Human TLR8 induces inflammatory bone marrow erythromyeloblastic islands and anemia in SLE-prone mice.

Anemia commonly occurs in systemic lupus erythematosus, a disease characterized by innate immune activation by nucleic acids. Overactivation of cytoplasmic sensors by self-DNA or RNA can cause erythroid cell death, while sparing other hematopoietic cell lineages. Whereas chronic inflammation is involved in this mechanism, less is known about the impact of systemic lupus erythematosus on the BM erythropoietic niche. We discovered that expression of the endosomal ssRNA sensor human TLR8 induces fatal anemia in Sle1.Yaa lupus mice. We observed that anemia was associated with a decrease in erythromyeloblastic islands and a block in differentiation at the CFU-E to proerythroblast transition in the BM. Single-cell RNAseq analyses of isolated BM erythromyeloblastic islands from human TLR8-expressing mice revealed that genes associated with essential central macrophage functions including adhesion and provision of nutrients were down-regulated. Although compensatory stress erythropoiesis occurred in the spleen, red blood cell half-life decreased because of hemophagocytosis. These data implicate the endosomal RNA sensor TLR8 as an additional innate receptor whose overactivation causes acquired failure of erythropoiesis via myeloid cell dysregulation.

Circulating tumour DNA in patients with advanced melanoma treated with dabrafenib or dabrafenib plus trametinib: a clinical validation study.

BACKGROUND: Melanoma lacks validated blood-based biomarkers for monitoring and predicting treatment efficacy. Cell-free circulating tumour DNA (ctDNA) is a promising biomarker; however, various detection methods have been used, and, to date, no large studies have examined the association between serial changes in ctDNA and survival after BRAF, MEK, or BRAF plus MEK inhibitor therapy. We aimed to evaluate whether baseline ctDNA concentrations and kinetics could predict survival outcomes. METHODS: In this clinical validation study, we used analytically validated droplet digital PCR assays to measure BRAFV600-mutant ctDNA in pretreatment and on-treatment plasma samples from patients aged 18 years or older enrolled in two clinical trials. COMBI-d (NCT01584648) was a double-blind, randomised phase 3 study of dabrafenib plus trametinib versus dabrafenib plus placebo in previously untreated patients with BRAFV600 mutation-positive unresectable or metastatic melanoma. Patients had an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. COMBI-MB (NCT02039947) was an open-label, non-randomised, phase 2 study evaluating dabrafenib plus trametinib in patients with BRAFV600 mutation-positive metastatic melanoma and brain metastases. Patients in cohort A of COMBI-MB had asymptomatic brain metastases, no previous local brain-directed therapy, and an ECOG performance status of 0 or 1. Biomarker analysis was a prespecified exploratory endpoint in both trials and performed in the intention-to-treat populations in COMBI-d and COMBI-MB. We investigated the association between mutant copy number (baseline or week 4 or zero conversion status) and efficacy endpoints (progression-free survival, overall survival, and best overall response). We used Cox models, Kaplan-Meier plots, and log-rank tests to explore the association of pretreatment ctDNA concentrations with progression-free survival and overall survival. The effect of additional prognostic variables such as lactate dehydrogenase was also investigated in addition to the mutant copy number. FINDINGS: In COMBI-d, pretreatment plasma samples were available from 345 (82%) of 423 patients and on-treatment (week 4) plasma samples were available from 224 (53%) of 423 patients. In cohort A of COMBI-MB, pretreatment and on-treatment samples were available from 38 (50%) of 76 patients with intracranial and extracranial metastatic melanoma. ctDNA was detected in pretreatment samples from 320 (93%) of 345 patients (COMBI-d) and 34 (89%) of 38 patients (COMBI-MB). When assessed as a continuous variable, elevated baseline BRAFV600 mutation-positive ctDNA concentration was associated with worse overall survival outcome (hazard ratio [HR] 1·13 [95% CI 1·09-1·18], p<0·0001 by univariate analysis), independent of treatment group and baseline lactate dehydrogenase concentrations (1·08 [1·03-1·13], p=0·0020), in COMBI-d. A ctDNA cutoff point of 64 copies per mL of plasma stratified patients enrolled in COMBI-d as high risk or low risk with respect to survival outcomes (HR 1·74 [95% CI 1·37-2·21], p<0·0001 for progression-free survival; 2·23 [1·73-2·87], p<0·0001 for overall survival) and was validated in the COMBI-MB cohort (3·20 [1·39-7·34], p=0·0047 for progression-free survival; 2·94 [1·18-7·32], p=0·016 for overall survival). In COMBI-d, undetectable ctDNA at week 4 was significantly associated with extended progression-free and overall survival, particularly in patients with elevated lactate dehydrogenase concentrations (HR 1·99 [95% CI 1·08-3·64], p=0·027 for progression-free survival; 2·38 [1·24-4·54], p=0·0089 for overall survival). INTERPRETATION: Pretreatment and on-treatment BRAFV600-mutant ctDNA measurements could serve as independent, predictive biomarkers of clinical outcome with targeted therapy. FUNDING: Novartis.

TERT, BRAF, and NRAS Mutational Heterogeneity between Paired Primary and Metastatic Melanoma Tumors.

Mutational heterogeneity can contribute to therapeutic resistance in solid cancers. In melanoma, the frequencies of intertumoral and intratumoral heterogeneity are controversial. We examined mutational heterogeneity within individual patients with melanoma using multiplatform analysis of commonly mutated driver and nonpassenger genes. We analyzed paired primary and metastatic tumors from 60 patients and multiple metastatic tumors from 39 patients whose primary tumors were unavailable (n = 271 tumors). We used a combination of multiplex SNaPshot assays, Sanger sequencing, mutation-specific PCR, or droplet digital PCR to determine the presence of BRAFV600, NRASQ61, TERT-124C>T, and TERT-146C>T mutations. Mutations were detected in BRAF (39%), NRAS (21%), and/or TERT (78%). Thirteen patients had TERTmutant discordant tumors; seven of these had a single tumor with both TERT-124C>T and TERT-146C>T mutations present at different allele frequencies. Two patients had both BRAF and NRAS mutations; one had different tumors and the other had a single tumor with both mutations. One patient with a BRAFmutant primary lacked mutant BRAF in at least one of their metastases. Overall, we identified mutational heterogeneity in 18 of 99 patients (18%). These results suggest that some primary melanomas may be composed of subclones with differing mutational profiles. Such heterogeneity may be relevant to treatment responses and survival outcomes.

Validation of Circulating Tumor DNA Assays for Detection of Metastatic Melanoma.

The detection of cell-free, circulating tumor DNA (ctDNA) in the blood of patients with solid tumors is often referred to as "liquid biopsy." ctDNA is particularly attractive as a candidate biomarker in the blood. It is relatively stable after blood collection, can be easily purified, and can be quantitatively measured with high sensitivity and specificity using advanced technologies. Current liquid biopsy research has focused on detecting and quantifying ctDNA to (1) diagnose and characterize mutations in a patient's cancer to help select the appropriate treatment; (2) predict clinical outcomes associated with different treatments; and (3) monitor the response and/or progression of a patient's disease. The diagnostic use of liquid biopsies is probably greatest in tumors where the difficulty and/or risk of obtaining a tissue specimen for molecular diagnostics is high (e.g., lung, colon). In metastatic melanoma, however, obtaining a tissue sample for molecular diagnostics is not typically a major obstacle to patient care plans; rather predicting treatment outcomes and monitoring a patient's disease course during therapy are considered the current priorities for this cancer type. In this chapter we describe an approach to the validation of ctDNA detection assays for melanoma, focusing primarily on analytical validation, and provide methods to guide the use of droplet digital PCR assays for measuring ctDNA levels in plasma samples.

Development of Novel Mutation-Specific Droplet Digital PCR Assays Detecting TERT Promoter Mutations in Tumor and Plasma Samples.

Detecting mutations in the plasma of patients with solid tumors is becoming a valuable method of diagnosing and monitoring cancer. The TERT promoter is mutated at high frequencies in multiple cancer types, most commonly at positions -124 and -146 (designated C228T and C250T, respectively). Detection of these mutations has been challenging because of the high GC content of this region (approximately 80%). We describe development of novel probe-based droplet digital PCR assays that specifically detect and quantify these two mutations, along with the less common 242-243 CC>TT mutation, and demonstrate their application using human tumor and plasma samples from melanoma patients. Assay designs and running conditions were optimized using cancer cell line genomic DNAs with the C228T or C250T mutations. The limits of detection were 0.062% and 0.051% mutant allele fraction for the C228T and C250T assays, respectively. Concordance of 100% was observed between droplet digital PCR and sequencing-based orthogonal methods in the detection of TERT mutant DNA in 32 formalin-fixed, paraffin-embedded melanoma tumors. TERTmutant DNA was also identified in 21 of 27 plasma samples (78%) from patients with TERTmutant tumors, with plasma mutant allele fractions ranging from 0.06% to 15.3%. There were no false positives in plasma. These data demonstrate the potential of these assays to specifically detect and quantify TERTmutant DNA in tumors and plasma of cancer patients.

Prediction of breast cancer risk based on flow-variant analysis of circulating peripheral blood B cells.

PURPOSE: Identifying women at high risk for breast cancer can trigger a personal program of annual mammograms and magnetic resonance imaging scans for early detection, prophylactic surgery, or chemoprevention to reduce the risk of cancer. Yet, current strategies to identify high-risk mutations based on sequencing panels of genes have significant false-positive and false-negative results, suggesting the need for alternative approaches. METHODS: Flow-variant assays (FVAs) that assess the effects of mutations in the double-strand break (DSB) repair genetic pathway in lymphoblastoid cells in response to treatment with radiomimetic agents were assessed for sensitivity, specificity, and accuracy both alone and as part of a logistic regression classification score. In turn, these assays were validated in circulating B cells and applied to individuals with personal and/or family history of breast and/or ovarian cancer. RESULTS: A three-FVA classification score based on logistic regression had 95% accuracy. Individuals from a breast cancer-positive cohort with affected family members had high-risk FVA classification scores. CONCLUSION: Application of a classification score based on multiple FVAs could represent an alternative to panel sequencing for identifying women at high risk for cancer.Genet Med advance online publication 16 March 2017.

Inhibition of Prostaglandin Transporter (PGT) Promotes Perfusion and Vascularization and Accelerates Wound Healing in Non-Diabetic and Diabetic Rats.

Peripheral ischemia, resulting from diminished arterial flow and defective local vascularization, is one of the main causes of impaired wound healing in diabetes. Vasodilatory prostaglandins (PGs), including PGE2 and PGI2, regulate blood flow in peripheral tissues. PGs also stimulate angiogenesis by inducing vascular endothelial growth factor. However, PG levels are reduced in diabetes mainly due to enhanced degradation. We hypothesized that inhibition of the prostaglandin transporter (PGT) (SLCO2A1), which mediates the degradation of PGs, would increase blood flow and stimulate vascularization, thereby mitigating peripheral ischemia and accelerating wound healing in diabetes. Here we report that inhibiting PGT with intravenously injected PGT inhibitor, T26A, increased blood flow in ischemic hind limbs created in non-diabetic rats and streptozotocin induced diabetic rats. Systemic, or combined with topical, T26A accelerated closure of cutaneous wounds. Immunohistochemical examination revealed that inhibition of PGT enhanced vascularization (marked by larger numbers of vessels formed by CD34+ cells), and accelerated re-epithelialization of cutaneous wounds. In cultured primary human bone marrow CD34+ cells and human epidermal keratinocytes (HEKs) either inhibiting or silencing PGT increased migration in both cell lines. Thus PGT directly regulates mobilization of endothelial progenitor cells (EPCs) and HEKs, which could contribute to PGT-mediated vascularization and re-epithelialization. At the molecular level, systemic inhibition of PGT raised circulating PGE2. Taken together, our data demonstrate that PGT modulates arterial blood flow, mobilization of EPCs and HEKs, and vascularization and epithelialization in wound healing by regulating vasodilatory and pro-angiogenic PGs.

Prostaglandin transporter modulates wound healing in diabetes by regulating prostaglandin-induced angiogenesis.

Prostaglandin transporter (PGT) mediates prostaglandin (PG) catabolism and PG signal termination. The prostanoid PGE(2), which induces angiogenesis and vasodilation, is diminished in diabetic skin, suggesting that PGT up-regulation could be important in wound healing deficiency, typified by diabetic foot ulcer. We hypothesized that up-regulation of PGT in hyperglycemia could contribute to weakened PGE(2) signaling, leading to impaired angiogenesis and wound healing. In human dermal microvascular endothelial cells (HDMECs), exposure to hyperglycemia increased PGT expression and activity up to threefold, accompanied by reduced levels of PGE(2). Hyperglycemia reduced HDMEC migration by 50% and abolished tube formation. Deficits in PGE(2) expression, HDMEC migration, and tube formation could be corrected by treatment with the PGT inhibitor T26A, consistent with the idea that PGT hyperactivity is responsible for impairments in angiogenesis mediated by PG signaling. In vivo, PGT expression was profoundly induced in diabetes and by wounding, correlating with diminished levels of proangiogenic factors PGE(2) and VEGF in cutaneous wounds of diabetic mice. Pharmacological inhibition of PGT corrected these deficits. PGT inhibition shortened cutaneous wound closure time in diabetic mice from 22 to 16 days. This effect was associated with increased proliferation, re-epithelialization, neovascularization, and blood flow. These data provide evidence that hyperglycemia enhances PGT expression and activity, leading to diminished angiogenic signaling, a possible key mechanism underlying defective wound healing in diabetes.