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.

Outcomes With Combination Pembrolizumab and Axitinib in Second and Further Line Treatment of Metastatic Renal Cell Carcinoma.

INTRODUCTION: Combination immune checkpoint inhibitors (ICI) and vascular endothelial growth factor receptor tyrosine kinase inhibitors (VEGF-R-TKI), including pembrolizumab/axitinib, are approved for first-line treatment of metastatic renal cell carcinoma (mRCC). Pembrolizumab/axitinib is associated with superior progression free survival (PFS), objective response rate (ORR), and overall survival over sunitinib. However, to date, the activity and safety of pembrolizumab/axitinib in later lines of therapy has not been reported. MATERIALS AND METHODS: Clinical data of consecutive patients receiving pembrolizumab/axitinib in the second-line or beyond for mRCC at Yale-New Haven Hospital were retrospectively collected. Best objective response was assessed using RECIST 1.1 criteria. Kaplan-Meier function was used to analyze survival. RESULTS: Thirty-eight patients were included. Median age was 64, 92.1% had clear cell mRCC, 18.4% had sarcomatoid dedifferentiation; 94.7% had prior ICI and 39.5% had prior VEGF-R-TKI. Pembrolizumab/axitinib was administered as second-line therapy in 21 (55.5%) patients, third-line in 5 (13.2%) and beyond in 12 (30.2%). Adverse events (AEs) occurred in 86.8% of patients. Grade 3-4 AEs attributed to pembrolizumab and axitinib were seen in 18.4% and 6.4% of patients, respectively. No grade 5 AEs occurred. At a median follow up of 17.1 months, median PFS was 9.7 months (95% CI, 4.1-15.3). Amongst 36 response evaluable patients, the ORR was 25.0% (all partial) and disease control rate (including stable disease for at least 6 months) was 66.6%. The most frequent treatment sequence was first-line nivolumab/ipilimumab followed by second-line pembrolizumab/axitinib (n = 17, 44.7%); among this cohort, median PFS with pembrolizumab/axitinib was 11.1 (95% CI, 8.4-13.7) months, with an ORR of 31.4%. CONCLUSION: Combination pembrolizumab/axitinib among previously treated mRCC patients has activity, with AE rates comparable to those reported in the first line. Prospective studies evaluating ICI-VEGF-R-TKI combinations beyond first-line are warranted to identify the most beneficial treatment sequencing in mRCC.

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.

Immune Checkpoint Inhibitor-Induced Hypophysitis and Patterns of Loss of Pituitary Function.

Background: Immune checkpoint inhibitors (ICI) are clinically active across multiple tumor types but the associated immune-related adverse events (irAEs) lead to treatment delays or discontinuation and negatively impact quality-of-life. Hypophysitis is often a permanent irAE that may affect multiple pituitary hormonal axes. Here we comprehensively characterize our institution's clinical experience with ICI-induced hypophysitis and the associated patterns of pituitary function loss. Methods: Patients with solid tumors, mostly melanoma and renal cell carcinoma (RCC), treated with ICI at Yale Cancer Center were prospectively enrolled from October 2016-May 2021. Demographics and clinical data were obtained from the medical record including type and timing of irAEs. Patients were included in this cohort if hypophysitis was diagnosed by pre-specified biochemical and clinical parameters. Results: The overall incidence of hypophysitis was 69/490 (14%) in patients with melanoma (n=58, 84%), RCC (n=10,14%), and merkel cell carcinoma (n=1, 1%) who received ipilimumab plus nivolumab (77%; 53/69), anti-PD-(L)1 (17%; 12/69), or ipilimumab monotherapy (6%; 4/69). Of the 69 patients analyzed, median time to hypophysitis on combination ICI versus anti-PD-1 was 2.8 vs. 4.1 months. The incidence of hypophysitis in patients with melanoma was 25% (46/187) with ipilimumab plus nivolumab and 5% (7/129) with anti-PD-(L)1 compared to 9% (7/77) and 8% (3/37), respectively, in patients with RCC. Patients who developed hypophysitis on combination ICI had a higher rate of headache (p=0.05) and co-occurring irAEs (p=0.01) compared anti-PD-(L1)1 monotherapy. At a median follow-up of 2.2 years, 77% of patients were alive. Objective response rates to ICI in melanoma patients were higher than previously reported for unselected populations. Central hypothyroidism and hypogonadism were the most common pituitary axes affected after the adrenal axis. In select cases, there was evidence of spontaneous rebound in free testosterone levels after an initial decline. Conclusions: We demonstrate a higher rate of ICI-induced hypophysitis than previously reported, which may be reflective of real-world practice due to increased awareness as experience with ICI has grown. In select cases, there was evidence of rebound in free testosterone and/or gonadotropins but not in adrenal axis hormones.

Inhibition of renalase drives tumour rejection by promoting T cell activation.

BACKGROUND: Although programmed cell death protein 1 (PD-1) inhibitors have revolutionised treatment for advanced melanoma, not all patients respond. We previously showed that inhibition of the flavoprotein renalase (RNLS) in preclinical melanoma models decreases tumour growth. We hypothesised that RNLS inhibition promotes tumour rejection by effects on the tumour microenvironment (TME). METHODS: We used two distinct murine melanoma models, studied in RNLS knockout (KO) or wild-type (WT) mice. WT mice were treated with the anti-RNLS antibody, m28, with or without anti-PD-1. 10X single-cell RNA-sequencing was used to identify transcriptional differences between treatment groups, and tumour cell content was interrogated by flow cytometry. Samples from patients treated with immunotherapy were examined for RNLS expression by quantitative immunofluorescence. RESULTS: RNLS KO mice injected with wild-type melanoma cells reject their tumours, supporting the importance of RNLS in cells in the TME. This effect was blunted by anti-cluster of differentiation 3. However, MØ-specific RNLS ablation was insufficient to abrogate tumour formation. Anti-RNLS antibody treatment of melanoma-bearing mice resulted in enhanced T cell infiltration and activation and resulted in immune memory on rechallenging mice with injection of melanoma cells. At the single-cell level, treatment with anti-RNLS antibodies resulted in increased tumour density of MØ, neutrophils and lymphocytes and increased expression of IFNγ and granzyme B in natural killer cells and T cells. Intratumoural Forkhead Box P3 + CD4 cells were decreased. In two distinct murine melanoma models, we showed that melanoma-bearing mice treated with anti-RNLS antibodies plus anti-PD-1 had superior tumour shrinkage and survival than with either treatment alone. Importantly, in pretreatment samples from patients treated with PD-1 inhibitors, high RNLS expression was associated with decreased survival (log-rank P = 0.006), independent of other prognostic variables. CONCLUSIONS: RNLS KO results in melanoma tumour regression in a T-cell-dependent fashion. Anti-RNLS antibodies enhance anti-PD-1 activity in two distinct aggressive murine melanoma models resistant to PD-1 inhibitors, supporting the development of anti-RNLS antibodies with PD-1 inhibitors as a novel approach for melanomas poorly responsive to anti-PD-1.

KDM5B promotes immune evasion by recruiting SETDB1 to silence retroelements.

Tumours use various strategies to evade immune surveillance1,2. Immunotherapies targeting tumour immune evasion such as immune checkpoint blockade have shown considerable efficacy on multiple cancers3,4 but are ineffective for most patients due to primary or acquired resistance5-7. Recent studies showed that some epigenetic regulators suppress anti-tumour immunity2,8-12, suggesting that epigenetic therapies could boost anti-tumour immune responses and overcome resistance to current immunotherapies. Here we show that, in mouse melanoma models, depletion of KDM5B-an H3K4 demethylase that is critical for melanoma maintenance and drug resistance13-15-induces robust adaptive immune responses and enhances responses to immune checkpoint blockade. Mechanistically, KDM5B recruits the H3K9 methyltransferase SETDB1 to repress endogenous retroelements such as MMVL30 in a demethylase-independent manner. Derepression of these retroelements activates cytosolic RNA-sensing and DNA-sensing pathways and the subsequent type-I interferon response, leading to tumour rejection and induction of immune memory. Our results demonstrate that KDM5B suppresses anti-tumour immunity by epigenetic silencing of retroelements. We therefore reveal roles of KDM5B in heterochromatin regulation and immune evasion in melanoma, opening new paths for the development of KDM5B-targeting and SETDB1-targeting therapies to enhance tumour immunogenicity and overcome immunotherapy resistance.

A Phase I Study of APX005M and Cabiralizumab with or without Nivolumab in Patients with Melanoma, Kidney Cancer, or Non-Small Cell Lung Cancer Resistant to Anti-PD-1/PD-L1.

PURPOSE: PD-1/PD-L1 inhibitors are approved for multiple tumor types. However, resistance poses substantial clinical challenges. PATIENTS AND METHODS: We conducted a phase I trial of CD40 agonist APX005M (sotigalimab) and CSF1R inhibitor cabiralizumab with or without nivolumab using a 3+3 dose-escalation design (NCT03502330). Patients were enrolled from June 2018 to April 2019. Eligibility included patients with biopsy-proven advanced melanoma, non-small cell lung cancer (NSCLC), or renal cell carcinoma (RCC) who progressed on anti-PD-1/PD-L1. APX005M was dose escalated (0.03, 0.1, or 0.3 mg/kg i.v.) with a fixed dose of cabiralizumab with or without nivolumab every 2 weeks until disease progression or intolerable toxicity. RESULTS: Twenty-six patients (12 melanoma, 1 NSCLC, and 13 RCC) were enrolled in six cohorts, 17 on nivolumab-containing regimens. Median duration of follow-up was 21.3 months. The most common treatment-related adverse events were asymptomatic elevations of lactate dehydrogenase (n = 26), creatine kinase (n = 25), aspartate aminotransferase (n = 25), and alanine aminotransferase (n = 19); periorbital edema (n = 17); and fatigue (n = 13). One dose-limiting toxicity (acute respiratory distress syndrome) occurred in cohort 2. The recommended phase 2 dose was APX005M 0.3 mg/kg, cabiralizumab 4 mg/kg, and nivolumab 240 mg every 2 weeks. Median days on treatment were 66 (range, 23-443). Median cycles were 4.5 (range, 2-21). One patient had unconfirmed partial response (4%), 8 stable disease (31%), 16 disease progression (62%), and 1 unevaluable (4%). Pro-inflammatory cytokines were upregulated 4 hours post-infusion. CD40 and MCSF increased after therapy. CONCLUSIONS: This first in-human study of patients with anti-PD-1/PD-L1-resistant tumors treated with dual macrophage-polarizing therapy, with or without nivolumab demonstrated safety and pharmacodynamic activity. Optimization of the dosing frequency and sequence of this combination is warranted.

Analysis of multispectral imaging with the AstroPath platform informs efficacy of PD-1 blockade.

Next-generation tissue-based biomarkers for immunotherapy will likely include the simultaneous analysis of multiple cell types and their spatial interactions, as well as distinct expression patterns of immunoregulatory molecules. Here, we introduce a comprehensive platform for multispectral imaging and mapping of multiple parameters in tumor tissue sections with high-fidelity single-cell resolution. Image analysis and data handling components were drawn from the field of astronomy. Using this "AstroPath" whole-slide platform and only six markers, we identified key features in pretreatment melanoma specimens that predicted response to anti-programmed cell death-1 (PD-1)-based therapy, including CD163+PD-L1- myeloid cells and CD8+FoxP3+PD-1low/mid T cells. These features were combined to stratify long-term survival after anti-PD-1 blockade. This signature was validated in an independent cohort of patients with melanoma from a different institution.

Clinical Significance of PDCD4 in Melanoma by Subcellular Expression and in Tumor-Associated Immune Cells.

Little is known about the subcellular localization and function of programmed cell death 4 (PDCD4) in melanoma. Our past studies suggest PDCD4 interacts with Pleckstrin Homology Domain Containing A5 (PLEKHA5) to influence melanoma brain metastasis outcomes, as high intracranial PDCD4 expression leads to improved survival. We aimed to define the subcellular distribution of PDCD4 in melanoma and in the tumor microenvironment during neoplastic progression and its impact on clinical outcomes. We analyzed multiple tissue microarrays with well-annotated clinicopathological variables using quantitative immunofluorescence and evaluated single-cell RNA-sequencing on a brain metastasis sample to characterize PDCD4+ immune cell subsets. We demonstrate differences in PDCD4 expression during neoplastic progression, with high tumor and stromal PDCD4 levels associated with improved survival in primary melanomas and in intracranial metastases, but not in extracranial metastatic disease. While the expression of PDCD4 is well-documented on CD8+ T cells and natural killer cells, we show that it is also found on B cells and mast cells. PDCD4 expression in the tumor microenvironment is associated with increased immune cell infiltration. Further studies are needed to define the interaction of PDCD4 and PLEKHA5 and to evaluate the utility of this pathway as a therapeutic target in melanoma brain metastasis.

Circulating clonally expanded T cells reflect functions of tumor-infiltrating T cells.

Understanding the relationship between tumor and peripheral immune environments could allow longitudinal immune monitoring in cancer. Here, we examined whether T cells that share the same TCRαß and are found in both tumor and blood can be interrogated to gain insight into the ongoing tumor T cell response. Paired transcriptome and TCRαß repertoire of circulating and tumor-infiltrating T cells were analyzed at the single-cell level from matched tumor and blood from patients with metastatic melanoma. We found that in circulating T cells matching clonally expanded tumor-infiltrating T cells (circulating TILs), gene signatures of effector functions, but not terminal exhaustion, reflect those observed in the tumor. In contrast, features of exhaustion are displayed predominantly by tumor-exclusive T cells. Finally, genes associated with a high degree of blood-tumor TCR sharing were overexpressed in tumor tissue after immunotherapy. These data demonstrate that circulating TILs have unique transcriptional patterns that may have utility for the interrogation of T cell function in cancer immunotherapy.

Mycophenolate as Primary Treatment for Immune Checkpoint Inhibitor Induced Acute Kidney Injury in a Patient with Concurrent Immunotherapy-Associated Diabetes: A Case Report.

Immune checkpoint inhibitors enhance T cell response against malignant cells and are standard of care in many tumor types. Disinhibition of cytotoxic T cells in normal organs and inhibition of regulatory T cells can lead to immune-related adverse events. Here we describe a 60-year-old man with metastatic melanoma treated with three cycles of nivolumab and ipilimumab. He subsequently presented with new-onset brittle diabetes, rash, fever, and acute kidney injury. After initiation of insulin and aggressive fluid resuscitation, his kidney functions transiently improved but then dramatically worsened. Due concerns regarding hyperglycemia, a steroid-sparing agent was necessary and he was successfully treated with front-line mycophenolate mofetil, leading to normalization of renal function. The patient went on to develop a complete response and remains disease-free four years later. We conclude that mycophenolate can serve as an effective frontline therapy for immune-mediated acute kidney injury when steroids are contraindicated.

Automated Assay of a Four-Protein Biomarker Panel for Improved Detection of Ovarian Cancer.

Background: Mortality from ovarian cancer remains high due to the lack of methods for early detection. The difficulty lies in the low prevalence of the disease necessitating a significantly high specificity and positive-predictive value (PPV) to avoid unneeded and invasive intervention. Currently, cancer antigen- 125 (CA-125) is the most commonly used biomarker for the early detection of ovarian cancer. In this study we determine the value of combining macrophage migration inhibitory factor (MIF), osteopontin (OPN), and prolactin (PROL) with CA-125 in the detection of ovarian cancer serum samples from healthy controls. Materials and Methods: A total of 432 serum samples were included in this study. 153 samples were from ovarian cancer patients and 279 samples were from age-matched healthy controls. The four proteins were quantified using a fully automated, multi-analyte immunoassay. The serum samples were divided into training and testing datasets and analyzed using four classification models to calculate accuracy, sensitivity, specificity, PPV, negative predictive value (NPV), and area under the receiver operating characteristic curve (AUC). Results: The four-protein biomarker panel yielded an average accuracy of 91% compared to 85% using CA-125 alone across four classification models (p = 3.224 × 10-9). Further, in our cohort, the four-protein biomarker panel demonstrated a higher sensitivity (median of 76%), specificity (median of 98%), PPV (median of 91.5%), and NPV (median of 92%), compared to CA-125 alone. The performance of the four-protein biomarker remained better than CA-125 alone even in experiments comparing early stage (Stage I and Stage II) ovarian cancer to healthy controls. Conclusions: Combining MIF, OPN, PROL, and CA-125 can better differentiate ovarian cancer from healthy controls compared to CA-125 alone.

Non-Small-cell Lung Cancer Patients With Adenocarcinoma Morphology Have a Better Outcome Compared With Patients Diagnosed With Non-Small-cell Lung Cancer Favor Adenocarcinoma.

BACKGROUND: Non-small-cell lung cancer (NSCLC) includes 2 major histologic subtypes: squamous cell carcinoma and non-squamous carcinoma, mainly adenocarcinoma, a distinction that carries significant clinical and therapeutic implications. NSCLC is diagnosed as adenocarcinoma or as squamous cell carcinoma on the basis of histologic parameters. However, when morphology is inconclusive, tumors with immunohistochemistry (IHC) findings characteristic of adenocarcinoma are referred to as "NSCLC favor adenocarcinoma" (NFA). Our aim was to evaluate whether pulmonary adenocarcinoma diagnosis on the basis of morphology had a similar prognosis compared with NFA. PATIENTS AND METHODS: Patients with advanced NSCLC non-squamous carcinoma who were treated with a platinum-pemetrexed doublet as first-line combination chemotherapy were identified. Demographic, clinical, laboratory, and pathological data including the method of pathological diagnosis (morphology or IHC) was extracted from the clinical charts. The correlation between the various parameters and overall survival was evaluated. RESULTS: Lack of adenocarcinoma morphology, male sex, smoking history, and negative thyroid transcription factor 1 IHC were associated with worse prognosis and shorter overall survival in multivariate analysis. High white blood cell count, absolute neutrophil count, neutrophil to lymphocyte ratio, and low albumin levels were associated with shorter overall survival only in univariate analysis. CONCLUSION: Pulmonary adenocarcinoma has a better prognosis than NFA, regarding advanced NSCLC treated with platinum-pemetrexed combination chemotherapy. This distinction should be a stratification factor in clinical trials and a prognostic factor to consider in analysis of previous trials.

Improving posttransplantation survival of human ovarian tissue by treating the host and graft.

OBJECTIVE: To improve posttransplantation survival of frozen-thawed human ovarian tissue in immunodeficient mice. DESIGN: Histologic study of transplanted human ovaries after treating the host and graft. SETTING: Infertility unit, university-affiliated tertiary medical center. PATIENT(S): Ovarian tissue from six girls/women (aged 5-23 years) who had undergone ovarian laparoscopy for fertility preservation. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Thawed ovarian samples were transplanted into the back muscle of immunodeficient mice divided into four groups: A) no treatment; B) host treatment with vitamin E and gonadotropins before and after grafting; C) graft incubation with vascular endothelial growth factor A (VEGF-A) and vitamin E before transplantation; and D) host as in B, graft as in C. Ungrafted thawed samples served as control. Assessment of graft survival was conducted by follicle counts, apoptosis evaluation, immunohistochemical stainings for proliferating cell nuclear antigen (PCNA) and VEGF-A expression. RESULT(S): Only grafts incubated before transplantation (groups C and D) retained their original size. Follicle number was low in all grafts. PCNA expression was found in most grafts. Apoptosis was significantly lower in the untreated and treated grafts transplanted into treated hosts (groups B and D) than in ungrafted-thawed samples and group A grafts. All grafted groups had significantly higher expression of VEGF-A than ungrafted-thawed samples. CONCLUSION(S): Survival of transplanted human ovarian tissue may be improved by treatment of the host and graft. Further studies to evaluate treatments with a potential benefit in human ovarian autotransplantation are needed.