Genetic and methylation profiles distinguish benign, malignant and spitzoid melanocytic tumors.

Accurate classification of melanocytic tumors is important for prognostic evaluation, treatment and follow-up protocols of patients. The majority of melanocytic proliferations can be classified solely based on clinical and pathological criteria, however in select cases a definitive diagnostic assessment remains challenging and additional diagnostic biomarkers would be advantageous. We analyzed melanomas, nevi, Spitz nevi and atypical spitzoid tumors using parallel sequencing (exons of 611 genes and 507 gene translocation analysis) and methylation arrays (850k Illumina EPIC). By combining detailed genetic and epigenetic analysis with reference-based and reference-free DNA methylome deconvolution we compared Spitz nevi to nevi and melanoma and assessed the potential for these methods in classifying challenging spitzoid tumors. Results were correlated with clinical and histologic features. Spitz nevi were found to cluster independently of nevi and melanoma and demonstrated a different mutation profile. Multiple copy number alterations and TERT promoter mutations were identified only in melanomas. Genome-wide methylation in Spitz nevi was comparable to benign nevi while the Leukocytes UnMethylation for Purity (LUMP) algorithm in Spitz nevi was comparable to melanoma. Histologically difficult to classify Spitz tumor cases were assessed which, based on methylation arrays, clustered between Spitz nevi and melanoma and in terms of genetic profile or copy number variations demonstrated worrisome features suggesting a malignant neoplasm. Comprehensive sequencing and methylation analysis verify Spitz nevi as an independent melanocytic entity distinct from both nevi and melanoma. Combined genetic and methylation assays can offer additional insights in diagnosing difficult to classify Spitzoid tumors.

Genome-wide miRNAs profiles of pearl millet under contrasting high vapor pressure deficit reveal their functional roles in drought stress adaptations.

Pearl millet (Pennisetum glaucum [L.] R. Br.) is an important crop capable of growing in harsh and marginal environments, with the highest degree of tolerance to drought and heat stresses among cereals. Diverse germplasm of pearl millet shows a significant phenotypic variation in response to abiotic stresses, making it a unique model to study the mechanisms responsible for stress mitigation. The present study focuses on identifying the physiological response of two pearl millet high-resolution cross (HRC) genotypes, ICMR 1122 and ICMR 1152, in response to low and high vapor pressure deficit (VPD). Under high VPD conditions, ICMR 1152 exhibited a lower transpiration rate (Tr), higher transpiration efficiency, and lower root sap exudation than ICMR 1122. Further, Pg-miRNAs expressed in the contrasting genotypes under low and high VPD conditions were identified by deep sequencing analysis. A total of 116 known and 61 novel Pg-miRNAs were identified from ICMR 1152, while 26 known and six novel Pg-miRNAs were identified from ICMR 1122 genotypes, respectively. While Pg-miR165, 168, 170, and 319 families exhibited significant differential expression under low and high VPD conditions in both genotypes, ICMR 1152 showed abundant expression of Pg-miR167, Pg-miR172, Pg-miR396 Pg-miR399, Pg-miR862, Pg-miR868, Pg-miR950, Pg-miR5054, and Pg-miR7527 indicating their direct and indirect role in root physiology and abiotic stress responses. Drought responsive Pg-miRNA targets showed upregulation in response to high VPD stress, further narrowing down the miRNAs involved in regulation of drought tolerance in pearl millet.

Role of miRNAs in the host-pathogen interaction between sugarcane and Colletotrichum falcatum, the red rot pathogen.

KEY MESSAGE: Sugarcane microRNAs specifically involved during compatible and incompatible interactions with red rot pathogen Colletotrichum falcatum were identified. We have identified how the miRNAs regulate their gene targets and elaborated evidently on the underlying molecular mechanism of sugarcane defense response to C. falcatum for the first time. Resistance against the fungal pathogen Colletotrichum falcatum causing red rot is one of the most desirable traits for sustainable crop cultivation in sugarcane. To gain new insight into the host defense mechanism against C. falcatum, we studied the role of sugarcane microRNAs during compatible and incompatible interactions by adopting the NGS platform. We have sequenced a total of 80 miRNA families that comprised 980 miRNAs, and the putative targets of the miRNAs include transcription factors, membrane-bound proteins, glutamate receptor proteins, lignin biosynthesis proteins, signaling cascade proteins, transporter proteins, mitochondrial proteins, ER proteins, defense-related, stress response proteins, translational regulation proteins, cell proliferation, and ubiquitination proteins. Further, qRT-PCR analyses of 8 differentially regulated miRNAs and 26 gene transcript targets expression indicated that these miRNAs have a regulatory effect on the expression of respective target genes in most of the cases. Also, the results suggest that certain miRNA regulates many target genes that are involved in inciting early responses to the pathogen infection, signaling pathways, endoplasmic reticulum stress, and resistance gene activation through feedback response from various cellular processes during the compatible and incompatible interaction with the red rot pathogen C. falcatum. The present study revealed the role of sugarcane miRNAs and their target genes during sugarcane-C. falcatum interaction and provided new insight into the miRNA-mediated defense mechanism in sugarcane for the first time.

Candidate drugs associated with sensitivity of cancer cell lines with DLST amplification or high mRNA levels.

Overexpression of the dihydrolipoamide S-succinyltransferase (DLST) is associated with poor outcome in neuroblastoma patients and triple-negative breast cancer (TNBC) and specifically with the oxidative phosphorylation (OXPHOS) pathway. Inhibitors of OXPHOS were previously suggested as a potential therapeutic strategy for a subset of patients with high-risk neuroblastoma. Here, we tested if cell lines with DLST amplifications or high mRNA levels were associated with sensitivity to 250 drugs from the Genomics of Drug Sensitivity in Cancer (GDSC) dataset by comparing them to cell lines without these changes. DLST-altered cell lines were more sensitive to 7 approved drugs, among these obatoclax mesylate, a BCL2 inhibitor that reduces OXPHOS in human leukemia stem cells. Moreover, several protein kinase inhibitors were identified to be efficient in cell lines with DLST amplifications or high mRNA levels, suggesting a vulnerability of DLST-altered cell lines for drugs targeting the ERK/MAPK pathway. Furthermore, increased DLST expression in cell lines with driver mutations in KRAS supported this relationship. We therefore conclude that, in addition to OXPHOS, protein kinases could be potential targets of therapy in the presence of DLST amplifications or high mRNA levels. The new drug candidates proposed here could serve in experimental testing on drug efficacy in knock-in cell lines and DLST-activated tumors.

Database for queries of melanoma cohorts with molecular and clinical data.

Large genome-scale studies are deposited in various public sequence repositories. However, their access and analysis can be non-trivial to infrequent users. Here, we present a new database connecting whole transcriptomes with clinical data for straight-forward access and analysis of patient-specific samples. Users can perform association tests of survival and gene expression across different cohorts, identify cell-type expressions, or correlate the presence of immune cells. In summary, we present a new data hub for bench scientists to perform replication and discovery studies.

HLA Class II Loss and JAK1/2 Deficiency Coevolve in Melanoma Leading to CD4 T-cell and IFNγ Cross-Resistance.

PURPOSE: Recent studies have demonstrated HLA class II (HLA-II)-dependent killing of melanoma cells by cytotoxic CD4 T cells. We investigated evolution of HLA-II-loss tumors that escape cytotoxic CD4 T-cell activity and contribute to immunotherapy resistance. EXPERIMENTAL DESIGN: Melanoma cells from longitudinal metastases were studied for constitutive and IFN-inducible HLA-II expression, sensitivity towards autologous CD4 T cells, and immune evasion by HLA-II loss. Clinical significance of HLA-II-low tumors was determined by analysis of transcriptomic data sets from patients with immune checkpoint blockade (ICB). RESULTS: Analysis of longitudinal samples revealed strong intermetastatic heterogeneity in melanoma cell-intrinsic HLA-II expression and subclonal HLA-II loss. Tumor cells from early lesions either constitutively expressed HLA-II, sensitizing to cytotoxic CD4 T cells, or induced HLA-II and gained CD4 T-cell sensitivity in the presence of IFNγ. In contrast, late outgrowing subclones displayed a stable CD4 T-cell-resistant HLA-II-loss phenotype. These cells lacked not only constitutive but also IFNγ-inducible HLA-II due to JAK1/2-STAT1 pathway inactivation. Coevolution of JAK1/2 deficiency and HLA-II loss established melanoma cross-resistance to IFNγ and CD4 T cells, as detected in distinct stage IV metastases. In line with their immune-evasive phenotype, HLA-II-low melanomas showed reduced CD4 T-cell infiltrates and correlated with disease progression under ICB. CONCLUSIONS: Our study links melanoma resistance to CD4 T cells, IFNγ, and ICB at the level of HLA-II, highlighting the significance of tumor cell-intrinsic HLA-II antigen presentation in disease control and calling for strategies to overcome its downregulation for improvement of patient outcome.

Genetic and Methylation Analysis of CTNNB1 in Benign and Malignant Melanocytic Lesions.

Melanocytic neoplasms have been genetically characterized in detail during the last decade. Recurrent CTNNB1 exon 3 mutations have been recognized in the distinct group of melanocytic tumors showing deep penetrating nevus-like morphology. In addition, they have been identified in 1-2% of advanced melanoma. Performing a detailed genetic analysis of difficult-to-classify nevi and melanomas with CTNNB1 mutations, we found that benign tumors (nevi) show characteristic morphological, genetic and epigenetic traits, which distinguish them from other nevi and melanoma. Malignant CTNNB1-mutant tumors (melanomas) demonstrated a different genetic profile, instead grouping clearly with other non-CTNNB1 melanomas in methylation assays. To further evaluate the role of CTNNB1 mutations in melanoma, we assessed a large cohort of clinically sequenced melanomas, identifying 38 tumors with CTNNB1 exon 3 mutations, including recurrent S45 (n = 13, 34%), G34 (n = 5, 13%), and S27 (n = 5, 13%) mutations. Locations and histological subtype of CTNNB1-mutated melanoma varied; none were reported as showing deep penetrating nevus-like morphology. The most frequent concurrent activating mutations were BRAF V600 (n = 21, 55%) and NRAS Q61 (n = 13, 34%). In our cohort, four of seven (58%) and one of nine (11%) patients treated with targeted therapy (BRAF and MEK Inhibitors) or immune-checkpoint therapy, respectively, showed disease control (partial response or stable disease). In summary, CTNNB1 mutations are associated with a unique melanocytic tumor type in benign tumors (nevi), which can be applied in a diagnostic setting. In advanced disease, no clear characteristics distinguishing CTNNB1-mutant from other melanomas were observed; however, studies of larger, optimally prospective, cohorts are warranted.

About the Role of Interfaces on the Fatigue Crack Propagation in Laminated Metallic Composites.

The influence of gradients in hardness and elastic properties at interfaces of dissimilar materials in laminated metallic composites (LMCs) on fatigue crack propagation is investigated experimentally for three different LMC systems: Al/Al-LMCs with dissimilar yield stress and Al/Steel-LMCs as well as Al/Ti/Steel-LMCs with dissimilar yield stress and Young's modulus, respectively. The damage tolerant fatigue behavior in Al/Al-LMCs with an alternating layer structure is enhanced significantly compared to constituent monolithic materials. The prevalent toughening mechanisms at the interfaces are identified by microscopical methods and synchrotron X-ray computed tomography. For the soft/hard transition, crack deflection mechanisms at the vicinity of the interface are observed, whereas crack bifurcation mechanisms can be seen for the hard/soft transition. The crack propagation in Al/Steel-LMCs was studied conducting in-situ scanning electron microscope (SEM) experiments in the respective low cycle fatigue (LCF) and high cycle fatigue (HCF) regimes of the laminate. The enhanced resistance against crack propagation in the LCF regime is attributed to the prevalent stress redistribution, crack deflection, and crack bridging mechanisms. The fatigue properties of different Al/Ti/Steel-LMC systems show the potential of LMCs in terms of an appropriate selection of constituents in combination with an optimized architecture. The results are also discussed under the aspect of tailored lightweight applications subjected to cyclic loading.

Molecular pathology as a diagnostic aid in difficult-to-classify melanocytic tumours with spitzoid morphology.

Accurate classification of melanocytic proliferations has important implications for prognostic prediction, treatment and follow-up. Although most melanocytic proliferations can be accurately classified using clinical and pathological criteria, classification (specifically distinction between nevus and melanoma) can be challenging in a subset of cases, including those with spitzoid morphology. Genetic studies have shown that mutation profiles differ between primary melanoma subtypes and Spitz nevi. These differences may aid in distinguishing benign from malignant in some melanocytic tumours. Here, we present a selection of melanocytic proliferations with equivocal histopathological criteria, wherein genetic analysis was requested to help guide classification. In two of four cases, the genetic results offered valuable insights, allowing a definitive diagnosis, indicating the diagnostic value of mutation profiling in a real-world routine clinical setting. Although histopathological assessment remains decisive in melanocytic proliferation classification, we recommend including genetic profiling in cases of borderline or atypical lesion to support accurate classification.

Clinical characteristics and therapy response in unresectable melanoma patients stage IIIB-IIID with in-transit and satellite metastases.

INTRODUCTION: Cutaneous melanoma is notorious for the development of in-transit metastases (ITM). For unknown biological reasons, ITM remain the leading tumour manifestation without progression to distant sites in some patients. METHODS: In total, 191 patients with initially unresectable stage III ITM and satellite metastases from 16 skin cancer centres were retrospectively evaluated for their tumour characteristics, survival and therapy response. Three groups according to disease kinetics (no distant progress, slow (>6 months) and fast (<6 months) distant progression) were analysed separately. RESULTS: Median follow-up time was 30.5 (range 0.8-154.0) months from unresectable ITM. Progression to stage IV was observed in 56.5% of cases. Patients without distant metastasis were more often female, older (>70 years) and presented as stage III with lymph node or ITM at initial diagnosis in 45.7% of cases. Melanoma located on the leg had a significantly better overall survival (OS) from time of initial diagnosis compared to non-leg localised primaries (hazard ratio [HR] = 0.61, 95% confidence interval [CI] 0.40-0.91; p = 0.017), but not from diagnosis of unresectable stage III (HR = 0.67, 95% CI 0.45-1.02; p = 0.06). Forty percent of patients received local therapy for satellite and ITM. Overall response rate (ORR) to all local first-line treatments was 38%; disease control rate (DCR) was 49%. In total, 72.3% of patients received systemic therapy for unresectable stage IIIB-D. ORR for targeted therapy (n = 19) was highest with 63.2% and DCR was 84.2% compared to an ORR of 31.4% and a DCR of 54.3% in PD-1 treated patients (n = 70). Patients receiving PD-1 and intralesional talimogene laherparepvec (n = 12) had an ORR of 41.7% and a DCR of 75%. CONCLUSION: Patients with unresectable ITM and without distant progression are more often female, older, and have a primary on the leg. Response to PD-1 inhibitors in this cohort was lower than expected, but further investigation is required to elucidate the biology of ITM development and the interplay with the immune system.

Interactome of miRNAs and transcriptome of human umbilical cord endothelial cells exposed to short-term simulated microgravity.

Adaptation of humans in low gravity conditions is a matter of utmost importance when efforts are on to a gigantic leap in human space expeditions for tourism and formation of space colonies. In this connection, cardiovascular adaptation in low gravity is a critical component of human space exploration. Deep high-throughput sequencing approach allowed us to analyze the miRNA and mRNA expression profiles in human umbilical cord vein endothelial cells (HUVEC), cultured under gravity (G), and stimulated microgravity (MG) achieved with a clinostat. The present study identified totally 1870 miRNAs differentially expressed in HUVEC under MG condition when compared to the cells subjected to unitary G conditions. The functional association of identified miRNAs targeting specific mRNAs revealed that miRNAs, hsa-mir-496, hsa-mir-151a, hsa-miR-296-3p, hsa-mir-148a, hsa-miR-365b-5p, hsa-miR-3687, hsa-mir-454, hsa-miR-155-5p, and hsa-miR-145-5p differentially regulated the genes involved in cell adhesion, angiogenesis, cell cycle, JAK-STAT signaling, MAPK signaling, nitric oxide signaling, VEGF signaling, and wound healing pathways. Further, the q-PCR based experimental studies of upregulated and downregulated miRNA and mRNAs demonstrate that the above reported miRNAs influence the cell proliferation and vascular functions of the HUVEC in MG conditions effectively. Consensus on the interactome results indicates restricted fluctuations in the transcriptome of the HUVEC exposed to short-term MG that could lead to higher levels of endothelial functions like angiogenesis and vascular patterning.