Radiomic analysis of patient and inter-organ heterogeneity in response to immunotherapies and BRAF targeted therapy in metastatic melanoma.

Background: Variability in treatment response may be attributable to organ-level heterogeneity in tumor lesions. Radiomic analysis of medical images can elucidate non-invasive biomarkers of clinical outcome. Organ-specific radiomic comparison across immunotherapies and targeted therapies has not been previously reported. Methods: We queried UPMC Hillman Cancer Center registry for patients with metastatic melanoma (MEL) treated with immune checkpoint inhibitors (ICI) (anti-PD1/CTLA4 [ipilimumab+nivolumab; I+N] or anti-PD1 monotherapy) or BRAF targeted therapy. Best overall response was measured using RECIST v1.1. Lesions were segmented into discrete volume-of-interest with 400 radiomics features extracted. Overall and organ-specific machine-learning models were constructed to predict disease control (DC) versus progressive disease (PD) using XGBoost. Results: 291 MEL patients were identified, including 242 ICI (91 I+N, 151 PD1) and 49 BRAF. 667 metastases were analyzed, including 541 ICI (236 I+N, 305 PD1) and 126 BRAF. Across cohorts, baseline demographics included 39-47% female, 24-29% M1C, 24-46% M1D, and 61-80% with elevated LDH. Among patients experiencing DC, the organs with the greatest reduction were liver (-88%±12%, I+N; mean±S.E.M.) and lung (-72%±8%, I+N). For patients with multiple same-organ target lesions, the highest inter-lesion heterogeneity was observed in brain among patients who received ICI while no intra-organ heterogeneity was observed in BRAF. 267 patients were kept for radiomic modeling, including 221 ICI (86 I+N, 135 PD1) and 46 BRAF. Models consisting of optimized radiomic signatures classified DC/PD across I+N (AUC=0.85) and PD1 (0.71) and within individual organ sites (AUC=0.72∼0.94). Integration of clinical variables improved the models' performance. Comparison of models between treatments and across organ sites suggested mostly non-overlapping DC or PD features. Skewness, kurtosis, and informational measure of correlation (IMC) were among the radiomic features shared between overall response models. Kurtosis and IMC were also utilized by multiple organ-site models. Conclusions: Differential organ-specific response was observed across BRAF and ICI with within organ heterogeneity observed for ICI but not for BRAF. Radiomic features of organ-specific response demonstrated little overlap. Integrating clinical factors with radiomics improves the prediction of disease course outcome and prediction of tumor heterogeneity.

GZ17-6.02 kills PDX isolates of uveal melanoma.

GZ17-6.02 has undergone phase I evaluation in patients with solid tumors (NCT03775525). The RP2D is 375 mg PO BID, with an uveal melanoma patient exhibiting a 15% reduction in tumor mass for 5 months at this dose. Studies in this manuscript have defined the biology of GZ17-6.02 in PDX isolates of uveal melanoma cells. GZ17-6.02 killed uveal melanoma cells through multiple convergent signals including enhanced ATM-AMPK-mTORC1 activity, inactivation of YAP/TAZ and inactivation of eIF2α. GZ17-6.02 significantly enhanced the expression of BAP1, predictive to reduce metastasis, and reduced the levels of ERBB family RTKs, predicted to reduce growth. GZ17-6.02 interacted with doxorubicin or ERBB family inhibitors to significantly enhance tumor cell killing which was associated with greater levels of autophagosome formation and autophagic flux. Knock down of Beclin1, ATG5 or eIF2α were more protective than knock down of ATM, AMPKα, CD95 or FADD, however, over-expression of FLIP-s provided greater protection compared to knock down of CD95 or FADD. Expression of activated forms of mTOR and STAT3 significantly reduced tumor cell killing. GZ17-6.02 reduced the expression of PD-L1 in uveal melanoma cells to a similar extent as observed in cutaneous melanoma cells whereas it was less effective at enhancing the levels of MHCA. The components of GZ17-6.02 were detected in tumors using a syngeneic tumor model. Our data support future testing GZ17-6.02 in uveal melanoma as a single agent, in combination with ERBB family inhibitors, in combination with cytotoxic drugs, or with an anti-PD1 immunotherapy.

Automated Quantitative CD8+ Tumor-Infiltrating Lymphocytes and Tumor Mutation Burden as Independent Biomarkers in Melanoma Patients Receiving Front-Line Anti-PD-1 Immunotherapy.

BACKGROUND: CD8+ tumor-infiltrating lymphocyte (TIL) predicts response to anti-PD-(L)1 therapy. However, there remains no standardized method to assess CD8+ TIL in melanoma, and developing a specific, cost-effective, reproducible, and clinically actionable biomarker to anti-PD-(L)1 remains elusive. We report on the development of automatic CD8+ TIL density quantification via whole slide image (WSI) analysis in advanced melanoma patients treated with front-line anti-PD-1 blockade, and correlation immunotherapy response. METHODS: Seventy-eight patients treated with PD-1 inhibitors in the front-line setting between January 2015 and May 2023 at the University of Pittsburgh Cancer Institute were included. CD8+ TIL density was quantified using an image analysis algorithm on digitized WSI. Targeted next-generation sequencing (NGS) was performed to determine tumor mutation burden (TMB) in a subset of 62 patients. ROC curves were used to determine biomarker cutoffs and response to therapy. Correlation between CD8+ TIL density and TMB cutoffs and response to therapy was studied. RESULTS: Higher CD8+ TIL density was significantly associated with improved response to front-line anti-PD-1 across all time points measured. CD8+ TIL density ≥222.9 cells/mm2 reliably segregated responders and non-responders to front-line anti-PD-1 therapy regardless of when response was measured. In a multivariate analysis, patients with CD8+ TIL density exceeding cutoff had significantly improved PFS with a trend toward improved OS. Similarly, increasing TMB was associated with improved response to anti-PD-1, and a cutoff of 14.70 Mut/Mb was associated with improved odds of response. The correlation between TMB and CD8+ TIL density was low, suggesting that each represented independent predictive biomarkers of response. CONCLUSIONS: An automatic digital analysis algorithm provides a standardized method to quantify CD8+ TIL density, which predicts response to front-line anti-PD-1 therapy. CD8+ TIL density and TMB are independent predictors of response to anti-PD-1 blockade.

Pembrolizumab Versus Placebo as Adjuvant Therapy in Resected Stage IIB or IIC Melanoma: Final Analysis of Distant Metastasis-Free Survival in the Phase III KEYNOTE-716 Study.

Clinical trials frequently include multiple end points that mature at different times. The initial report, typically based on the primary end point, may be published when key planned co-primary or secondary analyses are not yet available. Clinical Trial Updates provide an opportunity to disseminate additional results from studies, published in JCO or elsewhere, for which the primary end point has already been reported.Pembrolizumab adjuvant therapy was shown to significantly improve recurrence-free survival (RFS) and distant metastasis-free survival (DMFS) in patients with resected stage IIB or IIC melanoma in earlier analyses of the randomized, double-blind, phase III KEYNOTE-716 study (ClinicalTrials.gov identifier: NCT03553836). We report results of the protocol-specified final analysis of DMFS for KEYNOTE-716. Overall, 976 patients were randomly allocated to pembrolizumab (n = 487) or placebo (n = 489). As of January 4, 2023, median follow-up was 39.4 months (range, 26.0-51.4 months). The median DMFS was not reached in either treatment group, and the estimated 36-month DMFS was 84.4% for pembrolizumab and 74.7% for placebo (hazard ratio [HR], 0.59 [95% CI, 0.44 to 0.79]). The median RFS was not reached in either treatment group, and the estimated 36-month RFS was 76.2% for pembrolizumab and 63.4% for placebo (HR, 0.62 [95% CI, 0.49 to 0.79]). DMFS and RFS results were consistent across most prespecified subgroups, including stage IIB and stage IIC melanoma. The safety profile of pembrolizumab was manageable and consistent with previous reports. These results continue to support the use of pembrolizumab adjuvant therapy in patients with resected stage IIB or IIC melanoma.

Pembrolizumab versus placebo as adjuvant therapy in resected stage IIB or IIC melanoma: Outcomes in histopathologic subgroups from the randomized, double-blind, phase 3 KEYNOTE-716 trial.

BACKGROUND: Adjuvant pembrolizumab significantly improved recurrence-free survival (RFS) and distant metastasis-free survival (DMFS) versus placebo in the phase 3 KEYNOTE-716 study of resected stage IIB or IIC melanoma. At the prespecified third interim analysis (data cut-off, January 4, 2022), the HR for RFS in the overall population was 0.64 (95% CI, 0.50 to 0.84) and the HR for DMFS was 0.64 (95% CI, 0.47 to 0.88). We present a post hoc analysis of efficacy by subtypes defined by histopathologic characteristics. METHODS: Patients aged ≥12 years with newly diagnosed, resected stage IIB or IIC melanoma were randomly assigned (1:1) to pembrolizumab 200 mg every 3 weeks (2 mg/kg up to 200 mg for pediatric patients) or placebo. The primary end point was RFS per investigator review; DMFS per investigator review was secondary. Subgroups of interest were melanoma subtype (nodular vs non-nodular), tumor thickness (≤4 mm vs >4 mm), presence of ulceration (yes vs no), mitotic rate (<5 per mm2 (median) vs ≥5 per mm2), and presence of tumor-infiltrating lymphocytes (TILs; absent vs present). RESULTS: Between September 23, 2018, and November 4, 2020, 976 patients were assigned to pembrolizumab (n=487) or placebo (n=489). Median follow-up was 27.4 months (range, 14.0-39.4). The HR (95% CI) for RFS was 0.54 (0.37 to 0.79) for nodular and 0.77 (0.53 to 1.11) for non-nodular melanoma; 0.57 (0.37 to 0.89) for thickness ≤4 mm and 0.69 (0.50 to 0.96) for >4 mm; 0.66 (0.50 to 0.89) for ulceration and 0.57 (0.32 to 1.03) for no ulceration; 0.57 (0.35 to 0.92) for mitotic rate <5 per mm2 and 0.57 (0.40 to 0.80) for ≥5 per mm2; and 0.89 (0.52 to 1.54) for TILs absent and 0.51 (0.34 to 0.76) for TILs present. DMFS results were similar. In a Cox multivariate analysis, treatment arm, tumor thickness, and mitotic rate were significant independent factors for RFS, and treatment arm and mitotic rate were significant independent factors for DMFS. CONCLUSIONS: In this post hoc analysis, the benefit of pembrolizumab was largely consistent with the overall study population regardless of histopathologic characteristics. These results support the use of adjuvant pembrolizumab in patients with resected stage IIB or IIC melanoma. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov, NCT03553836.

Neoadjuvant Dual Checkpoint Inhibitors vs Anti-PD1 Therapy in High-Risk Resectable Melanoma: A Pooled Analysis.

Importance: Despite the clear potential benefits of neoadjuvant therapy, the optimal neoadjuvant regimen for patients with high-risk resectable melanoma (HRRM) is not known. Objective: To compare the safety and efficacy of dual checkpoint inhibitors with anti-programmed cell death protein-1 (anti-PD1) therapy in a neoadjuvant setting among patients with HRRM. Design, Setting, and Participants: In this pooled analysis of clinical trials, studies were selected provided they investigated immune checkpoint inhibitor treatment, were published between January 2018 and March 2023, and were phase 1, 2, or 3 clinical trials. Participant data included in the analysis were derived from trials evaluating the efficacy and safety of anti-PD1 monotherapy and the combination of anti-cytotoxic T lymphocyte-associated protein-4 with anti-PD1 in the neoadjuvant setting, specifically among patients with HRRM. Interventions: Patients were treated with either anti-PD1 monotherapy; dual checkpoint inhibition (DCPI) with a conventional dose of 3-mg/kg ipilimumab and 1-mg/kg nivolumab; or DCPI with an alternative-dose regimen of 1-mg/kg ipilimumab and 3-mg/kg nivolumab. Main Outcomes and Measures: The main outcomes were radiologic complete response (rCR), radiologic overall objective response (rOOR), and radiologic progressive disease. Also, pathologic complete response (pCR), the proportion of patients undergoing surgical resection, and occurrence of grade 3 or 4 immune-related adverse events (irAEs) were considered. Results: Among 573 patients enrolled in 6 clinical trials, neoadjuvant therapy with DCPI was associated with higher odds of achieving pCR compared with anti-PD1 monotherapy (odds ratio [OR], 3.16; P < .001). DCPI was associated with higher odds of grade 3 or 4 irAEs compared with anti-PD1 monotherapy (OR, 3.75; P < .001). When comparing the alternative-dose ipilimumab and nivolumab (IPI-NIVO) regimen with conventional-dose IPI-NIVO, no statistically significant difference in rCR, rOOR, radiologic progressive disease, or pCR was noted. However, the conventional-dose IPI-NIVO regimen was associated with increased grade 3 or 4 irAEs (OR, 4.76; P < .001). Conventional-dose IPI-NIVO was associated with greater odds of achieving improved rOOR (OR, 1.95; P = .046) and pCR (OR, 2.99; P < .001) compared with anti-PD1 monotherapy. The alternative dose of IPI-NIVO also was associated with higher odds of achieving rCR (OR, 2.55; P = .03) and pCR (OR, 3.87; P < .001) compared with anti-PD1 monotherapy. The risk for grade 3 or 4 irAEs is higher with both the conventional-dose (OR, 9.59; P < .001) and alternative-dose IPI-NIVO regimens (OR, 2.02; P = .02) compared with anti-PD1 monotherapy. Conclusion and Relevance: In this pooled analysis of 6 clinical trials, although DCPI was associated with increased likelihood of achieving pathological and radiologic responses, the associated risk for grade 3 or 4 irAEs was significantly lower with anti-PD1 monotherapy in the neoadjuvant setting for HRRM. Additionally, compared with alternative-dose IPI-NIVO, the conventional dose of IPI-NIVO was associated with increased risk for grade 3 or 4 irAEs, with no significant distinctions in radiologic or pathologic efficacy.

Plain language summary of the CheckMate 76K study results: nivolumab given after stage 2B/2C melanoma is removed by surgery.

WHAT IS THIS SUMMARY ABOUT?: In this article, we summarize results from the ongoing phase 3 CheckMate 76K clinical study published online in Nature Medicine in October 2023. The study goal was to learn whether nivolumab works as an adjuvant therapy (that is, helps to keep cancer from coming back when it is given after surgery) for stage 2 melanoma (skin cancer) that has not spread to other parts of the body. Nivolumab is an immunotherapy that activates a person's immune system so it can destroy cancer cells. In melanoma, staging describes the severity of the cancer. Melanoma staging ranges from 0 (very thin and confined to the upper layer of the skin) to 4 (spread to distant parts of the body), with earlier stages removed by surgery. The people in this study had stage 2 melanoma that had not spread to the lymph nodes or other organs in the body. HOW WAS THE STUDY DESIGNED?: People 12 years and older with stage 2 melanoma that had not spread and had been removed by surgery were included in CheckMate 76K. People were randomly assigned to receive either nivolumab (526 patients) or placebo (264 patients). A placebo resembles the test medicine but does not contain any active medicines. The researchers assessed whether people who received nivolumab lived longer without their cancer returning and/or spreading to other parts of their bodies (compared with placebo) and if nivolumab was well tolerated. WHAT WERE THE RESULTS?: Researchers found that people who received nivolumab were 58% less likely to have their cancer return and 53% less likely of having their cancer spread to distant parts of their body, compared with placebo. These reductions in risk with nivolumab were seen in different subgroups of people with a range of characteristics, and regardless of how deep the melanoma had gone into the skin. People taking nivolumab had more side effects than those taking placebo, but most were mild to moderate and manageable. WHAT DO THE RESULTS MEAN?: Results from CheckMate 76K support the benefit of using nivolumab as a treatment option for people with stage 2 melanoma post-surgery.

Real-world outcomes with immuno-oncology therapies in advanced melanoma: final results of the OPTIMIzE registry study.

Aim: The OPTIMIzE registry study evaluated real-world outcomes in patients with advanced melanoma receiving immuno-oncology therapies. Materials and methods: Data were collected for patients treated with anti-programmed death 1 (PD-1) monotherapy (nivolumab or pembrolizumab; n = 147) or nivolumab plus ipilimumab (n = 81) from 2015-2017 and followed for ≥3 years. Results: Nivolumab plus ipilimumab versus anti-PD-1 monotherapy was associated with a nonsignificantly lower risk of death (adjusted HR: 0.83; 95% CI: 0.54-1.28; p = 0.41), higher disease control rate (72 vs 56%; p = 0.04), and stable quality of life, but more grade 3-4 treatment-related adverse events (54 vs 26%; p < 0.0001). Conclusion: These results support the use of immuno-oncology therapy in advanced melanoma.

Melanoma is a serious form of skin cancer that develops from melanocytes, which are pigment cells that give the skin, hair, and other tissues their color. At advanced stages of spread, melanoma can be life-threatening. However, immunotherapy, a type of therapy that helps the body's immune system to destroy cancer cells, allows some patients with advanced melanoma to live longer. The OPTIMIzE study looked at how well patients with advanced melanoma did when treated with different immunotherapies. These patients were treated in a real-world setting, such as a doctor's office, and were not participating in a clinical trial. Compared with clinical trials, real-world studies like the OPTIMIzE study may include a more varied group of patients because of the less selective study enrollment requirements. In the OPTIMIzE study, patients were treated with either a single immunotherapy (nivolumab or pembrolizumab alone) or a combination of two immunotherapies (nivolumab plus ipilimumab). Both single and combination immunotherapies were effective and tolerable. Patients receiving nivolumab plus ipilimumab had greater tumor shrinkage than patients receiving nivolumab or pembrolizumab alone, but with more side effects from their treatment. Despite the occurrence of side effects with both single and combination immunotherapies, patients reported that their quality of life remained stable while being treated. The OPTIMIzE study shows that immunotherapy is effective and tolerable for patients with advanced melanoma in the real-world setting. This information may help doctors with selecting treatments for their patients with advanced melanoma. Clinical Trial Registration: NCT02780089 (ClinicalTrials.gov).

Combination of Itacitinib or Parsaclisib with Pembrolizumab in Patients with Advanced Solid Tumors: A Phase I Study.

PURPOSE: This phase Ib open-label, multicenter, platform study (NCT02646748) explored safety, tolerability, and preliminary activity of itacitinib (Janus kinase 1 inhibitor) or parsaclisib (phosphatidylinositol 3-kinase δ inhibitor) in combination with pembrolizumab [programmed death-1 (PD-1) inhibitor]. EXPERIMENTAL DESIGN: Patients with advanced or metastatic solid tumors with disease progression following all available therapies were enrolled and received itacitinib (Part 1 initially 300 mg once daily) or parsaclisib (Part 1 initially 10 mg once daily; Part 2 all patients 0.3 mg once daily) plus pembrolizumab (200 mg every 3 weeks). RESULTS: A total of 159 patients were enrolled in the study and treated with itacitinib (Part 1, n = 49) or parsaclisib (Part 1, n = 83; Part 2, n = 27) plus pembrolizumab. The maximum tolerated/pharmacologically active doses were itacitinib 300 mg once daily and parsaclisib 30 mg once daily. Most common itacitinib treatment-related adverse events (TRAE) were fatigue, nausea, and anemia. Most common parsaclisib TRAEs were fatigue, nausea, diarrhea, and pyrexia in Part 1, and fatigue, maculopapular rash, diarrhea, nausea, and pruritus in Part 2. In patients receiving itacitinib plus pembrolizumab, four (8.2%) achieved a partial response (PR) in Part 1. Among patients receiving parsaclisib plus pembrolizumab, 5 (6.0%) achieved a complete response and 9 (10.8%) a PR in Part 1; 5 of 27 (18.5%) patients in Part 2 achieved a PR. CONCLUSIONS: Although combination of itacitinib or parsaclisib with pembrolizumab showed modest clinical activity in this study, the overall response rates observed did not support continued development in patients with solid tumors. SIGNIFICANCE: PD-1 blockade combined with targeted therapies have demonstrated encouraging preclinical activity. In this phase I study, patients with advanced solid tumors treated with pembrolizumab (PD-1 inhibitor) and either itacitinib (JAK1 inhibitor) or parsaclisib (PI3Kδ inhibitor) experienced limited clinical activity beyond that expected with checkpoint inhibition alone and showed little effect on T-cell infiltration in the tumor. These results do not support continued development of these combinations.

Prognostic gene expression profile testing to inform use of adjuvant therapy: A survey of melanoma experts.

OBJECTIVES: To investigate current practices and attitudes regarding use of adjuvant immunotherapy and prognostic gene expression profile (GEP) testing among melanoma medical and surgical oncologists. METHODS: An anonymous RedCap-based survey was emailed to ~300 melanoma experts. RESULTS: Respondents generally favored adjuvant immunotherapy over observation (73% for all Stage IIIA, 50% for Stage IIB/IIC) and cited a minimum 10-year recurrence risk of 11%-20% (48%) or 21%-30% (33%) to justify treatment, but acknowledged that risks of serious adverse events may outweigh potential benefits for some Stage IIB/IIC patients. While GEP test results did not strongly influence decision-making regarding follow-up or intervention, most were receptive to randomized trials using GEP testing to identify subsets of Stage IIB/IIC (74%) and Stage IB/IIA (54%) patients who may not or may, respectively, benefit from adjuvant therapy. CONCLUSION: Although most respondents do not routinely use GEP testing, many would participate in clinical trials to determine clinical utility.

Three-Year Overall Survival with Tebentafusp in Metastatic Uveal Melanoma.

BACKGROUND: Tebentafusp, a T-cell receptor-bispecific molecule that targets glycoprotein 100 and CD3, is approved for adult patients who are positive for HLA-A*02:01 and have unresectable or metastatic uveal melanoma. The primary analysis in the present phase 3 trial supported a long-term survival benefit associated with the drug. METHODS: We report the 3-year efficacy and safety results from our open-label, phase 3 trial in which HLA-A*02:01-positive patients with previously untreated metastatic uveal melanoma were randomly assigned in a 2:1 ratio to receive tebentafusp (tebentafusp group) or the investigator's choice of therapy with pembrolizumab, ipilimumab, or dacarbazine (control group), with randomization stratified according to the lactate dehydrogenase level. The primary end point was overall survival. RESULTS: At a minimum follow-up of 36 months, median overall survival was 21.6 months in the tebentafusp group and 16.9 months in the control group (hazard ratio for death, 0.68; 95% confidence interval, 0.54 to 0.87). The estimated percentage of patients surviving at 3 years was 27% in the tebentafusp group and 18% in the control group. The most common treatment-related adverse events of any grade in the tebentafusp group were rash (83%), pyrexia (76%), pruritus (70%), and hypotension (38%). Most tebentafusp-related adverse events occurred early during treatment, and no new adverse events were observed with long-term administration. The percentage of patients who discontinued treatment because of adverse events continued to be low in both treatment groups (2% in the tebentafusp group and 5% in the control group). No treatment-related deaths occurred. CONCLUSIONS: This 3-year analysis supported a continued long-term benefit of tebentafusp for overall survival among adult HLA-A*02:01-positive patients with previously untreated metastatic uveal melanoma. (Funded by Immunocore; IMCgp100-202 ClinicalTrials.gov number, NCT03070392; EudraCT number, 2015-003153-18.).

Identification of tumor-intrinsic drivers of immune exclusion in acral melanoma.

Acral melanoma (AM) has distinct characteristics as compared with cutaneous melanoma and exhibits poor response to immune checkpoint inhibitors (ICIs). Tumor-intrinsic mechanisms of immune exclusion have been identified in many cancers but less studied in AM. We characterized clinically annotated tumors from patients diagnosed with AM at our institution in correlation with ICI response using whole transcriptome RNAseq, whole exome sequencing, CD8 immunohistochemistry, and multispectral immunofluorescence imaging. A defined interferon-γ-associated T cell-inflamed gene signature was used to categorize tumors into non-T cell-inflamed and T cell-inflamed phenotypes. In combination with AM tumors from two published studies, we systematically assessed the immune landscape of AM and detected differential gene expression and pathway activation in a non-T cell-inflamed tumor microenvironment (TME). Two single-cell(sc) RNAseq AM cohorts and 11 bulk RNAseq cohorts of various tumor types were used for independent validation on pathways associated with lack of ICI response. In total, 892 specimens were included in this study. 72.5% of AM tumors showed low expression of the T cell-inflamed gene signature, with 23.9% of total tumors categorized as the non-T cell-inflamed phenotype. Patients of low CD3+CD8+PD1+ intratumoral T cell density showed poor prognosis. We identified 11 oncogenic pathways significantly upregulated in non-T cell-inflamed relative to T cell-inflamed TME shared across all three acral cohorts (MYC, HGF, MITF, VEGF, EGFR, SP1, ERBB2, TFEB, SREBF1, SOX2, and CCND1). scRNAseq analysis revealed that tumor cell-expressing pathway scores were significantly higher in low versus high T cell-infiltrated AM tumors. We further demonstrated that the 11 pathways were enriched in ICI non-responders compared with responders across cancers, including AM, cutaneous melanoma, triple-negative breast cancer, and non-small cell lung cancer. Pathway activation was associated with low expression of interferon stimulated genes, suggesting suppression of antigen presentation. Across the 11 pathways, fatty acid synthase and CXCL8 were unifying downstream target molecules suggesting potential nodes for therapeutic intervention. A unique set of pathways is associated with immune exclusion and ICI resistance in AM. These data may inform immunotherapy combinations for immediate clinical translation.

Adjuvant nivolumab in resected stage IIB/C melanoma: primary results from the randomized, phase 3 CheckMate 76K trial.

Patients with resected stage IIB/C melanoma have high recurrence risk, similar to those with resected stage IIIA/B disease. The phase 3, double-blind CheckMate 76K trial assessed 790 patients with resected stage IIB/C melanoma randomized 2:1 (stratified by tumor category) to nivolumab 480 mg or placebo every 4 weeks for 12 months. The primary endpoint was investigator-assessed recurrence-free survival (RFS). Secondary endpoints included distant metastasis-free survival (DMFS) and safety. At 7.8 months of minimum follow-up, nivolumab significantly improved RFS versus placebo (hazard ratio (HR) = 0.42; 95% confidence interval (CI): 0.30-0.59; P < 0.0001), with 12-month RFS of 89.0% versus 79.4% and benefit observed across subgroups; DMFS was also improved (HR = 0.47; 95% CI: 0.30-0.72). Treatment-related grade 3/4 adverse events occurred in 10.3% (nivolumab) and 2.3% (placebo) of patients. One treatment-related death (0.2%) occurred with nivolumab. Nivolumab is an effective and generally well-tolerated adjuvant treatment in patients with resected stage IIB/C melanoma. ClinicalTrials.gov identifier: NCT04099251 .

On thresholds for controlling negative particle (PM2.5) readings in air quality reporting.

Ambient PM2.5 (particles less than 2.5 µm in diameter) is monitored in many countries including Australia. Occasionally PM2.5 instruments may report negative measurements, although in realty the ambient air can never contain negative amounts of particles. Some negative readings are caused by instrument faults or procedural errors, thus can be simply invalidated from air quality reporting. There are occasions, however, when negative readings occur due to other factors including technological or procedural limitations. Treatment of such negative data requires consideration of factors such as measurement uncertainty, instrument noise and risk for significant bias in air quality reporting. There is very limited documentation on handling negative PM2.5 data in the literature. This paper demonstrates how a threshold is determined for controlling negative hourly PM2.5 readings in the New South Wales (NSW) air quality data system. The investigation involved a review of thresholds used in different data systems and an assessment of instrument measurement uncertainties, zero air test data and impacts on key reporting statistics when applying different thresholds to historical datasets. The results show that a threshold of -10.0 µg/m3 appears optimal for controlling negative PM2.5 data in public reporting. This choice is consistent with the measurement uncertainty estimates and the zero air test data statistics calculated for the NSW Air Quality Monitoring Network, and is expected not to have significant impacts on key compliance reporting statistics such as data availability and annual average pollution levels. The analysis can be useful for air quality monitoring in other Australian jurisdictions or wider context.

Integrated BATF transcriptional network regulates suppressive intratumoral regulatory T cells.

Human regulatory T cells (Tregs) are crucial regulators of tissue repair, autoimmune diseases, and cancer. However, it is challenging to inhibit the suppressive function of Tregs for cancer therapy without affecting immune homeostasis. Identifying pathways that may distinguish tumor-restricted Tregs is important, yet the transcriptional programs that control intratumoral Treg gene expression, and that are distinct from Tregs in healthy tissues, remain largely unknown. We profiled single-cell transcriptomes of CD4+ T cells in tumors and peripheral blood from patients with head and neck squamous cell carcinomas (HNSCC) and those in nontumor tonsil tissues and peripheral blood from healthy donors. We identified a subpopulation of activated Tregs expressing multiple tumor necrosis factor receptor (TNFR) genes (TNFR+ Tregs) that is highly enriched in the tumor microenvironment (TME) compared with nontumor tissue and the periphery. TNFR+ Tregs are associated with worse prognosis in HNSCC and across multiple solid tumor types. Mechanistically, the transcription factor BATF is a central component of a gene regulatory network that governs key aspects of TNFR+ Tregs. CRISPR-Cas9-mediated BATF knockout in human activated Tregs in conjunction with bulk RNA sequencing, immunophenotyping, and in vitro functional assays corroborated the central role of BATF in limiting excessive activation and promoting the survival of human activated Tregs. Last, we identified a suite of surface molecules reflective of the BATF-driven transcriptional network on intratumoral Tregs in patients with HNSCC. These findings uncover a primary transcriptional regulator of highly suppressive intratumoral Tregs, highlighting potential opportunities for therapeutic intervention in cancer without affecting immune homeostasis.

Identification of tumor-intrinsic drivers of immune exclusion in acral melanoma.

Background: Acral melanoma (AM) has distinct characteristics as compared to cutaneous melanoma and exhibits poor response to immune checkpoint inhibitors (ICI). Tumor-intrinsic mechanisms of immune exclusion have been identified in many cancers but less studied in AM. Methods: We characterized clinically annotated tumors from patients diagnosed with AM at our institution in correlation with ICI response using whole transcriptome RNAseq, whole exome sequencing, CD8 immunohistochemistry, and multispectral immunofluorescence imaging. A defined interferon-γ-associated T cell-inflamed gene signature was used to categorize tumors into non-T cell-inflamed and T cell-inflamed phenotypes. In combination with AM tumors from two published studies, we systematically assessed the immune landscape of AM and detected differential gene expression and pathway activation in a non-T cell-inflamed tumor microenvironment (TME). Two single-cell(sc) RNAseq AM cohorts and 11 bulk RNAseq cohorts of various tumor types were used for independent validation on pathways associated with lack of ICI response. In total, 892 specimens were included in this study. Results: 72.5% of AM tumors showed low expression of the T cell-inflamed gene signature, with 23.9% of total tumors categorized as the non-T cell-inflamed phenotype. Patients of low CD3 + CD8 + PD1 + intratumoral T cell density showed poor prognosis. We identified 11 oncogenic pathways significantly upregulated in non-T cell-inflamed relative to T cell-inflamed TME shared across all three acral cohorts (MYC, HGF, MITF, VEGF, EGFR, SP1, ERBB2, TFEB, SREBF1, SOX2, and CCND1). scRNAseq analysis revealed that tumor cell-expressing pathway scores were significantly higher in low vs high T cell-infiltrated AM tumors. We further demonstrated that the 11 pathways were enriched in ICI non-responders compared to responders across cancers, including acral melanoma, cutaneous melanoma, triple-negative breast cancer, and non-small cell lung cancer. Pathway activation was associated with low expression of interferon stimulated genes, suggesting suppression of antigen presentation. Across the 11 pathways, fatty acid synthase and CXCL8 were unifying downstream target molecules suggesting potential nodes for therapeutic intervention. Conclusions: A unique set of pathways is associated with immune exclusion and ICI resistance in AM. These data may inform immunotherapy combinations for immediate clinical translation.

Dual Targeting of the PDZ1 and PDZ2 Domains of MDA-9/Syntenin Inhibits Melanoma Metastasis.

Genome-wide gene expression analysis and animal modeling indicate that melanoma differentiation associated gene-9 (mda-9, Syntenin, Syndecan binding protein, referred to as MDA-9/Syntenin) positively regulates melanoma metastasis. The MDA-9/Syntenin protein contains two tandem PDZ domains serving as a nexus for interactions with multiple proteins that initiate transcription of metastasis-associated genes. Although targeting either PDZ domain abrogates signaling and prometastatic phenotypes, the integrity of both domains is critical for full biological function. Fragment-based drug discovery and NMR identified PDZ1i, an inhibitor of the PDZ1 domain that effectively blocks cancer invasion in vitro and in vivo in multiple experimental animal models. To maximize disruption of MDA-9/Syntenin signaling, an inhibitor has now been developed that simultaneously binds and blocks activity of both PDZ domains. PDZ1i was joined to the second PDZ binding peptide (TNYYFV) with a PEG linker, resulting in PDZ1i/2i (IVMT-Rx-3) that engages both PDZ domains of MDA-9/Syntenin. IVMT-Rx-3 blocks MDA-9/Syntenin interaction with Src, reduces NF-κB activation, and inhibits MMP-2/MMP-9 expression, culminating in repression of melanoma metastasis. The in vivo antimetastatic properties of IVMT-Rx-3 are enhanced when combined with an immune-checkpoint inhibitor. Collectively, our results support the feasibility of engineering MDA-9 dual-PDZ inhibitors with enhanced antimetastatic activities and applications of IVMT-Rx-3 for developing novel therapeutic strategies effectively targeting melanoma and in principle, a broad spectrum of human cancers that also overexpress MDA-9/Syntenin.