A Genetic Model for Central Chondrosarcoma Evolution Correlates with Patient Outcome

The treatment options for central chondrosarcoma are limited, and prognoses are generally unreliable. The presence and absence of mutations in IDH1, and IDH2 are defining events, and TERT mutations have been recently been associated with poor outcome. Despite this, molecular biomarkers are lacking. Here, analysing data from 356 patients, comprising results from whole genome sequencing (n=68), digital droplet PCR (n=346), and methylation arrays (n=57), we present a comprehensive genetic analysis of chondrosarcoma and suggest its clinical utility. Methylation profiles, TERT promoter mutations, genome doubling with prior haploidisation, and age at diagnosis of high grade, distinguish IDH1-mutant, IDH2-mutant and IDH wildtype tumours. The majority of IDH2-mutant tumours harbour TERT mutations, though a significant reduction in survival is only found in the less common mutational combination of IDH1 and TERT. We suggest that diagnostic testing for IDH1, IDH2 and TERT mutations could guide clinical monitoring and prognostication.

Low-Coverage Whole Genome Sequencing Using Laser Capture Microscopy with Combined Digital Droplet PCR: An Effective Tool to Study Copy Number and Kras Mutations in Early Lung Adenocarcinoma Development

Defining detailed genomic characterization of early tumor progression is critical to identifying key regulators and pathways in carcinogenesis as potentially druggable targets. In human lung cancer, work to characterize early cancer development has mainly focused on squamous cancer, as the earliest lesions are more proximal in the airways and often accessible by repeated bronchoscopy. Adenocarcinomas are typically located distally in the lung, limiting accessibility for biopsy of pre-malignant and early stages. Mouse lung cancer models recapitulate many human genomic features and provide a model for tumorigenesis with pre-malignant atypical adenomatous hyperplasia and in situ adenocarcinomas often developing contemporaneously within the same animal. Here, we combined tissue characterization and collection by laser capture microscopy (LCM) with digital droplet PCR (ddPCR) and low-coverage whole genome sequencing (LC-WGS). ddPCR can be used to identify specific missense mutations in Kras (Kirsten rat sarcoma viral oncogene homolog, here focused on Kras Q61) and estimate the percentage of mutation predominance. LC-WGS is a cost-effective method to infer localized copy number alterations (CNAs) across the genome using low-input DNA. Combining these methods, the histological stage of lung cancer can be correlated with appearance of Kras mutations and CNAs. The utility of this approach is adaptable to other mouse models of human cancer.

Loss of heterozygosity in CHEK2-associated breast cancer

Background. CHEK2-associated neoplasms account for a significant proportion of hereditary breast cancer (BC) in Russia. The phenomenon of somatic deletion of the normal allele of a gene affected by a hereditary mutation, or loss of heterozygosity (LOH), is a frequent mechanism of complete inactivation of the corresponding protein, which is realized during the development of hereditary breast carcinomas. The contribution of the LOH phenomenon to the pathogenesis of CHEK2-dependent tumors is poorly understood, and almost all available data concern only one type of mutations - CHEK2 1100delC. The aim of the study was to characterize the frequency of LOH in breast tumor tissues from carriers of the three types of CHEK2 alterations: CHEK2 1000delC, CHEK2 IVS2+1G>A, and CHEK2 del5395. Materials and methods. LOH analysis was performed in a group of 50 breast cancer cases from women carrying CHEK2 1000delC (n = 19), CHEK2 IVS2+1G>A (n = 12), and CHEK2 del5395 (n = 19) mutations. Detection of LOH was carried out using a combination of methods that directly analyze the mutation locus (allele-specific PCR, Sanger sequencing, digital droplet PCR), and assess the status of single nucleotide polymorphisms surrounding the CHEK2 gene (digital droplet PCR).Results. The frequency of the LOH phenomenon in the studied cohort reached 27/50 (54%). Loss of heterozygosity was observed in 10/19 (52.6%) CHEK2 1000delC-associated, 6/12 (50%) CHEK2 IVS2+1G>A-associated, and 11/19 (57.9%) CHEK2 del5395-associated tumors. In one carcinoma from a carrier of the CHEK2 IVS2+1G>A alteration, the loss of mutated allele was confirmed. The main clinical and pathological characteristics were compared between tumors with loss and retention of heterozygosity. This comparison did not reveal any significant differences.Conclusion. Loss of heterozygosity is observed in about half of breast carcinomas arising in CHEK2 mutation carriers; the frequency of this phenomenon does not differ between three types of CHEK2 genetic defects.

An Improved Reference Gene for Detection of “Candidatus Liberibacter asiaticus” Associated with Citrus Huanglongbing by qPCR and Digital Droplet PCR Assays

Citrus huanglongbing (HLB) disease associated with the ‘Candidatus Liberibacter asiaticus’ (CLas) bacterium has caused significant financial damage to many citrus industries. Large-scale pathogen surveys are routinely conducted in California to detect CLas early in the disease cycle by lab-based qPCR assays. We have developed an improved reference gene for the sensitive detection of CLas from plants in diagnostic duplex qPCR and analytical digital droplet PCR (ddPCR) assays. The mitochondrial cytochrome oxidase gene (COX), widely used as a reference, is not ideal because its high copy number can inhibit amplification of small quantities of target genes. In ddPCRs, oversaturation of droplets complicates data normalization and quantification. The variable copy numbers of COX gene in metabolically active young tissue, greenhouse plants, and citrus relatives suggest the need for a non-variable, nuclear, low copy, universal reference gene for analysis of HLB hosts. The single-copy nuclear gene, malate dehydrogenase (MDH), developed here as a reference gene, is amenable to data normalization, suitable for duplex qPCR and ddPCR assays. The sequence of MDH fragment selected is conserved in most HLB hosts in the taxonomic group Aurantioideae. This study emphasizes the need to develop standard guidelines for reference genes in DNA-based PCR assays.

Digital Droplet PCR (ddPCR) for Detection and Quantitation of Hepatitis Delt Virus

Background & Aims The prevalence of hepatitis delt virus (HDV) far exceeds our expected level, there remains a lack of reliable quantitative assays for HDV RNA detection. We sought to develop a new method based on digital droplet PCR (ddPCR) for HDV RNA quantitative detection. Methods With plasmid (pMD19T) containing HDV full-genome, we determined the method for ddPCR-based HDV RNA quantification. To compare various assays for HDV detection, 30 cases diagnosed hepatitis D and 14 controls were examined by ELISA, RT-PCR and ddPCR. 728 HBV-related patients including 182 chronic hepatitis B (CHB), 182 liver cirrhosis (LC), 182 hepatocellular carcinoma (HCC) and 182 liver failure (LF) were screened for HDV infection. Results The limit of detection of ddPCR for HDV is significantly low, which lower limit of detection (LLoD) and lower limit of quantitation (LLoQ) to be 5.51 copies/reaction (95% CI: 1.15–6.4*105) and 0.18 copies/reaction (95% CI: 0.0012151- 0.76436), respectively. Among the 44 samples, ELISA detected 30 cases positive for anti-HDV, ddPCR reported 24 samples and RT-PCR reported 10 samples positive for HDV RNA. Moreover, the positive rates of anti-HDV IgG were 1.1%, 3.3%, 2.7% and 7.1% in patients with CHB, LC, HCC, and LF; the detection rates of RT-PCR in HDV RNA were 0%, 16.67%, 15.4% and 20%, however, the detection rates of ddPCR were 0%, 33.33%, 30.77% and 60%. Conclusion We establish a high sensitivity and high specificity quantitative HDV RNA detection method based on ddPCR compared to RT-PCR. HBV-related end-stage liver disease, especially liver failure, are associated with a remarkably high rate of HDV infection.

Serum miR371 in testicular germ cell cancer before and after orchiectomy, assessed by digital-droplet PCR in a prospective study

MicroRNA-371a-3p (miR371) has been suggested as a sensitive biomarker in testicular germ cell cancer (TGCC). We aimed to compare miR371 with the classical biomarkers α-fetoprotein (AFP) and β-human chorionic gonadotropin (hCGβ). Overall, 180 patients were prospectively enrolled in the study, with serum samples collected before and after orchiectomy. We compared the use of digital droplet PCR (RT-ddPCR) with the quantitative PCR used by others for detection of miR371. The novel RT-ddPCR protocol showed high performance in detection of miR371 in serum samples. In the study cohort, miR371 was measured using RT-ddPCR. MiR371 detected CS1 of the seminoma and the non-seminoma sub-types with a sensitivity of 87% and 89%, respectively. The total sensitivity was 89%. After orchiectomy, miR371 levels declined in 154 of 159 TGCC cases. The ratio of miR371 pre- and post-orchiectomy was 20.5 in CS1 compared to 6.5 in systemic disease. AFP and hCGβ had sensitivities of 52% and 51% in the non-seminomas. MiR371 is a sensitive marker that performs better than the classical markers in all sub-types and clinical stages. Especially for the seminomas CS1, the high sensitivity of miR371 in detecting TGCC cells may have clinical implications.