Monitoring Chronic Myeloid Leukemia: How Molecular Tools May Drive Therapeutic Approaches

More than 15 years ago, imatinib entered into the clinical practice as a "magic bullet"; from that point on, the prognosis of patients affected by chronic myeloid leukemia (CML) became comparable to that of aged-matched healthy subjects. The aims of treatment with tyrosine kinase inhibitors (TKIs) are for complete hematological response after 3 months of treatment, complete cytogenetic response after 6 months, and a reduction of the molecular disease of at least 3 logs after 12 months. Patients who do not reach their goal can switch to another TKI. Thus, the molecular monitoring of response is the main consideration of management of CML patients. Moreover, cases in deep and persistent molecular response can tempt the physician to interrupt treatment, and this "dream" is possible due to the quantitative PCR. After great international effort, today the BCR-ABL1 expression obtained in each laboratory is standardized and expressed as "international scale." This aim has been reached after the establishment of the EUTOS program (in Europe) and the LabNet network (in Italy), the platforms where biologists meet clinicians. In the field of quantitative PCR, the digital PCR is now a new and promising, sensitive and accurate tool. Some authors reported that digital PCR is able to better classify patients in precise "molecular classes," which could lead to a better identification of those cases that will benefit from the interruption of therapy. In addition, digital PCR can be used to identify a point mutation in the ABL1 domain, mutations that are often responsible for the TKI resistance. In the field of resistance, a prominent role is played by the NGS that enables identification of any mutation in ABL1 domain, even at sub-clonal levels. This manuscript reviews how the molecular tools can lead the management of CML patients, focusing on the more recent technical advances

The Q-LAMP Method Represents a Valid and Rapid Alternative for the Detection of the BCR-ABL1 Rearrangement in Philadelphia-Positive Leukemias

Molecular detection of the BCR-ABL1 fusion transcripts is necessary for the genetic confirmation of a chronic myeloid leukemia diagnosis and for the risk classification of acute lymphoblastic leukemia. BCR-ABL1 mRNAs are usually identified using a conventional RT-PCR technique according to the BIOMED-1 method. In this study, we evaluated 122 BCR-ABL1-positive samples with the Q-LAMP assay to establish if this technology may represent a valid alternative to the qualitative BIOMED-1 PCR technique usually employed for the detection and the discrimination of the common BCR-ABL1 transcripts (p190 and p210 isoforms). We found a 100% concordance rate between the two methods. Specifically, the p190- and p210-positive samples were amplified by Q-LAMP with a median threshold time (Tt) of 26.70 min (range: 24.45-31.80 min) and 20.26 min (range: 15.25-34.57 min), respectively. A median time of 19.63 was observed in samples displaying both (e13a2/e14a2) p210 isoforms. Moreover, the Q-LAMP assay allowed recognition of the BCR-ABL1 e13a2 and e14a2 isoforms (median Tts 18.48 for e13a2 vs. 26.08 min for e14a2; p < 0.001). Finally, 20 samples harboring rare BCR-ABL1 isoforms (e1a3, e13a3, e14a3, and e19a2) were correctly identified by the Q-LAMP assay. We conclude that the Q-LAMP assay may represent a faster and valid alternative to the qualitative BIOMED-1 RT-PCR for the diagnosis at BCR-ABL1-positive leukemias, especially when samples are analyzed in centers with restricted resources and/or limited technical expertise

Characterization Of The Bone Marrow Microenvironment That Provides Survival Signals Contributing To Therapy Failure In Patients With Leukemia.

The importance of tumor microenvironment for cancer progression is becoming widely recognized in recent years. The Bone Marrow (BM) is a dynamic microenvironment with high concentration of growth factors and cytokines necessary for haematopoiesis, making it a highly permissive zone for cancer haematopoietic staminal cell homing and survival. It is possible that the same factors that modulate hematopoiesis promote leukemogenesis, enhance blast survival and make them resistant to treatment within the BM microenvironment. In the era of molecular target therapy, whereas Imatinib has shown a cumulative best complete cytogenetic response rate of 82% and an estimated event free survival at 8 years of 85%, several in vitro data have confirmed that Ph+ CD34+ progenitor cells crammed in BM niches are resistant to TKI treatments. We attempted to define BM microenvironment markers that nurture and determine stem cell fate in leukemia associated-niches. We treated Ph+ K562 cell lines and primary CD34+ BM cells derived from untreated CML patients with a dose range of TKIs (0-100µM) in the presence of a monolayer of human BM mesenchimal stromal cell line (HS-5) or HS5 conditioned media (HCM), to assess the role of BM niche in the regulation of TKI responsiveness. We demonstrated that BM stroma environment significantly protects K562 cell line from TKI-induced apoptosis. Indeed, we demonstrated that Half maximal inhibitory concentration (IC50) value (calculated on cell viability) of Imatinib, Nilotinib and Dasatinib significantly increased when leukemic cells are exposed to HS5 or HCM. Moreover, we prove that a significant TKI-resistance could be achieved also by Ph+ CD34+ primary CML cells exposed to HS5 or HCM. Taken together, these findings indicate that BM-derived stroma cell line produces a strong effect on the regulation of TKI responsiveness in Ph+ CML cells by both a direct cell-to-cell contact and exposition to soluble factors. Moreover, the observed TKI resistance is associated to a BCR-ABL independent STAT-3 activation leading to a significant down-modulation of apoptosis when either Ph+ cell line or primary CD34+ progenitor cells derived from patients with CML are treated with TKIs in the presence of a direct mesenchymal stroma cell interaction or exposition to SCM. Finally, I proved that JAK inhibitor Ruxolitinib, that inhibits STAT3 phosphorylation (a marker of JAK activity), synergizes with TKIs in the induction of apoptosis in CML primary cells. Indeed, compared with single agent treatment, exposure of CML cells to the combination of TKI and JAK inhibitor Ruxolitinib significantly decreased viability of CML cells and increased their apoptosis in vitro. Taken together, our data show that the rational drug combination of TKI and Ruxolitinib may enhance the eradication of primary human Ph+ cells homed in BM stroma niche

FLUORESCENT IN SITU HYBRIDIZATION (FISH) ON PERIPHERAL BLOOD AND BONE MARROW SMARS: 120 MINUTES FOR DETECTION OF PML/RARa FUSION GENE

Acute promyelocytic leukemia (APL) is a clonal hematopoietic stem cell disorder characterized by a chromosomal translocation involving the retinoic acid receptor-a gene on chromosome 17 (RARA). In 95% of cases of APL, RARA gene is involved in a balanced reciprocal translocation with the promyelocytic leukemia gene (PML) on chromosome 15, a translocation denoted as t(15;17)(q22;q12). The long-term outlook is now favorable for the majority of APL patients, when treatment is instituted promptly, due to the availability of therapies such as all-trans retinoic acid (ATRA) with anthracycline-based chemotherapy (idarubicin) and arsenic trioxide (ATO). Therefore, rapid diagnosis of APL contributes to a highly effective therapy. The conventional cytogenetic analysis is an excellent method for detecting the t(15;17)(q22;q12) in APL but it is subject to limitations. Since only dividing cells can be analyzed, sometimes there is no useful results for some patients, because of poor chromosome morphology and/or an insufficient amounts of assessable metaphases; furthermore the conventional chromosome analysis is labour-intensive and time consuming. Fluorescence in situ hybridization (FISH) overcomes some of these limitations and enables the detection of chromosomal rearrangements even on interphase cells, avoiding the requirement of metaphase obtention. Typically, the technique involves multiple step of sample preparation and hybridization of the sample and the probe so taking about 24 hours to analyze the data. Given the need for a rapid diagnosis in patients with APL, we investigated the usefulness and the accuracy of FISH, performed with a commercial probe, to identify the PML-RAR fusion gene, using a quick method applied on peripheral blood and bone marrow smears. Using this 120 minutes lasting procedure, we obtained bright, distinct, compact and easily evaluable hybridization signals with low background and we were able to clearly and unambiguously detect the fusion gene PML/ RARa in 90% of the cases, due to the variable quality of the material available. This study suggests that FISH performed on peripheral blood and bone marrow smears is a reliable method for the rapid detection of PML/RARa rearrangement

MiR‐27a downregulates 14‐3‐3θ, RUNX1, AF4, and MLL‐AF4, crucial drivers of blast transformation in t(4;11) leukemia cells

The chromosomal translocation t(4;11)(q21;q23), a hallmark of an aggressive form of acute lymphoblastic leukemia (ALL), encodes mixed-lineage leukemia (MLL)-AF4 oncogenic chimera that triggers aberrant transcription of genes involved in lymphocyte differentiation, including HOXA9 and MEIS1. The scaffold protein 14-3-3θ, which promotes the binding of MLL-AF4 to the HOXA9 promoter, is a target of MiR-27a, a tumor suppressor in different human leukemia cell types. We herein study the role of MiR-27a in the pathogenesis of t(4;11) ALL. Reverse transcription quantitative PCR (qPCR) reveals that MiR-27a and 14-3-3θ expression is inversely correlated&nbsp;in t(4;11) ALL cell lines; interestingly, MiR-27a relative expression is significantly lower in patients affected by t(4;11) ALL than in patients affected by the less severe t(12;21) leukemia. In t(4;11) leukemia cells, ectopic expression of MiR-27a decreases protein level of 14-3-3θ and of the key transcription factor RUNX1. We show for the first time that MiR-27a also targets AF4 and MLL-AF4; in agreement, MiR-27a overexpression strongly reduces AF4 and MLL-AF4 protein levels in RS4;11 cells. Consequent to AF4 and MLL-AF4 downregulation, MiR-27a overexpression negatively affects transcription of HOXA9 and MEIS1&nbsp;in different t(4;11) leukemia cell lines. In agreement, we show through chromatin immunoprecipitation experiments that MiR-27a overexpression impairs the binding of MLL-AF4 to the&nbsp;HOXA9 promoter. Lastly, we found that MiR-27a overexpression decreases viability, proliferation, and clonogenicity of t(4;11) cells, whereas it enhances their apoptotic rate. Overall, our study identifies the first microRNAthat strikes in one hit four crucial drivers of blast transformation&nbsp;in t(4;11) leukemia. Therefore, MiR-27a emerges as a new promising therapeutic target for this aggressive and poorly curable form of leukemia

WT1-mediated repression of the proapoptotic transcription factor ZNF224 is triggered by the BCR-ABL oncogene.

The Kruppel-like protein ZNF224 is a co-factor of the Wilms' tumor 1 protein, WT1. We have previously shown that ZNF224 exerts a specific proapoptotic role in chronic myelogenous leukemia (CML) K562 cells and contributes to cytosine arabinoside-induced apoptosis, by modulating WT1-dependent transcription of apoptotic genes. Here we demonstrate that ZNF224 gene expression is down-regulated both in BCR-ABL positive cell lines and in primary CML samples and is restored after imatinib and second generation tyrosine kinase inhibitors treatment. We also show that WT1, whose expression is positively regulated by BCR-ABL, represses transcription of the ZNF224 gene. Finally, we report that ZNF224 is significantly down-regulated in patients with BCR-ABL positive chronic phase-CML showing poor response or resistance to imatinib treatment as compared to high-responder patients. Taken as a whole, our data disclose a novel pathway activated by BCR-ABL that leads to inhibition of apoptosis through the ZNF224 repression. ZNF224 could thus represent a novel promising therapeutic target in CML

A Novel Score to Predict Interferon-Alpha Therapy Responsiveness in Patients with Essential Thrombocythemia

Interferon alpha (IFN-α) is an attractive agent for the treatment of Essential Thrombocythemia (ET) due to its ability to induce clonal complete remission, sometimes lasting beyond treatment discontinuation, and to its recognized non-leukemogenicity. However, despite decades of clinical experience with IFN-α in patients with MPNs, optimal dose schedules, treatment duration and the ultimate molecular basis of the heterogeneous response still remain undefined. Hence, the early identification of IFN-sensitive patients may help limit IFN-α exposure to those who really benefit from treatment. Aim. Here we report the results of a trial involving 61 ET patients treated with IFN-α, aimed to identify the baseline molecular and clinical parameters able to predict response to treatment. Methods. IFN treatment schedule implied an initial induction phase with 3MU/five times a week; in patients who reached a platelet count 600x109/L or platelet reduction was <50% the baseline level, the patient was considered resistant to the IFN therapy (Bad-R). Careful medical history, main laboratory data and spleen volume, assessed by ultrasonography scan were recorded in all patients at presentation and during follow-up. Complete hematological response (CHR) is defined as the normalization of both platelet and WBC counts (<400x109/L and <10x109/L, respectively) and the absence of disease-related symptoms. mRNA levels of JAK1, JAK2, STAT1, STAT3, SOCS1, SOCS3 and TYK2 were assayed in pre-treatment bone marrow specimens by Real-Time PCR using the SYBR Green method. Results. After a median follow-up of 41.2 months, 72% of patients achieved CHR and were considered Good-Rs for subsequent analysis, whereas the remaining 17 were considered Bad-Rs. Among the Good-Rs, 24 (54%) are still on therapy with standard IFN-α doses (i.e. 3 MU 3 or 2 times a week), whereas 10 (23%) are maintained in CHR by the administration of very low doses of IFN-α-2b (3 MU every 7 or 15 days), and 3 (7%) have maintained CHR after therapy discontinuation (up to a median time of 31 months). The initial univariate analysis indicated that the mRNA levels of JAK1, STAT3, SOCS3 were significantly lower in Good-R than in Bad-R patients. Interestingly, among the different genes involved in the IFN-α receptor pathway, the expression levels of JAK1, together with spleen volume and platelet count, were selected by the stepwise multivariate analysis as the variables that independently correlate with IFN-α response. We used the relative HRs and the optimal cut-offs for response calculated for each variable by the ROC analysis to develop a prognostic score able to predict IFN-α response. This score has an overall 87% diagnostic efficiency in discriminating IFN-α response and unambiguously identifies the response to IFN-α in most patients, avoiding treatment in those with no probability of gaining benefit from this therapy. In addition, this score is able to identify unambiguously the response to IFN in a sizeable proportion of patients: an IFN-R score of 3 or 4 (31 patients, corresponding to 70.4% of the Good-R) indicates a 100% odd to obtain CHR, while a score of 0 indicates no chance of achieving a response. Conclusion. In conclusion, this study shows for the first time that the use of three simples parameters predicts the response to IFN in ET patients

MOLECULAR EVALUATION OF ZNF224 MRNA EXPRESSION IN CML PATIENTS AS A NOVEL DETERMINANT OF TKI RESPONSIVENESS

The transcription factor Wilms’ tumor gene 1, WT1, is implicated both in normal developmental processes and in the generation of a variety of solid tumors and hematological malignancies. WT1 is highly expressed in leukemia cells and its overexpression is associated with a poor response to therapy. Recently the Krüppel-like zinc-finger protein, ZNF224 was identified as a novel WT1-interacting factor involved in WT1 transcriptional regulation. ZNF224 itself could be modulated by cytosine arabinoside (ara-C), a drug widely used in the treatment of myeloid leukemia and that ZNF224 overexpression increases susceptibility to apoptosis of Ph+ K562 cell lines. In our retrospective analysis we evaluated the relative expression of ZNF224 mRNA in 30 adult patients with BCR-ABL–positive chronic phase chronic myeloid leukaemia (CP-CML) as a determinant of imatinib sensitivity. Methods: Response to tyrosine kinase inhibitor (TKI) imatinib is assessed with standardized real quantitative polymerase chain reaction and/or cytogenetics at 3, 6, and 12 months. Response to the therapy was classified as optimal, warning, and failure, according to the recent ELN criteria. We compared the ZNF224 expression at diagnosis with molecular response over the first 12 month of imatinib therapy. Sample have been selected, for retrospective analysis, for them interim molecular results a 12 month, showing 15 patients in optimal response (OR), 10 patients in a warning response (WR) and 5 patients in failure response (FR). 5 healthy donors (HDs) were included to the study. All patients signed informed consent in accordance with the Declaration of Helsinki. RT-qPCR results were normalized by the expression of ABL mRNA (Normalized mRNA copy Number: NCN).Results:ZNF224 mRNA were significantly up-regulated in PB samples at diagnosis of patients with OR compared to patients with WR/FR, (1.13±0.76 vs 0.62±0.25 NCN,respectively; p=0.05). Interesting the ZNF224 mRNA expression in HDs was significantly higher (2.11±0.98 NCN vs OR patients, p=0.05 and WR/FR patients; p=0.0005). The treatment for 12 month with imatinib increase the ZNF224 expression in both CML categories (2.91±1.72 NCN in OR and1.77±1.52 NCN in WR/FR; p=0.05). Conclusions:We observed that the OR patients express a significantly higher number of copies of the ZNF224 transcript than WR/FR. Furthermore, in both groups of patients at diagnosis, ZNF224 protein levels are lower than those after therapy with TKI at 12 month