Pancreatic Cyst Size Measurement on Magnetic Resonance Imaging Compared to Pathology.

BACKGROUND: While multiple cyst features are evaluated for stratifying pancreatic intraductal papillary mucinous neoplasms (IPMN), cyst size is an important factor that can influence treatment strategies. When magnetic resonance imaging (MRI) is used to evaluate IPMNs, no universally accepted sequence provides optimal size measurements. T2-weighted coronal/axial have been suggested as primary measurement sequences; however, it remains unknown how well these and maximum all-sequence diameter measurements correlate with pathology size. This study aims to compare agreement and bias between IPMN long-axis measurements on seven commonly obtained MRI sequences with pathologic size measurements. METHODS: This retrospective cohort included surgically resected IPMN cases with preoperative MRI exams. Long-axis diameter tumor measurements and the presence of worrisome features and/orhigh-risk stigmata were noted on all seven MRI sequences. MRI size and pathology agreement and MRI inter-observer agreement involved concordance correlation coefficient (CCC) and intraclass correlation coefficient (ICC), respectively. The presence of worrisome features and high-risk stigmata were compared to the tumor grade using kappa analysis. The Bland-Altman analysis assessed the systematic bias between MRI-size and pathology. RESULTS: In 52 patients (age 68 ± 13 years, 22 males), MRI sequences produced mean long-axis tumor measurements from 2.45-2.65 cm. The maximum MRI lesion size had a strong agreement with pathology (CCC = 0.82 (95% CI: 0.71-0.89)). The maximum IPMN size was typically observed on the axial T1 arterial post-contrast and MRCP coronal series and overestimated size versus pathology with bias +0.34 cm. The radiologist interobserver agreement reached ICCs 0.74 to 0.91 on the MRI sequences. CONCLUSION: The maximum MRI IPMN size strongly correlated with but tended to overestimate the length compared to the pathology, potentially related to formalin tissue shrinkage during tissue processing.

Combined BRAF, MEK, and heat-shock protein 90 inhibition in advanced BRAF V600-mutant melanoma.

BACKGROUND: Resistance to BRAF and MEK inhibitors in BRAF V600-mutant melanoma is common. Multiple resistance mechanisms involve heat-shock protein 90 (HSP90) clients, and a phase 1 study of vemurafenib with the HSP90 inhibitor XL888 in patients with advanced melanoma showed activity equivalent to that of BRAF and MEK inhibitors. METHODS: Vemurafenib (960 mg orally twice daily) and cobimetinib (60 mg orally once daily for 21 of 28 days) with escalating dose cohorts of XL888 (30, 45, 60, or 90 mg orally twice weekly) was investigated in a phase 1 trial of advanced melanoma, with a modified Ji dose-escalation design. RESULTS: Twenty-five patients were enrolled. After two dose-limiting toxicities (DLTs) (rash and acute kidney injury) in the first cohort, lower doses of vemurafenib (720 mg) and cobimetinib (40 mg) were investigated with the same XL888 doses. Three DLTs (rash) were observed in 12 patients in the XL888 60-mg cohort, and this was determined as the maximum tolerated dose. Objective responses were observed in 19 patients (76%), and the median progression-free survival was 7.6 months, with a 5-year progression-free survival rate of 20%. The median overall survival was 41.7 months, with a 5-year overall survival rate of 37%. Single-cell RNA sequencing was performed on baseline and on-treatment biopsies; treatment was associated with increased immune cell influx (CD4-positive and CD8-positive T cells) and decreased melanoma cells. CONCLUSIONS: Combined vemurafenib and cobimetinib plus XL888 had significant toxicity, requiring frequent dose reductions, which may have contributed to the relatively low progression-free survival despite a high tumor response rate. Given overlapping toxicities, caution must be used when combining HSP90 inhibitors with BRAF and MEK inhibitors.

Differential requirements for CD4+ T cells in the efficacy of the anti-PD-1+LAG-3 and anti-PD-1+CTLA-4 combinations in melanoma flank and brain metastasis models.

BACKGROUND: Although the anti-PD-1+LAG-3 and the anti-PD-1+CTLA-4 combinations are effective in advanced melanoma, it remains unclear whether their mechanisms of action overlap. METHODS: We used single cell (sc) RNA-seq, flow cytometry and IHC analysis of responding SM1, D4M-UV2 and B16 melanoma flank tumors and SM1 brain metastases to explore the mechanism of action of the anti-PD-1+LAG-3 and the anti-PD-1+CTLA-4 combination. CD4+ and CD8+ T cell depletion, tetramer binding assays and ELISPOT assays were used to demonstrate the unique role of CD4+T cell help in the antitumor effects of the anti-PD-1+LAG-3 combination. RESULTS: The anti-PD-1+CTLA-4 combination was associated with the infiltration of FOXP3+regulatory CD4+ cells (Tregs), fewer activated CD4+T cells and the accumulation of a subset of IFNγ secreting cytotoxic CD8+T cells, whereas the anti-PD-1+LAG-3 combination led to the accumulation of CD4+T helper cells that expressed CXCR4, TNFSF8, IL21R and a subset of CD8+T cells with reduced expression of cytotoxic markers. T cell depletion studies showed a requirement for CD4+T cells for the anti-PD-1+LAG-3 combination, but not the PD-1-CTLA-4 combination at both flank and brain tumor sites. In anti-PD-1+LAG-3 treated tumors, CD4+T cell depletion was associated with fewer activated (CD69+) CD8+T cells and impaired IFNγ release but, conversely, increased numbers of activated CD8+T cells and IFNγ release in anti-PD-1+CTLA-4 treated tumors. CONCLUSIONS: Together these studies suggest that these two clinically relevant immune checkpoint inhibitor (ICI) combinations have differential effects on CD4+T cell polarization, which in turn, impacted cytotoxic CD8+T cell function. Further insights into the mechanisms of action/resistance of these clinically-relevant ICI combinations will allow therapy to be further personalized.

HDAC8-mediated inhibition of EP300 drives a transcriptional state that increases melanoma brain metastasis.

Melanomas can adopt multiple transcriptional states. Little is known about the epigenetic drivers of these cell states, limiting our ability to regulate melanoma heterogeneity. Here, we identify stress-induced HDAC8 activity as driving melanoma brain metastasis development. Exposure of melanocytes and melanoma cells to multiple stresses increases HDAC8 activation leading to a neural crest-stem cell transcriptional state and an amoeboid, invasive phenotype that increases seeding to the brain. Using ATAC-Seq and ChIP-Seq we show that increased HDAC8 activity alters chromatin structure by increasing H3K27ac and enhancing accessibility at c-Jun binding sites. Functionally, HDAC8 deacetylates the histone acetyltransferase EP300, causing its enzymatic inactivation. This, in turn, increases binding of EP300 to Jun-transcriptional sites and decreases binding to MITF-transcriptional sites. Inhibition of EP300 increases melanoma cell invasion, resistance to stress and increases melanoma brain metastasis development. HDAC8 is identified as a mediator of transcriptional co-factor inactivation and chromatin accessibility that drives brain metastasis.

The Polyamine-Hypusine Circuit Controls an Oncogenic Translational Program Essential for Malignant Conversion in MYC-Driven Lymphoma.

The MYC oncoprotein is activated in a broad spectrum of human malignancies and transcriptionally reprograms the genome to drive cancer cell growth. Given this, it is unclear if targeting a single effector of MYC will have therapeutic benefit. MYC activates the polyamine-hypusine circuit, which posttranslationally modifies the eukaryotic translation factor eIF5A. The roles of this circuit in cancer are unclear. Here we report essential intrinsic roles for hypusinated eIF5A in the development and maintenance of MYC-driven lymphoma, where the loss of eIF5A hypusination abolishes malignant transformation of MYC-overexpressing B cells. Mechanistically, integrating RNA sequencing, ribosome sequencing, and proteomic analyses revealed that efficient translation of select targets is dependent upon eIF5A hypusination, including regulators of G1-S phase cell-cycle progression and DNA replication. This circuit thus controls MYC's proliferative response, and it is also activated across multiple malignancies. These findings suggest the hypusine circuit as a therapeutic target for several human tumor types. SIGNIFICANCE: Elevated EIF5A and the polyamine-hypusine circuit are manifest in many malignancies, including MYC-driven tumors, and eIF5A hypusination is necessary for MYC proliferative signaling. Not-ably, this circuit controls an oncogenic translational program essential for the development and maintenance of MYC-driven lymphoma, supporting this axis as a target for cancer prevention and treatment. See related commentary by Wilson and Klein, p. 248. This article is highlighted in the In This Issue feature, p. 247.

Quantifying Cell Heterogeneity and Subpopulations Using Single Cell Metabolomics.

Mass spectrometry (MS) has become an indispensable tool for metabolomics studies. However, due to the lack of applicable experimental platforms, suitable algorithm, software, and quantitative analyses of cell heterogeneity and subpopulations, investigating global metabolomics profiling at the single cell level remains challenging. We combined the Single-probe single cell MS (SCMS) experimental technique with a bioinformatics software package, SinCHet-MS (Single Cell Heterogeneity for Mass Spectrometry), to characterize changes of tumor heterogeneity, quantify cell subpopulations, and prioritize the metabolite biomarkers of each subpopulation. As proof of principle studies, two melanoma cancer cell lines, the primary (WM115; with a lower drug resistance) and the metastatic (WM266-4; with a higher drug resistance), were used as models. Our results indicate that after the treatment of the anticancer drug vemurafenib, a new subpopulation emerged in WM115 cells, while the proportion of the existing subpopulations was changed in the WM266-4 cells. In addition, metabolites for each subpopulation can be prioritized. Combining the SCMS experimental technique with a bioinformatics tool, our label-free approach can be applied to quantitatively study cell heterogeneity, prioritize markers for further investigation, and improve the understanding of cell metabolism in human diseases and response to therapy.

A Real-World Data Retrospective Cohort Study of HER2-positive, Early-Stage Breast Cancer in Patients 70 Years of Age or Older: Natural History, Treatment Patterns, and Outcomes.

PURPOSE: Early-stage human epidermal growth factor receptor 2-positive (HER2-positive) breast cancers (BCs) are routinely treated with intense perioperative chemotherapy combined with HER2-targeted agents. There is thus an unmet need for knowledge about treatment patterns and outcomes among patients 70 years of age or older, as this is an under-represented subset of patients in large clinical trials. METHODS: We used a deidentified cohort derived from a nationwide electronic health record database to conduct a retrospective cohort study of patients with HER2-positive BCs. Descriptive statistics were used to evaluate tumor characteristics, treatment patterns across age groups, and pathologic complete response rates. We used Kaplan-Meier survival curves to estimate recurrence-free and overall survivals; Cox proportional methods were used for adjustments with covariates of interest, including age as a categorical variable. RESULTS: We included 395 patients with HER2-positive stage I to III BCs who were 70 years of age or older. Most patients had tumors with high nuclear-grade T2 tumors, and received surgical treatment first. Most patients (61.7%) who received HER2 therapies underwent treatment in the adjuvant setting; paclitaxel and trastuzumab combination was the most commonly used adjuvant regimen. Older age was associated with increased hazard of recurrence or death. We did not detect significant evidence of decline in performance status, but there was modest weight drop after perioperative HER2 treatments. CONCLUSION: Findings suggest that patients in this older-age cohort were treated with de-escalated perioperative strategies and had poorer outcomes; our findings should be validated in future studies.

Dose-Limiting Pulmonary Toxicity in a Phase 1/2 Study of Radiation and Chemotherapy with Ipilimumab Followed by Nivolumab for Patients With Stage 3 Unresectable Non-Small Cell Lung Cancer.

PURPOSE: We hypothesized that concurrent ipilimumab with chemoradiationtherapy (chemoRT) followed by maintenance nivolumab would be safe for patients with unresectable stage III non-small cell lung cancer (NSCLC). We aimed to assess the safety (phase 1) and the 12-month progression-free survival (PFS) (phase 2) in a multi-institution prospective trial. METHODS AND MATERIALS: Eligible patients had unresectable stage III NSCLC. The treatment included platinum doublet chemotherapy with concurrent thoracic radiation therapy to 60 Gy in 30 fractions and ipilimumab (1 mg/kg) delivered during weeks 1 and 4. After chemoRT, maintenance nivolumab (480 mg) was given every 4 weeks for up to 12 cycles. Adverse events (AEs) were assessed according to the Common Terminology Criteria for Adverse Events, version 5.0. Survival analyses were performed with Kaplan Meier (KM) methods and log-rank tests. RESULTS: The trial was discontinued early after enrolling 19 patients without proceeding to the phase 2 component because of unacceptable toxicity. Sixteen patients (84%) had grade ≥3 (G3+) possible treatment-related toxicity, most commonly pulmonary AEs (n = 8, 42%). Fourteen patients (74%) discontinued study therapy early because of AEs (n = 12, 63%) or patient choice (n = 2, 11%). Eleven patients (58%) experienced G2+ pulmonary toxicity with median time to onset 4.1 months (95% CI 2.6-not reached [NR]), and 12-month freedom from G2+ pulmonary toxicity 37% (95% CI, 16-59). Five patients had G5 AEs, including 3 with G5 pulmonary AEs (1 respiratory failure with pneumonitis and pulmonary embolism, 1 pneumonia/chronic obstructive pulmonary disease exacerbation, 1 pulmonary fibrosis). Despite toxicities, the median PFS was 19.2 months (95% CI 6.1-NR) and the median overall survival was NR (95% CI 6.1-NR) with median follow-up of 30.1 months by the reverse KM method. CONCLUSIONS: Concurrent ipilimumab with chemoRT for unresectable stage III NSCLC is associated with pulmonary toxicity that may limit opportunities for improved outcomes. Future studies aiming to incorporate ipilimumab or other anti-CTLA4 therapies into management of unresectable stage III NSCLC should consider careful measures to minimize toxicity risk.

Ethnic and racial-specific differences in levels of centrosome-associated mitotic kinases, proliferative and epithelial-to-mesenchymal markers in breast cancers.

Molecular epidemiology evidence indicates racial and ethnic differences in the aggressiveness and survival of breast cancer. Hispanics/Latinas (H/Ls) and non-Hispanic Black women (NHB) are at higher risk of breast cancer (BC)-related death relative to non-Hispanic white (NHW) women in part because they are diagnosed with hormone receptor-negative (HR) subtype and at higher stages. Since the cell cycle is one of the most commonly deregulated cellular processes in cancer, we propose that the mitotic kinases TTK (or Mps1), TBK1, and Nek2 could be novel targets to prevent breast cancer progression among NHBs and H/Ls. In this study, we calculated levels of TTK, p-TBK1, epithelial (E-cadherin), mesenchymal (Vimentin), and proliferation (Ki67) markers through immunohistochemical (IHC) staining of breast cancer tissue microarrays (TMAs) that includes samples from 6 regions in the Southeast of the United States and Puerto Rico -regions enriched with NHB and H/L breast cancer patients. IHC analysis showed that TTK, Ki67, and Vimentin were significantly expressed in triple-negative (TNBC) tumors relative to other subtypes, while E-cadherin showed decreased expression. TTK correlated with all of the clinical variables but p-TBK1 did not correlate with any of them. TCGA analysis revealed that the mRNA levels of multiple mitotic kinases, including TTK, Nek2, Plk1, Bub1, and Aurora kinases A and B, and transcription factors that are known to control the expression of these kinases (e.g. FoxM1 and E2F1-3) were upregulated in NHBs versus NHWs and correlated with higher aneuploidy indexes in NHB, suggesting that these mitotic kinases may be future novel targets for breast cancer treatment in NHB women.

Drepmel-A Multi-Omics Melanoma Drug Repurposing Resource for Prioritizing Drug Combinations and Understanding Tumor Microenvironment.

Although substantial progress has been made in treating patients with advanced melanoma with targeted and immuno-therapies, de novo and acquired resistance is commonplace. After treatment failure, therapeutic options are very limited and novel strategies are urgently needed. Combination therapies are often more effective than single agents and are now widely used in clinical practice. Thus, there is a strong need for a comprehensive computational resource to define rational combination therapies. We developed a Shiny app, DRepMel to provide rational combination treatment predictions for melanoma patients from seventy-three thousand combinations based on a multi-omics drug repurposing computational approach using whole exome sequencing and RNA-seq data in bulk samples from two independent patient cohorts. DRepMel provides robust predictions as a resource and also identifies potential treatment effects on the tumor microenvironment (TME) using single-cell RNA-seq data from melanoma patients. Availability: DRepMel is accessible online.

A Real-World Data Retrospective Cohort Study of Low Estrogen Receptor-Positive Early Breast Cancer: Natural History and Treatment Outcomes.

Purpose: Estrogen receptor-positive (ER+) breast cancer (BC) is a heterogeneous disease, and there is an ongoing debate regarding the optimal cut point for clinically relevant ER expression. We used a real-world database to assess the prognostic and predictive values of lower ER expression levels on treatment outcomes with endocrine therapy. Methods: We used a nationwide electronic health record database. Descriptive statistics were used to evaluate the association between ER expression, tumor characteristics, and treatment patterns among patients with early-stage BC. We used Kaplan-Meier survival curves to estimate recurrence-free survival (RFS) and overall survival (OS). We assessed associations between an alternative ER expression-level cut point and clinical outcomes. Results: Among 4697 patients with early-stage HER2-negative BC, 83 (2.04%) had ER+-low BC (ER expression, 1-9.99%) and 36 (0.88%) had ER+-intermediate BC (10-19.9%). ER+-low tumors were associated with higher tumor grade, larger size, and higher axillary tumor burden than ER+-high tumors (≥20% ER expression). African Americans had a higher prevalence of both triple-negative BC (TNBC) and ER+-low BC than ER+-high BC. Patients with ER+-low and ER+-intermediate tumors had survival outcomes similar to patients with TNBC and worse survival outcomes than patients with ER+-high tumors (P < 0.001). Tumors with <20% ER expression were associated with worse outcomes. Conclusion: In our cohort, patients with BCs with ER expression levels <20% had poor clinical outcomes similar to those of patients with TNBC.

Adding Cyclooxygenase Inhibitors to Immune Checkpoint Inhibitors Did Not Improve Outcomes in Metastatic Renal Cell Carcinoma.

Modulating the cyclooxygenase 2 (COX-2) pathway has improved responses to immune checkpoint inhibitors (ICIs) in certain solid tumors, such as melanoma. Little is known about COX-2 inhibition in response to ICIs in metastatic renal cell carcinoma (mRCC). In this retrospective cohort study, we examined the effect of COX-2 inhibitors on the long-term outcomes of mRCC patients undergoing ICI therapies. Among 211 patients with mRCC, 23 patients were excluded due to loss to follow-up. Among 188 included patients, 120 patients received either an NSAID or aspirin for at least three weeks during ICI therapies. Clear cell histology was present in 96% of cases. The median overall survival (OS) was similar regardless of the COX inhibitor (COXi) (i.e., NSAID or aspirin) use (27 months for COXi vs. 33 months for no-COXi groups; p = 0.73). The no-COXi group showed a trend toward longer median progression-free survival (8 months for COXi vs. 13 months for no-COXi groups; p = 0.13). When looking specifically at NSAID use in a multivariate analysis, NSAID use was associated with a higher risk of progression (HR = 1.52 [95% CI, 1.04-2.22]) and death (HR = 1.60 [95% CI, 1.02-2.52]). In summary, COXis did not improve disease control or survival among patients with mRCC who were undergoing ICI therapies. Instead, the concurrent use of NSAIDs was associated with worse outcomes. Larger studies are needed to validate our observation.

T cell repertoire in peripheral blood as a potential biomarker for predicting response to concurrent cetuximab and nivolumab in head and neck squamous cell carcinoma.

BACKGROUND: T cell receptor (TCR) signaling profile is a fundamental property that underpins both adaptive and innate immunity in the host. Despite its potential clinical relevance, the TCR repertoire in peripheral blood has not been thoroughly explored for its value as an immunotherapy efficacy biomarker in head and neck squamous cell carcinoma (HNSCC). The purpose of the present study is to characterize and compare the TCR repertoire in peripheral blood mononuclear cells (PBMC) from patients with HNSCC treated with the combination of cetuximab and nivolumab. METHODS: We used the immunoSEQ assay to sequence the TCR beta (TCR-B) chain repertoire from serially obtained PBMC at baseline and during the treatments from a total of 41 patients who received the combination (NCT03370276). Key TCR repertoire metrics, including diversity and clonality, were calculated and compared between patients with different therapy responses and clinical characteristics (eg, human papillomavirus (HPV) status and smoking history). Patient survival outcomes were compared according to patient groups stratified by the TCR-B clonotyping. To confirm the observed patterns in TCR spectrum, samples from patients who achieved complete response (CR) and partial response (PR) were further profiled with the immunoSEQ deep resolution assay. RESULTS: Our data indicated that the patients who achieved CR and PR had an increased TCR sequence diversity in their baseline samples, this tendency being more pronounced in HPV-negative patients or those with a smoking history. Notably, the CR/PR group had the lowest proportion of patients with oligoclonal TCR clones (2 out of 8 patients), followed by the stable disease group (9 out of 20 patients) and lastly the progressive disease group (7 out of 10 patients). An overall trend toward favorable patient survival was also observed in the polyclonal group. Finally, we reported the shared TCR clones across patients within the same response group, as well as the shared clones by aligning immunoSEQ reads with TCR data retrieved from The Cancer Genome Atlas- head and neck squamous cell carcinoma (TCGA-HNSC) cohort. CONCLUSIONS: Our data suggest that, despite the great clinical heterogeneity of HNSCC and the limited responders in the present cohort, the peripheral TCR repertoires from pretreatment PBMC may be developed as biomarkers for the benefit of immunotherapy in HNSCC.

A pilot study to troubleshoot quality control metrics when assessing circulating miRNA expression data reproducibility across study sites.

BACKGROUND: Given the growing interest in using microRNAs (miRNAs) as biomarkers of early disease, establishment of robust protocols and platforms for miRNA quantification in biological fluids is critical. OBJECTIVE: The goal of this multi-center pilot study was to evaluate the reproducibility of NanoString nCounter™ technology when analyzing the abundance of miRNAs in plasma and cystic fluid from patients with pancreatic lesions. METHODS: Using sample triplicates analyzed across three study sites, we assessed potential sources of variability (RNA isolation, sample processing/ligation, hybridization, and lot-to-lot variability) that may contribute to suboptimal reproducibility of miRNA abundance when using nCounter™, and evaluated expression of positive and negative controls, housekeeping genes, spike-in genes, and miRNAs. RESULTS: Positive controls showed a high correlation across samples from each site (median correlation coefficient, r> 0.9). Most negative control probes had expression levels below background. Housekeeping and spike-in genes each showed a similar distribution of expression and comparable pairwise correlation coefficients of replicate samples across sites. A total of 804 miRNAs showed a similar distribution of pairwise correlation coefficients between replicate samples (p= 0.93). After normalization and selecting miRNAs with expression levels above zero in 80% of samples, 55 miRNAs were identified; heatmap and principal component analysis revealed similar expression patterns and clustering in replicate samples. CONCLUSIONS: Findings from this pilot investigation suggest the nCounter platform can yield reproducible results across study sites. This study underscores the importance of implementing quality control procedures when designing multi-center evaluations of miRNA abundance.

Phase II Multi-institutional Clinical Trial Result of Concurrent Cetuximab and Nivolumab in Recurrent and/or Metastatic Head and Neck Squamous Cell Carcinoma.

PURPOSE: A phase II multi-institutional clinical trial was conducted to determine overall survival (OS) in patients with recurrent and/or metastatic (R/M) head and neck squamous cell carcinoma (HNSCC) treated with a combination of cetuximab and nivolumab. PATIENTS AND METHODS: Patients with R/M HNSCC were treated with cetuximab 500 mg/m2 i.v. on day 14 as a lead-in followed by cetuximab 500 mg/m2 i.v. and nivolumab 240 mg i.v. on day 1 and day 15 of each 28-day cycle. Expression of p16 and programmed cell death-ligand 1 (PD-L1) in archived tumors were determined. Tumor-tissue-modified human papillomavirus (TTMV) DNA was quantified in plasma. RESULTS: Ninety-five patients were enrolled, and 88 patients were evaluable for OS with a median follow-up of 15.9 months. Median OS in the 45 patients who had prior therapy for R/M HNSCC (cohort A) was 11.4 months, with a 1 year OS 50% [90% confidence interval (CI), 0.43-0.57]. Median OS in the 43 patients who had no prior therapy (cohort B) was 20.2 months, with a 1-year OS 66% (90% CI, 0.59-0.71). In the combined cohorts, the p16-negative immunostaining was associated with higher response rate (RR; P = 0.02) but did not impact survival while higher PD-L1 combined positive score was associated with higher RR (P = 0.03) and longer OS (log-rank P = 0.04). In the p16-positive patients, lower median (1,230 copies/mL) TTMV DNA counts were associated with higher RR (P = 0.01) and longer OS compared with higher median (log-rank P = 0.05). CONCLUSIONS: The combination of cetuximab and nivolumab is effective in patients with both previously treated and untreated R/M HNSCC and warrants further evaluation.

Single-cell Characterization of the Cellular Landscape of Acral Melanoma Identifies Novel Targets for Immunotherapy.

PURPOSE: Acral melanoma is a rare subtype of melanoma that arises on the non-hair-bearing skin of the palms, soles, and nail beds. In this study, we used single-cell RNA sequencing (scRNA-seq) to map the transcriptional landscape of acral melanoma and identify novel immunotherapeutic targets. EXPERIMENTAL DESIGN: We performed scRNA-seq on nine clinical specimens (five primary, four metastases) of acral melanoma. Detailed cell type curation was performed, the immune landscapes were mapped, and key results were validated by analysis of The Cancer Genome Atlas (TCGA) and single-cell datasets. Cell-cell interactions were inferred and compared with those in nonacral cutaneous melanoma. RESULTS: Multiple phenotypic subsets of T cells, natural killer (NK) cells, B cells, macrophages, and dendritic cells with varying levels of activation/exhaustion were identified. A comparison between primary and metastatic acral melanoma identified gene signatures associated with changes in immune responses and metabolism. Acral melanoma was characterized by a lower overall immune infiltrate, fewer effector CD8 T cells and NK cells, and a near-complete absence of γδ T cells compared with nonacral cutaneous melanomas. Immune cells associated with acral melanoma exhibited expression of multiple checkpoints including PD-1, LAG-3, CTLA-4, V-domain immunoglobin suppressor of T cell activation (VISTA), TIGIT, and the Adenosine A2A receptor (ADORA2). VISTA was expressed in 58.3% of myeloid cells and TIGIT was expressed in 22.3% of T/NK cells. CONCLUSIONS: Acral melanoma has a suppressed immune environment compared with that of cutaneous melanoma from nonacral skin. Expression of multiple, therapeutically tractable immune checkpoints were observed, offering new options for clinical translation.

Improving Multi-site Interaction Through Remote Learning Technology: Report from a Training Program to Increase Underrepresented Undergraduate and Medical Students in Health Disparities Research.

Since 2018, we have evaluated the effectiveness of various teaching technologies for training young investigators on translational research in cancer health disparities. The Southeast Partnership for Improving Research and Training in Cancer Health Disparities (SPIRIT-CHD) unites Moffitt Cancer Center and the Louisiana State University Health Sciences Center. One of the main components of the SPIRIT-CHD is the Cancer Research Education Program (CREP) for training undergraduate and medical students from underrepresented backgrounds. The CREP utilizes a web-based didactic curriculum to engage students at both institutions in biobanking, precision medicine, and cancer health disparities topics. We report experiences from our cross-institutional cancer education program, specifically evaluating the cohorts' satisfaction and learning gains using various communication technologies and instructional approaches. Trainees completed a survey with questions evaluating the curriculum and technology. Trainees reported satisfaction with the flipped classroom model (FCM) content and overall program (mean score = 3.2, SD = 0.79), and would recommend the program to peers. Yet, despite improved program delivery, trainees felt interaction between the two sites (mean score = 1.5, SD = 0.85) and engagement with faculty (mean score = 2.80, SD = 1.14) could be improved. The technology with the highest reported use was e-mail, with a mean score of 4.6 (SD = 0.52). LinkedIn and Twitter had the lowest frequency of use with mean scores at 1.90 (SD = 0.99) and 1.30 (SD = 1.34). Our study highlights the successes and challenges of remote learning using technology to increase interaction and engagement among trainees and faculty in a multi-site cancer research training program.

Comparison of Radiomic Features in a Diverse Cohort of Patients With Pancreatic Ductal Adenocarcinomas.

BACKGROUND: Significant racial disparities in pancreatic cancer incidence and mortality rates exist, with the highest rates in African Americans compared to Non-Hispanic Whites and Hispanic/Latinx populations. Computer-derived quantitative imaging or "radiomic" features may serve as non-invasive surrogates for underlying biological factors and heterogeneity that characterize pancreatic tumors from African Americans, yet studies are lacking in this area. The objective of this pilot study was to determine if the radiomic tumor profile extracted from pretreatment computed tomography (CT) images differs between African Americans, Non-Hispanic Whites, and Hispanic/Latinx with pancreatic cancer. METHODS: We evaluated a retrospective cohort of 71 pancreatic cancer cases (23 African American, 33 Non-Hispanic White, and 15 Hispanic/Latinx) who underwent pretreatment CT imaging at Moffitt Cancer Center and Research Institute. Whole lesion semi-automated segmentation was performed on each slice of the lesion on all pretreatment venous phase CT exams using Healthmyne Software (Healthmyne, Madison, WI, USA) to generate a volume of interest. To reduce feature dimensionality, 135 highly relevant non-texture and texture features were extracted from each segmented lesion and analyzed for each volume of interest. RESULTS: Thirty features were identified and significantly associated with race/ethnicity based on Kruskal-Wallis test. Ten of the radiomic features were highly associated with race/ethnicity independent of tumor grade, including sphericity, volumetric mean Hounsfield units (HU), minimum HU, coefficient of variation HU, four gray level texture features, and two wavelet texture features. A radiomic signature summarized by the first principal component partially differentiated African American from non-African American tumors (area underneath the curve = 0.80). Poorer survival among African Americans compared to Non-African Americans was observed for tumors with lower volumetric mean CT [HR: 3.90 (95% CI:1.19-12.78), p=0.024], lower GLCM Avg Column Mean [HR:4.75 (95% CI: 1.44,15.37), p=0.010], and higher GLCM Cluster Tendency [HR:3.36 (95% CI: 1.06-10.68), p=0.040], and associations persisted in volumetric mean CT and GLCM Avg Column after adjustment for key clinicopathologic factors. CONCLUSIONS: This pilot study identified several textural radiomics features associated with poor overall survival among African Americans with PDAC, independent of other prognostic factors such as grade. Our findings suggest that CT radiomic features may serve as surrogates for underlying biological factors and add value in predicting clinical outcomes when integrated with other parameters in ongoing and future studies of cancer health disparities.

Single-Cell Characterization of the Immune Microenvironment of Melanoma Brain and Leptomeningeal Metastases.

PURPOSE: Melanoma brain metastases (MBM) and leptomeningeal melanoma metastases (LMM) are two different manifestations of melanoma CNS metastasis. Here, we used single-cell RNA sequencing (scRNA-seq) to define the immune landscape of MBM, LMM, and melanoma skin metastases. EXPERIMENTAL DESIGN: scRNA-seq was undertaken on 43 patient specimens, including 8 skin metastases, 14 MBM, and 19 serial LMM specimens. Detailed cell type curation was performed, the immune landscapes were mapped, and key results were validated by IHC and flow cytometry. Association analyses were undertaken to identify immune cell subsets correlated with overall survival. RESULTS: The LMM microenvironment was characterized by an immune-suppressed T-cell landscape distinct from that of brain and skin metastases. An LMM patient with long-term survival demonstrated an immune repertoire distinct from that of poor survivors and more similar to normal cerebrospinal fluid (CSF). Upon response to PD-1 therapy, this extreme responder showed increased levels of T cells and dendritic cells in their CSF, whereas poor survivors showed little improvement in their T-cell responses. In MBM patients, therapy led to increased immune infiltrate, with similar T-cell transcriptional diversity noted between skin metastases and MBM. A correlation analysis across the entire immune landscape identified the presence of a rare population of dendritic cells (DC3) that was associated with increased overall survival and positively regulated the immune environment through modulation of activated T cells and MHC expression. CONCLUSIONS: Our study provides the first atlas of two distinct sites of melanoma CNS metastases and defines the immune cell landscape that underlies the biology of this devastating disease.

Concurrent Cetuximab and Nivolumab as a Second-Line or beyond Treatment of Patients with Recurrent and/or Metastatic Head and Neck Squamous Cell Carcinoma: Results of Phase I/II Study.

We hypothesized the combination of cetuximab and nivolumab would improve survival in recurrent and/or metastatic (R/M) HNSCC by providing synergy in cancer control and evaluated toxicities and efficacy of the combination. Effects of sequential administration of cetuximab and anti-Programmed Cell Death-1 checkpoint inhibitors (CPI) were also explored. Patients who failed at least one line of palliative treatment for incurable HNSCC were treated with cetuximab 500 mg/m2 IV on Day (D)-14 as a lead-in followed by cetuximab 500 mg/m2 IV and nivolumab 240 mg/m2 IV on D1 and D15 every 28-D cycle. Electronic health record-derived real-world data (RWD) were used to explore sequential treatment effects of CPI and cetuximab. A total of 45 evaluable patients were analyzed, and 31/45 (69%) patients had prior exposure to either CPI or cetuximab. The only grade 4 treatment-related adverse event was cetuximab infusion reaction in one patient. The 1-year progression-free survival (PFS) and overall survival (OS) rates were 19% and 44%, respectively. Although patients with no prior CPI (23/45, 51%) showed a trend for more favorable PFS relative to patients with prior CPI (22/45, 49%), the improvement in the 1-year OS did not reach the statistical threshold. For evaluation of sequential CPI and cetuximab treatment effects, we selected RWD-cetuximab cohort with 173 patients and RWD-CPI cohort with 658 patients from 6862 R/M HNSCC. Our result suggested patients treated with RWD-cetuximab after RWD-CPI had worse OS compared to no prior RWD-CPI (HR 1.81, 95% CI 1.02-3.16). Our data suggest the combination of cetuximab and nivolumab is well tolerated. Optimal sequencing of cetuximab and CPI may have an impact in prognosis and requires further evaluation.