Patched homolog 1 gene (PTCH1) expression and the ratio of PTCH1 to Smoothened (SMO) expression have been proposed as prognostic markers of the response of chronic myeloid leukemia (CML) patients to ...imatinib. We compared these measurements in a realistic cohort of 101 patients with CML in chronic phase (CP) using a simplified qPCR method, and confirmed the prognostic power of each in a competing risk analysis. Gene expression levels were measured in peripheral blood samples at diagnosis. The PTCH1/SMO ratio did not improve PTCH1 prognostic power (area under the receiver operating characteristic curve 0.71 vs. 0.72). In order to reduce the number of genes to be analyzed, PTCH1 was the selected measurement. High and low PTCH1 expression groups had significantly different cumulative incidences of imatinib failure (IF), which was defined as discontinuation of imatinib due to lack of efficacy (5% vs. 25% at 4 years, P = 0.013), probabilities of achieving a major molecular response (81% vs. 53% at first year, P = 0.02), and proportions of early molecular failure (14% vs. 43%, P = 0.015). Every progression to an advanced phase (n = 3) and CML-related death (n = 2) occurred in the low PTCH1 group (P<0.001 for both comparisons). PTCH1 was an independent prognostic factor for the prediction of IF. We also validated previously published thresholds for PTCH1 expression. Therefore, we confirmed that PTCH1 expression can predict the imatinib response in CML patients in CP by applying a more rigorous statistical analysis. Thus, PTCH1 expression is a promising molecular marker for predicting the imatinib response in CML patients in CP.
Urothelial bladder cancer (UBC) is heterogeneous at the clinical, pathological and genetic levels. Tumor invasiveness (T) and grade (G) are the main factors associated with outcome and determine ...patient management. A discovery exome sequencing screen (n = 17), followed by a prevalence screen (n = 60), identified new genes mutated in this tumor coding for proteins involved in chromatin modification (MLL2, ASXL2 and BPTF), cell division (STAG2, SMC1A and SMC1B) and DNA repair (ATM, ERCC2 and FANCA). STAG2, a subunit of cohesin, was significantly and commonly mutated or lost in UBC, mainly in tumors of low stage or grade, and its loss was associated with improved outcome. Loss of expression was often observed in chromosomally stable tumors, and STAG2 knockdown in bladder cancer cells did not increase aneuploidy. STAG2 reintroduction in non-expressing cells led to reduced colony formation. Our findings indicate that STAG2 is a new UBC tumor suppressor acting through mechanisms that are different from its role in preventing aneuploidy.
Somatic mutations in the ten-eleven translocation methylcytosine dioxygenase 2 gene (
TET2)
have been associated to hematologic malignancies. More recently, biallelic, and monoallelic germline ...mutations conferring susceptibility to lymphoid and myeloid cancer have been described. We report two unrelated autoimmune lymphoproliferative syndrome-like patients who presented with T-cell lymphoma associated with novel germline biallelic or monoallelic mutations in the
TET2
gene. Both patients presented a history of chronic lymphoproliferation with lymphadenopathies and splenomegaly, cytopenias, and immune dysregulation. We identified the first compound heterozygous patient for
TET2
mutations (P1) and the first ALPS-like patient with a monoallelic
TET2
mutation (P2). P1 had the most severe form of autosomal recessive disease due to TET2 loss of function resulting in absent TET2 expression and profound increase in DNA methylation. Additionally, the immunophenotype showed some alterations in innate and adaptive immune system as inverted myeloid/plasmacytoid dendritic cells ratio, elevated terminally differentiated effector memory CD8 + T-cells re-expressing CD45RA, regulatory T-cells, and Th2 circulating follicular T-cells. Double-negative T-cells, vitamin B12, and IL-10 were elevated according to the ALPS-like suspicion. Interestingly, the healthy P1’s brother carried a TET2 mutation and presented some markers of immune dysregulation. P2 showed elevated vitamin B12, hypergammaglobulinemia, and decreased HDL levels. Therefore, novel molecular defects in TET2 confirm and expand both clinical and immunological phenotype, contributing to a better knowledge of the bridge between cancer and immunity.
Three monocytic neoplasms in a single patient Pérez-Sáenz, María Angeles; Rodriguez-Pinilla, Socorro Maria; Salgado, Rocío N. ...
Leukemia & lymphoma,
08/2020, Letnik:
61, Številka:
10
Journal Article
Introduction
Acute myeloid leukemia (AML) is a clonal disease with a reduced life expectancy due to a high relapse rate. One explanation is that leukemic stem cells (LSC) evade the action of ...conventional chemotherapy due to their quiescent state. Several mechanisms have been proposed that regulate their quiescence, however, by analogy with normal hematopoietic stem cells, a key role may be carried out by the signaling pathways Notch and Hedgehog (Hh). The objectives of this study are to analyze the role of Notch and Hh pathways in the quiescence of LSC and to verify if the pharmacological inhibition of the Notch and Hh pathways decreases the percentage of quiescent LSC. In this way, LSCs would be sensitized to chemotherapy treatments and the high relapse rate of AML could be reduced.
Methods
Expression of GLI1, a transcription factor of the Hh signaling pathway and NOTCH Internal Cleaved Domain (NICD) were analyzed in the hematopoietic stem and progenitor cells of four patients diagnosed with AML. The selection of quiescent fraction was performed by flow cytometry using anti-CD34-FITC, anti-CD117-PerCP, anti-CD45PE-Cy7, anti-CD38-APC-Cy7 and anti-KI67-BV510 antibodies. KI67 negative cells were considered quiescent. The activation of NOTCH and Hh pathways was studied using rabbit anti-GLI, anti-NICD primary antibodies and anti-rabbit BV421 secondary antibodies. Results were expressed in median (range) or mean ± standard deviation.
Dose-response curves of inhibitors of the Notch pathway (BMS-906024, inhibitor of γ-secretase), Hh pathway (BMS-833923, SMO inhibitor and GANT61, GLI inhibitor), and cytarabine (AraC) were made to study drug potency in the OCI-AML3 cell line. We also analyzed its synergistic behavior in combination with Arac by calculating the combination index (CI) of each of them. These experiments were conducted in triplicate and values were expressed as the mean ± standard deviation.
Finally, the effect of BMS-833923 and BMS-906024 on the quiescence of the CD34+CD38- cells of two patients diagnosed with AML was studied by flow cytometry.
The paired samples t-test was used in the statistical analysis of GLI and NICD expression between G0 and proliferating cells and in the statistical analysis of the decrease of quiescent cells due to Notch and Hh inhibitors.
Results
First of all, hematopoietic and progenitor cells were quantified in four AML patients: the median of the percentage of CD34+CD38- cells with respect to total cells in the bone marrow of the AML patient studied was 1.1% (range: 0.12%-9.05%), within which 71.50% (range: 64.30%-88.43%) are quiescent. Interestingly, we found a trend for a higher expression of NOTCH signaling pathway in the proliferating CD34+CD38- cells (relative median fluorescent intensity (MFI) = 1.91 (range: 1.51-3.34)) compared to the quiescent fraction (relative MFI=1.55 (range: 1.18-1.94); p=0.105). But no differences were found in expression of GLI1.
Before studying the effect on cellular quiescence of Notch and Hh inhibitors in monotherapy and in combination with AraC, we evaluated their effect on cell viability. The most potent drug studied was AraC (IC50 = 4.055 μM), followed by inhibitors of the Hh pathway (IC50 BMS-833923 = 5.041 μM; IC50 GANT61 = 7.042 μM) on the OCI-AML3 cell line. In contrast, the γ-secretase inhibitor (BMS-906024) showed no effect. Moreover, it was found that the combination 0.8 μM AraC plus 8 μM BMS-833923 was the most synergistic (CI = 0.53, 15% viability with respect to DMSO control).
Subsequently, the effect of the SMO inhibitor on the quiescent CD34+CD38- cells of two patients diagnosed with CD34+ AML was analyzed: BMS-833923 decreased the percentage of quiescent CD34+CD38- cells by 88.5±16.3% in monotherapy (p=0.083) and in presence of AraC by 85.8 ± 21.2% (p=0.113) (figure 1).
Conclusion
The use of SMO inhibitors for the treatment of AML is promising because it increases the sensitivity of leukemic cells to chemotherapy and facilitates their action by reducing the percentage of quiescent LSC. This could mean a decrease in the probability of relapse in patients with AML treated with Hh inhibitors. These results were derived from an ongoing project and more patients are being studied in order to confirm the explained results.
This work is partially funded by the Madrid Association of Hematology and Hemotherapy.
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No relevant conflicts of interest to declare.
Introduction
Chronic myeloid leukaemia (CML) is a clonal disorder of the pluripotent hematopoietic stem cell compartment, characterized by a reciprocal translocation between chromosomes 9 and 22, ...giving rise to the BCR-ABL1 oncogene. Hedgehog (Hh) signaling pathway seems to be an important role in physiopathology of CML, with a constitutive activation in progenitor cells. Moreover, an increased expression of PTCH1, a negative regulator of Hh signaling pathway, is a prognostic marker in CML patients associated with a low probability of discontinuation of treatment, higher rates of major molecular response and a lower probability of progression or death related to CML. We sought to test the effect of different tyrosine kinase inhibitors (TKI) on PTCH1 expression and to characterize the downstream effectors related to apoptosis controlled by Hh pathway in CML.
Methods
CD34+ cells from bone marrow samples of CML patients had been seeded at 7 x 104 -1 x 106 cell/mL on RPMI 1640, 10% fetal bovine serum with 700nM imatinib, 30nM or 200µM nilotinib. After 3-12 hours, mRNA was extracted and qPCR were performed to analyzed PTCH1 mRNA expression. mRNA expression of PTCH1 of CD34+ and CD34- cell from 4 bone marrow samples of CML patient had been compared using qPCR. Differences in expression values had been analyzed using T-Student or Wilcoxon test for paired samples.
K562 cell line seeded at 2 x 105 cell/ml had been treated with ciclopamine 2.5 or 5 µM. 300µg of protein had been used to perform a Proteome Profiler Human Apoptosis Array Kit (R&D). Differences in expression of 34 protein related to apoptosis were analyzed using One-way ANOVA test.
Results
Nilotinib did not show significative effect in PTCH1 mRNA expression at 3 hours neither with 200 µM (figure 1a, P= 0.71, n=3) nor 30 µM (figure 1a, P= 0.65, n=2). When the incubation time with drugs had been increased (12 hours), the differences were not significatives neither with Nilotinib (figure1b, p = 0.18) nor Imatinib (figure1b, p = 0.18). PTCH1 mRNA expression did not show significative difference between CD34+ and CD34- cells (P= 0.157). However, ciclopamine, antagonist drug of SMO and thus, inhibitor of Hedgehog pathway like PTCH1, decreased the expression of BCL2 protein, an antiapoptotic protein, in K562 cell line at 2.5 µM (56.3 %, P = 0.026) and 5 µM (62.1 %, P = 0.038) in comparison with this cell line without treatment.
Conclusion
We have not found statistically significant differences in PTCH1 expression under different TKI treatments but maybe due to lack of statistical potency. No differences in PTCH1 expression between CD34+ and CD34- had been previously reported. Our sample is very reduced but support those results. As previously described in other malignancies, BCL2 is a downstream effector of Hh pathway also in CML.
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No relevant conflicts of interest to declare.
Introduction
Acute myeloid leukemia (AML) is a clonal disease characterized by multiple genetic anomalies. The internal tandem duplications (FLT3-ITD mutations) in the juxtamembrane domain of the ...receptor occur in approximately 15-35% of de novo AML . This mutation is one of the most frequent genetic alterations, and confers an increased risk of treatment failure and a reduced disease-free survival (DFS). There are scarce data regarding the possible implication of the length of the FLT3-ITD fragment in the clinical outcome . We sought to shed light on the possible prognostic relevance of the length of the FLT3-ITD fragment in AML patients.
Methods
Twenty five patients (n=25) diagnosed with de novo AML in Hospital Universitario 12 de Octubre between 2005-2017 were included in the study. The median follow-up was 10 months from diagnosis (range 0.5-138 months). The median age was 65 years (range 1-85 years) with 72% of female patients. Bone marrow or blood samples were analyzed by fragment length analysis in an ABI3100 sequencer in order to detect the FLT3-ITD mutation.
A ROC curve was plotted to predict death for any reason. Overall survival (OS) was estimated by Kaplan-Meier curves and groups were compared by a stratified log-rank test based on allelic burden of FLT3 (≥ 0.5 vs <0.5). Additionally, Fisher test was employed in order to compare the proportion of patients with negative minimal residual disease.
Results
Twenty four percent of patients (n = 6) were acute leukemias secondary to previous hematologic neoplasms (n = 2) or prior therapies received (n = 4). In forty four percent of patients (n = 11), hyperleukocytosis was observed at diagnosis (median leukocytes 126.000/mm3, range 1400-209.000/mm3). Fifty six percent of patients (n = 14) showed a normal karyotype and only two of the individuals a complex karyotype. Thirty two percent of patients (n = 8) have achieved negative minimal residual disease at any time-point.med Seventy two percent of the patients died (n = 18). Sixty percent of the total (n=15) presented additional genetic alterations NPM1: 53% (n = 8), PML-RARA: 7% (n = 1), RUNX1/RUNXT1: 13% (n = 2), WT-1: 66% (n = 9).
The allelic burden of the ITD mutation expressed as a mutated/unmutated ratio oscillates between 0.05-1.96 with a median of 0.5. The length range of the ITD fragments analyzed ranged between 18-71 pairs of bases (pb) with a median of 38 pb.
The ROC curve showed an area under the curve of 0.58 (IC 95%: 0,37-0,78). A cutoff point of 33.5 pb was chosen with a sensitivity for prediction of death for any cause of 41.2%, specificity 87.5%, positive predictive value 87.5% and negative predictive value 41.2%.
Fifty two percent of patients (n = 13) had an allelic burden ≥ 0.5 of which 85% (n = 11) presented a length of the fragment affection ≥ 33.5 pb (total n = 17) and 48% of patients (n = 12) an allelic burden < 0.5 of whom 50% (n = 6) presented a length of the fragment affection < 33.5 pb (total n = 8). In terms of survival analysis, the P-value of the stratified test log-range was 0.3 (Figure 1). Fisher's exact test for MRD prediction was not significant either (P = 0.182).
Conclusion
To the best of our knowledge, the largest report on this topic included seventy three patients and showed that the length of the FLT3-ITD was clinically relevant. We have conducted this study and found only a trend towards significance probably due to the reduced number of patients analyzed. A multicenter collaboration is currently being carried out in order to gather a larger number of patients and try to shed light on this controversial topic.
Martinez Lopez:Celgene: Research Funding, Speakers Bureau; Jansen: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau; BMS: Research Funding, Speakers Bureau.
Introduction: Chronic myeloid leukemia (CML) is a neoplasm produced by the formation of the BCRABL fusion gene. Tyrosine kinase inhibitors (TKI) have changed dramatically the clinical course of the ...disease. Quantitative PCR (qPCR) is the reference technique for measuring response in CML patients. Droplet digital PCR (ddPCR) is, in brief, a technology based on partitioning of the sample in a high number of small fractions which, in a further step, are read as positive or negative by its fluorescence. ddPCR performs an absolute quantification of the number of transcripts and their results do not depend on PCR efficiency. In spite of these theoretical advantages over qPCR, ddPCR is not routinely applied for BCRABL monitoring. In this study, we sought to compare qPCR and ddPCR techniques in similar amplification conditions.
Methods: We included samples from 43 CML patients with different levels of response determined by qPCR: 23 patients with BCRABL/ABL1 levels <15%, 7 patients with MR3.0, 10 patients with MR4.0 and 3 patients who showed MR4.5 (according to European LeukemiaNet guidelines). Additionally, 3 non-CML patients were included. High-Capacity cDNA Reverse Transcription Kit was employed, following manufacturer instructions, in a unique reaction for both subsequent technologies utilized. qPCR was carried out with primers and probes for BCRABL and ABL1 from Europe Against Cancer report with 5 microL of non-measured cDNA in duplicates. The standards were BCR-ABL P210 ELITe Standard (ELITech Group) and the conversion factor was obtained with the Philadelphia P210 RNA Reference (Elitech Group). qPCR assys were run in an ABI 7500 platform in standard quantitation mode. EUTOS recommendations for BCRABL quantification were followed.
ddPCR reaction was performed with the same primers and probes. Median cDNA concentration was 4,485ng/µL (1,771-7,787ng/µL). In order to avoid saturation of the technology, 2microL of cDNA at a concentration of 600ng/microL (i.e. 1,200ng) were added and run in duplicates in a C1000 touch Thermal Cycler (Bio-RAD) with the same amplification conditions used in qPCR. The same RNA reference material was used to obtain the calibration factor for the ddPCR technique. Bio-Rad QX200 Droplet Digital PCR System was employed for analysis. We used the mode of analysis for rare event detection and thresholds were set individually in each reaction. Qualitative comparations were carried out using Kappa coefficient with quadratic weight. For quantitative comparations, we applied Pearson correlation, intraclass correlation coefficient for absolute agreement and Passing-Bablok tests. A subgroup analysis, including samples with MR3.0 or deeper, was also performed.
Results: All the samples showed ≥10000 ABL copies per well by qPCR. More than 10000 events were analyzed in every ddPCR reaction well. Good replicates were observed in qPCR and ddPCR. Adequate separation of positive and negative events could be performed and no saturation was observed in the ddPCR reations. No signal from BCRABL was detected in non-CML patients in ddPCR. The Kappa coefficient between qPCR and ddPCR measurements was 0.76 (P< 0.001, 95% confidence interval (CI): 0.63-0.88). In the 20 samples with MR 3.0 or deeper, kappa=0.13 (P >0.05 ) .
For quantitative comparison, 32 samples with detectable BCRABL were included. Pearson test yielded an R2=0.91 (P< 0.001). The intraclass correlation coefficient for absolute agreement showed a value of 0.96 (P <0.001). Passing-Bablok test provided a non significant constant difference (0.041, CI 95%: -0.04-0.05) and a significant proportional difference (0.8, CI 95%: 0.7-0.96) (i.e. ddPCR results= 0.041 + (0.8*qPCR results). There were only 9 samples with a BCRABL/ABL1 ratio and quantitative test were not performed.
Conclusion: Although a global excelent qualitative correlation is observed, in patients with MR3.0 or deeper which are the more important to classify correctly, qualitative correlation is absent. The R2 and intraclass correlation coefficient for absolute agreement showed a high concordance but Passing-Bablok test yielded a significant proportional difference among qPCR and ddPCR. We sought to test both technologies in similar conditions but different ddPCR strategies could be implemented in order to improve reproducibility of both techniques: higher amount of replicates or, given the lack of saturation, a higher cDNA concentration in the reactions.
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No relevant conflicts of interest to declare.
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