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

AST-487 Inhibits RET Kinase Driven TERT Expression in Bladder Cancer

Mutations in the promoter of the human Telomerase Reverse Transcriptase (hTERT) gene are common and associated with its elevated expression in bladder cancer, melanoma, and glioblastoma. Though these mutations and TERT overexpression are associated with aggressive disease and poor outcome, an incomplete understanding of mutant TERT regulation limits treatment options directed at this gene. Herein, we unravel a signaling pathway that leads to upregulated hTERT expression resulting from the −124 bp promoter mutation, the most frequent variant across human cancer. We employed engineered bladder cancer cells that harbor a GFP insertion at the TSS region on −124 hTERT promoter for high-content screening drug discovery using a focused library of ~800 kinase inhibitors. Studies using in vitro and in vivo models prioritized AST-487, an inhibitor of the wild-type, and mutant RET (rearranged during transfection) proto-oncogene as a novel drug inhibitor of both wild-type and mutant promoter-driven hTERT expression. We also identified the RET kinase pathway, targeted by AST-487, as a novel regulator of mutant hTERT promoter-driven transcription in bladder cancer cells. Collectively, our work provides new potential precision medicine approaches for cancer patients with upregulated hTERT expression, perhaps, especially those harboring mutations in both the RET gene and the hTERT promoter, such as in thyroid cancer

High-Content Drug Discovery Targeting Molecular Bladder Cancer Subtypes

Molecular subtypes of muscle-invasive bladder cancer (MIBC) display differential survival and drug sensitivities in clinical trials. To date, they have not been used as a paradigm for phenotypic drug discovery. This study aimed to discover novel subtype-stratified therapy approaches based on high-content screening (HCS) drug discovery. Transcriptome expression data of CCLE and BLA-40 cell lines were used for molecular subtype assignment in basal, luminal, and mesenchymal-like cell lines. Two independent HCSs, using focused compound libraries, were conducted to identify subtype-specific drug leads. We correlated lead drug sensitivity data with functional genomics, regulon analysis, and in-vitro drug response-based enrichment analysis. The basal MIBC subtype displayed sensitivity to HDAC and CHK inhibitors, while the luminal subtype was sensitive to MDM2 inhibitors. The mesenchymal-like cell lines were exclusively sensitive to the ITGAV inhibitor SB273005. The role of integrins within this mesenchymal-like MIBC subtype was confirmed via its regulon activity and gene essentiality based on CRISPR–Cas9 knock-out data. Patients with high ITGAV expression showed a significant decrease in the median overall survival. Phenotypic high-content drug screens based on bladder cancer cell lines provide rationales for novel stratified therapeutic approaches as a framework for further prospective validation in clinical trials

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

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

Inter-experimental variance for the seven pME-2 standard dilutions.

<p>Calculated were performed from 387 consecutive PCR experiments. Numbers on the x-axis correspond to the amounts of pME-2 plasmid molecules that serve as target within the respective PCR reactions. Decreasing numbers of target molecules inversely correlate with standard error of mean (SEM given in Ct values) due to pipetting inaccuracy.</p

The benefit of quality control charts (QCC) for routine quantitative <i>BCR-ABL1</i> monitoring in chronic myeloid leukemia - Fig 2

<p><b>Visualization of decay in the life span of five consecutive working solutions (A-E) of the pME-2 standard dilution 4000.</b> The Ct drift due to molecule degradation is shown by the increasing Ct values and is visualizes by the linear regression lines (red). For noise reduction and a more compact image size data point calculations were based on the mean of duplex PCR reactions.</p

Decay of plasmid working solutions.

<p>Decay of plasmid working solutions.</p

一次性レイノー現象の宿主・環境要因に関する研究 第1編 山間部と平野部における一般住民のレイノー現象の有症率の比較 第2編 レイノー現象発作と気温との関係 - 山間部と平野部におけるレイノー現象有症者の長期観察 - 第3編 健常者およびレイノー現象有症者における抗核抗体

第1編 山間部と平野部における一般住民のレイノー現象の有症率の比較第2編 レイノー現象発作と気温との関係 - 山間部と平野部におけるレイノー現象有症者の長期観察 -第3編 健常者およびレイノー現象有症者における抗核抗