Evolution Of Chronic Myeloid Leukemia Research Articles

The structure of the hematopoietic system can explain chronic myeloid leukemia progression

Almost all cancer types share the hallmarks of cancer and a similar tumor formation: fueled by stochastic mutations in somatic cells. In case of chronic myeloid leukemia (CML), this evolutionary process can be tracked from an asymptomatic long-lasting chronic phase to a final rapidly evolving blast phase. Somatic evolution in CML occurs in the context of healthy blood production, a hierarchical process of cell division; initiated by stem cells that self-renew and differentiate to produce mature blood cells. Here we introduce a general model of hierarchical cell division explaining the particular progression of CML as resulting from the structure of the hematopoietic system. Driver mutations confer a growth advantage to the cells carrying them, for instance, the BCR::ABL1 gene, which also acts as a marker for CML. We investigated the relation of the BCR::ABL1 mutation strength to the hematopoietic stem cell division rate by employing computer simulations and fitting the model parameters to the reported median duration for the chronic and accelerated phases. Our results demonstrate that driver mutations (additional to the BCR::ABL1 mutation) are necessary to explain CML progression if stem cells divide sufficiently slowly. We observed that the number of mutations accumulated by cells at the more differentiated levels of the hierarchy is not affected by driver mutations present in the stem cells. Our results shed light on somatic evolution in a hierarchical tissue and show that the clinical hallmarks of CML progression result from the structural characteristics of blood production.

Clinical significance of clonal evolution in chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a hematological malignancy characterized by the Philadelphia (Ph) chromosome, which is formed by a t (9;22)(q34;q11) translocation. The aberrant activation of the ABL1 tyrosine kinase is caused by the BCR::ABL1 fusion gene on the Ph chromosome, leading to significant leukemic cell proliferation. CML is typically diagnosed in the chronic phase with few clinical symptoms and progresses to a blast crisis within years. CML acquires additional genetic abnormalities on top of BCR::ABL1 fusion during clonal evolution. ASXL1 mutations are found in the chronic phase, with a frequency of approximately 20%, whereas other mutations are rare. Most blast crisis cases have additional genetic abnormalities, including frequent ASXL1 and RUNX1 mutations. Recent studies have revealed that a subset of these genetic mutations affects the sensitivity of tyrosine kinase inhibitors to leukemic cells as well as patient prognosis, indicating applications for patient stratification and individualized treatment.

The Impact of Cytogenetic Aberrations in the Clonal Evolution of Chronic Myeloid Leukemia: A Single-Center Experience Among 450 Turkish Patients (Cohort Study)

Chronic myeloid leukemia (CML) is a clonal hematologic disorder characterized by t(9;22) translocation, in which cytogenetic aberrations can occur in Ph(+) and (-) clones. These aberrations develop due to clonal evolution as well as treatment and they have prognostic significance. They are grouped as major and minor route anomalies in terms of their effects on prognostic parameters, such as treatment response, overall survival (OS), disease stage, complete cytogenetic response (CCyR), and major molecular response (MMR). It is stated that major route anomalies have unfavorable prognostic effects compared to minor route anomalies. We aimed to investigate the frequency and prognostic effects of cytogenetic anomalies detected in Ph(+) and (-) clones. In this study, we retrospectively analyzed the cytogenetic results of 450 patients diagnosed with CML between 2005 and 2020. We detected cytogenetic aberrations in Ph-positive and negative clones in 41 of 450 patients. The most common anomalies were trisomy 8 (+8), additional Ph chromosome (+Ph), and loss of chromosome Y. Rarely, aneuploidy of the Y chromosome, dup (22), +11, and +6 were seen in CML patients. We observed that these identified aberrations negatively affected MMR and CCyR, and generally resulted in changing imatinib treatment for second-generation tyrosine kinase activity inhibitors. Our results are compatible with the literature. We suggest that cytogenetic aberrations detected in Ph(+) and (-) clones should be a warning sign in terms of treatment and require close observation. The use of cytogenetic methods for the identification of these anomalies is also important.

Emergence of Chronic Myeloid Leukemia in Patients with Myeloproliferative Neoplasms: A Single-Institution Experience

Introduction: Chronic myeloid leukemia (CML) is characterized by the presence of the BCR-ABL1 translocation and is typically distinct from other myeloproliferative neoplasms (MPN) including polycythemia vera (PV), essential thrombocythemia (ET) and myelofibrosis (MF). Concurrent diagnosis of CML and MF is uncommon and infrequently described in case reports.1 Moreover, very few cases have been described of CML occurring after an established diagnosis of MPN.2-3 Due to the rarity of concurrent CML and MPN, management strategies in this population are not defined. In addition, the latency time from the emergence of BCR-ABL1 positive clones to a clinical diagnosis of CML in this setting is largely unknown. To address these questions, we interrogated our institutional experience and retrospectively analyzed available specimens. Methods: We collected information on patients seen at Massachusetts General Hospital (MGH) from January 1 2012 to January 1 2022 for CML occurring after an established diagnosis of MPN. Available stored samples were retrospectively tested for BCR-ABL1 with a fusion transcript assay (MGH Center for Integrated Diagnostics, Boston, MA). Results: We identified four patients with CML preceded by MPN (Table 1). Three patients had prior MF, diagnosed 4.3-7.6 years before the onset of CML; two of these had post-ET MF. Patient #4 had ET diagnosed 4 years prior to onset of CML, then went on to develop MF 4.9 years after CML diagnosis. None of the patients were on JAK inhibitors, hydroxyurea or interferon at the time of CML diagnosis. All patients had a leukocytosis with characteristic features on the differential prompting evaluation for CML. The median white blood cell count was 45.83 x 10^9/L (range 27.49-121.02). All four patients had an elevated basophil count (median 2.99 x 10^9/L, range 1.1-22.99) and two had an elevated eosinophil count (median 1.11 x 10^9/L, range 0.55-44.17). Regarding MPN driver mutations, one patient had a JAK2 V617F, one patient had a CALR exon 9 deletion, and two patients had no identifiable mutations on extended next-generation sequencing. For CML treatment, three patients received dasatinib (two with 100 mg daily, one with 50 mg daily), whereas one received nilotinib (150 mg twice daily). Two achieved a major molecular response (MMR) (at 7 and 8 months); one had an early molecular response at 6 months (last follow-up); one (with e13-a3 transcript) could not be quantified on a standard PCR assay but achieved a complete cytogenetic response by 4 months. At the time of last follow-up, two patients were in MMR on tyrosine kinase inhibitors (TKI), whereas two underwent allogeneic hematopoietic cell transplantation (alloHCT), primarily for progression of MF. We retrospectively interrogated available samples for evidence of the BCR-ABL1 translocation prior to a clinical diagnosis of CML. Patient #2 was followed at MGH for MF and thus had available samples. We identified BCR-ABL1 transcripts in the peripheral blood three months prior to his clinical diagnosis of CML (unable to be quantified due to e13-a3 transcript); there were no available samples from earlier in his disease course for testing (Figure 1). Discussion: CML can emerge in patients with MPN. Herein, we describe four cases of this phenomenon. Signs of CML evolution included marked leukocytosis, as well as basophilia and eosinophilia. Despite underlying MF, three patients tolerated full-dose TKI and achieved molecular responses early in their disease course. However, MPN progression remained a significant challenge, with two patients ultimately requiring an alloHCT. Of note, we found evidence of BCR-ABL1 in the peripheral blood three months prior to clinically apparent CML in one patient. Experiments using single cell techniques in patients with concurrent MPN and CML would provide insights into the clonal architecture and are in planning stages.

Progression, detection and remission: evolution of chronic myeloid leukemia using a three-stage probabilistic model

We present a three-stage probabilistic model for the progression of Chronic Myeloid Leukemia (CML), as manifested by the leukemic stem cells, progenitor cells and mature leukemic cells. This progression is captured through the process of cell division and cell mutation, with probabilities of occurrence being assigned to both of them. The key contributions of this study include, the determination of the expected number of the leukemic stem cells, progenitor cells, mature leukemic cells, as well as total number of these cells (in terms of probabilities, and contingent on the initial cell count), expected time to reach a threshold level of total and injurious leukemic cells, as well as the critical time when the disease changes its phases, the probability of extinction of CML, and the dynamics of CML evolution consequent to primary therapy. Finally, the results obtained are demonstrated with numerical illustrations.

Role of Somatic Mutations in Clonal Evolution of Chronic Myeloid Leukemia from Chronic Phase to Blast Phase

BackgroundRole of somatic mutations in Chronic Myeloid Leukemia (CML) is fairly underexplored. We hypothesize that clonal evolution of chronic phase CML (CP-CML) to blast phase CML (BP-CML) is molecularly driven thus augmenting on previous findings of ASXL1 being a commonly recurrent mutation in CML. Therefore, early detection of specific somatic mutations can predict progression of disease.MethodsWe performed a retrospective chart review of clinically and molecularly annotated 60 cases of CML from Moffitt Cancer Center. Genotyping was performed using next generation targeted DNA sequencing detecting for common recurrent mutations in hematological malignancies. Fisher's Exact and Student T-test were used to determine statistical significance (p-value of <0.05). All calculated p values were two sided.ResultsTotal 48 patients with CP-CML and 12 patients with BP-CML were included. Median age was 56 and 53.5 years in CP-CML and BP-CML cohort, respectively. Among CP-CML cohort, 37.5% (n=18) had at least 1 mutation with ASXL1 being the most frequently mutated gene (27.7%, n=5) where 80% (n=4) were isolated and only 20% (n=1) had comutation in RUNX1 (Figure 1A). In BP-CML patients, at least 1 mutation was detected in 100% of the cases (n=12) with ASXL1 being the most frequently mutated gene (67%, n=8) (Figure 1B). In contrast to the CP-CML cohort, ASXL1 mutation co-occurred with other mutations in 87.5% of the cases (n=7). The most frequent co-occurring mutations were TET2 (37.5%, n=3) and CUX1 (37.5%, n=3) followed by RUNX1 (25%, n=2), SETBP1 (25%, n=2), TP53 (25%, n=2) and PHF6 (12.5%, n=1). Majority of the BP-CML comprised of myeloid BP (83%) where ASXL1 mutations were exclusively found. Lymphoid BP (16%, n=2) cases revealed lymphoid specific mutations in CDKN2A and IKZF1 genes.In BP-CML compared to CP-CML, presence of ASXL1 mutation was more frequent (OR 17.2, 95% CI 3.77-78.32, p=0.002) with increased frequency of co-occurrence with at least one other mutation (OR 28.0, 95% CI 1.35-580.0, p=0.032). In BP-CML cohort, 83% (n=10) of the patients had additional cytogenetic abnormalities (ACAs) and only 16.5% (n=2) had isolated standard Philadelphia chromosome. In contrast to BP-CML, there was no correlation between presence of ACAs and presence of mutations in CP-CML cohort.ConclusionsAlthough this study is comprised of a small sample size, the findings suggest that the presence of ASXL1 in CP-CML may represent an early event or “first hit”, which then acquires an additional mutation or “second hit”, contributing to progression to advanced phase CML. These findings need to be validated in a large prospective cohort study. [Display omitted] DisclosuresChavez:Abbvie: Speakers Bureau; Janssen: Speakers Bureau; Kite: Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees. Pinilla-Ibarz:Pfizer: Honoraria, Speakers Bureau; ARIAD: Consultancy, Honoraria; BMS: Honoraria, Speakers Bureau.

Understanding and Monitoring Chronic Myeloid Leukemia Blast Crisis: How to Better Manage Patients.

Chronic myeloid leukemia (CML) is triggered primarily by the t(9;22) (q34.13; q11.23) translocation. This reciprocal chromosomal translocation leads to the formation of the BCR-ABL fusion gene. Patients in the chronic phase (CP) experience a good curative effect with tyrosine kinase inhibitors. However, cases are treatment refractory, with a dismal prognosis, when the disease has progressed to the accelerated phase (AP) or blast phase (BP). Until now, few reports have provided a comprehensive description of the mechanisms involved at different molecular levels. Indeed, the underlying pathogenesis of CML evolution comprises genetic aberrations, chromosomal translocations (except for the Philadelphia chromosome), telomere biology, and epigenetic anomalies. Herein, we provide knowledge of the biology responsible for blast transformation of CML at several levels, such as genetics, telomere biology, and epigenetic anomalies. Because of the limited treatment options available and poor outcomes, only the therapeutic response is monitored regularly, which involves BCR-ABL transcript level assessment and immunologic surveillance, with the optimal treatment strategy for patients in CP adapted to evaluate disease recurrence or progression. Overall, selecting optimal treatment endpoints to predict survival and successful TFR improves the quality of life of patients. Thus, identifying risk factors and developing risk-adapted therapeutic options may contribute to a better outcome for advanced-phase patients.

Involvement of Oxidative Stress in Resistance to Tyrosine-Kinase Inhibitors Therapy in Chronic Myeloid Leukemia.

Oxidative stress involves disruption of the cellular redox status through excessive production of reactive oxygen species or through deficiency in the cellular antioxidant capacity. It is involved in the pathogeny of multiple entities (hematological diseases, metabolic disorders, cardiovascular and renal pathology etc.), as well as in the pharmacokinetics of specific treatments for these pathologies. Chronic myeloid leukemia is a chronic myeloproliferative disease for which current standard treatment is BCR-ABL tyrosine kinase inhibitors. The innovation of this therapy has significantly improved life expectancy for patients with chronic myeloid leukemia, but in some cases, this treatment becomes ineffective, installing the resistance to tyrosine kinase inhibitors therapy. There were described two types of tyrosin kinase inhibitors resistance: primary and secondary resistance. In the present paper we proposed to evaluate the involvement of oxidative in the resistance to tyrosine kinase inhibitors therapy, in the clonal instability in chronic myeloid leukemia and in the progression of the disease to an advanced stage. We concluded that oxidative stress can play a dual role in the evolution of chronic myeloid leukemia: on the one hand it can promote genomic instability and accelerate the progression of the disease to advanced stages associated with tyrosin kinase inhibitors resistance and, on the other hand, it can contribute to leukemic cell apoptosis. It seems to be outlined a fragile balance between the pro- and anti-apoptotic effects of the reactive oxygen species, closely related to their level in the leukemic cells.

Assessment of Oxidative Stress in Patients with Chronic Myeloid Leukemia Depending on Associated Comorbidities.

Oxidative stress (OS) implies an imbalance between the amount of tissue level of prooxidant and antioxidant compounds. It is involved in the pathophysiology of multiple pathological entities (neoplasms, disorders of carbohydrate and lipid metabolism, cardiovascular and renal pathology etc.), as well as in the pharmacokinetics of specific treatments for these pathologies. Chronic myeloid leukemia (CML) is a chronic myeloproliferative disease for which current standard treatment is BCR-ABL tyrosine kinase inhibitors (TKIs). It is known that OS is involved in CML pathogenesis and response to TKIs therapy, but in reality, there are a number of additional factors (associated comorbidities, specific therapies) that modulate oxidative status, possibly affecting the evolution and prognosis of CML. In the present paper we proposed the evaluation of OS in a group of patients with CML following treatment with TKIs, depending on the presence of comorbidities and associated treatments. There were considered associated comorbidities: diabetes mellitus, dyslipidemia, arterial hypertension, heart failure, chronic kidney disease. The variability of the oxidative status was found depending on the type of associated comorbidity, but also according to the associated treatment, with the possibility of producing drug interactions between the standard treatment of CML and the associated specific therapies. Their impact on the prognosis of CML patients in treatment with TKIs is not negligible and may represent a future research topic.

This Is a Title in Title Case: Prediction of CML Evolution By Telomere Length Analysis at the Single-Chromosome Level

Telomeresplay a vital role in DNA repair activities and protecting chromosomes from degradation[1]. Telomeres are shortened during each cell division because of the end replication problem. After several cell division, telomeres are shortened to a critical length (&lt; 3 kb), and eventually lead to overall genomic instability and triggering the DNA damage response[2-3], which is related to the cancerization of numerous cancers. The commonly used method to estimate telomere length isterminal restriction fragment (TRF) basedon southern blot, which requires thousands of cells and provides only a crude estimate of the average telomere length of all cells analyzed. However, it is believed that the frequency of critically short telomeres, rather than the mean telomere length, is a crucial factor for telomere dysfunction. Therefore, analysis of telomere length at the single-chromosome level is necessary to determine the frequency of critically short telomeres. Here, we describe the development of a high-throughput method for telomere length analysis at the single-chromosome level by using a laboratory-built high-sensitivity flow cytometer (HSFCM)[4] combined with targeted fluorescent peptide nucleic acid (PNA) probes. The unambiguous detection of the telomere signalsfrom a single chromosome was achieved via HSFCM analysis. The fluorescence intensity of single chromosome was converted to the probe number by a calibration curve, and was further transformed to the base pair number of the telomere. Five representative cell lines were analyzed to compare their telomere length and the ratio of critically short telomeres at the single-chromosome level. The potential of using frequency of short telomere for disease treatment monitoring is examined by analyzing the telomere length in lymphocyte of leukemia patients. The abundance of short telomeres was compared between healthy donors and patients with chronic myeloid leukemia (CML) to see whether it can be used to predict the efficacy of therapeutics. Moreover, the quantity of short telomeres was compared among patients with acute leukemia, patients in different phases of CML and healthy donors to see whether it can be used as a marker for disease progression prediction in CML. Reference [1] Blackburn E. H., Epel E. S., Lin J., Science,2015, 350, 1193-1198. [2]Collado M., Blasco M. A., Serrano M., Cell, 2007, 130, 223-233. [3] Deng Y., Chan S. S., Chang S., Nat. Rev. Cancer, 2008, 8, 450-458. [4] Yang LL, Zhu SB, Hang W, Wu LN, Yan XM, Anal. Chem., 2009, 81, 2555-2563. Figure 1 Disclosures No relevant conflicts of interest to declare.

Impact of Additional Chromosomal Aberrations on the Disease Progression of Chronic Myelogenous Leukemia.

The emergence of additional chromosomal abnormalities (ACAs) in Philadelphia chromosome/BCR-ABL1 positive chronic myeloid leukemia (CML), is considered to be a feature of disease evolution. However, their frequency of incidence, impact on prognosis and treatment response effect in CML is not conclusive. In the present study, we performed a chromosome analysis of 489 patients in different clinical stages of CML, using conventional GTG-banding, Fluorescent in situ Hybridization and Spectral Karyotyping. Among the de novo CP cases, ACAs were observed in 30 patients (10.20%) with lowest incidence, followed by IM resistant CP (16.66%) whereas in AP and BC, the occurrence of ACAs were higher, and was about 40.63 and 50.98%, respectively. The frequency of occurrence of ACAs were compared between the study groups and it was found that the incidence of ACAs was higher in BC compared to de novo and IM resistant CP cases. Likewise, it was higher in AP patients when compared between de novo and IM resistant CP cases, mirroring the fact of cytogenetic evolution with disease progression in CML. In addition, we observed 10 novel and 10 rare chromosomal aberrations among the study subjects. This study pinpoints the fact that the genome of advanced phase patients was highly unstable, and this environment of genomic instability is responsible for the high occurrence of ACAs. Treatment response analysis revealed that compared to initial phases, ACAs were associated with an adverse prognostic effect during the progressive stages of CML. This study further portrayed the cytogenetic mechanism of disease evolution in CML.

Laboratory Monitoring of Chronic Myeloid Leukemia in Patients on Tyrosine Kinase Inhibitors.

Chronic Myeloid Leukemia (CML) is a myeloproliferative neoplasm characterized by translocation of genetic material from chromosome 9 to chromosome 22 to form a fusion gene (BCR-ABL1) that is responsible for abnormal tyrosine kinase activity and alteration of various downstream signaling pathways. In addition to morphological diagnosis of CML phase, it is essential to detect BCR-ABL1 fusion by either metaphase cytogenetics or reverse transcriptase polymerase chain reaction that also determines type of mRNA transcript. Once treatment begins, monitoring the response to Tyrosine Kinase Inhibitor (TKI) using standardized techniques and guidelines is important to check for failure of response and thus, plan timely intervention by increasing the dose of TKI or opting for second line TKIs. The goal is to stop evolution of CML to accelerated phase or blast crisis that has poor response to treatment. Also, it is desirable to achieve good outcomes and even treatment free remission in patients of CML on TKI. Thus, molecular monitoring by reverse transcriptase quantitative PCR (RT-qPCR) is done at regular intervals. There are international recommendations and quality control measures to standardize the reporting of fusion gene transcript levels by quantitative PCR (RT-qPCR) in CML to achieve and maintain sensitivity in molecular detection of CML disease burden. Various state-of-the-art molecular techniques have emerged to accurately determine the number of fusion-gene transcript levels. This review highlights various methodologies and their practical implications in management of CML patients on TKI.

Whole-genome analysis reveals unexpected dynamics of mutant subclone development in a patient with JAK2-V617F-positive chronic myeloid leukemia

We report here the first use of whole-genome sequencing (WGS) to examine the initial clonal dynamics in an unusual patient with chronic myeloid leukemia (CML), who presented in chronic phase (CP) with doubly marked BCR-ABL1+/JAK2V617F-mutant cells and, over a 9-year period, progressed into an accelerated phase (AP) and then terminal blast phase (BP). WGS revealed that the diagnostic cells also contained mutations in ASXL1, SEC23B, MAD1L1, and RREB1 as well as 12,000 additional uncommon DNA variants. WGS of endothelial cells generated from circulating precursors revealed many of these were shared with the CML clone. Surprisingly, WGS of induced pluripotent stem cells (iPSCs) derived from the AP cells revealed only six additional coding somatic mutations, despite retention by the hematopoietic progeny of the parental AP cell levels of BCR-ABL1 expression and sensitivity to imatinib and pimozide. Limited analysis of BP cells revealed independent subclonal progression to homozygosity of the MAD1L1 and RREB1 variants. MAD1L1 and SEC23B mutations were also identified in 2 of 101 cases of myeloproliferative neoplasms, but not in 42 healthy subjects. These findings challenge historic concepts of clonal evolution in CML.

Microsatellite Instability in Chronic Myeloid Leukemia using D17S261 and D3S643 markers: A Pilot Study in Gujarat Population.

Tumor progresses through a series of genetic alterations that involve proto-oncogenes and tumor suppressor genes - the gatekeeper, caretakers, and landscaper genes. Microsatellites are short tandem repeat sequences, present over the span of human genome and are known to be variable at multiple loci due to errors in DNA Mismatch Repair machinery. The present study was aimed to evaluate the association between Microsatellite Instability (MSI) and evolution of Chronic Myeloid Leukemia (CML) - genetically a rare event but profound in this pilot study. We explore the possibility of MSI in primary CML patients confirmed by t(9;22) using capillary electrophoresis. Fifteen CML patients and healthy individual samples, respectively, were used to study the markers D17S261 and D3S643. The DNA was amplified using tagged and nontagged primers and further subjected to bioanalysis and fragment analysis. While the results from bioanalyzer fluctuated, fragment analysis indicated the presence of microsatellite variability in 80% of the patients' samples as compared to no MSI in normal individuals for both the markers. These findings suggest that MSI is a genetic event that may have a role in CML progression or evolution. Further studies are warranted to understand the plausible underlying causes.

Prevalence and Dynamics of BCR-ABL Independent Gene Mutations in Patients with Chronic Myeloid Leukemia

Living without treatment has become a realistic target for patients with chronic myeloid leukemia (CML) who achieved durable deep molecular remission under treatment with tyrosine kinase inhibitors (TKI). Recently, we have identified novel BCR-ABL-independent gene mutations in newly diagnosed CML patients that may function as important cofactors in the evolution of CML and persistence of the disease (Schmidt M, Rinke J, et al. Leukemia 2014; 28(12): 2292-9). Here, we sought to investigate the dynamics of such mutations during a 2-year treatment with the second generation TKI nilotinib. To check if BCR-ABL independent mutations are an age associated phenomenon we also screened a cohort of children and adolescents with CML. A total of 51 chronic phase CML patients were analyzed including 31 adults (male, n=23; median age 57 years, range 42 to 73 years) and 20 children/adolescents (male, n= 14; median age 14 years, range 10 months to 27 years). For each adult patient, samples were analyzed at diagnosis, in subsequent complete cytogenetic remission (CCyR) after 3 months and in deep molecular remission (≤ MMR) after 24 months of nilotinib therapy. Children/adolescents samples were investigated at diagnosis only. Targeted deep next-generation sequencing (NGS) was used to analyze a panel of 30 commonly mutated genes in myeloid disorders. Constitutional DNA obtained from buccal swabs at diagnosis was investigated to check whether mutations are of somatic origin. BCR-ABL independent gene mutations were found in 8/31 (26%) adult CML patients at diagnosis affecting the genes ASXL1 (n=3), DNMT3A (n=3), RUNX1 (n=1) and RUNX1 plus TET2 (n=1). No mutation was recognized in corresponding constitutional DNA specimens indicating that all mutations were somatically acquired. Analysis of individual hematopoietic colonies from three adult patients revealed that most mutations were part of the BCR-ABL-positive clone. NGS of subsequent samples obtained after three months of TKI therapy identified two DNMT3A mutations in BCR-ABL-negative cells that were also present in BCR-ABL-positive cells at diagnosis, implying that this mutation preceded the BCR-ABL rearrangement. After 24 months of TKI treatment, NGS revealed these two DNMT3A mutations again as well as a novel low-level EZH2 mutation in a patient with deep molecular remission (MR4.5) that was neither detectable at diagnosis nor after 3 months of treatment indicating the emergence of a BCR-ABL-independent subclone in BCR-ABL-negative cells during long-term nilotinib treatment. Within the children/adolescents cohort BCR-ABL independent gene mutations were identified in 5/20 (25%) subjects all affecting the ASXL1 gene. All mutations were stop codon or frameshift mutations implying loss of ASXL1 gene function. We conclude that BCR-ABL independent gene mutations can be found frequently in CML patients and affect predominately epigenetic modifier genes. The finding of frequent mutations also in children/adolescents implicates that BCR-ABL independent gene mutations are not just age-related events. Such molecular aberrations may play an important role in the evolution and persistence of the disease and may affect therapy in both children and adults with CML. These findings may provide important novel genetic information regarding CML biology and for the design and performance of discontinuation trials. DisclosuresHochhaus:Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; BMS: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding.

Role of Shelterin Complex and Alternative Telomere Lengthening in Genomic Instability and Disease Progression in Chronic Myeloid Leukemia

Genomic instability has many sources, among others, shortening of telomeres, nucleoprotein complexes located at the ends of chromosomes. Tumor cells have aberrant mechanisms of telomere maintenance: their telomeres are shortened, no longer preventing chromosome end-to-end fusion and recombination, but frequently not short enough to lead to cell senescence. Both telomerase and shelterin complexes are involved in telomere homeostasis. Reduction in the telomere length is considered as one of the features of chronic myeloid leukemia (CML) similar to other human malignancies and telomere shortening is correlated with disease progression from the chronic phase (CML-CP) to the blastic phase (CML-BP)1. However, recent report shows that shorter telomeres can actually be detected in patients who discontinued imatinib and are in treatment-free remission as compared to those who relapsed2. Therefore, there is no agreement on the telomere length dynamics in CML evolution. Moreover, the precise role of telomere-associated proteins, including shelterin complex in BCR-ABL1-mediated genomic instability in CML progression and resistance to TKIs, is not fully elucidated.Initially, we confirmed that the telomere shortening was positively correlated with CML progression (CML-BP in comparison to CML-CP). However, in CD34+ samples from CML-CP TKI-resistant patients in comparison to CML-CP patients, an increase in telomere length was observed. This suggests that shortening of telomeres in CML progression may have a biphasic scenario. This can be explained by alternative telomere lengthening (ALT) mechanisms, since no significant changes in the expression of subunits of the telomerase complex and its enzymatic activity were observed at different phases of the disease; enzymatic activity of telomerase was measured immunoenzymatically, while length of telomeres was determined by Southern blotting. Then we decided to analyze possible involvement of shelterin complex and of ALT mechanisms in CML progression. Importantly, expression of the three members of the shelterin complex, Protection Of Telomeres 1 (POT1), Repressor Activator Protein 1 (RAP1) and Tankyrase 1 (TNKS1) was significantly upregulated in CML-BP (10 samples) as compared to CML-CP (15 samples) and was also positively correlated with BCR-ABL1 expression. Moreover, as determined by TKI treatment of CD34+ CML-BP primary cells, expression of POT1 was BCR-ABL1-dependent. No significant changes were observed in the expression of other members of the shelterin complex, namely TINT1-PTOP-PIP1 (TPP1), TRF1 interactor 2 (TIN2) and Tankyrase 2 (TNKS2). Also telomere repeat-binding factor 1 and 2 (TRF1 and TRF2), which are responsible for anchoring shelterin complex to the double stranded telomeric repeats remain stable in the course of the disease. Expression of subunits of telomerase and shelterin complexes was examined by RT-qPCR and Western blotting. This was confirmed in K562 and K562 imatinib-resistant cell line model.Somatic mutations in POT1 have been recently described in human tumors including chronic lymphocytic leukemia (CLL). In CLL, mutations in POT1 affect telomere stability and are associated with shorter survival in patients receiving chemotherapy as a frontline treatment. We have screened our NGS data from targeted sequencing in a cohort of patients who progressed to CML-BP (paired CP and BP samples, n=10 and BP samples, n=9) but we did not detect any somatic mutations in POT1. This is in accordance with our data on POT1 upregulated expression and suggests that dysregulation of shelterin complex during progression of CML differs significantly from CLL.In conclusion, we present the first comprehensive analysis of the expression of all members of the shelterin complex in the course of CML. We postulate that abnormal expression of selected members such as POT1, RAP1 and TNKS1 may be responsible for the aberrant telomere maintenance mechanisms in CML cells and may play an important role in genomic instability associated with CML progression.

Optimized Treatment Schedules for Chronic Myeloid Leukemia.

Over the past decade, several targeted therapies (e.g. imatinib, dasatinib, nilotinib) have been developed to treat Chronic Myeloid Leukemia (CML). Despite an initial response to therapy, drug resistance remains a problem for some CML patients. Recent studies have shown that resistance mutations that preexist treatment can be detected in a substantial number of patients, and that this may be associated with eventual treatment failure. One proposed method to extend treatment efficacy is to use a combination of multiple targeted therapies. However, the design of such combination therapies (timing, sequence, etc.) remains an open challenge. In this work we mathematically model the dynamics of CML response to combination therapy and analyze the impact of combination treatment schedules on treatment efficacy in patients with preexisting resistance. We then propose an optimization problem to find the best schedule of multiple therapies based on the evolution of CML according to our ordinary differential equation model. This resulting optimization problem is nontrivial due to the presence of ordinary different equation constraints and integer variables. Our model also incorporates drug toxicity constraints by tracking the dynamics of patient neutrophil counts in response to therapy. We determine optimal combination strategies that maximize time until treatment failure on hypothetical patients, using parameters estimated from clinical data in the literature.

Measurement of separase proteolytic activity in single living cells by a fluorogenic flow cytometry assay.

ESPL1/Separase, an endopeptidase, is required for centrosome duplication and separation of sister-chromatides in anaphase of mitosis. Overexpression and deregulated proteolytic activity of Separase as frequently observed in human cancers is associated with the occurrence of supernumerary centrosomes, chromosomal missegregation and aneuploidy. Recently, we have hypothesized that increased Separase proteolytic activity in a small subpopulation of tumor cells may serve as driver of tumor heterogeneity and clonal evolution in chronic myeloid leukemia (CML). Currently, there is no quantitative assay to measure Separase activity levels in single cells. Therefore, we have designed a flow cytometry-based assay that utilizes a Cy5- and rhodamine 110 (Rh110)-biconjugated Rad21 cleavage site peptide ([Cy5-D-R-E-I-M-R]2-Rh110) as smart probe and intracellular substrate for detection of Separase enzyme activity in living cells. As measured by Cy5 fluorescence the cellular uptake of the fluorogenic peptide was fast and reached saturation after 210 min of incubation in human histiocytic lymphoma U937 cells. Separase activity was recorded as the intensity of Rh110 fluorescence released after intracellular peptide cleavage providing a linear signal gain within a 90–180 min time slot. Compared to conventional cell extract-based methods the flow cytometric assay delivers equivalent results but is more reliable, bypasses the problem of vague loading controls and unspecific proteolysis associated with whole cell extracts. Especially suited for the investigaton of blood- and bone marrow-derived hematopoietic cells the flow cytometric Separase assay allows generation of Separase activity profiles that tell about the number of Separase positive cells within a sample i.e. cells that currently progress through mitosis and about the range of intercellular variation in Separase activity levels within a cell population. The assay was used to quantify Separase proteolytic activity in leukemic cell lines and peripheral blood samples from leukemia patients.

MDR1/ABCB1 gene polymorphisms in patients with chronic myeloid leukemia.

BackgroundTyrosine kinase inhibitors (TKIs) are the recommended treatment for patients with chronic myeloid leukemia (CML). The MDR1/ABCB1 gene plays a role in resistance to a wide spectrum of drugs, including TKIs. However, the association of MDR1/ABCB1 gene polymorphisms (SNPs) such as C1236T, G2677T/A, and C3435T with the clinical therapeutic evolution of CML has been poorly studied. We investigated these gene polymorphisms in CML-patients treated with imatinib, nilotinib and/or dasatinib.MethodsABCB1-SNPs were studied in 22 CML-patients in the chronic phase (CP) and 2 CML-patients in blast crisis (BC), all of whom were treated with TKIs, and compared with 25 healthy controls using nested-PCR and sequencing techniques.ResultsSeventeen different haplotypes were identified: 7 only in controls, 6 only in CML-patients, and the remaining 4 in both groups. The distribution ratios of homozygous TT-variants present on each exon between controls and CML-patients were 2.9 for exon 12, and 0.32 for the other 2 exons. Heterozygous T-variants were observed in all controls (100%) and 75% of CML-patients. Wt-haplotype (CC-GG-CC) was observed in 6 CML-patients (25%). In this wt-group, two were treated with nilotinib and reached a major molecular response. The remaining 4 cases had either a minimal or null molecular response, or developed bone marrow aplasia.ConclusionOur results suggest that SNPs of the MDR1/ABCB1 gene could help to characterize the prognosis and the clinical-therapeutic evolution of CML-patients treated with TKIs. Wt-haplotype could be associated with a higher risk of developing CML, and a worse clinical-therapeutic evolution.

Molecularly Defined Clonal Evolution in Patients with Chronic Myeloid Leukemia Independent of the BCR-ABL Status

Molecularly defined clonal evolution has been identified as a key phenomenon in the biology of acute myeloid leukemia. Molecular aberrations may be markers of individual subclones of the disease, but also responsible for treatment resistance. In most patients with chronic myeloid leukemia (CML), treatment with tyrosine kinase inhibitors (TKI) induces complete cytogenetic remissions (CCyR) characterized by polyclonal hematopoiesis. However, some CML patients show emergence of clonal cytogenetic abnormalities (CCA) in Philadelphia (Ph) negative cells during treatment indicating clonal hematopoiesis. We searched for BCR-ABL independent gene mutations in both Ph-negative and Ph-positive clones in 29 chronic phase CML patients (male, n=16; median age 58 years, range 29-73 years) using targeted deep next-generation sequencing of 25 genes frequently mutated in myeloid disorders: ASXL1, BRAF, CBL, DNMT3A, ETV6, EZH2, FLT3, IDH1, IDH2, JAK2, KIT, KRAS, LNK, MPL, NPM1, NRAS, RUNX1, SF3B1, SRSF2, TP53, TET2, U2AF1, UTX, WT, and ZRSR2. Ph-negative clones were analyzed in 14 CML patients who developed CCA in Ph-negative cells (trisomy 8, n=10; nonrecurrent reciprocal translocations, n=2; monosomy 7, n=2). At time of analysis, patients were in CCyR (n=7), partial CyR (n=6), or minimal CyR (n=1) after a median of 21 months (range 7-114 months) of TKI treatment with imatinib, and/or nilotinib, and/or dasatinib. After exclusion of known polymorphisms, mutations were detected in 6/14 patients (43%) affecting the genes DNMT3A, EZH2, RUNX1, TET2, TP53, U2AF1, and ZRSR2. These patients were in CCyR (n=2), partial CyR (n=3), or minimal CyR (n=1), respectively, indicating BCR-ABL independent mutations in both Ph-negative and Ph-positive subclones. In two patients, the mutations were also found in corresponding diagnostic samples at higher or lower mutation level. To further investigate BCR-ABL independent gene mutations in Ph-positive clones, 15 randomly selected CML patients at diagnosis were analyzed. Mutations additional to BCR-ABL were found in 5/15 CML patients (33%) affecting ASXL1, DNMT3A, RUNX1, and TET2. None of the mutations were recognized in corresponding constitutional DNA specimens indicating that all mutations had been somatically acquired. Deep-sequencing of subsequent samples obtained in early CCyR after three months of TKI treatment revealed one DNMT3A mutation in Ph-negative cells which was also present in Ph-positive cells at diagnosis. Follow-up investigation showed that the mutation persisted in Ph-negative cells throughout CCyR and deep molecular remission (MR4.5) up to month 36 thereby not significantly changing its mutation level of approximately 15% implying a clonal hematopoiesis before the acquisition of the BCR-ABL rearrangement. In summary, BCR-ABL independent gene mutations were frequently found in Ph-negative and Ph-positive clones of CML patients and may be considered as important cofactors in the evolution of CML. Additional mutations acquired in the Ph-positive clone may impact on response to TKI treatment. Mutations preexisting to the occurrence of the BCR-ABL rearrangement may predispose patients to secondary hematological neoplasms. Our findings provide novel genetic information regarding CML biology and warrant further studies and impact on the design and performance of discontinuation trials. DisclosuresSchnittger:MLL Munich Leukemia Laboratory: Other. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Hochhaus:Novartis: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; ARIAD: Honoraria, Research Funding; Pfizer: Consultancy, Research Funding.