Management and outcome of patients with chronic myeloid leukemia in blast phase in the tyrosine kinase inhibitor era - analysis of the European LeukemiaNet Blast Phase Registry.

Blast phase (BP) of chronic myeloid leukemia (CML) still represents an unmet clinical need with a dismal prognosis. Due to the rarity of the condition and the heterogeneity of the biology and clinical presentation, prospective trials and concise treatment recommendations are lacking. Here we present the analysis of the European LeukemiaNet Blast Phase Registry, an international collection of the clinical presentation, treatment and outcome of blast phases which had been diagnosed in CML patients after 2015. Data reveal the expected heterogeneity of the entity, lacking a clear treatment standard. Outcomes remain dismal, with a median overall survival of 23.8 months (median follow up 27.8 months). Allogeneic stem cell transplantation (alloSCT) increases the rate of deep molecular responses. De novo BP and BP evolving from a previous CML do show slightly different features, suggesting a different biology between the two entities. Data show that outside clinical trials and in a real-world setting treatment of blast phase is individualized according to disease- and patient-related characteristics, with the aim of blast clearance prior to allogeneic stem cell transplantation. AlloSCT should be offered to all patients eligible for this procedure.

Targeting FLT3-TAZ signaling to suppress drug resistance in blast phase chronic myeloid leukemia.

BACKGROUND: Although the development of BCR::ABL1 tyrosine kinase inhibitors (TKIs) rendered chronic myeloid leukemia (CML) a manageable condition, acquisition of drug resistance during blast phase (BP) progression remains a critical challenge. Here, we reposition FLT3, one of the most frequently mutated drivers of acute myeloid leukemia (AML), as a prognostic marker and therapeutic target of BP-CML. METHODS: We generated FLT3 expressing BCR::ABL1 TKI-resistant CML cells and enrolled phase-specific CML patient cohort to obtain unpaired and paired serial specimens and verify the role of FLT3 signaling in BP-CML patients. We performed multi-omics approaches in animal and patient studies to demonstrate the clinical feasibility of FLT3 as a viable target of BP-CML by establishing the (1) molecular mechanisms of FLT3-driven drug resistance, (2) diagnostic methods of FLT3 protein expression and localization, (3) association between FLT3 signaling and CML prognosis, and (4) therapeutic strategies to tackle FLT3+ CML patients. RESULTS: We reposition the significance of FLT3 in the acquisition of drug resistance in BP-CML, thereby, newly classify a FLT3+ BP-CML subgroup. Mechanistically, FLT3 expression in CML cells activated the FLT3-JAK-STAT3-TAZ-TEAD-CD36 signaling pathway, which conferred resistance to a wide range of BCR::ABL1 TKIs that was independent of recurrent BCR::ABL1 mutations. Notably, FLT3+ BP-CML patients had significantly less favorable prognosis than FLT3- patients. Remarkably, we demonstrate that repurposing FLT3 inhibitors combined with BCR::ABL1 targeted therapies or the single treatment with ponatinib alone can overcome drug resistance and promote BP-CML cell death in patient-derived FLT3+ BCR::ABL1 cells and mouse xenograft models. CONCLUSION: Here, we reposition FLT3 as a critical determinant of CML progression via FLT3-JAK-STAT3-TAZ-TEAD-CD36 signaling pathway that promotes TKI resistance and predicts worse prognosis in BP-CML patients. Our findings open novel therapeutic opportunities that exploit the undescribed link between distinct types of malignancies.

CD36 Drives Metastasis and Relapse in Acute Myeloid Leukemia.

Identifying mechanisms underlying relapse is a major clinical issue for effective cancer treatment. The emerging understanding of the importance of metastasis in hematologic malignancies suggests that it could also play a role in drug resistance and relapse in acute myeloid leukemia (AML). In a cohort of 1,273 AML patients, we uncovered that the multifunctional scavenger receptor CD36 was positively associated with extramedullary dissemination of leukemic blasts, increased risk of relapse after intensive chemotherapy, and reduced event-free and overall survival. CD36 was dispensable for lipid uptake but fostered blast migration through its binding with thrombospondin-1. CD36-expressing blasts, which were largely enriched after chemotherapy, exhibited a senescent-like phenotype while maintaining their migratory ability. In xenograft mouse models, CD36 inhibition reduced metastasis of blasts and prolonged survival of chemotherapy-treated mice. These results pave the way for the development of CD36 as an independent marker of poor prognosis in AML patients and a promising actionable target to improve the outcome of patients. SIGNIFICANCE: CD36 promotes blast migration and extramedullary disease in acute myeloid leukemia and represents a critical target that can be exploited for clinical prognosis and patient treatment.

[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 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.

Abnormal B-lymphoblasts in myelodysplastic syndromes and myeloproliferative neoplasms other than chronic myeloid leukemia.

BACKGROUND: Lineage infidelity is characteristic of mixed phenotype acute leukemia and is also seen in blast phase of chronic myeloid leukemia (CML), myeloid/lymphoid neoplasia with eosinophilia and gene rearrangements, and subtypes of acute myeloid leukemia. Driver genetic events often occur in multipotent progenitor cells in myeloid neoplasms, suggesting that multilineage output may be more common than appreciated. This phenomenon is not well studied in myelodysplastic syndrome (MDS) and non-CML myeloproliferative neoplasms (MPN). METHODS: We systematically evaluated phenotypic lineage infidelity by reviewing bone marrow pathology and flow cytometry (FC) studies of 1262 consecutive patients with a diagnosis of MDS and/or non-CML MPN. We assessed B- and T-cells in these patients by FC. When abnormal B-lymphoblast (ABLB) populations were detected, we additionally evaluated immature B-cells using a high sensitivity FC assay for B-lymphoblastic leukemia/lymphoma (B-ALL). RESULTS: We identified 9 patients (7 MDS, 7/713, 1%; 2 non-CML MPN, 2/312, 0.6%; 0 in MDS/MPN) with low-level ABLB populations (0.012%-3.6% of WBCs in marrow) with abnormal immunophenotypes. Genetic studies on flow sorted cell populations confirmed that some ABLB populations were clonally related to myeloid blasts (4/6, 67%). On follow-up, ABLB populations in 8/9 patients remained stable or disappeared. Only 1 case progressed to B-ALL. CONCLUSIONS: These findings demonstrate that phenotypically detectable abnormal immature B lineage output occurs in MDS and non-CML MPN, albeit rarely. While presence of ABLB does not necessarily reflect blast crisis, the underlying disease biology of our findings may ultimately be relevant to patient management and warrants further investigation.

The bioengineered HALOA complex induces anoikis in chronic myeloid leukemia cells by targeting the BCR-ABL/Notch/Ikaros/Redox/Inflammation axis.

Blast crisis (BC) is an outcome that arises during the treatment process of chronic myeloid leukemia (CML), which is possibly attained by the dysregulation of the Notch and Ikaros signaling pathways, BCR-ABL translocation, redox, and inflammatory factors. This study demonstrated that biotherapeutic agents target aberrant molecular axis in CML-BC cells. The HALOA complex was synthesized by simple mixing of apo α-lactalbumin with oleic acid, which manages to inhibit BCR-ABL (b3a2 in K562 cells) translocation. It elevates the production of reactive oxygen species (ROS), reactive nitrogen species (RNS), and protein carbonyl, which induces DNA fragmentation in K562 cells but not in NIH cells. The complex manages to reduce the toxicity surrounding apoptotic cells by enhancing the production of superoxide dismutase (SOD) and the total antioxidant level. The HALOA complex increases leptin to maintain normoxic conditions, ultimately preventing angiogenesis. This complex downregulates the expression of IL-8 and MMP-9 and elevates the expression levels of Notch 4, Ikaros, and integrin alpha-D/CD-11d (tumor-suppressive), which conjointly prevents inflammation, metastasis, and epithelial-mesenchymal transition (EMT) in CML cells. Meanwhile, the complex downregulates Notch 1 and 2 (oncogenic), consequently inducing anoikis in CML cells. Overall, the HALOA complex shows credibility by targeting the combined molecular factors responsible for the pathogenesis of the disease and will also help to overcome MDR conditions in leukemia.

Extramedullary early T-cell lymphoblastic crisis in a young pregnant chronic myeloid leukemia patient: Diagnosis with fine-needle aspiration cytology and flow cytometry.

Chronic myeloid leukemia (CML) most commonly presents in chronic phase. Blast crisis in CML is usually of myeloid phenotype, whereas among lymphoid lineage, B-cell lymphoblastic crisis is common. T lymphoblastic crisis is rare with near early T-cell precursor (ETP) immunophenotype being exceedingly rare and very little is known about its characteristics, treatment, and prognosis. Blast crisis can occur in extramedullary sites with lymph node being the most common site. CML is also less investigated and studied in pregnancy as it is considered a disease of older adults. We report a rare case of CML presenting in extramedullary site (lymph node) as extramedullary T-cell lymphoblastic crisis of near ETP immunophenotype in a young pregnant female, which was diagnosed on fine-needle aspiration cytology in combination with flow cytometry.

All-trans retinoic acid induces differentiation in primary acute myeloid leukemia blasts carrying an inversion of chromosome 16.

All-trans retinoic acid (ATRA)-based therapy for acute promyelocytic leukemia (APL), a subtype of acute myeloid leukemia (AML), is the most successful example of differentiation therapy. Although ATRA can induce differentiation in some non-APL AML cell lines and primary blasts, clinical results of adding ATRA to standard therapy in non-APL AML patients have been inconsistent, probably due to use of different regimens and lack of diagnostic tools for identifying which patients may be sensitive to ATRA. In this study, we exposed primary blasts obtained from non-APL AML patients to ATRA to test for differentiation potential in vitro. We observed increased expression of differentiation markers, indicating a response to ATRA, in four out of fifteen primary AML samples. Three samples in which CD11b increased in response to ATRA had an inversion of chromosome 16 as well as the CBFB-MYH11 fusion gene, and the fourth sample was from a patient with KMT2A-rearranged, therapy-related AML. In conclusion, we identified a subgroup of non-APL AML patients with inv(16) and CBFB-MYH11 as the most sensitive to ATRA-mediated differentiation in vitro, and our results can help identify patients who may benefit from ATRA treatment.

Aleukemic Extramedullary Blast Crisis as an Initial Presentation of Chronic Myeloid Leukemia with E1A3 BCR-ABL1 Fusion Transcript.

Right neck swelling and pain occurred in a 49-year-old man. A Blood count showed a slight increase in platelet count without leukemoid reaction. After a biopsy of the cervical mass and bone marrow aspiration, a diagnosis of extramedullary blast crisis (EBC) of chronic myeloid leukemia (CML) was made. Fluorescence in situ hybridization (FISH) analysis showed a BCR-ABL1 fusion signal, but results of real-time polymerase chain reaction (RT-PCR) for major and minor BCR-ABL1 transcripts were negative. We identified a rare e1a3 BCR-ABL1 fusion transcript. Administration of dasatinib resulted in disappearance of the extramedullary tumor. This is the first reported case of CML-EBC with e1a3 transcript. An aleukemic extramedullary tumor can be the initial presentation of CML.

Blastic plasmacytoid dendritic-cell neoplasia: a challenging case report.

Blastic plasmacytoid dendritic-cell neoplasm (BPDCN) is an extremely rare disease that originates from dendritic cells and is associated with a poor overall survival (OS). Diagnostic and therapeutic standards are less well-established in comparison to other leukemic conditions and standards of care are lacking. Morphologic and molecular similarities to acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML) are hard to distinguish. We here report a BPDCN patient with a long, challenging diagnostic period. While bone marrow biopsies initially failed to prove the correct diagnosis, a cutaneous biopsy finally identified a CD45+/CD56+/CD4+/CD123+/CD33+/MPO- population suggestive of BPDCN which was confirmed by flow cytometry. Molecular analysis revealed an ASXL-1, TET2 and SRSF2-mutation, cytogenetic analysis showed a normal karyotype. Treatment with the recently approved CD123-cytotoxin Tagraxofusp showed initially a very good response. This case reflects diagnostic and therapeutic difficulties in BPDCN as very rare, easily misdiagnosed neoplasia and the need for precise diagnostic care.

[De novo blast phase of chronic myeloid leukemia with 3q26 abnormality diagnosed by cytogenetic analysis of extramedullary lesion].

A 62-year-old woman was presented at our hospital with visual disturbance. An ocular examination revealed bilateral Roth spots. Laboratory data revealed leukocytosis (236,200 µl) with an excess blast (11%). Physical examination and computed tomography (CT) showed systemic lymphadenopathy. A bone marrow examination revealed a composition of 9.2% blast. Chromosomal analysis on bone marrow cells revealed 46,XX,t (3;12)(q26.2;p13),t (9;22)(q34.1;q11.2) in 80% of metaphases (16/20). Inguinal lymph node biopsy revealed diffuse proliferation of myeloperoxidase (MPO)-positive abnormal cells. Fluorescence in situ hybridization analysis was used to detect the BCR-ABL1 fusion gene and split the signals of MECOM and ETV6. She was diagnosed with de-novo chronic myeloid leukemia (CML) extramedullary blast crisis. She received tyrosine kinase inhibitor (TKI) combination chemotherapy and allogeneic hematopoietic stem cell transplantation and achieved a major molecular response. In this study, we reported a case of CML in blast-phase initially presenting as extramedullary, in which cytogenetic and molecular analyses were useful in the staging method.

Genetic Biomarkers in Chronic Myeloid Leukemia: What Have We Learned So Far?

Chronic Myeloid Leukemia (CML) is a rare malignant proliferative disease of the hematopoietic system, whose molecular hallmark is the Philadelphia chromosome (Ph). The Ph chromosome originates an aberrant fusion gene with abnormal kinase activity, leading to the buildup of reactive oxygen species and genetic instability of relevance in disease progression. Several genetic abnormalities have been correlated with CML in the blast phase, including chromosomal aberrations and common altered genes. Some of these genes are involved in the regulation of cell apoptosis and proliferation, such as the epidermal growth factor receptor (EGFR), tumor protein p53 (TP53), or Schmidt-Ruppin A-2 proto-oncogene (SRC); cell adhesion, e.g., catenin beta 1 (CTNNB1); or genes associated to TGF-ß, such as SKI like proto-oncogene (SKIL), transforming growth factor beta 1 (TGFB1) or transforming growth factor beta 2 (TGFB2); and TNF-α pathways, such as Tumor necrosis factor (TNFA) or Nuclear factor kappa B subunit 1 (NFKB1). The involvement of miRNAs in CML is also gaining momentum, where dysregulation of some critical miRNAs, such as miRNA-451 and miRNA-21, which have been associated to the molecular modulation of pathogenesis, progression of disease states, and response to therapeutics. In this review, the most relevant genomic alterations found in CML will be addressed.

MTH1 Inhibitor TH1579 Induces Oxidative DNA Damage and Mitotic Arrest in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy, exhibiting high levels of reactive oxygen species (ROS). ROS levels have been suggested to drive leukemogenesis and is thus a potential novel target for treating AML. MTH1 prevents incorporation of oxidized nucleotides into the DNA to maintain genome integrity and is upregulated in many cancers. Here we demonstrate that hematologic cancers are highly sensitive to MTH1 inhibitor TH1579 (karonudib). A functional precision medicine ex vivo screen in primary AML bone marrow samples demonstrated a broad response profile of TH1579, independent of the genomic alteration of AML, resembling the response profile of the standard-of-care treatments cytarabine and doxorubicin. Furthermore, TH1579 killed primary human AML blast cells (CD45+) as well as chemotherapy resistance leukemic stem cells (CD45+Lin-CD34+CD38-), which are often responsible for AML progression. TH1579 killed AML cells by causing mitotic arrest, elevating intracellular ROS levels, and enhancing oxidative DNA damage. TH1579 showed a significant therapeutic window, was well tolerated in animals, and could be combined with standard-of-care treatments to further improve efficacy. TH1579 significantly improved survival in two different AML disease models in vivo. In conclusion, the preclinical data presented here support that TH1579 is a promising novel anticancer agent for AML, providing a rationale to investigate the clinical usefulness of TH1579 in AML in an ongoing clinical phase I trial. SIGNIFICANCE: The MTH1 inhibitor TH1579 is a potential novel AML treatment, targeting both blasts and the pivotal leukemic stem cells while sparing normal bone marrow cells.

Blast cells surviving acute myeloid leukemia induction therapy are in cycle with a signature of FOXM1 activity.

BACKGROUND: Disease relapse remains common following treatment of acute myeloid leukemia (AML) and is due to chemoresistance of leukemia cells with disease repopulating potential. To date, attempts to define the characteristics of in vivo resistant blasts have focused on comparisons between leukemic cells at presentation and relapse. However, further treatment responses are often seen following relapse, suggesting that most blasts remain chemosensitive. We sought to characterise in vivo chemoresistant blasts by studying the transcriptional and genetic features of blasts from before and shortly after induction chemotherapy using paired samples from six patients with primary refractory AML. METHODS: Leukemic blasts were isolated by fluorescence-activated cell sorting. Fluorescence in situ hybridization (FISH), targeted genetic sequencing and detailed immunophenotypic analysis were used to confirm that sorted cells were leukemic. Sorted blasts were subjected to RNA sequencing. Lentiviral vectors expressing short hairpin RNAs were used to assess the effect of FOXM1 knockdown on colony forming capacity, proliferative capacity and apoptosis in cell lines, primary AML cells and CD34+ cells from healthy donors. RESULTS: Molecular genetic analysis revealed early clonal selection occurring after induction chemotherapy. Immunophenotypic characterisation found leukemia-associated immunophenotypes in all cases that persisted following treatment. Despite the genetic heterogeneity of the leukemias studied, transcriptional analysis found concerted changes in gene expression in resistant blasts. Remarkably, the gene expression signature suggested that post-chemotherapy blasts were more proliferative than those at presentation. Resistant blasts also appeared less differentiated and expressed leukemia stem cell (LSC) maintenance genes. However, the proportion of immunophenotypically defined LSCs appeared to decrease following treatment, with implications for the targeting of these cells on the basis of cell surface antigen expression. The refractory gene signature was highly enriched with targets of the transcription factor FOXM1. shRNA knockdown experiments demonstrated that the viability of primary AML cells, but not normal CD34+ cells, depended on FOXM1 expression. CONCLUSIONS: We found that chemorefractory blasts from leukemias with varied genetic backgrounds expressed a common transcriptional program. In contrast to the notion that LSC quiescence confers resistance to chemotherapy we find that refractory blasts are both actively proliferating and enriched with LSC maintenance genes. Using primary patient material from a relevant clinical context we also provide further support for the role of FOXM1 in chemotherapy resistance, proliferation and stem cell function in AML.