Detection of Emerging Ibrutinib Resistance by Flow Cytometry in Patients with Chronic Lymphocytic Leukemia

Purpose: Bruton’s tyrosine kinase inhibitor ibrutinib has revolutionized the treatment of chronic lymphocytic leukemia (CLL). Although ibrutinib is a highly effective drug, during the treatment acquired ibrutinib resistance may occur and its early detection is an important issue. Our aim was to investigate several phenotypic markers on CLL cells to reveal changes in their expression during ibrutinib treatment.Methods: In our study 28 (treatment naive, ibrutinib sensitive, clinically ibrutinib resistant) peripheral blood (PB), and 6 paired PB and bone marrow (BM) samples were examined. The expression of several surface markers (CD69, CD184, CD86, CD185, CD27) was assessed by flow cytometry in each sample. Furthermore, the presence of the BTKC481S resistance mutation was tested using digital droplet PCR. In addition, we investigated the changes of the phenotype of CLL cells during ibrutinib treatment in one patient with acquired ibrutinib resistance.Results: The expression of CD27 decreased during ibrutinib therapy but increased again at the onset of clinical resistance. Expressions of CD69 and CD86 were also elevated at the onset of clinical ibrutinib resistance. The expression of CD86 showed correlation between PB and BM samples. Relapsed cases with high CD86 expression were positive for BTKC481S mutation. Our prospective study showed that the increases in the expression of CD27, CD69 and CD86 were detectable up to several months before the onset of clinical resistance.Conclusion: Our research suggests that the flow cytometric measurements of certain markers, especially CD86, may predict development of ibrutinib resistance, however, confirmatory experiments are still required.

Osteosarcoma: novel prognostic biomarkers using circulating and cell-free tumour DNA

Osteosarcoma (OS) is the most common primary bone tumour in children and adolescents. Despite treatment with curative-intent, many patients die of this disease. Biomarkers for assessment of disease burden and prognoses for osteosarcoma are not available. Circulating-free (cfDNA) and -tumour DNA (ctDNA) are promising biomarkers for disease surveillance in several major cancer types, however only two such studies are reported for OS. In this combined discovery and validation study, we identified four novel methylation-based biomarkers in 171 OS tumours (test set) and comprehensively validated our findings in silico in two independent osteosarcoma sample datasets (n= 162, n=107) and experimentally using digital droplet PCR (ddPCR, n=20 OS tumours). Custom ddPCR assays for these biomarkers were able to detect ctDNA in 40% of pre-operative plasma samples (n=72). ctDNA was detected in 5/17 (29%) post-operative plasma samples from patients who experienced a subsequent relapse post-operatively. Both cfDNA levels and ctDNA detection independently correlated with overall survival, p=0.0015, p=0.0096, respectively. Combining both assays increased the prognostic value of the data. Our findings illustrate the utility of mutation-independent methylation-based markers, broadly applicable ctDNA assays for tumour surveillance and prognostication. This study lays the foundation for multi-institutional collaborative studies to explore the utility of plasma-derived biomarkers for predicting clinical outcome of OS.

An efficient and adaptable workflow for editing disease-relevant single nucleotide variants using CRISPR/Cas9

Single nucleotide variants are the commonest genetic alterations in the human genome. At least 60,000 have been reported to be associated with disease. The CRISPR/Cas9 system has transformed genetic research, making it possible to edit single nucleotides and study the function of genetic variants in vitro. While significant advances have improved the efficiency of CRISPR/Cas9, the editing of single nucleotides remains challenging. There are two major obstacles: low efficiency of accurate editing and the isolation of these cells from a pool of cells with other editing outcomes. We present data from 85 transfections of induced pluripotent stem cells and an immortalised cell line, comparing the effects of altering CRISPR/Cas9 design and experimental conditions on rates of single nucleotide substitution. We targeted variants in TP53, which predispose to several cancers, and in TBXT which is implicated in the pathogenesis of the bone cancer, chordoma. We describe a scalable and adaptable workflow for single nucleotide editing that incorporates contemporary techniques including Illumina MiSeq sequencing, TaqMan qPCR and digital droplet PCR for screening transfected cells as well as quality control steps to mitigate against common pitfalls. This workflow can be applied to CRISPR/Cas9 and other genome editing systems to maximise experimental efficiency.

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.