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Merkel cell carcinoma is among the most aggressive and lethal of primary skin cancers, with a high rate of distant metastasis. Anti-programmed death receptor 1 (anti-PD-1) and programmed death ligand 1 (PD-L1) monotherapy is currently standard of care for unresectable, recurrent, or metastatic Merkel cell carcinoma. We assessed treatment with combined nivolumab plus ipilimumab, with or without stereotactic body radiotherapy (SBRT) in patients with advanced Merkel cell carcinoma as a first-line therapy or following previous treatment with anti-PD-1 and PD-L1 monotherapy.In this randomised, open label, phase 2 trial, we randomly assigned adults from two cancer sites in the USA (one in Florida and one in Ohio) to group A (combined nivolumab and ipilimumab) or group B (combined nivolumab and ipilimumab plus SBRT) in a 1:1 ratio. Eligible patients were aged at least 18 years with histologically proven advanced stage (unresectable, recurrent, or stage IV) Merkel cell carcinoma, a minimum of two tumour lesions measureable by CT, MRI or clinical exam, and tumour tissue available for exploratory biomarker analysis. Patients were stratified by previous immune-checkpoint inhibitor (ICI) status to receive nivolumab 240 mg intravenously every 2 weeks plus ipilimumab 1 mg/kg intravenously every 6 weeks (group A) or the same schedule of combined nivolumab and ipilimumab with the addition of SBRT to at least one tumour site (24 Gy in three fractions at week 2; group B). Patients had to have at least two measurable sites of disease so one non-irradiated site could be followed for response. The primary endpoint was objective response rate (ORR) in all randomly assigned patients who received at least one dose of combined nivolumab and ipilimumab. ORR was defined as the proportion of patients with a complete response or partial response per immune-related Response Evaluation Criteria in Solid Tumours. Response was assessed every 12 weeks. Safety was assessed in all patients. This trial is registered with ClinicalTrials.gov, NCT03071406.50 patients (25 in both group A and group B) were enrolled between March 14, 2017, and Dec 21, 2021, including 24 ICI-naive patients (13 [52%] of 25 group A patients and 11 [44%] of 25 group B patients]) and 26 patients with previous ICI (12 [48%] of 25 group A patients and 14 [56%] of 25 group B patients]). One patient in group B did not receive SBRT due to concerns about excess toxicity. Median follow-up was 14·6 months (IQR 9·1-26·5). Two patients in group B were excluded from the analysis of the primary endpoint because the target lesions were irradiated and so the patients were deemed non-evaluable. Of the ICI-naive patients, 22 (100%) of 22 (95% CI 82-100) had an objective response, including nine (41% [95% CI 21-63]) with complete response. Of the patients who had previously had ICI exposure, eight (31%) of 26 patients (95% CI 15-52) had an objective response and four (15% [5-36]) had a complete response. No significant differences in ORR were observed between groups A (18 [72%] of 25 patients) and B (12 [52%] of 23 patients; p=0·26). Grade 3 or 4 treatment-related adverse events were observed in 10 (40%) of 25 patients in group A and 8 (32%) of 25 patients in group B.First-line combined nivolumab and ipilimumab in patients with advanced Merkel cell carcinoma showed a high ORR with durable responses and an expected safety profile. Combined nivolumab and ipilimumab also showed clinical benefit in patients with previous anti-PD-1 and PD-L1 treatment. Addition of SBRT did not improve efficacy of combined nivolumab and ipilimumab. The combination of nivolumab and ipilimumab represents a new first-line and salvage therapeutic option for advanced Merkel cell carcinoma.Bristol Myers Squibb Rare Population Malignancy Program.

Anti-PD1 therapy demonstrated impressive, prolonged responses in advanced cutaneous squamous cell carcinoma (CSCC). Therapy for ICI-refractory/ineligible disease remains unclear. We performed a retrospective analysis in locally-advanced/metastatic CSCC using cetuximab across three cohorts: immediately after ICI failure (A), not immediately following ICI failure (B), or without prior ICI (C). The primary endpoint was the overall response rate (ORR). Secondary endpoints included disease-control rate (DCR), progression-free survival (PFS), overall survival (OS), time-to-response (TTR) and toxicity. Twenty-three patients were included. In cohort A (n = 11), the ORR was 64% and DCR was 91%, with six ongoing responses at data cutoff. In cohort B (n = 2), all patients had progression as the best response. At a median follow-up of 21 months for A and B, TTR and PFS were 2.0 and 17.3 months, respectively. The median OS was not reached. In cohort C (n = 10), the ORR and DCR were 80%, including five ongoing responses at the data cutoff. At a median follow-up of 22.4 months, the TTR, PFS and OS were 2.5, 7.3 and 23.1 months, respectively. Cetuximab was well tolerated in all cohorts. In summary, cetuximab is effective in patients with failure/contraindications to ICI. Cetuximab immediately after ICI failure yielded particularly fast, durable responses. If confirmed, this could be the preferred therapy following ICI failure.

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PDF file 186K, Loss of heterozygosity on Chromosome 11p15.5

Immunohistochemistry H-score changes for PDGFRα and PDGFRβ.

CTC enumeration patterns in all population CTCs by best overall response rate.

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Background Cancer of unknown primary (CUP) comprises a heterogeneous collection of malignancies that are typically associated with a poor prognosis and a lack of effective treatment options. We retrospectively evaluated the clinical utility of targeted next-generation sequencing (NGS) among CUP patients to assist with diagnosis and identify opportunities for molecularly guided therapy. Patients and Methods Patients with a CUP at Moffitt Cancer Center who underwent NGS between January 1, 2014 and December 31, 2019, were eligible for study inclusion. Next-generation sequencing results were assessed to determine the frequency of clinically actionable molecular alterations, and chart reviews were performed to ascertain the number of patients receiving molecularly guided therapy. Results Ninety-five CUP patients were identified for analysis. Next-generation sequencing testing identified options for molecularly guided therapy for 55% (n = 52) of patients. Among patients with molecularly guided therapy options, 33% (n = 17) were prescribed a molecularly guided therapy. The median overall survival for those receiving molecularly guided therapy was 23.6 months. Among the evaluable patients, the median duration of treatment for CUP patients (n = 7) receiving molecular-guided therapy as a first-line therapy was 39 weeks. The median duration of treatment for CUP patients (n = 8) treated with molecularly guided therapy in the second- or later-line setting was 13 weeks. Next-generation sequencing results were found to be suggestive of a likely primary tumor type for 15% (n = 14) of patients. Conclusion Next-generation sequencing results enabled the identification of treatment options in a majority of patients and assisted with the identification of a likely primary tumor type in a clinically meaningful subset of patients.

Using transcriptome data for expression profiling of tumors. Heatmap of transcriptome data allowing for expression profiling and clustering of diagnoses based on the 2000 most highly differentially expressed genes in comparison to the other cohort samples to validate tumor diagnosis. The diagnostic abbreviations are the same as in Figure 1 with the addition of EWLS for Ewing-like sarcoma.

Supplementary Method

Imaging from two clinical vignettes highlighting the importance of sequencing at diagnosis and relapse. A. An epithelioid inflammatory myofibroblastic sarcoma (Patient NCI0244) positive for a RANBP2-ALK fusion leading to activation of ALK was treated with targeted monotherapy. PET scans are shown in the top row. At initial diagnosis, abdominal tumors were located in the left upper quadrant with extensive FDG-avid peritoneal lesions. Clinical response after 8 months of targeted therapy with crizotinib was noted. First relapse with development of a recurrent nodule noted in between the right kidney and duodenum with pulmonary and hepatic metastases was observed after 14 months of crizotinib therapy. A secondary clinical response after treatment with ceritinib was obtained after 1 month of therapy, but was not maintained. The primary tumor sample derived cell line, labeled Tumor.1, did not contain an ALK I1171T mutation. Sanger validation confirmed the presence of an ALK p.I1171T mutation after targeted therapy with crizotinib and ceritinib. The patient's tumor samples all contained a RANBP2-ALK fusion, which was not present in the germline. B. A GNAQ Q209L mutation-positive melanoma (Patient NCI0155), treated with trametinib. The melanoma originated from the left frontotemporal scalp and left orbit, and was metastatic to regional lymph nodes, dura mater, liver, bone and lung as shown on CT (first column, first row) with PET avidity in metastases (first column, second row). WES and WTS of her tumor revealed a GNAQ Q209L mutation. The patient was treated with trametinib and had an initial mixed response, with loss of PET avidity of several liver metastases (second column, second row). However, progressive disease of the primary tumor was noted 5 months after initiating therapy, and PET/CT scans showed increased size of the primary tumor in the scalp (third column, first row)

Figure S4

Flowcharts of filtering criteria. A. Flowchart of filtering criteria for germline variants, B. Flowchart of filtering criteria for somatic variants

Purpose: We undertook a multidimensional clinical genomics study of children and adolescent young adults with relapsed and refractory cancers to determine the feasibility of genome-guided precision therapy.Experimental Design: Patients with non-central nervous system solid tumors underwent a combination of whole exome sequencing (WES), whole transcriptome sequencing (WTS), and high-density single-nucleotide polymorphism array analysis of the tumor, with WES of matched germline DNA. Clinically actionable alterations were identified as a reportable germline mutation, a diagnosis change, or a somatic event (including a single nucleotide variant, an indel, an amplification, a deletion, or a fusion gene), which could be targeted with drugs in existing clinical trials or with FDA-approved drugs.Results: Fifty-nine patients in 20 diagnostic categories were enrolled from 2010 to 2014. Ages ranged from 7 months to 25 years old. Seventy-three percent of the patients had prior chemotherapy, and the tumors from these patients with relapsed or refractory cancers had a higher mutational burden than that reported in the literature. Thirty patients (51% of total) had clinically actionable mutations, of which 24 (41%) had a mutation that was currently targetable in a clinical trial setting, 4 patients (7%) had a change in diagnosis, and 7 patients (12%) had a reportable germline mutation.Conclusions: We found a remarkably high number of clinically actionable mutations in 51% of the patients, and 12% with significant germline mutations. We demonstrated the clinical feasibility of next-generation sequencing in a diverse population of relapsed and refractory pediatric solid tumors. Clin Cancer Res; 22(15); 3810–20. ©2016 AACR.

Tab 1: Patient Demographics at Enrollment. Tab 2: Details of Multiple Samples from Individual Patients , Tab 3: Sample Data of Patients and Sequencing Data of Each Patient Sample, Tab 4: Sample Depth and Coverage, Tab 5: Somatic SNVs in Tumor Samples, Tab 6: Somatic Indels in Tumor Samples, Tab 7: Germline SNVs and Indels in Normal Samples, and Tab 8: Fusion Genes in Defuse, and Tab 9: Fusion Genes in TopHat

Schema for planned genomics guided CLIA sequencing program (ClinOmics) for the Center for Cancer Research of the intramural National Cancer Institute.

American College of Medical Genetics (ACMG) Reportable Criteria and Tab2: NCI-Adult MATCH Criteria for Matching Mutation to Drug

An adrenocortical carcinoma in Patient NCI0226 with a de-novo germline TP53 mutation. Germline DNA samples from brother and parents were not noted to have the mutation. The mutation was a novel two base substitution at codon c.358_359delCGinsAA, leading to an amino acid change, A159K. The mutation was heterozygous in the germline, and homozygous in tumor exome and transcriptome due to loss of heterozygosity of the wild-type allele.

Background Immune checkpoint inhibitors (ICIs) have shown modest antitumor activity in unselected advanced sarcomas. Histology driven approach to patient selection is the current standard for off-label anti-programmed cell death 1 (PD1) immunotherapy use. Methods We retrospectively reviewed the clinical characteristics and outcomes of patients with advanced sarcoma who were treated with off label anti-PD1 immunotherapy at our center. Results A total of 84 patients with 25 histological subtypes were included. Nineteen patients (23%) had a cutaneous primary tumor site. Eighteen patients (21%) were classified as having clinical benefit, including 1 patient with complete response, 14 with partial response, and 3 with stable disease lasting over 6 months with previously progressive disease. Cutaneous primary site location was associated with higher clinical benefit rate (58% vs. 11%, p . 2.5 months, p = 0.003) and OS (19.0 vs. 9.2 months, p = 0.011), compared to non-cutaneous primary. Patients with histological subtypes that pembrolizumab is indicated per current National Comprehensive Cancer Network guidelines had modestly higher rate of clinical benefit versus other histologies, however, the difference was statistically insignificant (29% vs. 15%, p = 0.182) and no statistically significant difference in PFS or OS was observed between these groups. Immune-related adverse events were more frequently seen among patients with clinical benefit (72% vs. 35%, p = 0.007). Conclusions Anti-PD1-based immunotherapy is highly efficacious in advanced sarcomas of cutaneous primary site. Cutaneous primary site location is a stronger predictor of ICI response than histologic subtype and should be accounted for in treatment guidelines and clinical trial design.

BackgroundAdjuvant therapy for high-risk resected melanoma with programmed cell-death 1 blockade results in a median relapse-free survival (RFS) of 5 years. The addition of low dose ipilimumab (IPI) to a regimen of adjuvant nivolumab (NIVO) in CheckMate-915 did not result in increased RFS. A pilot phase II adjuvant study of either standard dose or low dose IPI with NIVO was conducted at two centers to evaluate RFS with correlative biomarker studies.MethodsPatients with resected stages IIIB/IIIC/IV melanoma received either IPI 3 mg/kg and NIVO 1 mg/kg (cohort 4) or IPI 1 mg/kg and NIVO 3 mg/kg (cohorts 5 and 6) induction therapy every 3 weeks for 12 weeks, followed by maintenance NIVO. In an amalgamated subset of patients across cohorts, peripheral T cells at baseline and on-treatment were assessed by flow cytometry and RNA sequencing for exploratory biomarkers.ResultsHigh rates of grade 3–4 adverse events precluded completion of induction therapy in 50%, 35% and 7% of the patients in cohorts 4, 5 and 6, respectively. At a median of 63.9 months of follow-up, 16/56 patients (29%) relapsed. For all patients, at 5 years, RFS was 71% (95% CI: 60 to 84), and overall survival was 94% (95% CI: 88 to 100). Expansion of CD3+CD4+CD38+CD127−GARP− T cells, an on-treatment increase in CD39 expression in CD8+ T cells, and T-cell expression of phosphorylated signal-transducer-and-activator-of-transcription (STAT)2 and STAT5 were associated with relapse.ConclusionsAdjuvant IPI/NIVO at the induction doses used resulted in promising relapse-free and overall survival, although with a high rate of grade 3–4 adverse events. Biomarker analyses highlight an association of ectoenzyme-expressing T cells and STAT signaling pathways with relapse, warranting future validation.Trial registration numberNCT01176474andNCT02970981.

Background Soft tissue sarcomas (STS) are rare and heterogeneous tumors making chemotherapy use controversial. Our goal was to identify a subset of patients with primary STS that benefit with the addition of chemotherapy. Methods A retrospective chart review included intermediate to high-grade localized primary STS of the extremity/trunk, and tumor size > 5 cm. The effect of chemotherapy was evaluated for local control (LC), distant control (DC), progression free survival (PFS), and overall survival (OS). Results In this cohort (n = 273), patients were treated with surgery (98%), radiation (81%), and chemotherapy (24.5%). With a median follow-up of 51 months, the entire cohort’s 5-year LC, DC, PFS, and OS are 79.1%, 59.9%, 43.8%, and 68.7%, respectively. The addition of chemotherapy did not provide a DC benefit (p = 0.238) for the entire cohort. High-grade disease (n = 210) experienced a 5-year benefit in DC (68% vs. 54.4%, p = 0.04), which was more pronounced with MAI (Mesna, Adriamycin, Ifosfamide) based regimens (74.2%, p = 0.016), and a 5-year PFS (50.8% vs 45%, p = 0.025) and OS benefit (76.2% vs 70%, p = 0.067) vs. no chemotherapy. On multivariate analysis of the high-grade subset, chemotherapy independently predicted for a DC benefit (HR 0.48 95% CI 0.26–89, p = 0.019). The benefit of chemotherapy was more pronounced with MAI, showing a significant benefit in DC (HR 0.333 95% CI 0.145–0.767, p = 0.01) and PFS (HR 0.52 95% CI 0.28–0.99, p = 0.047). Conclusion In patients with localized STS > 5 cm, the high-grade subset had a distant control benefit with the addition of chemotherapy, leading to improved progression free survival. This is more pronounced with the use of MAI and should be considered in patients eligible for this regimen.

BackgroundThe combination of antiangiogenic agents with immune checkpoint inhibitors could potentially overcome immune suppression driven by tumor angiogenesis. We report results from a phase IB study of ziv-aflibercept plus pembrolizumab in patients with advanced solid tumors.MethodsThis is a multicenter phase IB dose-escalation study of the combination of ziv-aflibercept (at 2–4 mg/kg) plus pembrolizumab (at 2 mg/kg) administered intravenously every 2 weeks with expansion cohorts in programmed cell death protein 1 (PD-1)/programmed death-ligand 1(PD-L1)-naïve melanoma, renal cell carcinoma (RCC), microsatellite stable colorectal cancer (CRC), and ovarian cancer. The primary objective was to determine maximum tolerated dose (MTD) and recommended dose of the combination. Secondary endpoints included overall response rate (ORR) and overall survival (OS). Exploratory objectives included correlation of clinical efficacy with tumor and peripheral immune population densities.ResultsOverall, 33 patients were enrolled during dose escalation (n=3) and dose expansion (n=30). No dose-limiting toxicities were reported in the initial dose level. Ziv-aflibercept 4 mg/kg plus pembrolizumab 2 mg/kg every 2 weeks was established as the MTD. Grade ≥3 adverse events occurred in 19/33 patients (58%), the most common being hypertension (36%) and proteinuria (18%). ORR in the dose-expansion cohort was 16.7% (5/30, 90% CI 7% to 32%). Complete responses occurred in melanoma (n=2); partial responses occurred in RCC (n=1), mesothelioma (n=1), and melanoma (n=1). Median OS was as follows: melanoma, not reached (NR); RCC, 15.7 months (90% CI 2.5 to 15.7); CRC, 3.3 months (90% CI 0.6 to 3.4); ovarian, 12.5 months (90% CI 3.8 to 13.6); other solid tumors, NR. Activated tumor-infiltrating CD8 T cells at baseline (CD8+PD1+), high CD40L expression, and increased peripheral memory CD8 T cells correlated with clinical response.ConclusionThe combination of ziv-aflibercept and pembrolizumab demonstrated an acceptable safety profile with antitumor activity in solid tumors. The combination is currently being studied in sarcoma and anti-PD-1-resistant melanoma.Trial registration numberNCT02298959.

SUMMARY We perform an immunogenomics analysis utilizing whole-transcriptome sequencing of 657 pediatric extra-cranial solid cancer samples representing 14 diagnoses, and additionally utilize transcriptomes of 131 pediatric cancer cell lines and 147 normal tissue samples for comparison. We describe patterns of infiltrating immune cells, T cell receptor (TCR) clonal expansion, and translationally relevant immune checkpoints. We find that tumor-infiltrating lymphocytes and TCR counts vary widely across cancer types and within each diagnosis, and notably are significantly predictive of survival in osteosarcoma patients. We identify potential cancer-specific immunotherapeutic targets for adoptive cell therapies including cell-surface proteins, tumor germline antigens, and lineage-specific transcription factors. Using an orthogonal immunopeptidomics approach, we find several potential immunotherapeutic targets in osteosarcoma and Ewing sarcoma and validated PRAME as a bona fide multi-pediatric cancer target. Importantly, this work provides a critical framework for immune targeting of extracranial solid tumors using parallel immuno-transcriptomic and -peptidomic approaches., Graphical Abstract, In brief Brohl et al. perform immunogenomics analysis of a cohort of 788 pediatric extracranial solid tumors and cell lines, representing 14 diagnoses, utilizing RNA sequencing. They broadly describe prognostically relevant immunophenotypes and provide proof of concept of a transcriptomics-informed adoptive cellular therapy approach, validating PRAME as a multi-pediatric cancer immunotherapy target.

BackgroundAngiogenic factors play a role in regulating immune suppression in the tumor microenvironment and driving resistance to immune checkpoint inhibitor therapy.1 Ziv-aflibercept is a soluble decoy receptor that ”traps” endogenous vascular endothelial growth factor (VEGF) with 100-fold increased binding affinity compared to Bevacizumab.2 The combination of ziv-aflibercept with either cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) or programmed cell death protein 1 (PD-1) blockade has shown promising antitumor efficacy in mouse models.3 4 We hypothesized that a novel combination of ziv-aflibercept and anti-PD-1 would be tolerable and lead to clinical benefits in tumors that traditionally do not respond to checkpoint blockade.MethodsThis is a multicenter phase 1B dose escalation study (NCT02298959) of the combination of ziv-aflibercept (at 2–4 mg/kg) plus pembrolizumab (at 2 mg/kg) administered intravenously every 2 weeks with expansion cohorts in PD-1/PD-L1 naïve melanoma, renal cell carcinoma (RCC), microsatellite stable colorectal cancer (MSS CRC), and ovarian cancer (figure 1). The primary objective was to determine the maximum tolerated dose (MTD) and recommended dose of the combination. Secondary endpoints included overall response rate (ORR) and overall survival (OS). Exploratory objectives included correlation of clinical efficacy and immune population densities in the tissue and periphery.ResultsOverall, 33 patients were enrolled during dose escalation (n=3) and dose expansion (n=30). No dose-limiting toxicities (DLTs) were reported in the initial dose level. Ziv-aflibercept 4 mg/kg plus pembrolizumab 2 mg/kg every 2 weeks was established as the MTD. Grade ≥3 treatment-related adverse events occurred in 19/33 patients (58%), the most common being hypertension (36%) and proteinuria (18%). ORR in the dose expansion cohort was 16.7% (5/30; 95% CI, 7–32%). Complete responses occurred in melanoma (n=2), partial responses occurred in RCC (n=1), mesothelioma (n=1), and melanoma (n=1). Efficacy outcomes by tumor type are shown in table 1 and figure 2. Median OS was as follows: melanoma, not reached; RCC, 15.7 months (90% CI, 2.5–15.7); CRC, 3.3 months (90% CI, 0.6–3.4), ovarian, 12.5 months (90% CI, 3.8–13.6), other solid tumors, not reached (figure 3). Activated tumor infiltrating CD8 T cells at baseline (CD8+PD1+), high CD40L expression (figure 4), and increased memory CD8 T cells in the periphery (figure 5) correlated with clinical response to the combination therapy.Abstract 374 Figure 1Study Schema of dose escalation and dose expansion. (A) Cohort 1 included the following tumors: clear cell sarcoma, triple negative breast cancer (TNBC), and mesothelioma. (B) Other solid tumors in Cohort 2 were: epithelioid mesothelioma (2) and TNBC (1).Abstract 374 Table 1Efficacy outcomes by dose level and tumor typea. Solid tumors included clear cell sarcoma (1), breast cancer (2), mesothelioma (3).b. Both patients with CR had melanoma and were in Cohort 2 (DL2).Abstract 374 Figure 2Waterfall plot of best RECIST response. Waterfall plot of maximum change from baseline in sum of target lesions for 28 patients with tumor measurements over time. Plot is color-coded by tumor type. Triangles indicate patients who developed new lesions, yellow circles indicate the 3 patients who received DL1.Abstract 374 Figure 3Progression and overall survival by tumor type. Kaplan-Meier curves for (A) progression-free survival and (B) overall survival based on tumor type.Abstract 374 Figure 4Luminex assay analysis of clinical response. Luminex analysis of baseline biomarkers. Patients were analyzed by clinical response (complete response [CR] and partial response [PR]) and durable clinical benefit (DCB) which includes patients with CR, PR, and stable disease (SD). (A) Baseline levels of IL-6 were lower in responders vs. non-responders (median 2.605 vs. 9.847 pg/mL, p = 0.009). Baseline CD40L was increased in responders (median 2,840 vs. 2,267 pg/mL, p = 0.06). (B) Baseline levels of GroB (median 1,272 vs. 592 pg/mL, p = 0.006), CXCL5 (median 547.5 vs. 296.2 pg/mL, p = 0.02), and CD40L (median 2,807 vs. 1,595 pg/mL, p = 0.001) were higher in patients with DCB vs. no DCB. P-values for baseline comparisons were obtained through Wilcoxon rank-sum test. The solid black line indicates median. Violins show range and kernel density estimate distributions of each group. (*) p < 0.05, (**) p < 0.01.Abstract 374 Figure 5Flow cytometry analysis. Flow cytometry analysis comparing T cell populations and monocytes between patients with clinical response (CR or PR, n = 5) and non-responders (n = 18) and patients with disease control (CR, PR, or SD, n = 12) and no disease control (n = 11). (A) CD4+ populations were increased in responders vs. non-responders at all time points. (B) CD8+ populations were decreased in responders vs. non-responders at all time points. (C) Treg CD4+/CD25+/FoxP3+ was decreased at baseline in responders. (D) Treg CD4+/CD25+/FoxP3+ were decreased at baseline and 1-month in patients with disease control vs. no disease control. (E) TCM CD8+/CD45RO+/CCR7+ was increased at all time points in responders vs. non-responders. (F) TEMRA CD8+/CD45RO-/CCR7- was decreased in responders at baseline. (G) Non-classical TIE2 was increased at baseline in non-responders. (H) Classical monocytes were increased at all time points in non-responders.ConclusionsThe combination of ziv-aflibercept and pembrolizumab demonstrated an acceptable safety profile with antitumor activity in solid tumors. The combination is currently being studied in sarcoma and anti-PD-1 resistant melanoma.AcknowledgementsThis trial was supported by the National Cancer Institute (NCI) and by Merck, Sharpe, and Dohme and Sanofi via Cooperative Research and Development Agreements with the NCI. PLB was supported by NCI UM1 Grant CA186644.Trial RegistrationNCT02298959ReferencesRahma OE, Hodi FS. The intersection between tumor angiogenesis and immune suppression. Clin Cancer Res September 15 2019;25(18):5449–5457. doi:10.1158/1078-0432.CCR-18-1543Holash J, Davis S, Papadopoulos N, et al. VEGF-trap: a VEGF blocker with potent antitumor effects. Proceedings of the National Academy of Sciences 2002;99(17):11393–11398. doi:10.1073/pnas.172398299Burova E, Ioffe E, Taduriyasas C, et al. Abstract 5035: blockade of VEGF with ziv-aflibercept (VEGF Trap) enhances anti-tumor efficacy of CTLA-4 blocking antibody in an Fc dependent manner. Cancer Research 2014;74(19 Supplement):5035–5035. doi:10.1158/1538-7445.am2014-5035Di Tacchio M, Macas J, Weissenberger J, et al. Tumor vessel normalization, immunostimulatory reprogramming, and improved survival in glioblastoma with combined inhibition of PD-1, angiopoietin-2, and VEGF. Cancer Immunology Research 2019;7(12):1910–1927. doi:10.1158/2326-6066.cir-18-0865Ethics ApprovalThis trial (NCT02298959) was approved by all participating IRBs.

BackgroundAvelumab (anti-programmed death ligand 1 (PD-L1)) is approved in multiple countries for the treatment of metastatic Merkel cell carcinoma (mMCC), a rare and aggressive skin cancer. We report efficacy and safety data and exploratory biomarker analyses from a cohort of patients with mMCC treated with first-line avelumab in a phase II trial.MethodsPatients with treatment-naive mMCC received avelumab 10 mg/kg intravenously every 2 weeks. The primary endpoint was durable response, defined as objective response (complete or partial response; assessed by independent review) lasting ≥6 months. Additional assessments included progression-free survival (PFS), overall survival (OS), safety, and biomarker analyses.ResultsIn 116 patients treated with avelumab, median follow-up was 21.2 months (range: 14.9–36.6). Thirty-five patients had a response lasting ≥6 months, giving a durable response rate of 30.2% (95% CI: 22.0% to 39.4%). The objective response rate was 39.7% (95% CI: 30.7% to 49.2%). Median PFS was 4.1 months (95% CI: 1.4 to 6.1) and median OS was 20.3 months (95% CI: 12.4 to not estimable). Response rates were numerically higher in patients with PD-L1+ tumors, Merkel cell polyomavirus (MCPyV)-negative tumors, and tumors with increased intratumoral CD8+ T-cell density. Exploratory analyses did not identify a biomarker that could reliably predict a response to first-line treatment with avelumab; however, a novel gene expression signature to identify the presence of MCPyV+ tumors was derived. Treatment-related adverse events (any grade) occurred in 94 (81.0%) patients, including grade 3/4 events in 21 (18.1%) patients; no treatment-related deaths occurred.ConclusionIn patients with mMCC, first-line treatment with avelumab led to responses in 40% and durable responses in 30%, and was associated with a low rate of grade 3/4 treatment-related adverse events.

Metastasis is a pivotal event that changes the course of cancers from benign and treatable to malignant and difficult to treat, resulting in the demise of patients. Understanding the genetic control of metastasis is thus crucial to develop efficient and sustainable targeted therapies. Here we discuss the alterations in epigenetic mechanisms, transcription, chromosomal instability, chromosome imprinting, non-coding RNAs, coding RNAs, mutant RNAs, enhancers, G-quadruplexes, and copy number variation to dissect the genetic control of metastasis. We conclude that the genetic control of metastasis is predominantly executed through epithelial to mesenchymal transition and evasion of cell death. We discuss how genetic regulatory mechanisms can be harnessed for therapeutic purposes to achieve sustainable control over cancer metastasis.

Purpose: Merkel cell carcinoma (MCC) is a rare, aggressive cutaneous malignancy, which has demonstrated sensitivity to immune checkpoint inhibitor therapy. Here, we perform the largest genomics study in MCC to date to characterize the molecular landscape and evaluate for clinical and molecular correlates to immune checkpoint inhibitor response. Experimental Design: Comprehensive molecular profiling was performed on 317 tumors from patients with MCC, including the evaluation of oncogenic mutations, tumor mutational burden (TMB), mutational signatures, and the Merkel cell polyomavirus (MCPyV). For a subset of 57 patients, a retrospective analysis was conducted to evaluate for clinical and molecular correlates to immune checkpoint inhibitor response and disease survival. Results: Genomic analyses revealed a bimodal distribution in TMB, with 2 molecularly distinct subgroups. Ninety-four percent (n = 110) of TMB-high specimens exhibited an ultraviolet light (UV) mutational signature. MCPyV genomic DNA sequences were not identified in any TMB-high cases (0/117), but were in 63% (110/175) of TMB-low cases. For 36 evaluable patients treated with checkpoint inhibitors, the overall response rate was 44% and response correlated with survival at time of review (100% vs. 20%, P < 0.001). Response rate was 50% in TMB-high/UV-driven and 41% in TMB-low/MCPyV-positive tumors (P = 0.63). Response rate was significantly correlated with line of therapy: 75% in first-line, 39% in second-line, and 18% in third-line or beyond (P = 0.0066). PD-1, but not PD-L1, expression was associated with immunotherapy response (77% vs. 21%, P = 0.00598, for PD-1 positive and negative, respectively). Conclusions: We provide a comprehensive genomic landscape of MCC and demonstrate clinicogenomic associates of immunotherapy response.

Background Response rates are primary endpoints in many oncology trials; however, correlation with overall survival (OS) is not uniform across cancer types, treatments, or lines of therapy. This study explored the association between objective response (OR) and OS in patients with chemotherapy-refractory metastatic Merkel cell carcinoma who received avelumab (anti-PD-L1). Methods Eighty-eight patients enrolled in JAVELIN Merkel 200 (part A; NCT02155647) received i.v. avelumab 10 mg/kg every 2 weeks until confirmed progression, unacceptable toxicity, or withdrawal. Using conditional landmark analyses, we compared OS in patients with and without confirmed OR (RECIST v1.1). We applied a Cox model that included OR as a time-varying covariate and adjusted for age, visceral disease, and number of previous therapies. Results Twenty-nine patients had confirmed OR; 20 by study week 7 and 7 more between study weeks 7 and 13. Survival probabilities 18 months after treatment initiation were 90% [95% confidence interval (CI) 65.6–97.4] in patients with OR at week 7 and 26.2% (95% CI 15.7–37.8) in patients without OR but who were alive at week 7. Median OS was not reached in patients with OR and was 8.8 months (95% CI 6.4–12.9) in patients without. Similar results were observed for the week 13 landmark. The adjusted Cox model showed OR was associated with a 95% risk reduction of death [hazard ratio 0.052 (95% CI 0.018–0.152)] compared with a nonresponse. Conclusions Patients with OR by 7 or 13 weeks had significantly longer OS than patients without, confirming that early OR is an endpoint of major importance.

A subset of hepatocellular carcinoma (HCC) overexpresses the chromosome 19 miRNA cluster (C19MC) and is associated with an undifferentiated phenotype marked by overexpression of cancer testis antigens (CTAs) including anti-apoptotic melanoma-A antigens (MAGEAs). However, the regulation of C19MC miRNA and MAGEA expression in HCCs are not understood. Here we show that, C19MC overexpression is tightly linked to a sub-set of HCCs with transcription-incompetent p53. Using next-generation and Sanger sequencing we found that, p53 in Hep3B cells is impaired by TP53-FXR2 fusion, and that overexpression of the C19MC miRNA-520G in Hep3B cells promotes the expression of MAGEA-3, 6 and 12 mRNAs. Furthermore, overexpression of p53-R175H and p53-R273H mutants promote miR-520G and MAGEA RNA expression and cellular transformation. Moreover, IFN-γ co-operates with miR-520G to promote MAGEA expression. On the other hand, metals such as nickel and zinc promote miR-526B but not miR-520G, to result in the suppression of MAGEA mRNA expression, and evoke cell death through mitochondrial membrane depolarization. Therefore our study demonstrates that a MAGEA-promoting network involving miR-520G, p53-defects and IFN-γ that govern cellular transformation and cell survival pathways, but MAGEA expression and survival are counteracted by nickel and zinc combination.

Angiosarcoma is a rare sarcoma subtype with a poor outcome. Carotuximab plus pazopanib produced a median progression-free survival (PFS) of 7.8 months in pazopanib-naive patients with chemotherapy-refractory angiosarcoma in a phase 1/2 trial.To determine whether carotuximab plus pazopanib improves PFS compared with pazopanib alone in patients with advanced angiosarcoma.The TAPPAS Trial: An Adaptive Enrichment Phase 3 Trial of TRC105 and Pazopanib vs Pazopanib Alone in Patients With Advanced Angiosarcoma was a multinational, multicenter, open-label, parallel-group, phase 3 randomized clinical trial of 123 patients 18 years or older with advanced angiosarcoma that was conducted between February 16, 2017, and April 12, 2019, at 31 sites in the US and the European Union. Patients were randomized 1:1 to receive pazopanib alone or carotuximab plus pazopanib. The trial incorporated an adaptive enrichment design. Inclusion criteria were no more than 2 prior lines of systemic therapy and an Eastern Cooperative Oncology Group performance status of 0 or 1. The efficacy analysis used the intent-to-treat population; the safety analysis included all patients who received a dose of either study drug.Oral pazopanib, 800 mg/d, or intravenous carotuximab, 10 mg/kg, administered weekly, plus oral pazopanib, 800 mg/d, with dose modification allowed per patient tolerance or until disease progression.The primary end point was PFS, assessed by blinded independent radiographic and cutaneous photographic review per Response Evaluation Criteria in Solid Tumors (RECIST) guidelines, version 1.1. Secondary end points included the objective response rate and overall survival. An interim analysis to determine the final sample size was conducted after enrollment of 123 patients. PFS in the group receiving pazopanib alone was compared with PFS in the group receiving carotuximab plus pazopanib using the log rank test.Of 114 patients with evaluable data (53 in the pazopanib arm and 61 in the carotuximab plus pazopanib arm), 69 (61%) were female and the median age was 68 years (range, 24-82 years); 57 (50%) had cutaneous disease and 32 (28%) had had no prior treatment. The primary end point (PFS) was not reached (hazard ratio [HR], 0.98; 95% CI, 0.52-1.84; P = .95), with a median of 4.3 months (95% CI, 2.9 months to not reached) for pazopanib and 4.2 months (95% CI, 2.8-8.3 months) for the combination arm. The most common all-grade adverse events in the single-agent pazopanib arm vs the combination arm were fatigue (29 patients [55%] vs 37 [61%]), headache (12 patients [23%] vs 39 [64%]), diarrhea (27 patients [51%] vs 35 [57%]), nausea (26 patients [49%] vs 29 [48%]), vomiting (12 patients [23%] vs 23 [38%]), anemia (5 patients [9%] vs 27 [44%]), epistaxis (2 patients [4%] vs 34 [56%]), and hypertension (29 patients [55%] vs 22 [36%]).In this phase 3 randomized clinical trial, carotuximab plus pazopanib did not improve PFS compared with pazopanib alone in patients with advanced angiosarcoma.ClinicalTrials.gov Identifier: NCT02979899.

Background NKTR-262 is a small-molecule agonist of toll-like receptors (TLR) 7/8. Given by intratumoral (IT) injection, NKTR-262 is retained within the tumor microenvironment (TME) and promotes an immunostimulatory milieu and tumor antigen release. Bempegaldesleukin (BEMPEG) is a CD122-preferential IL-2 pathway agonist, which increases proliferation and tumor infiltration of CD8+ T cells and natural killer (NK) cells. Preclinically, NKTR-262 plus BEMPEG combined innate immune signaling and enhanced antigen presentation, with sustained T-cell activation, resulting in tumor growth inhibition of treated and abscopal lesions. Methods This phase 1 dose-escalation study enrolled patients with relapsed/refractory, advanced/metastatic solid tumors (REVEAL; NCT03435640). Patients received escalating doses of NKTR-262 (0.03 mg to 3.84 mg IT) followed 3 weeks‘ later by BEMPEG (0.006 mg/kg IV) q3wk utilizing a 3+3 design. The primary endpoint was safety and tolerability, including definition of the recommended phase 2 dose (RP2D). Other endpoints included antitumor activity, pharmacodynamics, and pharmacokinetics. Results As of June 15, 2020, 36 patients were enrolled. One dose-limiting toxicity, transient transaminase elevation, was observed at the highest NKTR-262 dose (3.84 mg). The most frequent treatment-related adverse events were flu-like symptoms, fatigue, nausea, and pruritus, consistent with the known profile of BEMPEG. Early evidence of clinical activity was observed in patients with metastatic melanoma, with a disease control rate (partial response [PR] + stable disease) of 41.2% (7/17 patients), including two patients with PRs after progression on two prior immunotherapy regimens. Preliminary analyses showed dose-dependent induction of CXCL10 and type 1 interferon genes, consistent with TLR7/8 engagement. CD11c+ target cells were significantly more abundant in baseline melanoma biopsies than other tumor types (p Conclusions NKTR-262 plus BEMPEG led to engagement of the entire immune activation cascade required for systemic tumor clearance. Robust TLR7/8 engagement supported the NKTR-262 mechanism of action, while the minimal toxicity profile underscored the benefit of local delivery of NKTR-262, and the BEMPEG combination induced systemic activation of T and NK cells. These data support the RP2D of NKTR-262 (3.84 mg IT) plus BEMPEG (0.006 mg IV) q3w, and the initiation of the phase 1b dose-expansion phase, which is exploring concurrent dosing, with or without nivolumab, in relapsed/refractory metastatic melanoma patients. Trial Registration NCT03435640 Ethics Approval The study was approved by the institutional review board of each participating site.