Exosomal miR-200c and miR-141 as cerebrospinal fluid biopsy biomarkers for the response to chemotherapy in primary central nervous system lymphoma.

BACKGROUND: To improve early diagnosis and chemotherapy efficacy monitoring in primary central nervous system lymphoma (PCNSL), cerebrospinal fluid (CSF) exosomal microRNA (miRNA) studies were performed. METHOD: Small RNA sequencing was performed to identify candidate exosomal miRNAs as CSF biopsy biomarkers from two patients with de novo PCNSL and two patients in remission after chemotherapy. miR-200c and miR-141 expression in CSF exosomes was further validated using relative quantitative real-time polymerase chain reaction in patients with PCNSL (n = 20), patients with other neurological diseases (n = 10), and normal subjects (n = 10). Receiver operating characteristic (ROC) curve analyses of miR-200c and miR-141 in the diagnosis and prediction of chemotherapy efficacy in PCNSL were performed in patients treated with methotrexate. Additionally, bioinformatics tools were utilized to predict the potential targets of miR-200c and miR-141. RESULTS: Exosomal miR-200c and miR-141 levels in CSF from patients with PCNSL were significantly lower than those in control subjects. Importantly, miR-200c and miR-141 were upregulated in patients with PCNSL after chemotherapy (P = 0.002). There was a significant correlation between the levels of miR-141 and IL-10 in CSF (P = 0.04). The combination of miR-200c and miR-141 yielded an area under the ROC curve of 0.761 for distinguishing PCNSL with sensitivity and specificity of 60.0% and 96.7%, respectively. The potential target genes of miR-200c and miR-141 in PCNSL included ATP1B3, DYNC1H1, MATR3, NUCKS1, ZNF638, NUDT4, RCN2, GNPDA1, ZBTB38, and DOLK. CONCLUSION: Collectively, miR-200c and miR-141 are likely to be upregulated in CSF exosomes after chemotherapy in patients with PCNSL, highlighting their potential as reliable liquid biopsy biomarkers for PCNSL diagnosis and chemotherapy efficacy monitoring.

Single-cell transcriptome analysis of diffuse large B cells in cerebrospinal fluid of central nervous system lymphoma.

Diffuse large B cells in the cerebrospinal fluid (CSF-DLBCs) have offered great promise for the diagnostics and therapeutics of central nervous system lymphoma (CNSL) leptomeningeal involvement. To explore the phenotypic states of CSF-DLBCs, we analyzed the transcriptomes of more than one thousand CSF-DLBCs from six patients with CNSL diffuse large B-cell lymphoma (DLBCL) using Smart-seq2 single-cell RNA sequencing. CSF-DLBCs were defined based on abundant expression of B-cell markers, the active cell proliferation and energy metabolism properties, and immunoglobulin light chain restriction. We identified inherent heterogeneity of CSF-DLBCs, which were mainly manifested in cell cycle state, cancer-testis antigen expression, and classification based on single-cell germinal center B-cell signature. In addition, the 16 upregulated genes in CSF-DLBCs compared to various normal B cells showed great possibility in the homing effect of the CNS-DLBCL for the leptomeninges. Our results will provide insight into the mechanism research and diagnostic direction of CNSL-DLBCL leptomeningeal involvement.

Circulating tumor cell characterization of lung cancer brain metastases in the cerebrospinal fluid through single-cell transcriptome analysis.

BACKGROUND: Brain metastases explain the majority of mortality associated with lung cancer, which is the leading cause of cancer death. Cytology analysis of the cerebrospinal fluid (CSF) remains the diagnostic gold standard, however, the circulating tumor cells (CTCs) in CSF (CSF-CTCs) are not well defined at the molecular and transcriptome levels. METHODS: We established an effective CSF-CTCs collection procedure and isolated individual CSF cells from five lung adenocarcinoma leptomeningeal metastases (LUAD-LM) patients and three controls. Three thousand seven hundred ninety-two single-cell transcriptomes were sequenced, and single-cell RNA sequencing (scRNA-seq) gene expression analysis was used to perform a comprehensive characterization of CSF cells. RESULTS: Through clustering and expression analysis, we defined CSF-CTCs at the transcriptome level based on epithelial markers, proliferation markers, and genes with lung origin. The metastatic-CTC signature genes are enriched for metabolic pathway and cell adhesion molecule categories, which are crucial for the survival and metastases of tumor cells. We discovered substantial heterogeneity in patient CSF-CTCs. We quantified the degree of heterogeneity and found significantly greater among-patient heterogeneity compared to among-cell heterogeneity within a patient. This observation could be explained by spatial heterogeneity of metastatic sites, cell-cycle gene, and cancer-testis antigen (CTA) expression profiles as well as the proportion of CTCs displaying mesenchymal and cancer stem cell properties. In addition, our CSF-CTCs transcriptome profiling allowed us to determine the biomarkers during the progression of an LM patient with cancer of unknown primary site (CUP). CONCLUSIONS: Our results will provide candidate genes for an RNA-based digital detection of CSF-CTCs from LUAD-LM and CUP-LM cases, and shed light on the therapy and mechanism of LUAD-LM.

A triplex probe-based TaqMan qPCR assay for Calreticulin type I and II mutation detection.

BACKGROUND: Calreticulin (CALR) exon 9 frameshift mutations have recently been identified in 30-40% of patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF) without JAK2 or MPL mutations. We aimed to develop a qPCR assay to screen type I and II mutations of CALR. METHODS: Three different fluorescent-labeled hydrolysis probes and one pair of primers in a closed-tube system were developed to detect CALR type I and II mutations and distinguish them from wild-type. The sensitivity and specificity were validated using TA-cloning plasmids containing CALR wild-type and type I and II mutants, respectively. Fifty-nine ET and PMF specimens were screened by TaqMan qPCR and sequenced by Sanger sequencing. For intra-assay validation, 20 replicates of the assay were performed with each sample. For inter-assay validation, four replications of each sample were carried out and repeated continuously for 5 days. RESULTS: We found that triplex probe-based TaqMan qPCR was reliable in detecting CALR type I and II mutants within DNA that was diluted to 1% of total DNA with the wild-type DNA as background. In 59 patient specimens, six of the observed mutations of CALR were type I and five were type II. Genotyping results obtained from TaqMan qPCR were 100% concordant with Sanger sequencing. The intra- and inter-assay CVs of TaqMan qPCR were less than 3%, respectively. CONCLUSIONS: Triplex probe-based TaqMan qPCR is an accurate and sensitive method for screening ET or PMF patients with type I and II mutations in CALR.

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.

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.

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.

Association of an intronic SNP of SLC2A9 gene with serum uric acid levels in the Chinese male Han population by high-resolution melting method.

SLC2A9 is a novel identified urate transporter influencing uric acid metabolism. It has been suggested that the single-nucleotide polymorphisms in SLC2A9 may affect the serum UA levels. The present study was designed to investigate rs6855911 polymorphism in intron 7 of SLC2A9 in a total of 372 Chinese male subjects. We examined 166 gout patients, as well as 206 healthy male volunteers in this study. DNA was purified from peripheral blood, and the rs6855911 polymorphism was evaluated using high-resolution melting (HRM) analysis and direct sequencing. Demographic and clinical data obtained from the patients and controls among the genotype groups were analyzed. A/A and A/G genotypes were unambiguously distinguished with HRM technology. The occurrence of the homozygous type (G/G) was completely absent among the study population. The prevalence of the A/A and A/G genotype was 96.0% and 4.0%, respectively. Genotyping based on HRM was fully concordant with sequencing. The G allele frequency was significantly higher in the low-uric-acid group than in the high-uric-acid group. The genotype distribution and allele frequencies were not significantly different between gout and control subjects (p = 0.04). However, serum uric acid levels in the A/G genotype subjects were significantly lower than those with the A/A genotypes (p < 0.01). Rapid and accurate genotyping analysis of SLC2A9 can be done with HRM. The polymorphism rs6855911 in SLC2A9 may be a genetic marker to assess risk of hyperuricemia among Chinese male Han population.

Rapid determination of AKAP12 promoter methylation levels in peripheral blood using methylation-sensitive high resolution melting (MS-HRM) analysis: application in colorectal cancer.

BACKGROUND: Colorectal cancer is the third most common form of cancer and hypermethylation has been shown to increase the risk of developing this disease. DNA hypermethylation in the A kinase anchor protein 12 (AKAP12/Gravin) promoter region and the accompanied underexpression of it has been noted in a variety of human cancers. METHODS: We applied methylation-specific high resolution melting (MS-HRM) technology to detect quantitatively A kinase anchor protein 12 (AKAP12/Gravin) methylation in peripheral blood from 100 colorectal cancer patients and 50 healthy volunteers and in 3 colorectal cancer cell lines. RESULTS: In this study 48 of the 100 colorectal cancer samples (48%) were found to be methylated at the AKAP12 promoter region. AKAP12 methylation was significantly higher in the colorectal cancer samples with differentiation (p=0.03). We also compared the results generated by MS-HRM with a traditional methylation-specific PCR (MSP) assay. We found that intra-assay variability ranged from 6.14 to 9.90% and inter-assay variability ranged from 14.5 to 17.2%. The AKAP12 MS-HRM assay was able to reproducibly detect 1% methylated DNA, whereas the MSP method was unable to detect less than 5% methylation. CONCLUSIONS: We demonstrate the utility of quantitative AKAP12 MS-HRM analysis of promoter methylation in peripheral blood samples. AKAP12 MS-HRM quantitative methods with excellent detection capabilities have many promising applications in the research and diagnosis of colorectal cancer.