An algorithm for standardization of tumor Infiltrating lymphocyte evaluation in head and neck cancers.

PURPOSE: The prognostic and predictive significance of pathologist-read tumor infiltrating lymphocytes (TILs) in head and neck cancers have been demonstrated through multiple studies over the years. TILs have not been broadly adopted clinically, perhaps due to substantial inter-observer variability. In this study, we developed a machine-based algorithm for TIL evaluation in head and neck cancers and validated its prognostic value in independent cohorts. EXPERIMENTAL DESIGN: A network classifier called NN3-17 was trained to identify and calculate tumor cells, lymphocytes, fibroblasts and "other" cells on hematoxylin-eosin stained sections using the QuPath software. These measurements were used to construct three predefined TIL variables. A retrospective collection of 154 head and neck squamous cell cancer cases was used as the discovery set to identify optimal association of TIL variables and survival. Two independent cohorts of 234 cases were used for validation. RESULTS: We found that electronic TIL variables were associated with favorable prognosis in both the HPV-positive and -negative cases. After adjusting for clinicopathologic factors, Cox regression analysis demonstrated that electronic total TILs% (p = 0.025) in the HPV-positive and electronic stromal TILs% (p < 0.001) in the HPV-negative population were independent markers of disease specific outcomes (disease free survival). CONCLUSIONS: Neural network TIL variables demonstrated independent prognostic value in validation cohorts of HPV-positive and HPV-negative head and neck cancers. These objective variables can be calculated by an open-source software and could be considered for testing in a prospective setting to assess potential clinical implications.

A bedside to bench study of anti-PD-1, anti-CD40, and anti-CSF1R indicates that more is not necessarily better.

BACKGROUND: Stimulating inflammatory tumor associated macrophages can overcome resistance to PD-(L)1 blockade. We previously conducted a phase I trial of cabiralizumab (anti-CSF1R), sotigalimab (CD40-agonist) and nivolumab. Our current purpose was to study the activity and cellular effects of this three-drug regimen in anti-PD-1-resistant melanoma. METHODS: We employed a Simon's two-stage design and analyzed circulating immune cells from patients treated with this regimen for treatment-related changes. We assessed various dose levels of anti-CSF1R in murine melanoma models and studied the cellular and molecular effects. RESULTS: Thirteen patients were enrolled in the first stage. We observed one (7.7%) confirmed and one (7.7%) unconfirmed partial response, 5 patients had stable disease (38.5%) and 6 disease progression (42.6%). We elected not to proceed to the second stage. CyTOF analysis revealed a reduction in non-classical monocytes. Patients with prolonged stable disease or partial response who remained on study for longer had increased markers of antigen presentation after treatment compared to patients whose disease progressed rapidly. In a murine model, higher anti-CSF1R doses resulted in increased tumor growth and worse survival. Using single-cell RNA-sequencing, we identified a suppressive monocyte/macrophage population in murine tumors exposed to higher doses. CONCLUSIONS: Higher anti-CSF1R doses are inferior to lower doses in a preclinical model, inducing a suppressive macrophage population, and potentially explaining the disappointing results observed in patients. While it is impossible to directly infer human doses from murine studies, careful intra-species evaluation can provide important insight. Cabiralizumab dose optimization is necessary for this patient population with limited treatment options. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03502330.

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.

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.

Immune dysfunction revealed by digital spatial profiling of immuno-oncology markers in progressive stages of renal cell carcinoma and in brain metastases.

BACKGROUND: The tumor microenvironment (TME) contributes to cancer progression and treatment response to therapy, including in renal cell carcinoma (RCC). Prior profiling studies, including single-cell transcriptomics, often involve limited sample sizes and lack spatial orientation. The TME of RCC brain metastases, a major cause of morbidity, also remains largely uncharacterized. METHODS: We performed digital spatial profiling on the NanoString GeoMx platform using 52 validated immuno-oncology markers on RCC tissue microarrays representing progressive stages of RCC, including brain metastases. We profiled 76 primary tumors, 27 adjacent histologically normal kidney samples, and 86 metastases, including 24 brain metastases. RESULTS: We observed lower immune checkpoint (TIM-3 and CTLA-4), cytolytic (GZMA and GZMB), and T cell activation (CD25) protein expression in metastases compared with primary tumors in two separate cohorts. We also identified changes in macrophages in metastases, with brain metastases-susceptible patients showing less M1-like, inflammatory macrophage markers (HLA-DR and CD127) in metastatic samples. A comparison of brain metastases to extracranial metastases revealed higher expression of the anti-apoptotic, BCL-2-family protein BCL-XL and lower expression of the innate immune activator STING in brain metastases. Lower TIM-3 and CD40 in the TME of brain metastases appear to be associated with longer survival, a finding that requires further validation. CONCLUSIONS: Compared with primary tumors, RCC metastases, including brain metastases, express lower levels of numerous markers of immune activation and current or investigational therapeutic targets. Our findings may have important implications for designing future biomarker and treatment studies and may aid in development of brain metastases-specific therapies.

Multiplex Immunohistochemistry and Immunofluorescence: A Practical Update for Pathologists.

Our understanding of the biology and management of human disease has undergone a remarkable evolution in recent decades. Improved understanding of the roles of complex immune populations in the tumor microenvironment has advanced our knowledge of antitumor immunity, and immunotherapy has radically improved outcomes for many advanced cancers. Digital pathology has unlocked new possibilities for the assessment and discovery of the tumor microenvironment, such as quantitative and spatial image analysis. Despite these advances, tissue-based evaluations for diagnosis and prognosis continue to rely on traditional practices, such as hematoxylin and eosin staining, supplemented by the assessment of single biomarkers largely using chromogenic immunohistochemistry (IHC). Such approaches are poorly suited to complex quantitative analyses and the simultaneous evaluation of multiple biomarkers. Thus, multiplex staining techniques have significant potential to improve diagnostic practice and immuno-oncology research. The different approaches to achieve multiplexed IHC and immunofluorescence are described in this study. Alternatives to multiplex immunofluorescence/IHC include epitope-based tissue mass spectrometry and digital spatial profiling (DSP), which require specialized platforms not available to most clinical laboratories. Virtual multiplexing, which involves digitally coregistering singleplex IHC stains performed on serial sections, is another alternative to multiplex staining. Regardless of the approach, analysis of multiplexed stains sequentially or simultaneously will benefit from standardized protocols and digital pathology workflows. Although this is a complex and rapidly advancing field, multiplex staining is now technically feasible for most clinical laboratories and may soon be leveraged for routine diagnostic use. This review provides an update on the current state of the art for tissue multiplexing, including the capabilities and limitations of different techniques, with an emphasis on potential relevance to clinical diagnostic practice.

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.

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.

Location matters: LAG3 levels are lower in renal cell carcinoma metastatic sites compared to primary tumors, and expression at metastatic sites only may have prognostic importance.

While great strides have been made in the treatment of advanced renal cell carcinoma (RCC) with the emergence of immune checkpoint inhibitors (ICIs) and VEGFR-targeting drugs, sizable proportions of patients still do not respond to upfront therapy and long-term responses only occur in a minority of patients. There is therefore a great need for the development of better predictors of response and an increased understanding of mechanisms of resistance to these therapies. Alternative immune checkpoints outside the PD-1/PD-L1 axis, such as LAG3, have been implicated as one mechanism of resistance to ICIs. These checkpoints thus represent attractive therapeutic targets, and indeed the LAG3 inhibitor relatlimab was recently approved for the treatment of metastatic melanoma in combination with anti-PD-1 therapy. LAG3 inhibitors are being evaluated for RCC as well. In this context, a better understanding of LAG3 expression patterns in RCC and how they relate to clinicopathologic features of disease and response to immunotherapy may give insight into mechanisms of resistance to PD-1 inhibitors and aid in the identification of subgroups of patients more likely to benefit from certain drug regimens. In this study, we assessed LAG3 protein levels in leukocytes in normal kidney adjacent to RCC, primary RCC tumors, and matched metastatic tumors, including large numbers of brain metastases. We found that LAG3 protein levels are on average lower at metastatic sites compared to matched primary tumors, and that the difference was more pronounced in patients with high-risk clinical characteristics, including those with larger primary tumor size, grade 4 tumors, IMDC poor-risk disease, and initial presentation with brain metastases. We further saw that the prognostic value of LAG3 levels varies depending on the tissue site queried (i.e., primary tumor versus metastases), and that relatively higher LAG3 levels at metastatic sites may predict a better response to immunotherapy and longer overall survival after the development of metastatic disease. These findings may have important implications for the design of future studies involving LAG3 or other immunotherapies in RCC.

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.

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.

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.

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.

Quantitative measurement of HER2 expression to subclassify ERBB2 unamplified breast cancer.

The efficacy of the antibody drug conjugate (ADC) Trastuzumab deruxtecan (T-DXd) in HER2 low breast cancer patients suggests that the historical/conventional assays for HER2 may need revision for optimal patient care. Specifically, the conventional assay is designed to distinguish amplified HER2 from unamplified cases but is not sensitive enough to stratify the lower ranges of HER2 expression. Here we determine the optimal dynamic range for unamplified HER2 detection in breast cancer and then redesign an assay to increase the resolution of the assay to stratify HER2 expression in unamplified cases. We used the AQUA™ method of quantitative immunofluorescence to test a range of antibody concentrations to maximize the sensitivity within the lower range of HER2 expression. Then, using a cell line microarray with HER2 protein measured by mass spectrometry we determined the amount of HER2 protein in units of attomols/mm2. Then by calculation of the limits of detection, quantification, and linearity of this assay we determined that low HER2 range expression in unamplified cell lines is between 2 and 20 attomol/mm2. Finally, application of this assay to a serial collection of 364 breast cancer cases from Yale shows 67% of the population has HER2 expression above the limit of quantification and below the levels seen in HER2 amplified breast cancer. In the future, this assay could be used to determine the levels of HER2 required for response to T-DXd or similar HER2 conjugated ADCs.

Histone Deacetylase (HDAC) Inhibitors: A Promising Weapon to Tackle Therapy Resistance in Melanoma.

Melanoma is an aggressive malignant tumor, arising more commonly on the skin, while it can also occur on mucosal surfaces and the uveal tract of the eye. In the context of the unresectable and metastatic cases that account for the vast majority of melanoma-related deaths, the currently available therapeutic options are of limited value. The exponentially increasing knowledge in the field of molecular biology has identified epigenetic reprogramming and more specifically histone deacetylation (HDAC), as a crucial regulator of melanoma progression and as a key driver in the emergence of drug resistance. A variety of HDAC inhibitors (HDACi) have been developed and evaluated in multiple solid and hematologic malignancies, showing promising results. In melanoma, various experimental models have elucidated a critical role of histone deacetylases in disease pathogenesis. They could, therefore, represent a promising novel therapeutic approach for advanced disease. A number of clinical trials assessing the efficacy of HDACi have already been completed, while a few more are in progress. Despite some early promising signs, a lot of work is required in the field of clinical studies, and larger patient cohorts are needed in order for more valid conclusions to be extracted, regarding the potential of HDACi as mainstream treatment options for melanoma.

Quantitative assessment of the immune microenvironment in African American Triple Negative Breast Cancer: a case-control study.

PURPOSE: Triple negative breast cancer (TNBC) is more common in African American (AA) than Non-AA (NAA) population. We hypothesize that tumor microenvironment (TME) contributes to this disparity. Here, we use multiplex quantitative immunofluorescence to characterize the expression of immunologic biomarkers in the TME in both populations. PATIENTS AND METHODS: TNBC tumor resection specimen tissues from a 100-patient case: control cohort including 49 AA and 51 NAA were collected. TME markers including CD45, CD14, CD68, CD206, CD4, CD8, CD20, CD3, Ki67, GzB, Thy1, FAP, aSMA, CD34, Col4, VWF and PD-L1 we quantitatively assessed in every field of view. Mean expression levels were compared between cases and controls. RESULTS: Although no significant differences were detected in individual lymphoid and myeloid markers, we found that infiltration with CD45+ immune cells (p = 0.0102) was higher in TNBC in AA population. AA TNBC tumors also had significantly higher level of lymphocytic infiltration defined as CD45+ CD14- cells (p = 0.0081). CD3+ T-cells in AA tumors expressed significantly higher levels of Ki67 (0.0066) compared to NAAs, indicating that a higher percentage of AA tumors contained activated T-cells. All other biomarkers showed no significant differences between the AA and NAA group. CONCLUSIONS: While the TME in TNBC is rich in immune cells in both racial groups, there is a numerical increase in lymphoid infiltration in AA compared to NAA TNBC. Significantly, higher activated T cells seen in AA patients raises the possibility that there may be a subset of AA patients with improved response to immunotherapy.

Alpha-smooth Muscle Actin Expression in the Stroma Predicts Resistance to Trastuzumab in Patients with Early-stage HER2-positive Breast Cancer.

PURPOSE: The companion diagnostic test for trastuzumab has not changed much in the last 25 years. We used high-plex digital spatial profiling to identify biomarkers besides HER2 that can help predict response to trastuzumab in HER2-positive breast cancer. EXPERIMENTAL DESIGN: Fifty-eight protein targets were measured in three different molecularly defined compartments by the NanoString GeoMx Digital Spatial Profiler (DSP) in a tissue microarray containing 151 patients with breast cancer that received adjuvant trastuzumab as part of the Hellenic Cooperative Oncology Group 10/05 clinical trial. Promising candidate biomarkers were orthogonally validated with quantitative immunofluorescence (QIF). RNA-sequencing data from the Neoadjuvant Lapatinib and/or Trastuzumab Treatment Optimisation Study (NeoALTTO) were accessed to provide independent cohort validation. Disease-free survival (DFS) was the main outcome assessed. Statistical analyses were performed using a two-sided test (α = 0.05) and multiple testing correction (Benjamini-Hochberg method, FDR < 0.1). RESULTS: By DSP, high expression of alpha-smooth muscle actin (α-SMA), both in the leukocyte and stromal compartments, was associated with shorter DFS in univariate analysis (P = 0.002 and P = 0.023, respectively). High α-SMA expression in the stroma was validated by QIF after controlling for estrogen receptor and progesterone receptor status [HR, 3.12; 95% confidence interval (CI), 1.12-8.68; P = 0.029] showing recurrence on trastuzumab in the same cohort. In the NeoALTTO cohort, elevated levels of ACTA2 were predictive for shorter DFS in the multivariate analysis (HR, 3.21; 95% CI, 1.14-9.05; P = 0.027). CONCLUSIONS: This work identifies α-SMA as a novel, easy-to-implement biomarker of resistance to trastuzumab that may be valuable in settings where trastuzumab is combined with other therapies.

Putting the Microenvironment into the Immunotherapy Companion Diagnostic.

A post hoc analysis in advanced gastric cancer showed stronger association of programmed cell death-ligand 1 (PD-L1) with immunotherapy efficacy measured by combined positive score (CPS) compared with tumor cells (TC%). This finding suggests expression in immune cells in the tumor microenvironment increases the value of the companion diagnostic test.See related article by Lei et al., p. 3926.