Reverse Transcription Can Critically Impact the Diagnostic Outcome of BCR::ABL1 Quantitative Real-Time RT-PCR.

Reverse transcriptases (RT) are essential tools in BCR::ABL1 fusion transcript monitoring in chronic myeloid leukemia (CML). The RT type and cDNA priming method may impair the stoichiometry of cDNA synthesis, thereby potentially introducing a bias in BCR::ABL1 qRT-PCR data. Using the Acrometrix™ BCR::ABL1 reference panel and 37 clinical specimens, we have comparatively investigated the performance of the RTs MLV and SuperScript IV with random hexamer vs. target-specific priming. Quantitative RT-PCR results identified the priming type and RT type as major factors for diagnostic data variation, mainly due to the different efficacies of processing BCR::ABL1 low-copy-numbers (<50) compared to GUSB or ABL1 high-copy targets. The impairment of SuperScript IV in processing low- and high-copy-number RNA targets equally was not reflected by the diagnostically relevant Log (BCR::ABL1/GUSB%) values. Therefore, the correct representation of housekeeping and BCR::ABL1 target genes should have priority when aiming at as high a number of housekeeping gene copies as possible. Our data suggest that for improving BCR::ABL1 assay sensitivity, increased RNA/cDNA amounts and the use of distinct RT/priming combinations are advantageous. However, for inter-laboratory harmonization, the proper conversion factor according to the CML international standard (IS) has to be reevaluated each time the grade of RT is changed.

Gene Expression Pattern of ESPL1, PTTG1 and PTTG1IP Can Potentially Predict Response to TKI First-Line Treatment of Patients with Newly Diagnosed CML.

The achievement of major molecular response (MMR, BCR::ABL1 ≤ 0.1% IS) within the first year of treatment with tyrosine kinase inhibitors (TKI) is a milestone in the therapeutic management of patients with newly diagnosed chronic myeloid leukemia (CML). We analyzed the predictive value of gene expression levels of ESPL1/Separase, PTTG1/Securin and PTTG1IP/Securin interacting protein for MMR achievement within 12 months. Relative expression levels (normalized to GUSB) of ESPL1, PTTG1 and PTTG1IP in white blood cells of patients (responders n = 46, non-responders n = 51) at the time of diagnosis were comparatively analyzed by qRT-PCR. 3D scatter plot analysis combined with a distance analysis performed with respect to a commonly calculated centroid center resulted in a trend to larger distances for non-responders compared to the responder cohort (p = 0.0187). Logistic regression and analysis of maximum likelihood estimates revealed a positive correlation of distance (cut-off) with non-achieving MMR within 12 months (p = 0.0388, odds ratio 1.479, 95%CI: 1.020 to 2.143). Thus, 10% of the tested non-responders (cut-off ≥ 5.9) could have been predicted already at the time of diagnosis. Future scoring of ESPL1, PTTG1 and PTTG1IP transcript levels may be a helpful tool in risk stratification of CML patients before initiation of TKI first = line treatment.

A new aberrantly spliced BCR-ABL1 transcript variant (e13a1) identified in routine monitoring using different quantitative reverse transcription polymerase chain reaction techniques in a patient with chronic myeloid leukemia.

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) of BCR-ABL1 transcript level is an essential part of routine disease monitoring in patients with chronic myeloid leukemia. One patient sample (e13a2 transcript detected by nested PCR) attracted attention by revealing an aberrantly spliced BCR-ABL1 transcript variant e13a1. The last 38 base pairs (bp) of BCR exon 13 were replaced by a 37 bp insertion of the ABL1 intron 1-2/exon 1 sequence. The rare aberrant BCR-ABL1 fusion transcript can cause discrepancies in molecular diagnostics. This scenario highlights the importance of an individual characterization of the BCR-ABL1 fusion sequence in case of unclear qRT-PCR results.

Evidence for Recombinant GRP78, CALR, PDIA3 and GPI as Mediators of Genetic Instability in Human CD34+ Cells.

Soluble factors released from irradiated human mesenchymal stromal cells (MSC) may induce genetic instability in human CD34+ cells, potentially mediating hematologic disorders. Recently, we identified four key proteins in the secretome of X-ray-irradiated MSC, among them three endoplasmic reticulum proteins, the 78 kDa glucose-related protein (GRP78), calreticulin (CALR), and protein disulfide-isomerase A3 (PDIA3), as well as the glycolytic enzyme glucose-6-phosphate isomerase (GPI). Here, we demonstrate that exposition of CD34+ cells to recombinant GRP78, CALR, PDIA3 and GPI induces substantial genetic instability. Increased numbers of γH2AX foci (p < 0.0001), centrosome anomalies (p = 0.1000) and aberrant metaphases (p = 0.0022) were detected in CD34+ cells upon incubation with these factors. Specifically, γH2AX foci were found to be induced 4−5-fold in response to any individual of the four factors, and centrosome anomalies by 3−4 fold compared to control medium, which contained none of the recombinant proteins. Aberrant metaphases, not seen in the context of control medium, were detected to a similar extent than centrosome anomalies across the four factors. Notably, the strongest effects were observed when all four factors were collectively provided. In summary, our data suggest that specific components of the secretome from irradiated MSC act as mediators of genetic instability in CD34+ cells, thereby possibly contributing to the pathogenesis of radiation-induced hematologic disorders beyond direct radiation-evoked DNA strand breaks.

Proteins Marking the Sequence of Genotoxic Signaling from Irradiated Mesenchymal Stromal Cells to CD34+ Cells.

Non-targeted effects (NTE) of ionizing radiation may initiate myeloid neoplasms (MN). Here, protein mediators (I) in irradiated human mesenchymal stromal cells (MSC) as the NTE source, (II) in MSC conditioned supernatant and (III) in human bone marrow CD34+ cells undergoing genotoxic NTE were investigated. Healthy sublethal irradiated MSC showed significantly increased levels of reactive oxygen species. These cells responded by increasing intracellular abundance of proteins involved in proteasomal degradation, protein translation, cytoskeleton dynamics, nucleocytoplasmic shuttling, and those with antioxidant activity. Among the increased proteins were THY1 and GNA11/14, which are signaling proteins with hitherto unknown functions in the radiation response and NTE. In the corresponding MSC conditioned medium, the three chaperones GRP78, CALR, and PDIA3 were increased. Together with GPI, these were the only four altered proteins, which were associated with the observed genotoxic NTE. Healthy CD34+ cells cultured in MSC conditioned medium suffered from more than a six-fold increase in γH2AX focal staining, indicative for DNA double-strand breaks, as well as numerical and structural chromosomal aberrations within three days. At this stage, five proteins were altered, among them IQGAP1, HMGB1, and PA2G4, which are involved in malign development. In summary, our data provide novel insights into three sequential steps of genotoxic signaling from irradiated MSC to CD34+ cells, implicating that induced NTE might initiate the development of MN.

Genotoxic Bystander Signals from Irradiated Human Mesenchymal Stromal Cells Mainly Localize in the 10-100 kDa Fraction of Conditioned Medium.

Genotoxic bystander signals released from irradiated human mesenchymal stromal cells (MSC) may induce radiation-induced bystander effects (RIBEs) in human hematopoietic stem and progenitor cells (HSPC), potentially causing leukemic transformation. Although the source of bystander signals is evident, the identification and characterization of these signals is challenging. Here, RIBEs were analyzed in human CD34+ cells cultured in distinct molecular size fractions of medium, conditioned by 2 Gy irradiated human MSC. Specifically, γH2AX foci (as a marker of DNA double-strand breaks) and chromosomal instability were evaluated in CD34+ cells grown in approximate (I) < 10 kDa, (II) 10-100 kDa and (III) > 100 kDa fractions of MSC conditioned medium and un-/fractionated control medium, respectively. Hitherto, significantly increased numbers of γH2AX foci (p = 0.0286) and aberrant metaphases (p = 0.0022) were detected in CD34+ cells grown in the (II) 10-100 kDa fraction (0.67 ± 0.10 γH2AX foci per CD34+ cell ∨ 3.8 ± 0.3 aberrant metaphases per CD34+ cell sample; mean ± SEM) when compared to (I) < 10 kDa (0.19 ± 0.01 ∨ 0.3 ± 0.2) or (III) > 100 kDa fractions (0.23 ± 0.04 ∨ 0.4 ± 0.4) or un-/fractionated control medium (0.12 ± 0.01 ∨ 0.1 ± 0.1). Furthermore, RIBEs disappeared after heat inactivation of medium at 75 °C. Taken together, our data suggest that RIBEs are mainly mediated by the heat-sensitive (II) 10-100 kDa fraction of MSC conditioned medium. We postulate proteins as RIBE mediators and in-depth proteome analyses to identify key bystander signals, which define targets for the development of next-generation anti-leukemic drugs.

Correction: High-risk additional chromosomal abnormalities at low blast counts herald death by CML.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

High-risk additional chromosomal abnormalities at low blast counts herald death by CML.

Blast crisis is one of the remaining challenges in chronic myeloid leukemia (CML). Whether additional chromosomal abnormalities (ACAs) enable an earlier recognition of imminent blastic proliferation and a timelier change of treatment is unknown. One thousand five hundred and ten imatinib-treated patients with Philadelphia-chromosome-positive (Ph+) CML randomized in CML-study IV were analyzed for ACA/Ph+ and blast increase. By impact on survival, ACAs were grouped into high risk (+8, +Ph, i(17q), +17, +19, +21, 3q26.2, 11q23, -7/7q abnormalities; complex) and low risk (all other). The presence of high- and low-risk ACAs was linked to six cohorts with different blast levels (1%, 5%, 10%, 15%, 20%, and 30%) in a Cox model. One hundred and twenty-three patients displayed ACA/Ph+ (8.1%), 91 were high risk. At low blast levels (1-15%), high-risk ACA showed an increased hazard to die compared to no ACA (ratios: 3.65 in blood; 6.12 in marrow) in contrast to low-risk ACA. No effect was observed at blast levels of 20-30%. Sixty-three patients with high-risk ACA (69%) died (n = 37) or were alive after progression or progression-related transplantation (n = 26). High-risk ACA at low blast counts identify end-phase CML earlier than current diagnostic systems. Mortality was lower with earlier treatment. Cytogenetic monitoring is indicated when signs of progression surface or response to therapy is unsatisfactory.

Separase activity distribution can be a marker of major molecular response and proliferation of CD34+ cells in TKI-treated chronic myeloid leukemia patients.

Separase, a cysteine endopeptidase, is a key player in mitotic sister chromatid separation, replication fork dynamics, and DNA repair. Aberrant expression and/or altered separase proteolytic activity are associated with aneuploidy, tumorigenesis, and disease progression. Since genomic instability and clonal evolution are hallmarks of progressing chronic myeloid leukemia (CML), we have comparatively examined separase proteolytic activity in TKI-treated chronic phase CML. Separase proteolytic activity was analyzed on single cell level in 88 clinical samples and in 14 healthy controls by a flow cytometric assay. In parallel, BCR-ABL1 gene expression and replication fork velocity were measured by qRT-PCR and DNA fiber assays, respectively. The separase activity distribution (SAD) value indicating the occurrence of MNCs with elevated separase proteolytic activity within samples was found to positively correlate with BCR-ABL1 gene expression levels and loss of MMR (relapse) throughout routine BCR-ABL1 monitoring. Analyses of CD34+ cells and MNCs fractionized by flow cytometric cell sorting according to their separase activity levels (H- and L-fractions) revealed that CD34+ cells with elevated separase activity levels (H-fractions) displayed enhanced proliferation/viability when compared with cells with regular (L-fraction) separase activity (mean 3.3-fold, p = 0.0011). BCR-ABL1 gene expression positivity prevailed in MNC H-fractions over L-fractions (42% vs. 8%, respectively). Moreover, expanding CD34+ cells of H-fractions showed decreased replication fork velocity compared with cells of L-fractions (p < 0.0001). Our data suggests an association between high separase activity, residual BCR-ABL1 gene expression, and enhanced proliferative capacity in hematopoietic cells within the leukemic niche of TKI-treated chronic phase CML.

DNA Damage and DNA Damage Response in Chronic Myeloid Leukemia.

DNA damage and alterations in the DNA damage response (DDR) are critical sources of genetic instability that might be involved in BCR-ABL1 kinase-mediated blastic transformation of chronic myeloid leukemia (CML). Here, increased DNA damage is detected by γH2AX foci analysis in peripheral blood mononuclear cells (PBMCs) of de novo untreated chronic phase (CP)-CML patients (n = 5; 2.5 γH2AX foci per PBMC ± 0.5) and blast phase (BP)-CML patients (n = 3; 4.4 γH2AX foci per PBMC ± 0.7) as well as CP-CML patients with loss of major molecular response (MMR) (n = 5; 1.8 γH2AX foci per PBMC ± 0.4) when compared to DNA damage in PBMC of healthy donors (n = 8; 1.0 γH2AX foci per PBMC ± 0.1) and CP-CML patients in deep molecular response or MMR (n = 26; 1.0 γH2AX foci per PBMC ± 0.1). Progressive activation of erroneous non-homologous end joining (NHEJ) repair mechanisms during blastic transformation in CML is indicated by abundant co-localization of γH2AX/53BP1 foci, while a decline of the DDR is suggested by defective expression of (p-)ATM and (p-)CHK2. In summary, our data provide evidence for the accumulation of DNA damage in the course of CML and suggest ongoing DNA damage, erroneous NHEJ repair mechanisms, and alterations in the DDR as critical mediators of blastic transformation in CML.

Antileukemic Efficacy in Vitro of Talazoparib and APE1 Inhibitor III Combined with Decitabine in Myeloid Malignancies.

Malignant hematopoietic cells of myelodysplastic syndromes (MDS)/chronic myelomonocytic leukemias (CMML) and acute myeloid leukemias (AML) may be vulnerable to inhibition of poly(ADP ribose) polymerase 1/2 (PARP1/2) and apurinic/apyrimidinic endonuclease 1 (APE1). PARP1/2 and APE1 are critical enzymes involved in single-strand break repair and base excision repair, respectively. Here, we investigated the cytotoxic efficacy of talazoparib and APE1 inhibitor III, inhibitors of PARP1/2 and APE1, in primary CD34+ MDS/CMML cell samples (n = 8; 4 MDS and 4 CMML) and in primary CD34+ or CD34- AML cell samples (n = 18) in comparison to healthy CD34+ donor cell samples (n = 8). Strikingly, talazoparib and APE1 inhibitor III demonstrated critical antileukemic efficacy in selected MDS/CMML and AML cell samples. Low doses of talazoparib and APE1 inhibitor III further increased the cytotoxic efficacy of decitabine in MDS/CMML and AML cells. Moreover, low doses of APE1 inhibitor III increased the cytotoxic efficacy of talazoparib in MDS/CMML and AML cells. In summary, talazoparib and APE1 inhibitor III demonstrated substantial antileukemic efficacy as single agents, in combination with decitabine, and combined with each other. Hence, our findings support further investigation of these agents in sophisticated clinical trials.

Diagnostic performance of the molecular BCR-ABL1 monitoring system may impact on inclusion of CML patients in stopping trials.

In chronic myeloid leukemia (CML), the duration of deep molecular response (MR) before treatment cessation (MR4 or deeper, corresponding to BCR-ABL1 ≤ 0.01% on the International Scale (IS)) is considered as a prognostic factor for treatment free remission in stopping trials. MR level determination is dependent on the sensitivity of the monitoring technique. Here, we compared a newly established TaqMan (TM) and our so far routinely used LightCycler (LC) quantitative reverse transcription (qRT)-PCR systems for their ability to achieve the best possible sensitivity in BCR-ABL1 monitoring. We have comparatively analyzed RNA samples from peripheral blood mononuclear cells of 92 randomly chosen patients with CML resembling major molecular remission (MMR) or better and of 128 CML patients after treatment cessation (EURO-SKI stopping trial). While our LC system utilized ABL1, the TM system is based on GUSB as reference gene. We observed 99% concordance with respect to achievement of MMR. However, we found that 34 of the 92 patients monitored by TM/GUSB were re-classified to the next inferior MR log level, especially when LC/ABL1-based results were borderline to thresholds. Thirteen patients BCR-ABL1 negative in LC/ABL1 became positive after TM/GUSB analysis. In the 128 patients included in the EURO-SKI trial identical molecular findings were achieved for 114 patients. However, 14 patients were re-classified to the next inferior log-level by the TM/GUSB combination. Eight of these patients relapsed after treatment cessation; two of them were re-classified from MR4 to MMR and therefore did not meet inclusion criteria anymore. In conclusion, we consider both methods as comparable and interchangeable in terms of achievement of MMR and of longitudinal evaluation of clinical courses. However, in LC/ABL1 negative samples, slightly enhanced TM/GUSB sensitivity may lead to inferior classification of clinical samples in the context of TFR.

Human Endogenous Retroviruses: Residues of Ancient Times Are Differentially Expressed in Crohn's Disease.

BACKGROUND: Eight percent of the human genome consists of human endogenous retroviruses (HERV). These genetic elements are remnants of ancient retroviral germ-line infections. Altered HERV expression is associated with several chronic inflammatory diseases. A physiological role of the HERV-derived proteins syncytin-1 and -2 has been described for the integrity of the human placental cell layer in terms of maintaining feto-maternal tolerance. The aim of this project was to investigate HERV expression in Crohn's disease (CD) with a further focus on syncytins in the gut. MATERIAL AND METHODS: Seventy-four ileal and colonic tissue samples of CD patients and healthy controls have been investigated for mRNA expression of major HERV groups by a comprehensive microarray screening. The most prominent differences have been validated by qRT-PCR. Immunohistochemistry (IHC), Western Blot (WB) and qRT-PCR were performed for syncytin-1 and -2. RESULTS: HERV microarray screening revealed a distinct expression profile in ileal and colonic tissue, as well as differential expression in CD compared to healthy controls. qRT-PCR validated differential expression of at least 3 HERV-groups in CD. qRT-PCR, IHC and WB showed a tissue-dependent diminished epithelial expression of syncytins in inflamed CD. CONCLUSION: For the first time, HERV expression has been comprehensively studied in the gut. Between CD and healthy controls we could show a tissue dependent differential HERV expression profile. Notably, we could show that syncytin-1 and -2 are expressed in the epithelial layer in ileal and colonic tissue samples, whereas their diminished tissue-dependent expression in inflamed CD might modulate inflammatory processes at the gut barrier.

Accumulation of DNA damage and alteration of the DNA damage response in monoclonal B-cell lymphocytosis and chronic lymphocytic leukemia.

Accumulation of DNA damage and alteration of the DNA damage response (DDR) are critical features of genetic instability that is presumed to be implicated in the pathogenesis of monoclonal B-cell lymphocytosis (MBL) and chronic lymphocytic leukemia (CLL). Here, we show increased numbers of γH2AX foci, a marker of DNA double-strand breaks (DSB), in CD19+ cells of CLL patients as compared to CD19+ cells of MBL patients and healthy individuals. Furthermore, numerous γH2AX/53BP1 foci in CLL cells suggest activation of error-prone non-homologous end-joining repair mechanisms. Signatures of DDR proteins further indicate alterations of the DDR in CLL in contrast to a largely regular activation in MBL and healthy controls. In summary, our results provide evidence for the stepwise accumulation of DNA damage in the progression of MBL towards CLL and suggest increased DNA damage, error-prone DNA repair and altered DDR signaling to be critical mechanisms of clonal evolution in MBL and CLL.

The benefit of quality control charts (QCC) for routine quantitative BCR-ABL1 monitoring in chronic myeloid leukemia.

Quantitative real-time polymerase chain reaction (qRT-PCR) is state of the art in molecular monitoring of minimal residual disease in chronic myeloid leukemia (CML). In this context, maintenance of assay fidelity and detection of technical inaccuracy are crucial. Beside multiple common negative controls for the clinical sample preparations, quality control charts (QCC) are a common validation tool to sustain high process quality by continuously recording of qRT-PCR control parameters. Here, we report on establishment and benefit of QCC in qRT-PCR-based CML diagnostics. The absolute quantification of BCR-ABL1 fusion transcripts in patient samples is based on coamplification of a serially diluted reference plasmid (pME-2). For QCC establishment the measured Ct values of each pME-2 standard dilution (4-400,000) of a test set resembling 21 sequential qRT-PCR experiments were recorded and statistically evaluated. Test set data were used for determination of warning limits (mean +/- 2-fold standard deviation) and control (intervention) limits (mean +/- 3-fold standard deviation) to allow rapid detection of defined out-of-control situations which may require intervention. We have retrospectively analyzed QCC data of 282 sequential qRT-PCR experiments (564 reactions). Data evaluation using QCCs revealed three out-of-control situations that required intervention like experiment repeats, renewal of pME-2 standards, replacement of reagents or personnel re-training. In conclusion, with minimal more effort and hands-on time QCC rank among the best tools to grant high quality and reproducibility in CML routine molecular diagnosis.

Effect of ABCG2, OCT1, and ABCB1 (MDR1) Gene Expression on Treatment-Free Remission in a EURO-SKI Subtrial.

INTRODUCTION: Tyrosine kinase inhibitors (TKIs) can safely be discontinued in chronic myeloid leukemia (CML) patients with sustained deep molecular response. ABCG2 (breast cancer resistance protein), OCT1 (organic cation transporter 1), and ABCB1 (multidrug resistance protein 1) gene products are known to play a crucial role in acquired pharmacogenetic TKI resistance. Their influence on treatment-free remission (TFR) has not yet been investigated. MATERIALS AND METHODS: RNA was isolated on the last day of TKI intake from peripheral blood leukocytes of 132 chronic phase CML patients who discontinued TKI treatment within the European Stop Tyrosine Kinase Inhibitor Study trial. Plasmid standards were designed including subgenic inserts of OCT1, ABCG2, and ABCB1 together with GUSB as reference gene. For expression analyses, quantitative real-time polymerase chain reaction was used. Multiple Cox regression analysis was performed. In addition, gene expression cutoffs for patient risk stratification were investigated. RESULTS: The TFR rate of 132 patients, 12 months after TKI discontinuation, was 54% (95% confidence interval [CI], 46%-62%). ABCG2 expression (‰) was retained as the only significant variable (P = .02; hazard ratio, 1.04; 95% CI, 1.01-1.07) in multiple Cox regression analysis. Only for the ABCG2 efflux transporter, a significant cutoff was found (P = .04). Patients with an ABCG2/GUSB transcript level >4.5‰ (n = 93) showed a 12-month TFR rate of 47% (95% CI, 37%-57%), whereas patients with low ABCG2 expression (≤4.5‰; n = 39) had a 12-month TFR rate of 72% (95% CI, 55%-82%). CONCLUSION: In this study, we investigated the effect of pharmacogenetics in the context of a CML treatment discontinuation trial. The transcript levels of the efflux transporter ABCG2 predicted TFR after TKI discontinuation.

Increased separase activity and occurrence of centrosome aberrations concur with transformation of MDS.

ESPL1/separase, a cysteine endopeptidase, is a key player in centrosome duplication and mitotic sister chromatid separation. Aberrant expression and/or altered separase proteolytic activity are associated with centrosome amplification, aneuploidy, tumorigenesis and disease progression. Since centrosome alterations are a common and early detectable feature in patients with myelodysplastic syndrome (MDS) and cytogenetic aberrations play an important role in disease risk stratification, we examined separase activity on single cell level in 67 bone marrow samples obtained from patients with MDS, secondary acute myeloid leukemia (sAML), de novo acute myeloid leukemia (AML) and healthy controls by a flow cytometric separase activity assay. The separase activity distribution (SAD) value, a calculated measure for the occurrence of cells with prominent separase activity within the analyzed sample, was tested for correlation with the centrosome, karyotype and gene mutation status. We found higher SAD values in bone marrow cells of sAML patients than in corresponding cells of MDS patients. This concurred with an increased incidence of aberrant centrosome phenotypes in sAML vs. MDS samples. No correlation was found between SAD values and the karyotype/gene mutation status. During follow-up of four MDS patients we observed increasing SAD values after transformation to sAML, in two patients SAD values decreased during azacitidine therapy. Cell culture experiments employing MDS-L cells as an in vitro model of MDS revealed that treatment with rigosertib, a PLK1 inhibitor and therapeutic drug known to induce G2/M arrest, results in decreased SAD values. In conclusion, the appearance of cells with unusual high separase activity levels, as indicated by increased SAD values, concurs with the transformation of MDS to sAML and may reflect separase dysregulation potentially contributing to clonal evolution during MDS progression. Separase activity measurement may therefore be useful as a novel additional molecular marker for disease monitoring.

Leukocyte DNA damage after reduced and conventional absorbed radiation doses using 3rd generation dual-source CT technology.

PURPOSE: Computed tomography (CT) scans are an important source of ionizing irradiation (IR) in medicine that can induce a variety of DNA damage in human tissues. With technological improvements CT scans at reduced absorbed doses became feasible presumably lowering genotoxic side effects. MATERIALS AND METHODS: For measuring DNA damage we performed γH2AX foci microscopy in peripheral blood mononuclear cells (PBMC) after exposure to reduced and conventional absorbed radiation doses using 3rd generation dual-source CT (DSCT) technology. RESULTS: CT scans performed at reduced absorbed doses of 3 mGy induced significant lower levels (p < 0.0001) of DNA damage (0.05 focus per cell ± 0.01 [mean ± standard error of mean]) at 5 min after IR compared to conventional absorbed doses of 15 mGy (0.30 focus per cell ± 0.03). With ongoing DNA repair background γH2AX foci levels (0.05 focus per cell) were approached at 24 h after CT with both protocols. CONCLUSION: Our results provide evidence that reduced absorbed doses mediated by adjusted tube current in 3rd generation DSCT induce lower levels of DNA damage in PBMC compared to conventional absorbed doses suggesting a lower genotoxic risk for state-of-the-art tube current reduced CT protocols.

c-MYB is a transcriptional regulator of ESPL1/Separase in BCR-ABL-positive chronic myeloid leukemia.

BACKGROUND: Genomic instability and clonal evolution are hallmarks of progressing chronic myeloid leukemia (CML). Recently, we have shown that clonal evolution and blast crisis correlate with altered expression and activity of Separase, a cysteine endopeptidase that is a mitotic key player in chromosomal segregation and centriole duplication. Hyperactivation of Separase in human hematopoietic cells has been linked to a feedback mechanism that posttranslationally stimulates Separase proteolytic activity after imatinib therapy-induced reduction of Separase protein levels. METHODS AND RESULTS: In search for potential therapy-responsive transcriptional mechanisms we have investigated the role of the transcription factor c-MYB for Separase expression in CML cell lines (LAMA-84, K562, BV-173) and in clinical samples. Quantitative RT-PCR and Western blot immunostaining experiments revealed that c-MYB expression levels are decreased in an imatinib-dependent manner and positively correlate with Separase expression levels in cell lines and in clinical CML samples. RNA silencing of c-MYB expression in CML cell lines resulted in reduced Separase protein levels. Gelshift and ChIP assays confirmed that c-MYB binds to a putative c-MYB binding sequence located within the ESPL1 promoter. CONCLUSIONS: Our data suggest that ESPL1/Separase is a regulatory target of c-MYB. Therefore, c-MYB, known to be required for BCR-ABL-dependent transformation of hematopoietic progenitors and leukemogenesis, may also control the Separase-dependent fidelity of mitotic chromosomal segregation and centriole duplication essential for maintenance of genomic stability.

Impact of unbalanced minor route versus major route karyotypes at diagnosis on prognosis of CML.

Major route additional cytogenetic aberrations (ACA) at diagnosis of chronic myeloid leukaemia (CML) indicate an increased risk of progression and shorter survival. Since major route ACA are almost always unbalanced, it is unclear whether other unbalanced ACA at diagnosis also confer an unfavourable prognosis. On the basis of 1348 Philadelphia chromosome-positive chronic phase patients of the randomized CML study IV, we examined the impact of unbalanced minor route ACA at diagnosis versus major route ACA on prognosis. At diagnosis, 1175 patients (87.2 %) had a translocation t(9;22)(q34;q11) and 74 (5.5 %) a variant translocation t(v;22) only, while a loss of the Y chromosome (-Y) was present in addition in 44 (3.3 %), balanced or unbalanced minor route ACA each in 17 (1.3 %) and major route ACA in 21 (1.6 %) cases. Patients with unbalanced minor route ACA had no significantly different cumulative incidences of complete cytogenetic remission or major molecular remission and no significantly different progression-free survival (PFS) or overall survival (OS) than patients with t(9;22), t(v;22), -Y and balanced minor route karyotypes. In contrast, patients with major route ACA had a shorter OS and PFS than all other groups (all pairwise comparisons to each of the other groups: p ≤ 0.015). Five-year survival probabilities were for t(9;22) 91.4 % (95 % CI 89.5-93.1), t(v; 22) 87 % (77.2-94.3), -Y 89.0 % (76.7-97.0), balanced 100 %, unbalanced minor route 92.3 % (72.4-100) and major route 52.2 % (28.2-75.5). We conclude that only major route, but not balanced or unbalanced minor route ACA at diagnosis, has a negative impact on prognosis of CML.