Treatment resistance to melanoma therapeutics on a single cell level.

Therapy targeting the BRAF-MEK cascade created a treatment revolution for patients with BRAF mutant advanced melanoma. Unfortunately, 80% patients treated will progress by 5 years follow-up. Thus, it is imperative we study mechanisms of melanoma progression and therapeutic resistance. We created a scRNA (single cell RNA) atlas of 128,230 cells from 18 tumors across the treatment spectrum, discovering melanoma cells clustered strongly by transcriptome profiles of patients of origins. Our cell-level investigation revealed gains of 1q and 7q as likely early clonal events in metastatic melanomas. By comparing patient tumors and their derivative cell lines, we observed that PD1 responsive tumor fraction disappears when cells are propagated in vitro. We further established three anti-BRAF-MEK treatment resistant cell lines using three BRAF mutant tumors. ALDOA and PGK1 were found to be highly expressed in treatment resistant cell populations and metformin was effective in targeting the resistant cells. Our study suggests that the investigation of patient tumors and their derivative lines is essential for understanding disease progression, treatment response and resistance.

Functional analysis of recurrent CDC20 promoter variants in human melanoma.

Small nucleotide variants in non-coding regions of the genome can alter transcriptional regulation, leading to changes in gene expression which can activate oncogenic gene regulatory networks. Melanoma is heavily burdened by non-coding variants, representing over 99% of total genetic variation, including the well-characterized TERT promoter mutation. However, the compendium of regulatory non-coding variants is likely still functionally under-characterized. We developed a pipeline to identify hotspots, i.e. recurrently mutated regions, in melanoma containing putatively functional non-coding somatic variants that are located within predicted melanoma-specific regulatory regions. We identified hundreds of statistically significant hotspots, including the hotspot containing the TERT promoter variants, and focused on a hotspot in the promoter of CDC20. We found that variants in the promoter of CDC20, which putatively disrupt an ETS motif, lead to lower transcriptional activity in reporter assays. Using CRISPR/Cas9, we generated an indel in the CDC20 promoter in human A375 melanoma cell lines and observed decreased expression of CDC20, changes in migration capabilities, increased growth of xenografts, and an altered transcriptional state previously associated with a more proliferative and less migratory state. Overall, our analysis prioritized several recurrent functional non-coding variants that, through downregulation of CDC20, led to perturbation of key melanoma phenotypes.

Autologous humanized PDX modeling for immuno-oncology recapitulates features of the human tumor microenvironment.

BACKGROUND: Interactions between immune and tumor cells are critical to determining cancer progression and response. In addition, preclinical prediction of immune-related drug efficacy is limited by interspecies differences between human and mouse, as well as inter-person germline and somatic variation. To address these gaps, we developed an autologous system that models the tumor microenvironment (TME) from individual patients with solid tumors. METHOD: With patient-derived bone marrow hematopoietic stem and progenitor cells (HSPCs), we engrafted a patient's hematopoietic system in MISTRG6 mice, followed by transfer of patient-derived xenograft (PDX) tissue, providing a fully genetically matched model to recapitulate the individual's TME. We used this system to prospectively study tumor-immune interactions in patients with solid tumor. RESULTS: Autologous PDX mice generated innate and adaptive immune populations; these cells populated the TME; and tumors from autologously engrafted mice grew larger than tumors from non-engrafted littermate controls. Single-cell transcriptomics revealed a prominent vascular endothelial growth factor A (VEGFA) signature in TME myeloid cells, and inhibition of human VEGF-A abrogated enhanced growth. CONCLUSIONS: Humanization of the interleukin 6 locus in MISTRG6 mice enhances HSPC engraftment, making it feasible to model tumor-immune interactions in an autologous manner from a bedside bone marrow aspirate. The TME from these autologous tumors display hallmarks of the human TME including innate and adaptive immune activation and provide a platform for preclinical drug testing.

Breast Cancer in Jamaica: Stage, Grade and Molecular Subtype Distributions Across Age Blocks, the Implications for Screening and Treatment.

BACKGROUND: Breast cancer is the most commonly diagnosed and leading cause of cancer-related morbidity and mortality in females worldwide. Significant disparities exist in breast cancer incidence and mortalities between low- to middle- and high-income countries. The purpose of this study was to analyze the distribution of prognostic and predictive clinicopathological features of invasive breast cancer at a single institution in Jamaica across three age groups. METHODS: Data from patients diagnosed with invasive breast cancer who underwent definitive surgery between August 2017 and September 2018 were identified. The patients were divided into three age groups (< 50, 50 - 59 and > 59 years) and the distribution of tumor size, grade, molecular subtype, nodal status and anatomic stage were determined and compared with the US population registry. Comparisons of the various characteristics were performed using the Fisher's exact test. RESULTS: Ninety-nine definitive operations were performed and met the criteria for analysis. Average age at the time of diagnosis was 54 years compared to 62 years reported in the US databases. Thirty-six percent of the patients presented below age 50 years, which was twice the corresponding rate reported for Caucasian females (18%) in the USA. Fifty percent of patients in our registry had axillary lymph node metastases at presentation and they were younger than patients with negative axillary nodes (95% confidence interval (CI) -12.06 to -1.93, P = 0.007). Patients in the age group less than age 50 years were more likely to have advanced stage, high histological grade cancers compared to the older age blocks (95% CI 0.039 - 0.902, P = 0.033). CONCLUSION: Invasive breast cancer presents at an earlier age in Jamaican women and is associated with poor prognostic features such as high rates of axillary lymph node metastases, high histological grade, advanced stage, triple-negative subtypes and low luminal A subtypes.