Colorimetric assessment of BCR-ABL1 transcripts in clinical samples via gold nanoprobes.

Gold nanoparticles functionalized with thiolated oligonucleotides (Au-nanoprobes) have been used in a range of applications for the detection of bioanalytes of interest, from ions to proteins and DNA targets. These detection strategies are based on the unique optical properties of gold nanoparticles, in particular, the intense color that is subject to modulation by modification of the medium dieletric. Au-nanoprobes have been applied for the detection and characterization of specific DNA sequences of interest, namely pathogens and disease biomarkers. Nevertheless, despite its relevance, only a few reports exist on the detection of RNA targets. Among these strategies, the colorimetric detection of DNA has been proven to work for several different targets in controlled samples but demonstration in real clinical bioanalysis has been elusive. Here, we used a colorimetric method based on Au-nanoprobes for the direct detection of the e14a2 BCR-ABL fusion transcript in myeloid leukemia patient samples without the need for retro-transcription. Au-nanoprobes directly assessed total RNA from 38 clinical samples, and results were validated against reverse transcription-nested polymerase chain reaction (RT-nested PCR) and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The colorimetric Au-nanoprobe assay is a simple yet reliable strategy to scrutinize myeloid leukemia patients at diagnosis and evaluate progression, with obvious advantages in terms of time and cost, particularly in low- to medium-income countries where molecular screening is not routinely feasible. Graphical abstract Gold nanoprobe for colorimetric detection of BCR-ABL1 fusion transcripts originating from the Philadelphia chromosome.

What is the role of JAK2(V617F) mutation in leukemic transformation of myeloproliferative neoplasms?

BACKGROUND AND OBJECTIVES: The role of the Janus kinase 2 V617F (JAK2(V617F)) mutation in the pathogenesis of the various BCR-ABL1-negative myeloproliferative neoplasms (MPNs) remains unclear. Its significance in leukemic transformation is a matter of even greater controversy. The aim of this study was to evaluate both the JAK2(V617F) mutational status of the rare cases in which blast crisis occurred in our institution and the response after intensive treatment. MATERIALS AND METHODS: Between 1999 and 2009, 778 patients received diagnoses of BCR-ABL1-negative MPNs in our center (395 polycythemia vera, 329 essential thrombocythemia, and 45 primary myelofibrosis cases, as well as 9 MPN cases not otherwise classifiable). Of these patients, 7 developed leukemic transformation. The genotyping of the JAK2(V617F) mutation was performed by the amplification-refractory mutation system. RESULTS: Six of the 7 patients were tested for JAK2(V617F) in the chronic phase of their disease, and 3 of these patients were positive for JAK2(V617F). These patients, 2 with polycythemia vera and 1 with essential thrombocythemia, also harbored JAK2(V617F) in the heterozygous state during blast crisis and even after intensive treatment in one of these patients. The other cases that evolved to blast crisis did not harbor the JAK2(V617F) mutation before and after transformation. All 7 patients died despite conventional or supportive treatment. CONCLUSIONS: The transformation of MPNs into acute leukemia is by itself a very rare phenomenon, and so is the persistence of the JAK2(V617F) mutation after blast crisis. In our series, all JAK2(V617F)-positive patients remained positive for this mutation after leukemic transformation, although in the heterozygous state, suggesting that JAK2(V617F) is not essential for transformation in these cases. The fact that all JAK2(V617F)-negative cases remained negative after blast crisis reinforces the theory that other molecular event(s) may play a role in the clonal heterogeneity of MPNs. Owing to the poor outcome of acute myeloid leukemia secondary to MPN, patients should be included in clinical trials of the novel JAK2 inhibitors.

A novel BCR-ABL1 mutation in a patient with Philadelphia chromosome-positive B-cell acute lymphoblastic leukemia.

Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) represents the most common genetic subtype of adult ALL (20%-30%) and accounts for approximately 50% of all cases in the elderly. It has been considered the subgroup of ALL with the worst outcome. The introduction of tyrosine kinase inhibitors (TKIs) allows complete hematologic remission virtually in all patients, with improved disease-free survival and overall survival. Nevertheless, the emergence of resistant mutations in BCR-ABL1 may require different TKI strategies to overcome the patient's resistance and disease relapse. Here, we report a Ph+B-ALL case with persistent minimal residual disease (MRD) after treatment with dasatinib. The patient expressed the P190BCR-ABL1 isoform and a novel BCR-ABL1 mutation, p.Y440C. The latter is in the C-terminal lobe of the kinase domain, which likely induces deviations in the protein structure and activity and destabilizes its inactive conformation. The treatment was substituted by bosutinib, which binds to the active conformation of the protein, prior to allogeneic bone marrow transplant to overcome the lack of a complete response to dasatinib. These findings strengthen the importance of BCR-ABL1 mutational screening in Ph+ patients, particularly for those who do not achieve complete molecular remission.

A novel membrane-bound respiratory complex from Desulfovibrio desulfuricans ATCC 27774.

In the anaerobic respiration of sulfate, performed by sulfate-reducing prokaryotes, reduction of the terminal electron acceptor takes place in the cytoplasm. The membrane-associated electron transport chain that feeds electrons to the cytoplasmic reductases is still very poorly characterized. In this study we report the isolation and characterization of a novel membrane-bound redox complex from Desulfovibrio desulfuricans ATCC 27774. This complex is formed by three subunits, and contains two hemes b, two FAD groups and several iron-sulfur centers. The two hemes b are low-spin, with macroscopic redox potentials of +75 and -20 mV at pH 7.6. Both hemes are reduced by menadiol, a menaquinone analogue, indicating a function for this complex in the respiratory electron-transport chain. EPR studies of the as-isolated and dithionite-reduced complex support the presence of a [3Fe-4S](1+/0) center and at least four [4Fe-4S](2+/1+) centers. Cloning of the genes coding for the complex subunits revealed that they form a putative transcription unit and have homology to subunits of heterodisulfide reductases (Hdr). The first and second genes code for soluble proteins that have homology to HdrA, whereas the third gene codes for a novel type of membrane-associated protein that contains both a hydrophobic domain with homology to the heme b protein HdrE and a hydrophilic domain with homology to the iron-sulfur protein HdrC. Homologous operons are found in the genomes of other sulfate-reducing organisms and in the genome of the green-sulfur bacterium Chlorobium tepidum TLS. The isolated complex is the first example of a new family of respiratory complexes present in anaerobic prokaryotes.

A novel iron centre in the split-Soret cytochrome c from Desulfovibrio desulfuricans ATCC 27774.

The facultative sulfate/nitrate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774 harbours a split-Soret cytochrome c. This cytochrome is a homodimeric protein, having two bis-histidinyl c-type haems per monomer. It has an unique architecture at the haem domain: each haem has one of the coordinating histidines provided by the other monomer, and in each monomer the haems are parallel to each other, almost in van der Waals contact. This work reports the cloning and sequencing of the gene encoding for this cytochrome and shows, by transcriptional analysis, that it is more expressed in nitrate-grown cells than in sulfate-grown ones. In addition, the gene-deduced amino acid sequence revealed two new cysteine residues that could be involved in the binding of a non-haem iron centre. Indeed, the presence of a novel type of an iron-sulfur centre (possibly of the [2Fe-2S] type) was demonstrated by EPR spectroscopy, and putative models for its localization and structure in the cytochrome molecule are proposed on the basis of the so-far-known 3D crystallographic structure of the aerobically purified split-Soret cytochrome, which lacks this centre.