in clinical trials of NSCLC. The International Association for the Study of Lung Cancer (IASLC) published recommendations for pathologic evaluation of resected lung cancers after neoadjuvant therapy. The aim of this study was to assess pathologic response interobserver reproducibility using IASLC criteria. METHODS: An international panel of 11 pulmonary pathologists reviewed hematoxylin and eosin-stained slides from the lung tumors of resected NSCLC from 84 patients who received neoadjuvant immune checkpoint inhibitors in six clinical trials. Pathologic response was assessed for percent viable tumor, necrosis, and stroma. For each slide, tumor bed area was measured microscopically, and pre-embedded formulas calculated unweighted and weighted major pathologic response (MPR) averages to reflect variable tumor bed proportion. RESULTS: Unanimous agreement among pathologists for MPR was observed in 68 patients (81%), and inter-rater agreement (IRA) was 0.84 (95% confidence interval [CI]: 0.76-0.92) and 0.86 (95% CI: 0.79-0.93) for unweighted and weighted averages, respectively. Overall, unweighted and weighted methods did not reveal significant differences in the classification of MPR. The highest concordance by both methods was observed for cases with more than 95% viable tumor (IRA = 0.98, 95% CI: 0.96-1) and 0% viable tumor (IRA = 0.94, 95% CI: 0.89-0.98). The most common reasons for discrepancies included interpretations of tumor bed, presence of prominent stromal inflammation, distinction between reactive and neoplastic pneumocytes, and assessment of invasive mucinous adenocarcinoma. CONCLUSIONS: Our study revealed excellent reliability in cases with no residual viable tumor and good reliability for MPR with the IASLC recommended less than or equal to 10% cutoff for viable tumor after neoadjuvant therapy.

The ß1-adrenergic receptor links sympathetic nerves to T cell exhaustion.

CD8+ T cells are essential components of the immune response against viral infections and tumours, and are capable of eliminating infected and cancerous cells. However, when the antigen cannot be cleared, T cells enter a state known as exhaustion1. Although it is clear that chronic antigen contributes to CD8+ T cell exhaustion, less is known about how stress responses in tissues regulate T cell function. Here we show a new link between the stress-associated catecholamines and the progression of T cell exhaustion through the ß1-adrenergic receptor ADRB1. We identify that exhausted CD8+ T cells increase ADRB1 expression and that exposure of ADRB1+ T cells to catecholamines suppresses their cytokine production and proliferation. Exhausted CD8+ T cells cluster around sympathetic nerves in an ADRB1-dependent manner. Ablation of ß1-adrenergic signalling limits the progression of T cells towards the exhausted state in chronic infection and improves effector functions when combined with immune checkpoint blockade (ICB) in melanoma. In a pancreatic cancer model resistant to ICB, ß-blockers and ICB synergize to boost CD8+ T cell responses and induce the development of tissue-resident memory-like T cells. Malignant disease is associated with increased catecholamine levels in patients2,3, and our results establish a connection between the sympathetic stress response, tissue innervation and T cell exhaustion. Here, we uncover a new mechanism by which blocking ß-adrenergic signalling in CD8+ T cells rejuvenates anti-tumour functions.

Neuroendocrine lineage commitment of small cell lung cancers can be leveraged into p53-independent non-cytotoxic therapy.

Small cell lung cancers (SCLCs) rapidly resist cytotoxic chemotherapy and immune checkpoint inhibitor (ICI) treatments. New, non-cross-resistant therapies are thus needed. SCLC cells are committed into neuroendocrine lineage then maturation arrested. Implicating DNA methyltransferase 1 (DNMT1) in the maturation arrests, we find (1) the repression mark methylated CpG, written by DNMT1, is retained at suppressed neuroendocrine-lineage genes, even as other repression marks are erased; (2) DNMT1 is recurrently amplified, whereas Ten-Eleven-Translocation 2 (TET2), which functionally opposes DNMT1, is deleted; (3) DNMT1 is recruited into neuroendocrine-lineage master transcription factor (ASCL1, NEUROD1) hubs in SCLC cells; and (4) DNMT1 knockdown activated ASCL1-target genes and released SCLC cell-cycling exits by terminal lineage maturation, which are cycling exits that do not require the p53/apoptosis pathway used by cytotoxic chemotherapy. Inhibiting DNMT1/corepressors with clinical compounds accordingly extended survival of mice with chemorefractory and ICI-refractory, p53-null, disseminated SCLC. Lineage commitment of SCLC cells can hence be leveraged into non-cytotoxic therapy able to treat chemo/ICI-refractory SCLC.

Quantitative DNA Repair Biomarkers and Immune Profiling for Temozolomide and Olaparib in Metastatic Colorectal Cancer.

Purpose: O6-methylguanine DNA methyltransferase (MGMT)-silenced tumors reveal sensitivity to temozolomide (TMZ), which may be enhanced by PARP inhibitors. Approximately 40% of colorectal cancer has MGMT silencing and we aimed to measure antitumoral and immunomodulatory effects from TMZ and olaparib in colorectal cancer. Experimental Design: Patients with advanced colorectal cancer were screened for MGMT promoter hypermethylation using methylation-specific PCR of archival tumor. Eligible patients received TMZ 75 mg/m2 days 1-7 with olaparib 150 mg twice daily every 21 days. Pretreatment tumor biopsies were collected for whole-exome sequencing (WES), and multiplex quantitative immunofluorescence (QIF) of MGMT protein expression and immune markers. Results: MGMT promoter hypermethylation was detected in 18/51 (35%) patients, 9 received study treatment with no objective responses, 5/9 had stable disease (SD) and 4/9 had progressive disease as best response. Three patients had clinical benefit: carcinoembryonic antigen reduction, radiographic tumor regression, and prolonged SD. MGMT expression by multiplex QIF revealed prominent tumor MGMT protein from 6/9 patients without benefit, while MGMT protein was lower in 3/9 with benefit. Moreover, benefitting patients had higher baseline CD8+ tumor-infiltrating lymphocytes. WES revealed 8/9 patients with MAP kinase variants (7 KRAS and 1 ERBB2). Flow cytometry identified peripheral expansion of effector T cells. Conclusions: Our results indicate discordance between MGMT promoter hypermethylation and MGMT protein expression. Antitumor activity seen in patients with low MGMT protein expression, supports MGMT protein as a predictor of alkylator sensitivity. Increased CD8+ TILs and peripheral activated T cells, suggest a role for immunostimulatory combinations. Significance: TMZ and PARP inhibitors synergize in vitro and in vivo in tumors with MGMT silencing. Up to 40% of colorectal cancer is MGMT promoter hypermethylated, and we investigated whether TMZ and olaparib are effective in this population. We also measured MGMT by QIF and observed efficacy only in patients with low MGMT, suggesting quantitative MGMT biomarkers more accurately predict benefit to alkylator combinations.

NCI 7977: A Phase I Dose-Escalation Study of Intermittent Oral ABT-888 (Veliparib) plus Intravenous Irinotecan Administered in Patients with Advanced Solid Tumors.

Purpose: Veliparib is a PARP inhibitor (PARPi) with activity in BRCA 1/2/PALB2-deficient tumors. Preclinical observations reveal topoisomerase inhibitors like irinotecan are synergistic with PARPi irrespective of homologous recombination deficiency (HRD), potentially expanding the role for PARPi. Experimental Design: NCI 7977 was a multicohort phase I clinical trial evaluating the safety and efficacy of multiple dose schedules of veliparib with irinotecan for solid tumors. In the intermittent veliparib cohort, escalating doses of veliparib were given twice daily at dose level (DL) 1 (50 mg) and DL 2 (100 mg) days 1-4 and 8-11 with irinotecan 100 mg/m2 days 3 and 10 in 21-day cycles. Results: Fifteen patients enrolled, 8 of 15 (53%) received ≥4 prior systemic treatments. At DL1, 1 of 6 patients experienced a dose-limiting toxicity (DLT) of diarrhea. At DL2, 9 patients were treated, with 3 unevaluable for DLT, and 2 of 6 evaluable patients experienced a DLT of grade 3 neutropenia. Irinotecan 100 mg/m2 and veliparib 50 mg twice daily was the MTD. No objective responses were observed, although 4 patients had progression-free survival >6 months. Conclusions: The MTD of intermittent veliparib is 50 mg twice daily days 1-4 and 8-11 with weekly irinotecan 100 mg/m2 days 3 and 10 every 21 days. Multiple patients experienced prolonged stable disease irrespective of HRD and prior irinotecan. However, due to the toxicities with higher dose intermittent veliparib and irinotecan, this schedule was determined too toxic for further development and the arm was closed prematurely. Significance: The combination of intermittent veliparib with weekly irinotecan was deemed too toxic for further development. Future PARPi combinations should focus on agents with nonoverlapping toxicities to improve tolerability. The treatment combination showed limited efficacy with prolonged stable disease observed in multiple heavily pretreated patients, but no objective responses were seen.

Developing a definition of immune exclusion in cancer: results of a modified Delphi workshop.

Checkpoint inhibitors represent an effective treatment approach for a variety of cancers through their inhibition of immune regulatory pathways within the tumor microenvironment (TME). Unfortunately only a minority of patients with cancer achieve clinical benefit from immunotherapy, with the TME emerging as an important predictor of outcomes and sensitivity to therapy. The extent and pattern of T-cell infiltration can vary prominently within/across tumors and represents a biological continuum. Three immune profiles have been identified along this continuum: 'immune-desert' or 'T-cell cold' phenotype, 'immune-active', 'inflamed', or 'T-cell hot' phenotype, and 'immune excluded' phenotype. Of the three profiles, immune excluded remains the most ill-defined with no clear, universally accepted definition even though it is commonly associated with lack of response to immune checkpoint inhibitors and poor clinical outcomes. To address this, 16 multidisciplinary cancer experts from around the world were invited to participate in a symposium using a three-round modified Delphi approach. The first round was an open-ended questionnaire distributed via email and the second was an in-person discussion of the first round results that allowed for statements to be revised as necessary to achieve a maximum consensus (75% agreement) among the rating committee (RC). The final round questionnaire was distributed to the RC via email and had a 100% completion rate. The Delphi process resulted in moving us closer to a consensus definition for immune exclusion that is practical, clinically pertinent, and applicable across a wide range of cancer histologies. A general consensus of the role of immune exclusion in resistance to checkpoint therapy and five research priorities emerged from this process. Together, these tools could help efforts designed to address the underlying mechanisms of immune exclusion that span cancer types and, ultimately, aid in the development of treatments to target these mechanisms to improve patient outcomes.

Deep computational image analysis of immune cell niches reveals treatment-specific outcome associations in lung cancer.

The tumor immune composition influences prognosis and treatment sensitivity in lung cancer. The presence of effective adaptive immune responses is associated with increased clinical benefit after immune checkpoint blockers. Conversely, immunotherapy resistance can occur as a consequence of local T-cell exhaustion/dysfunction and upregulation of immunosuppressive signals and regulatory cells. Consequently, merely measuring the amount of tumor-infiltrating lymphocytes (TILs) may not accurately reflect the complexity of tumor-immune interactions and T-cell functional states and may not be valuable as a treatment-specific biomarker. In this work, we investigate an immune-related biomarker (PhenoTIL) and its value in associating with treatment-specific outcomes in non-small cell lung cancer (NSCLC). PhenoTIL is a novel computational pathology approach that uses machine learning to capture spatial interplay and infer functional features of immune cell niches associated with tumor rejection and patient outcomes. PhenoTIL's advantage is the computational characterization of the tumor immune microenvironment extracted from H&E-stained preparations. Association with clinical outcome and major non-small cell lung cancer (NSCLC) histology variants was studied in baseline tumor specimens from 1,774 lung cancer patients treated with immunotherapy and/or chemotherapy, including the clinical trial Checkmate 057 (NCT01673867).

Genomic and transcriptomic analysis of checkpoint blockade response in advanced non-small cell lung cancer.

Anti-PD-1/PD-L1 agents have transformed the treatment landscape of advanced non-small cell lung cancer (NSCLC). To expand our understanding of the molecular features underlying response to checkpoint inhibitors in NSCLC, we describe here the first joint analysis of the Stand Up To Cancer-Mark Foundation cohort, a resource of whole exome and/or RNA sequencing from 393 patients with NSCLC treated with anti-PD-(L)1 therapy, along with matched clinical response annotation. We identify a number of associations between molecular features and outcome, including (1) favorable (for example, ATM altered) and unfavorable (for example, TERT amplified) genomic subgroups, (2) a prominent association between expression of inducible components of the immunoproteasome and response and (3) a dedifferentiated tumor-intrinsic subtype with enhanced response to checkpoint blockade. Taken together, results from this cohort demonstrate the complexity of biological determinants underlying immunotherapy outcomes and reinforce the discovery potential of integrative analysis within large, well-curated, cancer-specific cohorts.

Human Immunodeficiency Virus Is Associated With Poor Overall Survival Among Patients With Head and Neck Cancer.

BACKGROUND: Head and neck squamous cell cancer (HNSCC) occurs at higher rates among persons with HIV (PWH). This study compares the impact of sociodemographic and clinicopathologic characteristics on outcomes among PWH-HNSCC compared with HNSCC patients without HIV. METHODS: Patient data from HNSCC individuals were collected at a single academic hospital center between 2002 and 2018. Forty-eight patients with HIV (HIV-HNSCC) and 2894 HNSCC patients without HIV were included. Multivariate analysis determined predictors of survival using Cox proportional hazards regression model. HIV-positive and -negative tumors were analyzed by quantitative immunofluorescence for expression of CD4, CD8, CD20 and PD-L1. RESULTS: HIV-HNSCC patients had a lower median overall survival than HNSCC patients without HIV (34 [18-84] vs 94 [86-103] months; P < .001). In multivariate analysis that included age, sex, race/ethnicity, stage, site, tobacco use, time to treatment initiation, and insurance status, HIV was an independent predictor of poorer survival, with a hazard ratio of 1.98 (95% CI: 1.32-2.97; P < .001). PWH with human papillomavirus (HPV)-positive oropharyngeal tumors also had worse prognosis than HPV-positive oropharyngeal tumors in the population without HIV (P < .001). The tumor microenvironment among HIV-HNSCC patients revealed lower intratumoral CD8 infiltration among HIV+ HPV+ tumors compared with HIV- HPV+ tumors (P = .04). CONCLUSIONS: HIV-HNSCC patients had worse prognosis than the non-HIV population, with HIV being an independent predictor of poor clinical outcomes when accounting for important sociodemographic and clinicopathologic factors. Our findings highlight differences in tumor biology that require further detailed characterization in large cohorts and increased inclusion of PWH in immunotherapy trials.

Predictive Biomarkers for Immunotherapy in Lung Cancer: Perspective From the International Association for the Study of Lung Cancer Pathology Committee.

Immunotherapy including immune checkpoint inhibitors (ICIs) has become the backbone of treatment for most lung cancers with advanced or metastatic disease. In addition, they have increasingly been used for early stage tumors in neoadjuvant and adjuvant settings. Unfortunately, however, only a subset of patients experiences meaningful response to ICIs. Although programmed death-ligand 1 (PD-L1) protein expression by immunohistochemistry (IHC) has played a role as the principal predictive biomarker for immunotherapy, its performance may not be optimal, and it suffers multiple practical issues with different companion diagnostic assays approved. Similarly, tumor mutational burden (TMB) has multiple technical issues as a predictive biomarker for ICIs. Now, ongoing research on tumor- and host immune-specific factors has identified immunotherapy biomarkers that may provide better response and prognosis prediction, in particular in a multimodal approach. This review by the International Association for the Study of Lung Cancer Pathology Committee provides an overview of various immunotherapy biomarkers, including updated data on PD-L1 IHC and TMB, and assessments of neoantigens, genetic and epigenetic signatures, immune microenvironment by IHC and transcriptomics, and microbiome and pathologic response to neoadjuvant immunotherapies. The aim of this review is to underline the efficacy of new individual or combined predictive biomarkers beyond PD-L1 IHC and TMB.

Spatially resolved proteomic profiling identifies tumor cell CD44 as a biomarker associated with sensitivity to PD-1 axis blockade in advanced non-small-cell lung cancer.

BACKGROUND: Most patients with advanced non-small-cell lung cancer (NSCLC) fail to derive significant benefit from programmed cell death protein-1 (PD-1) axis blockade, and new biomarkers of response are needed. In this study, we aimed to discover and validate spatially resolved protein markers associated with sensitivity to PD-1 axis inhibition in NSCLC. METHODS: We initially assessed a discovery cohort of 56 patients with NSCLC treated with PD-1 axis inhibitors at Yale Cancer Center. Using the GeoMx Digital Spatial Profiling (DSP) system, 71 proteins were measured in spatial context on each spot in a tissue microarray. We used the AQUA method of quantitative immunofluorescence (QIF) to orthogonally validate candidate biomarkers. For external independent validation, we assessed whole tissue sections derived from 128 patients with NSCLC treated with single-agent PD-1 axis inhibitors at the 12 de Octubre Hospital (Madrid) using DSP. We further analyzed two immunotherapy untreated cohorts to address prognostic significance (n=252 from Yale Cancer Center; n=124 from University Clinic of Navarra) using QIF and DSP, respectively. RESULTS: Using continuous log-scaled data, we identified CD44 expression in the tumor compartment (pan-cytokeratin (CK)+) as a novel predictor of prolonged progression-free survival (PFS) (multivariate HR=0.68, p=0.043) in the discovery set. We validated by QIF that tumor CD44 levels assessed as continuous QIF scores were associated with longer PFS (multivariate HR=0.31, p=0.022) and overall survival (multivariate HR=0.29, p=0.038). Using DSP in an independent immunotherapy treated cohort, we validated that CD44 levels in the tumor compartment, but not in the immune compartment (panCK-/CD45+), were associated with clinical benefit (OR=1.22, p=0.018) and extended PFS under PD-1 axis inhibition using the highest tertile cutpoint (multivariate HR=0.62, p=0.03). The effect of tumor cell CD44 in predicting PFS remained significant after correcting for programmed death-ligand 1 (PD-L1) Tumor Proportion Score (TPS) in both cohorts. High tumor cell CD44 was not prognostic in the absence of immunotherapy. Using DSP data, intratumoral regions with elevated tumor cell CD44 expression showed prominent (fold change>1.5, adjusted p<0.05) upregulation of PD-L1, TIM-3, ICOS, and CD40 in two independent cohorts. CONCLUSIONS: This work highlights CD44 as a novel indicative biomarker of sensitivity to PD-1 axis blockade that might help to improve immunotherapy strategies for NSCLC.

Quantitative tissue analysis and role of myeloid cells in non-small cell lung cancer.

BACKGROUND: Despite the prominent role of innate immunity in the antitumor response, little is known about the myeloid composition of human non-small cell lung cancer (NSCLC) with respect to histology and molecular subtype. We used multiplexed quantitative immunofluorescence (QIF) to measure the distribution and clinical significance of major myeloid cell subsets in large retrospective NSCLC collections. METHODS: We established a QIF panel to map major myeloid cell subsets in fixed human NSCLC including 4',6-Diamidino-2-Phenylindole for all cells, pancytokeratin for tumor-epithelial cells, CD68 for M1-like macrophages; and CD11b plus HLA-DR to interrogate mature and immature myeloid cell populations such as myeloid derived suppressor cells (MDSCs). We interrogated 793 NSCLCs represented in four tissue microarray-based cohorts: #1 (Yale, n=379) and #2 (Greece, n=230) with diverse NSCLC subtypes; #3 (Yale, n=138) with molecularly annotated lung adenocarcinomas (ADC); and #4 (Yale, n=46) with patient-matched NSCLC and morphologically-normal lung tissue. We examined associations between marker levels, myeloid cell profiles, clinicopathologic/molecular variables and survival. RESULTS: The levels of CD68+ M1 like macrophages were significantly lower and the fraction of CD11b+/HLA-DR- MDSC-like cells was prominently higher in tumor than in matched non-tumor lung tissues. HLA-DR was consistently higher in myeloid cells from tumors with elevated CD68 expression. Stromal CD11b was significantly higher in squamous cell carcinomas (SCC) than in ADC across the cohorts and EGFR-mutated lung ADCs displayed lower CD11b levels than KRAS-mutant tumors. Increased stromal CD68- and HLA-DR-expressing cells was associated with better survival in ADCs from two independent NSCLC cohorts. In SCC, increased stromal CD11b or HLA-DR expression was associated with a trend towards shorter 5-year survival. CONCLUSIONS: NSCLCs display an unfavorable myeloid immune contexture relative to non-tumor lung and exhibit distinct myeloid-cell profiles across histologies and presence of major oncogenic driver-mutations. Elevated M1-like stromal proinflammatory myeloid cells are prognostic in lung ADC, but not in SCC.