Population-based BRCA germline mutation screening in the Han Chinese identifies individuals at risk of BRCA mutation-related cancer: experience from a clinical diagnostic center from greater Shanghai area.

BACKGROUND: Deleterious BRCA1/2 (BRCA) mutation raises the risk for BRCA mutation-related malignancies, including breast, ovarian, prostate, and pancreatic cancer. Germline variation of BRCA exhibits substantial ethnical diversity. However, there is limited research on the Chinese Han population, constraining the development of strategies for BRCA mutation screening in this large ethnic group. METHODS: We profile the BRCA mutational spectrum, including single nucleotide variation, insertion/deletion, and large genomic rearrangements in 2,080 apparently healthy Chinese Han individuals and 522 patients with BRCA mutation-related cancer, to determine the BRCA genetic background of the Chinese Han population, especially of the East Han. Incident cancer events were monitored in 1,005 participants from the healthy group, comprising 11 BRCA pathogenic/likely pathogenic (PLP) variant carriers and 994 PLP-free individuals, including 3 LGR carriers. RESULTS: Healthy Chinese Han individuals demonstrated a distinct BRCA mutational spectrum compared to cancer patients, with a 0.53% (1 in 189) prevalence of pathogenic/likely pathogenic (PLP) variant, alongside a 3 in 2,080 occurrence of LGR. BRCA1 c. 5470_5477del demonstrated high prevalence (0.44%) in the North Han Chinese and penetrance for breast cancer. None of the 3 LGR carriers developed cancer during the follow-up. We calculated a relative risk of 135.55 (95% CI 25.07 to 732.88) for the development of BRCA mutation-related cancers in the BRCA PLP variant carriers (mean age 42.91 years, median follow-up 10 months) compared to PLP-free individuals (mean age 48.47 years, median follow-up 16 months). CONCLUSION: The unique BRCA mutational profile in the Chinese Han highlights the potential for standardized population-based BRCA variant screening to enhance BRCA mutation-related cancer prevention and treatment.

Long noncoding RNA CCAL transferred from fibroblasts by exosomes promotes chemoresistance of colorectal cancer cells.

Long noncoding RNAs (lncRNAs) are involved in the pathology of colorectal cancer (CRC). Current efforts to eradicate CRC predominantly focused on targeting the proliferation of rapidly growing cancer epithelial cells. This is largely ineffective with resistance arising in most tumors after exposure to chemotherapy. Despite the long-standing recognition of the crosstalk between carcinoma-associated fibroblasts (CAFs) and cancer cells in the tumor microenvironment, how CAFs may contribute to drug resistance in neighboring cancer cells is not well characterized. Here, we show that lncRNA CCAL (colorectal cancer-associated lncRNA) promotes oxaliplatin (Oxa) resistance of CRC cells. RNA-ISH shows higher CCAL expressed in the tumor stroma compared to cancer nests of CRC tissues. Functional studies reveal that CCAL is transferred from CAFs to the cancer cells via exosomes, where it suppresses CRC cell apoptosis, confers chemoresistance and activates ß-catenin pathway in vitro and in vivo. Mechanistically, CCAL interacts directly with mRNA stabilizing protein HuR (human antigen R) to increase ß-catenin mRNA and protein levels. Our findings indicate that CCAL expressed by CAFs of the colorectal tumor stroma contributes to tumor chemoresistance and CCAL may serve as a potential therapeutic target for Oxa resistance.

MicroRNA-296-5p promotes healing of diabetic wound by targeting sodium-glucose transporter 2 (SGLT2).

BACKGROUND: Diabetic wounds are refractory and very difficult to heal. We aimed to use miRNA to identify novel and specific molecular markers for diabetes mellitus (DM) diagnosis and treatment. METHODS: The expression level of miR-296-5p was determined in tissue samples of 12 DM patients. The effect of miR-296-5p on proliferation of ß-cells was examined using Cell Counting Kit-8 (CCK-8) and colony formation assay. The effect of miR-296-5p on cell cycle progression was analysed using flow cytometry. The target gene was verified using luciferase reporter assay. A rat diabetes model was used to assess the effect of miR-296-5p in vivo. RESULTS: Overexpression of miR-296-5p suppressed cell proliferation, arrested cell cycle progression, and increased the healing rate of diabetic wounds both in vivo and in vitro. TargetScan analysis results showed that miR-296-5p is a direct regulator of SGLT2. CONCLUSIONS: miR-296-5p can increase the healing rate of diabetic wounds and may be an effective molecular tool in DM diagnosis and therapy.

Elevated expression of the EZH2 gene in CALR-mutated patients with primary myelofibrosis.

Primary myelofibrosis (PMF) is one of the BCR/ABL-negative myeloproliferative neoplasms (MPNs), characterized by the diffuse fibrous hyperproliferation, bone marrow osteosclerosis, extramedullary hematopoiesis, and marked splenomegaly. The patients with PMF have an insidious onset, a long duration of clinical course, and the deteriorated quality of life. It has been reported that the CALR gene 9 exon mutations were detected in 25-30% PMF patients, particularly as high as 80% in the JAK2/MPL-negative ones. As the second most common mutation in BCR/ABL-negative MPNs, CALR mutation has been included in the latest World Health Organization (WHO) classification criteria as one of the main diagnostic criteria for both essential thrombocythemia (ET) and PMF. Moreover, the CALR mutations indicated a favorable prognosis, which the mechanism is still under investigation. It was demonstrated that a characterized high expression of EZH2 and SUZ12 in CALR-mutated patients. Taking EZH2 as the research entry point, we initially discussed the mechanism that the CALR-positive patients with PMF exhibited a better prognosis in the current study.

Biology of MiR-17-92 Cluster and Its Progress in Lung Cancer.

MicroRNAs, a class of short endogenous RNAs, acting as post-transcriptional regulators of gene expression, mostly silence gene expression via binding imperfectly matched sequences in the 3'UTR of target mRNA. MiR-17-92, a highly conserved gene cluster, has 6 members including miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1 and miR-92a. The miR-17-92 cluster, regarded as oncogene, is overexpressed in human cancers. Lung cancer is the leading cause of death all over the world. The molecular mechanism of lung cancer has been partly known at the levels of genes and proteins in last decade. However, new prognosis biomarkers and more target drugs should be developed in future. Therefore, noncoding RNAs, especially miRNAs, make them as new potentially clinical biomarkers for diagnosis and prognosis. In this review, we focus the current progress of miR-17-92 cluster in lung cancer.

Clinical evaluation of a substitute of HLA-B*58:01 in different Chinese ethnic groups.

The goal of this research was to investigate the linkage disequilibrium between rs9263726 and HLA-B*58:01 in different Chinese ethnic groups (Han, Tibet, and Hui) and to study the feasibility of rs9263726 replacing HLA-B*58:01 as an efficient indicator of potential allopurinol hypersensitivity syndrome. In this study, rs9263726 and HLA-B*58:01 were detected in all samples. For samples of individuals whose rs9263726 genotypes were not consistent with HLA-B*58:01, we did high-resolution typing of HLA-B gene to further confirm the correlation of rs9263726 genotype and special HLA-B alleles. We confirmed that the linkage disequilibrium between rs9263726 and HLA-B*58:01 was more significant in the Han ethnic group (r2=0.886, D'=1.0) than in the Tibet and Hui ethnic groups (for Tibetan, r2=0.606, D'=0.866; for Hui, r2=0.622, D'=0.924). For Han Chinese, samples with the GG genotype of rs9263726 did not carry HLA-B*58:01, while AA genotype samples were homozygous carriers of HLA-B*58:01. However, GA genotype samples of rs9263726 required a more sophisticated HLA-B genotyping assay before it was possible to identify whether they were HLA-B*58:01 carriers or not. For Tibetan and Hui, the linkage disequilibrium between rs9263726 and HLA-B*58:01 was not significant. Therefore, rs9263726 cannot replace HLA-B*58:01 in these two groups.

External magnetic field promotes homing of magnetized stem cells following subcutaneous injection.

BACKGROUND: Mesenchymal stem cells (MSCs) are multipotent stromal cells that have the ability to self-renew and migrate to sites of pathology. In vivo tracking of MSCs provides insights into both, the underlying mechanisms of MSC transformation and their potential as gene delivery vehicles. The aim of our study was to assess the ability of superparamagnetic iron oxide nanoparticles (SPIONs)-labeled Wharton's Jelly of the human umbilical cord-derived MSCs (WJ-MSCs) to carry the green fluorescent protein (GFP) gene to cutaneous injury sites in a murine model. METHODS: WJ-MSCs were isolated from a fresh umbilical cord and were genetically transformed to carry the GFP gene using lentiviral vectors with magnetically labeled SPIONs. The SPIONs/GFP-positive WJ-MSCs expressed multipotent cell markers and demonstrated the potential for osteogenic and adipogenic differentiation. Fifteen skin-injured mice were divided into three groups. Group I was treated with WJ-MSCs, group II with SPIONs/GFP-positive WJ-MSCs, and group III with SPIONs/GFP-positive WJ-MSCs exposed to an external magnetic field (EMF). Magnetic resonance imaging and optical molecular imaging were performed, and images were acquired 1, 2, and 7 days after cell injection. RESULTS: The results showed that GFP could be intensively detected around the wound in vivo 24 h after the cells were injected. Furthermore, we observed an accumulation of WJ-MSCs at the wound site, and EMF exposure increased the speed of cell transport. In conclusion, our study demonstrated that SPIONs/GFP function as cellular probes for monitoring in vivo migration and homing of WJ-MSCs. Moreover, exposure to an EMF can increase the transportation efficiency of SPIONs-labeled WJ-MSCs in vivo. CONCLUSIONS: Our findings could lead to the development of a gene carrier system for the treatment of diseases.

Diagnostic and prognostic value of tissue and circulating levels of Ephrin-A2 in prostate cancer.

Ephrin-A2, a member of the Eph/ephrin family, is associated with tumorigenesis and tumor progression. This study aimed to assess the diagnostic and prognostic value of both serum and tissue levels of Ephrin-A2 in prostate cancer (PCa) management. One hundred and forty-five frozen prostate tissues, 55 paraffin-embedded prostate tissues, 88 serum samples, and seven prostate cell lines (RWPE-1, LNCaP, LNCaP-LN3, PC-3, PC-3M, PC-3M-LN4, and DU145) were examined via quantitative reverse transcription-PCR (qRT-PCR), immunohistochemistry, enzyme-linked immunosorbent assay, and western blotting. Induced Ephrin-A2 messenger RNA (mRNA) or protein expression was detected in 8.6 % (5/58) benign prostatic hyperplasia (BPH), 59.8 % (52/87) PCa, and five prostate cancer cell lines. Ephrin-A2 immunostaining was present in 6.7 % (1/15) patients with BPHs and 62.5 % (25/40) clinically localized PCa. Accordingly, serum Ephrin-A2 was significantly higher in PCa patients compared to those in the BPH patients and controls (P < 0.001). The expression of Ephrin-A2 was higher in tumor patients with an elevated Gleason score or T3-T4 staging. Ephrin-A2 expression was correlated with Ki-67 expression in PCa patients, both at the gene scale and protein level. Our data indicate that Ephrin-A2 is a potential diagnostic and prognostic biomarker and a promising molecular therapeutic target to attenuate prostate cancer progression.

Downregulation of EphA5 by promoter methylation in human prostate cancer.

BACKGROUND: EphA5 is a member of the Eph/ephrin family and plays a critical role in the regulation of carcinogenesis. A significant reduction of EphA5 transcripts in high-grade prostate cancer tissue was shown using a transcriptomic analysis, compared to the low-grade prostate cancer tissue. As less is known about the mechanism of EphA5 downregulation and the function of EphA5, here we investigated the expression and an epigenetic change of EphA5 in prostate cancer and determined if these findings were correlated with clinicopathologic characteristics of prostate cancer. METHODS: Seven prostate cell lines (RWPE-1, LNCap, LNCap-LN3, CWR22rv-1, PC-3, PC-3M-LN4, and DU145), thirty-nine BPH, twenty-two primary prostate carcinomas, twenty-three paired noncancerous and cancerous prostate tissues were examined via qRT-PCR, methylation-specific PCR, bisulfite sequencing, immunohistochemistry and western blotting. The role of EphA5 in prostate cancer cell migration and invasion was examined by wound healing and transwell assay. RESULTS: Downregulation or loss of EphA5 mRNA or protein expression was detected in 28 of 45 (62.2%) prostate carcinomas, 2 of 39 (5.1%) hyperplasias, and all 6 prostate cancer cell lines. Methylation of the EphA5 promoter region was present in 32 of 45 (71.1%) carcinoma samples, 3 of 39 (7.7%) hyperplasias, and the 6 prostate cancer cell lines. Among 23 paired prostate carcinoma tissues, 16 tumor samples exhibited the hypermethylation of EphA5, and 15 of these 16 specimens (93.8%) shown the downregulation of EphA5 expression than that of their respectively matched noncancerous samples. Immunostaining analysis demonstrated that the EphA5 protein was absent or down-regulated in 10 of 13 (76.9%) available carcinoma samples, and 8 of these 10 samples (80.0%) exhibited hypermethylation. The frequency of EphA5 methylation was higher in cancer patients with an elevated Gleason score or T3-T4 staging. Following the treatment of 6 prostate cancer cell lines with 5-aza-2'-deoxycytidine, the levels of EphA5 mRNA were significantly increased. Prostate cancer cells invasion and migration were significantly suppressed by ectopic expression of EphA5 in vitro. CONCLUSION: Our study provides evidence that EphA5 is a potential target for epigenetic silencing in primary prostate cancer and is a potentially valuable prognosis predictor and thereapeutic marker for prostate cancer.

Detection of HLA-B*58:01 with TaqMan assay and its association with allopurinol-induced sCADR.

BACKGROUND: The HLA-B*58:01 allele is associated with allopurinol-induced severe cutaneous adverse drug reactions (sCADR) in certain geographic regions, but the diversity of the correlation is large. In addition, the currently available HLA-B*58:01 testing methods are too laborious for use in routine clinical detection. The objective of this study was to develop a new, convenient method for the detection of HLA-B*58:01 and to investigate the association of HLA-B*58:01 with allopurinol-induced sCADR in a Han Chinese population. METHODS: A new method combining sequence-specific primers (SSP) and TaqMan probe amplification was developed in this study and was used to detect the HLA-B*58:01 in 48 allopurinol-induced sCADR, 133 allopurinol-tolerant, and 280 healthy individuals. The accuracy, sensitivity, and specificity were assessed by a commercial PCR-SSP HLA-B typing kit. The low limit of detection was detected by serial dilution of an HLA-B*58:01-positive DNA template. RESULTS: The new method successfully identified HLA-B*58:01 in thousands of HLA-B alleles, and the results for 344 DNA samples were perfectly concordant with the results of the commercial PCR-SSP HLA-B kit. The analytical sensitivity is 100% and the specificity is over 99%. The low limit of detection of this assay is 100 pg DNA, which was 10 times more sensitive than the commercial PCR-SSP kit. HLA-B*58:01 was present in 93.8% of the patients with sCADR, 7.5% of the allopurinol-tolerant patients, and 12.1% of the healthy controls. The frequency of HLA-B*58:01 was significantly higher in the sCADR group than in the control group (p<0.0001). However, there was no significant difference between the allopurinol-tolerant and control groups (p=0.1547). CONCLUSIONS: HLA-B*58:01 has a strong association with allopurinol-induced sCADR in Han Chinese. The newly developed method is reliable for HLA-B*58:01 detection prior to allopurinol therapy.

Functional polymorphisms of the ABCG2 gene are associated with gout disease in the Chinese Han male population.

BACKGROUND: Gout is a common type of arthritis that is characterized by hyperuricemia, tophi and joint inflammation. Genetic variations in the ABCG2 gene have been reported to influence serum uric acid levels and to participate in the pathogenesis of gout, but no further data have been reported in the Han Chinese population. METHODS: Peripheral blood DNA was isolated from 352 male patients with gout and 350 gout-free normal male controls. High-resolution melting analysis and Sanger sequencing were performed to identify the genetic polymorphisms V12M, Q141K and Q126X in the ABCG2 gene. Genotype and haplotype analyses were utilized to determine the disease odds ratios (ORs). A prediction model for gout risk using ABCG2 protein function was established based on the genotype combination of Q126X and Q141K. RESULTS: For Q141K, the A allele frequency was 49.6% in the gout patients and 30.9% in the controls (OR 2.20, 95% confidence interval (CI): 1.77-2.74, p=8.99×10⁻¹³). Regarding Q126X, the T allele frequency was 4.7% in the gout patients and 1.7% in the controls (OR 2.91, 95% CI: 1.49-5.68, p=1.57×10⁻³). The A allele frequency for V12M was lower (18.3%) in the gout patients than in the controls (29%) (OR 0.55, 95% CI 0.43-0.71, p=2.55×10⁻6). In the order of V12M, Q126X and Q141K, the GCA and GTC haplotypes indicated increased disease risk (OR=2.30 and 2.71, respectively). Patients with mild to severe ABCG2 dysfunction accounted for 78.4% of gout cases. CONCLUSION: The ABCG2 126X and 141K alleles are associated with an increased risk of gout, whereas 12M has a protective effect on gout susceptibility in the Han Chinese population. ABCG2 dysfunction can be used to evaluate gout risk.

Oncogenic mutations in extramammary Paget's disease and their clinical relevance.

Extramammary Paget's disease (EMPD) is a rare cutaneous malignant neoplasm. The genetic alterations underlying its pathogenesis have less been described. Therefore, we analyzed the possible mutations in the KRAS, HRAS, NRAS, BRAF, ARAF, RAF1, PIK3CA, AKT1, CTNNB1 and APC genes as well as methylation and expression of CDH1 in 144 EMPD cases and 42 matched normal skin tissues. A distinct mutation profile was identified in EMPDs with 27 (19%) cases mutant for RAS and RAF genes and 50 (35%) cases harboring oncogenic mutations in PIK3CA and AKT1. Moreover, a mutually exclusive pattern was observed in the genetic variants in these two signaling pathways. No mutation was detected in CTNNB1 and APC genes. High prevalence of low expression and hypermethylation of CDH1 gene was detected in 33 and 48% of the EMPD cases, respectively. Furthermore, PIK3CA and AKT1 mutations were significantly correlated with CDH1 hypermethylation which could explain why the majority of EMPD cases with mutant PIK3CA and AKT1 were invasive. Our study demonstrates that genetic variants associated with constitutive activation of RAS/RAF and PI3K/AKT pathways are involved in the pathogenesis of EMPD. This may represent novel therapeutic targets for this skin cancer.

The m6A-related gene signature stratifies poor prognosis patients and characterizes immunosuppressive microenvironment in hepatocellular carcinoma.

Background: N6-methyladenosine (m6A) is the most abundant epitranscriptomic modification of RNA, which can affect RNA metabolism and protein translation. The m6A modification plays a critical role in cancer development, including hepatocellular carcinoma (HCC). Despite several m6A-related signatures in HCC, most of them lack the necessary validation and the reliability is still elusive. Methods: Differentially expressed genes (DEGs) in the Cancer Genome Atlas were comprehensively analyzed to identify m6A signature associated with HCC prognosis. Gene set enrichment analysis, tumor mutation burden (TMB), immune infiltration, and therapeutic response were evaluated. Importantly, mass spectrometry proteomics and multiplex immunofluorescence assays were performed for validation. Results: The m6A-related protein-coding gene signature was established, which can divide HCC into high-/low-risk subgroups with markedly different overall survival (OS) and clinical stages. Furthermore, we validated its reliability and robustness in our 101 independent HCC specimens using proteomic detection and confirmed that our signature readily identified high-risk HCC patients with 3-year survival rates of 44.1% vs. 71.8% in the low-risk group. Functional analysis indicated that the high-risk group might stimulate the cell cycle and activate oncogenic pathways such as MAPK, mTOR, and VEGF, whereas the low-risk group mainly regulated amino acid, fatty acid, and drug metabolism. Additionally, the high-risk group had more TMB, upregulated immune checkpoint molecule expression, including PD-1, CTLA4, TIM3, and LAG3, and preferentially formed an immunosuppressive microenvironment. Accordingly, potential therapeutic responses showed that high-risk patients were potentially sensitive to inhibitors targeting the cell cycle and MAPK signaling, with patients possibly benefiting from immunotherapy. Moreover, multiplex immunofluorescence assays indicated that high-risk HCC samples displayed distinct immunosuppressive features, with abundant M2-polarized macrophages and T-regulatory cell infiltration. Conclusion: The m6A signature had a prominent capacity to evaluate OS and characterize the tumor immune microenvironment of HCC, which may serve as a useful approach for risk stratification management and provide a valuable clue to choosing rational therapeutic strategies.

A rapid multiplex assay of human malaria parasites by digital PCR.

BACKGROUND: Blood smear examination through traditional optical microscopy is the gold standard for malaria diagnosis. However, it imposes strict requirements for operational staff and its sensitivity cannot perfectly satisfy the needs of clinical requirements. More sensitive and accurate modern technologies should be applied to this field. Digital PCR (dPCR), as an absolute quantification detection method, can serve as an effective tool to facilitate the diagnosis and classification of different malaria species. OBJECTIVE: We aimed to establish a new multiplex dPCR detection system for four main Plasmodium species: P. vivax, P. falciparum, P. ovale and P. malariae, which can distinguish exact species of malaria by one PCR reaction. METHODS: A total of 39 patients were identified as malaria-positive by microscopic examination in Huashan Hospital from 2016 to 2021; seventy blood samples from these patients were collected. Additionally, 20 healthy individuals, 20 patients with fever and 6 patients with other types of blood parasites infection were also included in this study. Each blood sample was subjected to examination by both blood smears and dPCR. By optimizing four different fluorescence-labeled probes in one reaction system, dPCR permitted the performance of accurate quantitation and working out the exact number of copies of malaria DNA per microliter in whole blood. Rapid diagnostic tests were also conducted to verify part of the results obtained by dPCR. RESULTS: The dPCR system was able to make rapid diagnosis and quantification of malaria DNA samples. The analytical sensitivity of multiplex dPCR was as low as 0.557 copies/µL (95% CI 0.521 to 0.607), and it had a sensitivity of 98.0% and a specificity of 100% in clinical samples. Additionally, three multiple malaria co-infection samples have been detected by this dPCR system, including one triple malaria infection case. By testing consecutive daily blood samples of Patient 39, dPCR facilitated monitoring the efficacy of drug treatment. It showed that the DNA concentrations of P. falciparum ranged from 5474 copies/µL to 0 copies/µL, which can reflect the efficacy of antimalarials in real time. This study also found that haemocyte samples (plasma removed) rather than whole blood had higher malaria detection capability and an enhanced positive rate. CONCLUSION: The multiplex dPCR system newly established here made a substantial contribution in detecting malaria infection at low concentrations. It is suitable for mixed-infection diagnosis and multi-sample continuous monitoring, and presents a promising candidate as an absolute quantitative tool in clinical practice.

Identification of three lncRNA-related prognostic signatures in gastric cancer by integrated multi-omics analysis.

Aims: The systematic identification of molecular features correlated with the clinical status of gastric cancer (GC) in patients is significant, although such investigation remains insufficient. Methods: GC subtyping based on RNA sequencing, copy number variation and DNA methylation data were derived from The Cancer Genome Atlas program. Prognostics lncRNA biomarkers for GC were identified by univariate Cox, LASSO and SVM-RFE analysis. Results: Three molecular subtypes with significant survival discrepancies, and their specific DEmRNAs and DElncRNAs were identified. Three reliable prognostic-associated lncRNA, including LINC00670, LINC00452 and LINC00160, were selected for GC. Conclusion: Our findings expanded the understanding on the regulatory network of lncRNAs in GC, providing potential targets for prognosis and treatment of GC patients.

Correlation of DLC1 gene methylation with oncogenic PIK3CA mutations in extramammary Paget's disease.

Extramammary Paget's disease is a rare cutaneous malignant neoplasm. The genetic and epigenetic mechanisms underlying its pathology remain unknown. In this study, we investigated the expression levels, and mutation and methylation status of a common tumor suppressor gene, deleted in liver cancer 1 (DLC1), and an oncogene, PIK3CA, in tumor (n=132) and normal tissues (n=20) from unrelated patients. The presence of epigenetic and genetic lesions was then correlated to the patient pathology data to determine the potential role of these genes in extramammary Paget's disease etiology and progression. The DLC1 gene was found to be downregulated in 43 (33%) tumors, as compared with immunohistochemistry results from normal tissues. Methylation-sensitive, high-resolution melting analysis indicated that the DLC1 promoter was hypermethylated in 51 (39%) extramammary Paget's disease tumors. This hypermethylation was associated with significantly decreased DLC1 levels (P=0.011), and had a strong positive correlation with advanced age (P=0.002). PIK3CA mutations were detected by direct sequencing in 32 (24%) tumors, the majority of which were invasive. Furthermore, PIK3CA mutations significantly correlated with DLC1 hypermethylation. Thus, aberrant DLC1 methylation and PIK3CA mutations may have important roles in extramammary Paget's disease pathogenesis, and may represent potential molecular targets for therapy.

Unlabeled-probe high-resolution melting to detect KRAS codon 12 and 13 mutations in pancreatic adenocarcinoma tissues.

BACKGROUND: The aim of our study was to establish an unlabeled-probe high-resolution melting (HRM) approach to the detection of Kirsten RAS (KRAS) codon 12 and 13 mutations in pancreatic adenocarcinoma (PA) tissues as a novel and effective diagnostic technique. METHODS: We tested the sensitivity and specificity of this genotyping approach in cell lines with known KRAS mutations using 166 bp amplicons and 37 bp wild-type probe to detect KRAS codon 12 and 13 mutations. We screened 49 PA tissues to be subsequently sequenced to confirm the mutations. Simultaneously, we tested the specimens using Sanger sequencing and then used target-DNA cloning and sequencing for verification. RESULTS: It was found that unlabeled-probe HRM was reliable in detecting 3% of mutant cell lines DNA diluted with that of the wild-type, whereas Sanger sequencing could only discriminate 20% mutant cell ratios. In detecting 49 specimens, the former was capable of detecting 23 mutations (46.9%); and the latter could observe 15 (30.6%). For further verification, T-A DNA cloning and sequencing was applied to the differences, with the results matching those of the unlabeled-probe HRM. CONCLUSIONS: It was concluded that the unlabeled-probe HRM approach can be a sensitive and accurate screening technique to detect KRAS codon 12 and 13 mutations in diagnosing and treating PA.

Downregulated miR-18a and miR-92a synergistically suppress non-small cell lung cancer via targeting Sprouty 4.

As a novel noncoding RNA cluster, miR-17-92 cluster include six members: miR-17, miR-18a, miR-19a, miR-19b-1, miR-20a, and miR-92a. Dysregulation of miR-17-92 has been proved to be connected with the advancement of a series of human diseases, but the roles of miR-17-92 cluster in non-small cell lung cancer (NSCLC) have not been absolutely elaborated. Herein, we determined that miR-17-92 cluster were upregulated significantly in NSCLC tissues, and the cell proliferation, migration and cycle progression of NSCLC were also facilitated under the function of miR-17-92 cluster. Sprouty 4 (SPRY4) was a direct target of miR-92a, and its overexpression restrained the exacerbation of NSCLC induced by miR-92a. Furthermore, the tumor xenograft assay showed that miR-92a facilitated tumor growth by inhibiting the expression of SPRY4 and mediating Epithelial-Mesenchymal Transition (EMT) in vivo. Finally, we looked into the synergistic effects of miR-92a and miR-18a on NSCLC, and found that antagomiR-18a treatment arrested the tumor growth rate of xenografted mice markedly.

Pan-cancer analysis reveals homologous recombination deficiency score as a predictive marker for immunotherapy responders.

The immune context of the tumor microenvironment (TME) is critical for effective immunotherapy. Nonetheless, DNA-based biomarkers for the immune-sensitive TME and the identification of immune checkpoint inhibitor (ICI) responders are under-explored. This study aims to comprehensively landscape the homologous recombination deficiency (HRD) score, an emerging hallmark for tumor genome instability that triggers immune responsiveness across major cancer types, and to unveil their link to the TME and immunotherapeutic response. The HRD-associated genomic scars were characterized in 9088 tumor samples across 32 cancer types from TCGA. We evaluated the HRD score's performance in classifying ICI responders using an independent breast cancer cohort (GSE87049) and 11 in vivo murine mammary tumor models treated with anti-PD1/CTLA4 regimen (GSE124821). This study revealed a broad association between HRD-high genotype and neoantigenesis in the major cancer types including bladder cancer, breast cancer, head and neck squamous carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, ovarian cancer, and sarcoma. Tumors with high HRD score bears increased leukocyte infiltration and lymphocyte fraction and demonstrated immune-sensitive microenvironment. The tumor immune dysfunction and exclusion (TIDE) model further confirmed HRD score-high genotype as a potential predictor for ICI immunotherapy responders in breast cancer. In conclusion, tumors with high HRD score exhibit an immune-sensitive TME. The HRD-high genotype is a promising marker for identifying ICI therapy responders among breast cancer patients.

Identification of BANK1 polymorphisms by unlabelled probe high resolution melting: association with systemic lupus erythematosus susceptibility and autoantibody production in Han Chinese.

OBJECTIVES: The three functional SNPs of BANK1 (rs10516487, rs17266594 and rs3733197) have been shown to be associated with SLE in Caucasian populations. The aim of this study was to investigate whether the association of BANK1 polymorphisms with SLE could be replicated in a Chinese population and whether the autoantibody production is relevant to BANK1 polymorphisms. METHODS: Genotyping of three variants in BANK1 was carried out by unlabelled probe high resolution melting (HRM) assay in 264 SLE cases and 268 controls in a Chinese Han population living in Shanghai region. The genotype frequencies of the detected polymorphisms were analysed in relation to the production of autoantibodies (ANA, anti-dsDNA, anti-RNP, anti-SSA, anti-SSB and anti-Smith) in SLE patients. RESULTS: Samples with the target genotypes were accurately detected and easily distinguishable by unlabelled probe HRM assay. The frequencies of the rs10516487 C allele and the rs17266594 T allele were significantly increased compared with the controls (C allele: 88.6 vs 83.2%, P = 0.011; T allele: 88.3 vs 83.2%, P = 0.019). However, the frequencies of the rs3733197 G allele were not associated with SLE (G allele: 79.9 vs 79.1%, P = 0.741). The rs10516487 and rs17266594 polymorphisms were significantly associated with high-titre ANA (≥1 : 320) and production of anti-SSA antibodies in SLE patients compared with the control subjects. CONCLUSIONS: Genotyping using unlabelled probes is a rapid, accurate and cost-effective closed-tube method. This study implies that rs10516487 and rs17266594 polymorphisms might contribute to individual susceptibility to SLE and influence the ANA/SSA autoantibody response in SLE patients in Chinese population.