Spatially informed gene signatures for response to immunotherapy in melanoma.

PURPOSE: We aim to improve the prediction of response or resistance to immunotherapies in melanoma patients. This goal is based on the hypothesis that current gene signatures predicting immunotherapy outcomes show only modest accuracy due to the lack of spatial information about cellular functions and molecular processes within tumors and their microenvironment. EXPERIMENTAL DESIGN: We collected gene expression data spatially from three cellular compartments defined by CD68+macrophages, CD45+leukocytes and S100B+tumor cells in 55-immunotherapy-treated melanoma specimens using Digital Spatial Profiling-Whole Transcriptome Atlas (DSP-WTA). We developed a computational pipeline to discover compartment-specific gene signatures and determine if adding spatial information can improve patient stratification. RESULTS: We achieved robust performance of compartment-specific signatures in predicting the outcome to ICI in the discovery cohort. Of the three signatures, S100B signature showed the best performance in the validation cohort (N=45). We also compared our compartment-specific signatures with published bulk signatures and found the S100B tumor spatial signature outperformed previous signatures. Within the 8-gene S100B signature, 5 genes (PSMB8, TAX1BP3, NOTCH3, LCP2, NQO1) with positive coefficients predict the response and 3 genes (KMT2C, OVCA2, MGRN1) with negative coefficients predict the resistance to treatment. CONCLUSION: We conclude that the spatially defined compartment signatures utilize tumor and TME-specific information, leading to more accurate prediction of treatment outcome, and thus merit prospective clinical assessment.

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.

Emerging Prognostic and Predictive Significance of Stress Keratin 17 in HPV-Associated and Non HPV-Associated Human Cancers: A Scoping Review.

A growing body of literature suggests that the expression of cytokeratin 17 (K17) correlates with inferior clinical outcomes across various cancer types. In this scoping review, we aimed to review and map the available clinical evidence of the prognostic and predictive value of K17 in human cancers. PubMed, Web of Science, Embase (via Scopus), Cochrane Central Register of Controlled Trials, and Google Scholar were searched for studies of K17 expression in human cancers. Eligible studies were peer-reviewed, published in English, presented original data, and directly evaluated the association between K17 and clinical outcomes in human cancers. Of the 1705 studies identified in our search, 58 studies met criteria for inclusion. Studies assessed the prognostic significance (n = 54), predictive significance (n = 2), or both the prognostic and predictive significance (n = 2). Altogether, 11 studies (19.0%) investigated the clinical relevance of K17 in cancers with a known etiologic association to HPV; of those, 8 (13.8%) were focused on head and neck squamous cell carcinoma (HNSCC), and 3 (5.1%) were focused on cervical squamous cell carcinoma (SCC). To date, HNSCC, as well as triple-negative breast cancer (TNBC) and pancreatic cancer, were the most frequently studied cancer types. K17 had prognostic significance in 16/17 investigated cancer types and 43/56 studies. Our analysis suggests that K17 is a negative prognostic factor in the majority of studied cancer types, including HPV-associated types such as HNSCC and cervical cancer (13/17), and a positive prognostic factor in 2/17 studied cancer types (urothelial carcinoma of the upper urinary tract and breast cancer). In three out of four predictive studies, K17 was a negative predictive factor for chemotherapy and immune checkpoint blockade therapy response.

Stress Keratin 17 Is a Predictive Biomarker Inversely Associated with Response to Immune Check-Point Blockade in Head and Neck Squamous Cell Carcinomas and Beyond.

Low response rates in immune check-point blockade (ICB)-treated head and neck squamous cell carcinoma (HNSCC) drive a critical need for robust, clinically validated predictive biomarkers. Our group previously showed that stress keratin 17 (CK17) suppresses macrophage-mediated CXCL9/CXCL10 chemokine signaling involved in attracting activated CD8+ T cells into tumors, correlating with decreased response rate to pembrolizumab-based therapy in a pilot cohort of ICB-treated HNSCC (n = 26). Here, we performed an expanded analysis of the predictive value of CK17 in ICB-treated HNSCC according to the REMARK criteria and investigated the gene expression profiles associated with high CK17 expression. Pretreatment samples from pembrolizumab-treated HNSCC patients were stained via immunohistochemistry using a CK17 monoclonal antibody (n = 48) and subjected to spatial transcriptomic profiling (n = 8). Our findings were validated in an independent retrospective cohort (n = 22). CK17 RNA expression in pembrolizumab-treated patients with various cancer types was investigated for predictive significance. Of the 48 patients (60% male, median age of 61.5 years), 21 (44%) were CK17 high, and 27 (56%) were CK17 low. A total of 17 patients (35%, 77% CK17 low) had disease control, while 31 patients (65%, 45% CK17 low) had progressive disease. High CK17 expression was associated with a lack of disease control (p = 0.037), shorter time to treatment failure (p = 0.025), and progression-free survival (PFS, p = 0.004), but not overall survival (OS, p = 0.06). A high CK17 expression was associated with lack of disease control in an independent validation cohort (p = 0.011). PD-L1 expression did not correlate with CK17 expression or clinical outcome. CK17 RNA expression was predictive of PFS and OS in 552 pembrolizumab-treated cancer patients. Our findings indicate that high CK17 expression may predict resistance to ICB in HNSCC patients and beyond.

Phase II Window Study of Olaparib Alone or with Cisplatin or Durvalumab in Operable Head and Neck Cancer.

Purpose: We conducted a phase II randomized noncomparative window of opportunity (WOO) trial to evaluate the inhibition of cellular proliferation and the modulation of immune microenvironment after treatment with olaparib alone or in combination with cisplatin or durvalumab in patients with operable head and neck squamous cell carcinoma (HNSCC). Experimental Design: Forty-one patients with HNSCC were randomized to cisplatin plus olaparib (arm A), olaparib alone (arm B), no treatment (arm C) or durvalumab plus olaparib (arm D). The primary endpoint was to evaluate the percentage of patients in each arm that achieved a reduction of at least 25% in Ki67. Secondary endpoints included objective response rate (ORR), safety, and pathologic complete response (pCR) rate. Paired baseline and resection tumor biopsies and blood samples were evaluated for prespecified biomarkers. Results: A decrease in Ki67 of at least 25% was observed in 44.8% of treated patients, as measured by quantitative immunofluorescence. The ORR among treated patients was 12.1%. pCR was observed in 2 patients. Two serious adverse events occurred in 2 patients.Programmed death ligand 1 (PD-L1) levels [combined positive score (CPS)] were significantly higher after treatment in arms A and D. Expression of CD163 and colony-stimulating factor 1 receptor (CSF1R) genes, markers of M2 macrophages, increased significantly posttreatment whereas the expression of CD80, a marker of M1 macrophages, decreased. Conclusion: Preoperative olaparib with cisplatin or alone or with durvalumab was safe in the preoperative setting and led to decrease in Ki67 of at least 25% in 44.8% of treated patients. Olaparib-based treatment modulates the tumor microenvironment leading to upregulation of PD-L1 and induction of protumor features of macrophages. Significance: HNSCC is characterized by defective DNA repair pathways and immunosuppressive tumor microenvironment. PARP inhibitors, which promote DNA damage and "reset" the inflammatory tumor microenvironment, can establish an effective antitumor response. This phase II WOO trial in HNSCC demonstrated the immunomodulatory effects of PARP inhibitor-induced DNA damage. In this chemo-naïve population, PARP inhibitor-based treatment, reduced tumor cell proliferation and modulated tumor microenvironment. After olaparib upregulation of PD-L1 and macrophages, suggests that combinatorial treatment might be beneficial. Synopsis: Our WOO study demonstrates that preoperative olaparib results in a reduction in Ki67, upregulation of PD-L1 CPS, and induction of protumor features of macrophages in HNSCC.

Digital Spatial Profiling Links Beta-2-microglobulin Expression with Immune Checkpoint Blockade Outcomes in Head and Neck Squamous Cell Carcinoma.

Programmed cell death protein-1 (PD-1)-targeted immunotherapy is approved for recurrent or metastatic head and neck squamous cell carcinoma (R/M HNSCC) treatment. Although its efficacy correlates with PD-L1 expression, response is limited even among positive cases. We employed digital spatial profiling (DSP) to discover potential biomarkers of immunotherapy outcomes in HNSCC. Fifty prospectively collected, pretreatment biopsy samples from patients with anti-PD-1-treated R/M HNSCC, were assessed using DSP, for 71 proteins in four molecularly defined compartments (tumor, leukocyte, macrophage, and stroma). Markers were evaluated for associations with progression-free (PFS) and overall survival (OS). High beta-2 microglobulin (B2M), LAG-3, CD25, and 4-1BB in tumor; high B2M, CD45, CD4 in stroma, and low fibronectin in the macrophage compartment, correlated with prolonged PFS. Improved PFS and OS were observed for cases with high B2M by quantitative and mRNA. Findings were validated in an independent cohort for PFS (HR, 0.41; 95% confidence interval, 0.19-0.93; P = 0.034). B2M-high tumors showed enrichment with immune cell and immune checkpoint markers. Our study illustrates B2M expression is associated with improved survival for immune checkpoint inhibitor (ICI)-treated HNSCC. Significance: In the current study, DSP revealed the positive association of B2M expression in the tumor compartment with immunotherapy outcomes in R/M HNSCC.

Spatial characterization and quantification of CD40 expression across cancer types.

BACKGROUND: CD40, a TNF receptor family member, is expressed by a variety of immune cells and is involved in the activation of both adaptive and innate immune responses. Here, we used quantitative immunofluorescence (QIF) to evaluate CD40 expression on the tumor epithelium of solid tumors in large patient cohorts of lung, ovarian, and pancreatic cancers. METHODS: Tissue samples from nine different solid tumors (bladder, breast, colon, gastric, head and neck, non-small cell lung cancer (NSCLC), ovarian, pancreatic and renal cell carcinoma), constructed in tissue microarray format, were initially assessed for CD40 expression by QIF. CD40 expression was then evaluated on the large available patient cohorts for three of the tumor types demonstrating high CD40 positivity rate; NSCLC, ovarian and pancreatic cancer. The prognostic impact of CD40 expression on tumor cells was also investigated. RESULTS: CD40 expression on tumor cells was found to be common, with 80% of the NSCLC population, 40% of the ovarian cancer population, and 68% of the pancreatic adenocarcinoma population displaying some degree of CD40 expression on cancer cells. All of three of these cancer types displayed considerable intra-tumoral heterogeneity of CD40 expression, as well as partial correlation between expression of CD40 on tumor cells and on surrounding stromal cells. CD40 was not found to be prognostic for overall survival in NSCLC, ovarian cancer, or pancreatic adenocarcinoma. CONCLUSIONS: The high percentage of tumor cells expressing CD40 in each of these solid tumors should be considered in the development of therapeutic agents designed to target CD40.

Quantitative, Spatially Defined Expression of Leukocyte-associated Immunoglobulin-like Receptor in Non-small Cell Lung Cancer.

Targeting the interaction of leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) and its ligands has been shown to reinstate antitumor immunity. In addition, the introduction of the LAIR-1 decoy protein, LAIR-2, sensitizes previously resistant lung tumors to programmed death-1 (PD-1) blockade, indicating the potential of LAIR-1 as an alternative marker for anti-PD-1 resistance in lung cancer. Here, we assessed LAIR-1 as compared with programmed death-ligand 1 (PD-L1) expression in various tumors, with a focus on non-small cell lung cancer (NSCLC) and its histologic subtypes using multiplexed quantitative immunofluorescence (mQIF) in 287 (discovery cohort) and 144 (validation cohort) patients with NSCLC. In addition, using multispectral imaging technology on mQIF images, we evaluated the localization of LAIR-1 on various cell types. We observed that CD14+, CD68+, and CD163+ monocytes and CK+ tumor cells predominantly expressed LAIR-1 more than other cell types. Furthermore, LAIR-1 expression in the tumor compartment was significantly higher in patients with lung adenocarcinoma (LUAD) than those with lung squamous cell carcinoma subtype (**, P = 0.003). Our results indicated that high tumor LAIR-1 expression in patients with LUAD is negatively associated with OS (overall survival, HR = 2.4; *, P = 0.02) highlighting its prognostic value in LUAD but not in other subtypes. The Pearson correlation between LAIR-1 and PD-L1 is 0.31; however, mutual exclusive staining pattern (i.e., several cases were positive for LAIR-1 and negative for PD-L1) was observed. Altogether, our data suggest that the combination therapy of anti-PD-1/PD-L1 with anti-LAIR-1 or the anti-LAIR-1 monotherapy alone may be promising cancer immunotherapeutic strategies. Significance: The spatial, quantitative assessment of LAIR-1 in NSCLC shows positive association of OS with high LAIR-1+/CD68+ cell densities and negative association of OS with high LAIR-1 expression in LUAD tumor subtype.

Next-generation sequencing (NGS) profiling of matched tumor and circulating tumor DNA (ctDNA) in head and neck squamous cell carcinoma (HNSCC).

OBJECTIVES: The aim of this pilot study was to evaluate the presence of somatic mutations in matched tumor and circulating DNA (ctDNA) samples from patients with primary head and neck squamous cell carcinoma (HNSCC) and assess the association of changes in ctDNA levels with survival. MATERIALS AND METHODS: Our study included 62 patients with stage I-IVB HNSCC treated with surgery or radical chemoradiotherapy with curative intent. Plasma samples were obtained at baseline, at the end of treatment (EOT), and at disease progression. Tumor DNA was extracted from plasma (ctDNA) and tumor tissue (tDNA). The Safe Sequencing System was used assess the presence of pathogenic variants in four genes (TP53, CDKN2A, HRAS and PI3KCA) in both ctDNA and tDNA. RESULTS: Forty-five patients had available tissue and plasma samples. Concordance of genotyping results between tDNA and ctDNA at baseline was 53.3%. TP53 mutations were most commonly identified at baseline in both ctDNA (32.6%) and tDNA (40%). The presence of mutations in this restricted set of 4 genes in tissue samples at baseline was associated with decreased overall survival (OS) [median 58.3 months for patients with mutations vs. 89 months for patients without mutations, p < 0.013]. Similarly, patients presenting with mutations in ctDNA had shorter OS [median 53.8 vs. 78.6 months, p < 0.037]. CtDNA clearance at EOT did not show any association with PFS or OS. CONCLUSIONS: Liquid biopsy enables real-time molecular characterization of HNSCC and might predict survival. Larger studies are needed to validate the utility of ctDNA as a biomarker in HNSCC.

Baseline gene expression profiling determines long-term benefit to programmed cell death protein 1 axis blockade.

Treatment with immune checkpoint inhibitors has altered the course of malignant melanoma, with approximately half of the patients with advanced disease surviving for more than 5 years after diagnosis. Currently, there are no biomarker methods for predicting outcome from immunotherapy. Here, we obtained transcriptomic information from a total of 105 baseline tumor samples comprising two cohorts of patients with advanced melanoma treated with programmed cell death protein 1 (PD-1)-based immunotherapies. Gene expression profiles were correlated with progression-free survival (PFS) within consecutive clinical benefit intervals (i.e., 6, 12, 18, and 24 months). Elastic net binomial regression models with cross validation were utilized to compare the predictive value of distinct genes across time. Lasso regression was used to generate a signature predicting long-term benefit (LTB), defined as patients who remain alive and free of disease progression at 24 months post treatment initiation. We show that baseline gene expression profiles were consistently able to predict long-term immunotherapy outcomes with high accuracy. The predictive value of different genes fluctuated across consecutive clinical benefit intervals, with a distinct set of genes defining benefit at 24 months compared to earlier outcomes. A 12-gene signature was able to predict LTB following anti-PD-1 therapy with an area under the curve (AUC) equal to 0.92 and 0.74 in the training and validation set, respectively. Evaluation of LTB, via a unique signature may complement objective response classification and characterize the logistics of sustained antitumor immune responses.

Programmed Cell Death Protein 1 Axis Inhibition in Viral Infections: Clinical Data and Therapeutic Opportunities.

A vital function of the immune system is the modulation of an evolving immune response. It is responsible for guarding against a wide variety of pathogens as well as the establishment of memory responses to some future hostile encounters. Simultaneously, it maintains self-tolerance and minimizes collateral tissue damage at sites of inflammation. In recent years, the regulation of T-cell responses to foreign or self-protein antigens and maintenance of balance between T-cell subsets have been linked to a distinct class of cell surface and extracellular components, the immune checkpoint molecules. The fact that both cancer and viral infections exploit similar, if not the same, immune checkpoint molecules to escape the host immune response highlights the need to study the impact of immune checkpoint blockade on viral infections. More importantly, the process through which immune checkpoint blockade completely changed the way we approach cancer could be the key to decipher the potential role of immunotherapy in the therapeutic algorithm of viral infections. This review focuses on the effect of programmed cell death protein 1/programmed death-ligand 1 blockade on the outcome of viral infections in cancer patients as well as the potential benefit from the incorporation of immune checkpoint inhibitors (ICIs) in treatment of viral infections.

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.

Immune Checkpoint Blockade in Hormone Receptor-Positive Breast Cancer: Resistance Mechanisms and Future Perspectives.

Anti-programmed cell death protein 1 immunotherapy has been incorporated in the treatment algorithm of triple-negative breast cancer (TNBC). However, clinical trial results for patients with hormone receptor (HR)-positive disease appear less compelling. HR-positive tumors exhibit lower levels of programmed death-ligand 1 expression in comparison with their triple-negative counterparts. Moreover, signaling through estrogen receptor alters the immune microenvironment, rendering such tumors immunologically "cold." To explain differential responses to immune checkpoint blockade, this review interrogates differences between HR-positive and TNBC. Starting from distinct genomic features, we further present disparities concerning the tumor microenvironment and finally, we summarize early-phase clinical trial results on promising novel immunotherapy combinations.

Objective assessment of tumor infiltrating lymphocytes as a prognostic marker in melanoma using machine learning algorithms.

BACKGROUND: The prognostic value of tumor-infiltrating lymphocytes (TILs) assessed by machine learning algorithms in melanoma patients has been previously demonstrated but has not been widely adopted in the clinic. We evaluated the prognostic value of objective automated electronic TILs (eTILs) quantification to define a subset of melanoma patients with a low risk of relapse after surgical treatment. METHODS: We analyzed data for 785 patients from 5 independent cohorts from multiple institutions to validate our previous finding that automated TIL score is prognostic in clinically-localized primary melanoma patients. Using serial tissue sections of the Yale TMA-76 melanoma cohort, both immunofluorescence and Hematoxylin-and-Eosin (H&E) staining were performed to understand the molecular characteristics of each TIL phenotype and their associations with survival outcomes. FINDINGS: Five previously-described TIL variables were each significantly associated with overall survival (p<0.0001). Assessing the receiver operating characteristic (ROC) curves by comparing the clinical impact of two models suggests that etTILs (electronic total TILs) (AUC: 0.793, specificity: 0.627, sensitivity: 0.938) outperformed eTILs (AUC: 0.77, specificity: 0.51, sensitivity: 0.938). We also found that the specific molecular subtype of cells representing TILs includes predominantly cells that are CD3+ and CD8+ or CD4+ T cells. INTERPRETATION: eTIL% and etTILs scores are robust prognostic markers in patients with primary melanoma and may identify a subgroup of stage II patients at high risk of recurrence who may benefit from adjuvant therapy. We also show the molecular correlates behind these scores. Our data support the need for prospective testing of this algorithm in a clinical trial. FUNDING: This work was also supported by a sponsored research agreements from Navigate Biopharma and NextCure and by grants from the NIH including the Yale SPORE in in Skin Cancer, P50 CA121974, the Yale SPORE in Lung Cancer, P50 CA196530, NYU SPORE in Skin Cancer P50CA225450 and the Yale Cancer Center Support Grant, P30CA016359.

Programmed Death-Ligand 1 and Programmed Death-Ligand 2 mRNAs Measured Using Closed-System Quantitative Real-Time Polymerase Chain Reaction Are Associated With Outcome and High Negative Predictive Value in Immunotherapy-Treated NSCLC.

INTRODUCTION: Immune checkpoint inhibitors (ICIs) have become standard of care in lung cancer management, but only a relatively small percentage of patients treated respond. Current predictive biomarkers, including immunohistochemical detection of programmed death-ligand 1 (PD-L1), are insufficient for determining who will respond or, more importantly in the adjuvant setting, who will not respond to ICI therapy. Here, we investigate an alternative method of assessment of PD-L1 to predict nonresponse. METHODS: This study uses a research use only quantitative real-time reverse transcription polymerase chain reaction assay on the GeneXpert system, to test for the association between four target immune genes, CD274 (PD-L1), PDCD1LG2 (programmed death-ligand 2 [PD-L2]), CD8A, and IRF1, and response to ICI therapy. Tissues were collected from 122 patients with advanced NSCLC before ICI therapy in a retrospective cohort, macrodissected, and analyzed using the GeneXpert. RESULTS: Both high PD-L1 and PD-L2 mRNA expression levels were associated with improved long-term benefit at 24 months (p = 0.047 for both PD-L1 and PD-L2) and overall survival (PD-L1, p = 0.048; PD-L2, p = 0.049). Both PD-L1 and PD-L2 mRNA levels were higher in patients with KRAS mutations. Most importantly, low PD-L1 mRNA level had a high negative predictive value of 0.92 for absence of long-term benefit. CONCLUSIONS: With further validation of this assay in low-stage patients, an assessment of PD-L1 mRNA rather than protein, could be a method to determine which low-stage patients that should not be treated with ICIs in the adjuvant setting. This approach may also be a useful objective method for selecting patients for treatment in the advanced setting.

Quantitative assessment of Siglec-15 expression in lung, breast, head, and neck squamous cell carcinoma and bladder cancer.

Immune checkpoint blockade with programmed cell death (PD-1)/programmed death-ligand 1 (PD-L1) inhibitors has resulted in significant progress in the treatment of various cancer types. However, not all patients respond to PD-1/PD-L1 blockade, underscoring the importance of identifying new potential targets for immunotherapy. One promising target is the immune system modulator Siglec-15. In this study, we assess Siglec-15 expression in solid tumors, with a focus on lung, breast, head and neck squamous and bladder cancers. Using quantitative immunofluorescence (QIF) with a previously validated antibody, we found increased Siglec-15 expression in both tumor and immune cells in all the four cancer types. Siglec-15 was seen to be predominantly expressed by the stromal immune cells (83% in lung, 70.1% in breast, 95.2% in head and neck squamous cell and 89% in bladder cancers). Considerable intra-tumoral heterogeneity was noted across cancer types. As previously described for non-small cell lung cancer (NSCLC), Siglec-15 expression was seen to be mutually exclusive to PD-L1 in all the four cancer types, although this differential expression was maintained but somewhat diminished in head and neck squamous cell carcinoma (HNSCC). Siglec-15 was not prognostic either for overall survival (OS) or progression-free survival (PFS). In summary, we show broad expression of this potential immune modulatory target in a wide range of cancer types. These data suggest potential future clinical trials in these tumor types.

Discovery of Biomarkers of Resistance to Immune Checkpoint Blockade in NSCLC Using High-Plex Digital Spatial Profiling.

INTRODUCTION: Despite the clinical efficacy of immune checkpoint inhibitors (ICIs) in NSCLC, only approximately 20% of patients remain disease-free at 5 years. Here, we use digital spatial profiling to find candidate biomarker proteins associated with ICI resistance. METHODS: Pretreatment samples from 56 patients with NSCLC treated with ICI were analyzed using the NanoString GeoMx digital spatial profiling method. A panel of 71 photocleavable oligonucleotide-labeled primary antibodies was used for protein detection in four molecular compartments (tumor, leukocytes, macrophages, and immune stroma). Promising candidates were orthogonally validated with quantitative immunofluorescence. Available pretreatment samples from 39 additional patients with NSCLC who received ICI and 236 non-ICI-treated patients with operable NSCLC were analyzed to provide independent cohort validation. RESULTS: Biomarker discovery using the protein-based molecular compartmentalization strategy allows 284 protein variables to be assessed for association with ICI resistance by univariate analysis using continuous log-scaled data. Of the 71 candidate protein biomarkers, CD66b in the CD45+CD68 molecular compartment (immune stroma) predicted significantly shorter overall survival (OS) (hazard ratio [HR] 1.31, p = 0.016) and was chosen for validation. Orthogonal validation by quantitative immunofluorescence illustrated that CD66b was associated with resistance to ICI therapy but not prognostic for poor outcomes in untreated NSCLC (discovery cohort [OS HR 2.49, p = 0.026], validation cohort [OS HR 2.05, p = 0.046], non-ICI-treated cohort [OS HR 1.67, p = 0.06]). CONCLUSIONS: Using the technique, we have discovered that CD66b expression is indicative of resistance to ICI therapy in NSCLC. Given that CD66b identifies neutrophils, further studies are warranted to characterize the role of neutrophils in ICI resistance.

Development of an immunohistochemical assay for Siglec-15.

Siglec-15, a member of sialic-acid binding immunoglobulin type lectins, is normally expressed by myeloid cells and upregulated in some human cancers and represents a promising new target for immunotherapy. While PD-L1 blockade is an important strategy for immunotherapy, its effectiveness is limited. The expression of Siglec-15 has been demonstrated to be predominantly mutually exclusive to PD-L1 in certain cancer histologies. Thus, there is significant opportunity for Siglec-15 as an immunotherapeutic target for patients that do not respond to PD-1/PD-L1 inhibition. The aim of this study was to prospectively develop an immunohistochemical (IHC) assay for Siglec-15 to be used as a companion diagnostic for future clinical trials. Here, we create and validate an IHC assay with a novel recombinant antibody to the cytoplasmic domain of Siglec-15. To find an enriched target, this antibody was first used in a quantitative fluorescence (QIF) assay to screen a broad range of tumor histologies to determine tumor types where Siglec-15 demonstrated high expression. Based on this and previous data, we focused on development of a chromogenic IHC assay for lung cancer. Then we developed a scoring system for this assay that has high concordance amongst pathologist readers. We then use this chromogenic IHC assay to test the expression of Siglec-15 in two cohorts of NSCLC. We found that this assay shows a higher level of staining in both tumor and immune cells compared to previous QIF assays utilizing a polyclonal antibody. However, similar to that study, only a small percentage of positive Siglec-15 cases showed high expression for PD-L1. This validated assay for Siglec-15 expression may support development of a companion diagnostic assay to enrich for patients expressing the Siglec-15 target for therapy.

Association of PD-1/PD-L1 Co-location with Immunotherapy Outcomes in Non-Small Cell Lung Cancer.

PURPOSE: Programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) interaction suppresses local T-cell responses and promotes peripheral tolerance. In the current study, we focus on PD-1/PD-L1 co-location as a surrogate for this interaction and assess its association with immunotherapy outcomes in patients with non-small cell lung cancer (NSCLC). EXPERIMENTAL DESIGN: Pretreatment biopsies from a retrospective cohort of 154 immunotherapy-treated patients with advanced NSCLC were analyzed. Expression of PD-1 and PD-L1 was assessed by multiplexed quantitative immunofluorescence (QIF) and PD-1 expression in the same pixels as PD-L1 (called a co-location score) was measured using an algorithm to define overlapping expression areas. Co-location scores were correlated with immunotherapy outcomes and PD-L1 tumor proportion score. RESULTS: PD-1/PD-L1 co-location score was associated with best overall response (P = 0.0012), progression-free survival (P = 0.0341), and overall survival after immunotherapy (P = 0.0249). The association was driven by patients receiving immune checkpoint inhibitors in the second or subsequent line of treatment. PD-L1 tumor proportion score by IHC was also correlated with best overall response and progression-free survival. PD-L1 measured within the tumor compartment by QIF did not show any significant association with either best overall response or overall survival. Finally, co-location score was not associated with PD-L1 expression by either method. CONCLUSIONS: On the basis of our findings, co-location score shows promise as a biomarker associated with outcome after immunotherapy. With further validation, it could have value as a predictive biomarker for the selection of patients with NSCLC receiving treatment with immune checkpoint inhibitors.