European LeukemiaNet laboratory recommendations for the diagnosis and management of chronic myeloid leukemia

From the laboratory perspective, effective management of patients with chronic myeloid leukemia (CML) requires accurate diagnosis, assessment of prognostic markers, sequential assessment of levels of residual disease and investigation of possible reasons for resistance, relapse or progression. Our scientific and clinical knowledge underpinning these requirements continues to evolve, as do laboratory methods and technologies. The European LeukemiaNet convened an expert panel to critically consider the current status of genetic laboratory approaches to help diagnose and manage CML patients. Our recommendations focus on current best practice and highlight the strengths and pitfalls of commonly used laboratory tests

Unraveling the complexity of tyrosine kinase inhibitor-resistant populations by ultra-deep sequencing of the BCR-ABL kinase domain

In chronic myeloid leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia, tyrosine kinase inhibitor (TKI) therapy may select for drug-resistant BCR-ABL mutants. We used an ultra-deep sequencing (UDS) approach to resolve qualitatively and quantitatively the complexity of mutated populations surviving TKIs and to investigate their clonal structure and evolution over time in relation to therapeutic intervention. To this purpose, we performed a longitudinal analysis of 106 samples from 33 patients who had received sequential treatment with multiple TKIs and had experienced sequential relapses accompanied by selection of 1 or more TKI-resistant mutations. We found that conventional Sanger sequencing had misclassified or underestimated BCR-ABL mutation status in 55% of the samples, where mutations with 1% to 15% abundance were detected. A complex clonal texture was uncovered by clonal analysis of samples harboring multiple mutations and up to 13 different mutated populations were identified. The landscape of these mutated populations was found to be highly dynamic. The high degree of complexity uncovered by UDS indicates that conventional Sanger sequencing might be an inadequate tool to assess BCR-ABL kinase domain mutation status, which currently represents an important component of the therapeutic decision algorithms. Further evaluation of the clinical usefulness of UDS-based approaches is warranted

How Do Microbial Pathogens Make CENs?

This article does not have an abstract

Small chromosomes among Danish Candida glabrata isolates originated through different mechanisms

We analyzed 192 strains of the pathogenic yeast Candida glabrata from patients, mainly suffering from systemic infection, at Danish hospitals during 1985-1999. Our analysis showed that these strains were closely related but exhibited large karyotype polymorphism. Nine strains contained small chromosomes, which were smaller than 0.5 Mb. Regarding the year, patient and hospital, these C. glabrata strains had independent origin and the analyzed small chromosomes were structurally not related to each other (i.e. they contained different sets of genes). We suggest that at least two mechanisms could participate in their origin: (i) through a segmental duplication which covered the centromeric region, or (ii) by a translocation event moving a larger chromosome arm to another chromosome that leaves the centromere part with the shorter arm. The first type of small chromosomes carrying duplicated genes exhibited mitotic instability, while the second type, which contained the corresponding genes in only one copy in the genome, was mitotically stable. Apparently, in patients C. glabrata chromosomes are frequently reshuffled resulting in new genetic configurations, including appearance of small chromosomes, and some of these resulting "mutant" strains can have increased fitness in a certain patient "environment"

Deletion of Cryptococcus neoformans AIF Ortholog Promotes Chromosome Aneuploidy and Fluconazole-Resistance in a Metacaspase-Independent Manner

Apoptosis is a form of programmed cell death critical for development and homeostasis in multicellular organisms. Apoptosis-like cell death (ALCD) has been described in several fungi, including the opportunistic human pathogen Cryptococcus neoformans. In addition, capsular polysaccharides of C. neoformans are known to induce apoptosis in host immune cells, thereby contributing to its virulence. Our goals were to characterize the apoptotic signaling cascade in C. neoformans as well as its unique features compared to the host machinery to exploit the endogenous fungal apoptotic pathways as a novel antifungal strategy in the future. The dissection of apoptotic pathways revealed that apoptosis-inducing factor (Aif1) and metacaspases (Mca1 and Mca2) are independently required for ALCD in C. neoformans. We show that the apoptotic pathways are required for cell fusion and sporulation during mating, indicating that apoptosis may occur during sexual development. Previous studies showed that antifungal drugs induce ALCD in fungi and that C. neoformans adapts to high concentrations of the antifungal fluconazole (FLC) by acquisition of aneuploidy, especially duplication of chromosome 1 (Chr1). Disruption of aif1, but not the metacaspases, stimulates the emergence of aneuploid subpopulations with Chr1 disomy that are resistant to fluconazole (FLCR) in vitro and in vivo. FLCR isolates in the aif1 background are stable in the absence of the drug, while those in the wild-type background readily revert to FLC sensitivity. We propose that apoptosis orchestrated by Aif1 might eliminate aneuploid cells from the population and defects in this pathway contribute to the selection of aneuploid FLCR subpopulations during treatment. Aneuploid clinical isolates with disomies for chromosomes other than Chr1 exhibit reduced AIF1 expression, suggesting that inactivation of Aif1 might be a novel aneuploidy-tolerating mechanism in fungi that facilitates the selection of antifungal drug resistance

Global Analysis of the Evolution and Mechanism of Echinocandin Resistance in Candida glabrata

The evolution of drug resistance has a profound impact on human health. Candida glabrata is a leading human fungal pathogen that can rapidly evolve resistance to echinocandins, which target cell wall biosynthesis and are front-line therapeutics for Candida infections. Here, we provide the first global analysis of mutations accompanying the evolution of fungal drug resistance in a human host utilizing a series of C. glabrata isolates that evolved echinocandin resistance in a patient treated with the echinocandin caspofungin for recurring bloodstream candidemia. Whole genome sequencing identified a mutation in the drug target, FKS2, accompanying a major resistance increase, and 8 additional non-synonymous mutations. The FKS2-T1987C mutation was sufficient for echinocandin resistance, and associated with a fitness cost that was mitigated with further evolution, observed in vitro and in a murine model of systemic candidemia. A CDC6-A511G(K171E) mutation acquired before FKS2-T1987C(S663P), conferred a small resistance increase. Elevated dosage of CDC55, which acquired a C463T(P155S) mutation after FKS2-T1987C(S663P), ameliorated fitness. To discover strategies to abrogate echinocandin resistance, we focused on the molecular chaperone Hsp90 and downstream effector calcineurin. Genetic or pharmacological compromise of Hsp90 or calcineurin function reduced basal tolerance and resistance. Hsp90 and calcineurin were required for caspofungin-dependent FKS2 induction, providing a mechanism governing echinocandin resistance. A mitochondrial respiration-defective petite mutant in the series revealed that the petite phenotype does not confer echinocandin resistance, but renders strains refractory to synergy between echinocandins and Hsp90 or calcineurin inhibitors. The kidneys of mice infected with the petite mutant were sterile, while those infected with the HSP90-repressible strain had reduced fungal burden. We provide the first global view of mutations accompanying the evolution of fungal drug resistance in a human host, implicate the premier compensatory mutation mitigating the cost of echinocandin resistance, and suggest a new mechanism of echinocandin resistance with broad therapeutic potential

Association Between TAS2R38 Gene Polymorphisms and Colorectal Cancer Risk: A Case-Control Study in Two Independent Populations of Caucasian Origin

Molecular sensing in the lingual mucosa and in the gastro-intestinal tract play a role in the detection of ingested harmful drugs and toxins. Therefore, genetic polymorphisms affecting the capability of initiating these responses may be critical for the subsequent efficiency of avoiding and/or eliminating possible threats to the organism. By using a tagging approach in the region of Taste Receptor 2R38 (TAS2R38) gene, we investigated all the common genetic variation of this gene region in relation to colorectal cancer risk with a case-control study in a German population (709 controls and 602 cases) and in a Czech population (623 controls and 601 cases). We found that there were no significant associations between individual SNPs of the TAS2R38 gene and colorectal cancer in the Czech or in the German population, nor in the joint analysis. However, when we analyzed the diplotypes and the phenotypes we found that the non-taster group had an increased risk of colorectal cancer in comparison to the taster group. This association was borderline significant in the Czech population, (OR = 1.28, 95% CI 0.99–1.67; Pvalue = 0.058) and statistically significant in the German population (OR = 1.36, 95% CI 1.06–1.75; Pvalue = 0.016) and in the joint analysis (OR = 1.34, 95% CI 1.12–1.61; Pvalue = 0.001). In conclusion, we found a suggestive association between the human bitter tasting phenotype and the risk of CRC in two different populations of Caucasian origin