BCR-ABL Translocation Research Articles

CircPCMTD1 : A protein-coding circular RNA that regulates DNA damage response in BCR/ABL -positive leukemias.

Circular RNAs are a novel class of RNA transcripts, which regulate important cellular functions in health and disease. Herein, we report on the functional relevance of the circPCMTD1 transcript in acute leukemias. In screening experiments, we found that circPCMTD1 depletion strongly inhibited the proliferative capacity of leukemic cells with BCR-ABL translocations. Mass cytometry experiments identified the aberrant activation of the DNA damage response as an early downstream event of circPCMTD1 depletion. In in vivo experiments, circPCMTD1 targeting prolonged the survival of mice engrafted with leukemic blasts harboring the Philadelphia chromosome. Mechanistically, we found that circPCMTD1 was enriched in the cytoplasm and associated with the ribosomes of the leukemic cells. We detected a cryptic open reading frame within the circPCMTD1 sequence and found that circPCMTD1 could generate a peptide product. The circPCMTD 1-derived peptide interacted with proteins of the BTR complex and enhanced BTR complex formation, thereby increasing tolerance to genotoxic stress.

Juvenile Myelomonocytic Leukemia – Experience from a Tertiary Care Hospital in Eastern India

Background: Juvenile myelomonocytic leukemia (JMML), previously known as juvenile chronic myeloid leukaemia, is a rare, unique, and aggressive myeloproliferative neoplasm of early childhood. Making a diagnosis of JMML is challenging because of the overlapping clinical and haematological features with other myeloproliferative neoplasms (MPN). However, some unique features like monocytosis, the absence of BCR-ABL translocation, and the presence of specific mutations (PTPN-11, K-RAS, N-RAS, CBL, or NF1) clinch the correct diagnosis.Methods: A prospective analysis of six JMML patients with variable clinical features treated with injection azacytidine as frontline therapy during the study period of 2 years. Results: The median age was 4.5 years with male:female ratio 2:4. Pallor and splenomegaly were the most common presenting signs. Four patients (66.67%) achieved complete remission (CR), two patients (33.33%) had partial remission (PR), and one patient (16.67%) had progressive disease (PD). The overall survival rate was 66.67% (four out of six), and the mortality rate was 33.33%.Conclusion: Azacitidine is an effective treatment option as upfront therapy for JMML, especially in resource poor developing countries.

The Tetraspanin CD9 Shapes Glucocorticoid Sensitivity in Pediatric B-Precursor Acute Lymphoblastic Leukemia

Background: De novo or acquired resistance to glucocorticoids (GCs), the common therapeutic regimens for remission-induction in pediatric B-precursor acute lymphoblastic leukemia, confronts treatment success and is a strong predictor for disease relapse. Discovery of predictive markers and underlying mechanisms of GC resistance could lead to new treatment and prognostic modalities. Aims: This study aims: (1) to demonstrate the association between CD9 expression and GC sensitivity in childhood BCP-ALL; (2) to dissect the underlying mechanisms driving GC resistance; and (3) to discover the possible therapeutic modalities for restoring GC susceptibility. Methods: Ex vivo drug sensitivity profiling to prednisone (Pred) and dexamethasone (Dex) was performed on 6 BCP-ALL cell lines and 18 patient samples with differential CD9 expression. Association of CD9 expression with patient responses to Pred prophase was analyzed in a pediatric BCP-ALL cohort (n=182). Functional impact of CD9 on GC susceptibility was dissected by gain- and loss-of-function experiments in BCP-ALL cells. Mechanistic basis of CD9-driven GC response was investigated through genomics, transcriptomics, and proteomics. Reversal of GC resistance by pathway-targeted agents were explored on BCP-ALL cells and xenograft models. Results: Distinctive differences in the drug sensitivity pattern were found between CD9- and CD9+ BCP-ALL cell lines, with the former showing resistance to GCs but not other chemotherapeutic agents. Concurrently, CD9- primary lymphoblasts exhibited resistance to Dex (IC50s: 2,761 vs. 31 nM) and Pred (IC50s: 8,185 vs. 268 nM) compared with CD9+ samples (positivity defined by the presence of ≥ 20% CD9+ blasts). More Pred poor responders (defined by the presence of ≥ 1×10 9/L circulating lymphoblasts on day 8) were found in CD9- than CD9+ patients (19.4% vs. 6.2%, P=0.018). Subgroup analyses revealed more poor responders with CD9- phenotype were enriched in patients with older age (60% vs. 3.7%, P=0.008), male gender (21.7% vs. 6.7%, P=0.045), higher white blood cell count (66.7% vs. 15.4%, P=0.004), and in those with BCR-ABL1 translocation (100% vs. 12.5%, P=0.024), or not otherwise cytogenetically specified BCP-ALL (30.8% vs. 5.8%, P=0.019). Univariate and multivariate analyses further confirmed CD9 negativity as an independent predictor for GC resistance (OR=5.1, P=0.009). Overexpression of CD9 in CD9- cells (SEM, KOPN-8) by lentiviral transduction significantly enhanced GC sensitivity by 2.8-62.3-fold ( P<0.024), whereas CD9 knockout in CD9+ cells (697) using CRISPR-Cas9 decreased GC susceptibility ( P<0.003). No significant differences in mRNA or protein expression of the GC receptor NR3C1 were identified between CD9+ and CD9- cells, as well as its isoforms or mutants. The cytoplasm-to-nucleus translocation of GC receptor was consistent among cell lines regardless of CD9 expression status, but CD9 was physically interacted with NR3C1. Upon GC stimulation, the presence of CD9 increased the number of DEGs (110 vs. 82 DEGs) and magnitude of GC-responsive genes exclusively detected in the background of CD9 (1.3-18.6-fold; e.g. AKAP13, STAT5A, EZR). CD9- cells showed constitutive activation of the MAPK pathway and could be preferentially restored for GC susceptibility by the MEK1/2 inhibitor trametinib in vitro. BCP-ALL xenograft modeling revealed that, compared to single agent Dex treatment, combination with trametinib significantly reduced the leukemic load in animal transplanted with CD9- SEM but not CD9+ BV-173 cell lines. Ex vivo drug testing further showed that the resistance to GCs in some CD9+ cases could be partially reversed by combination of trametinib and the JAK inhibitor ruxolitinib due to the activation of STAT5. Conclusions: This study provides a definitive linkage of CD9 with GC sensitivity in childhood BCP-ALL mediated through a NR3C1-independent mechanism. CD9- cases could be prone to trametinib combination therapy, whereas CD9+ cases might need a third drug such as ruxolitinib to counteract GC resistance.

Calr+ Myelofibrosis/BCR-ABL Chronic Myeloid Leukemia Overlap Syndrome Treated with Asciminib

Introduction Myeloproliferative neoplasms are considered mutually exclusive, and reports of co-occurring mutations are rare. Over the past 10 years, approximately two dozen case reports have been published of co-occurring BCR-ABL positive and CALR positive patients. Here we present the case of a patient who was reported to have myelofibrosis (MF)/myeloproliferative neoplasm (MPN), later developed chronic myeloid leukemia (CML), and ultimately progressed to acute myeloid leukemia (AML). This is unique in the literature reviewed, and may be the first reported case of this overlap syndrome being treated with asciminib. Case presentation A 40-year old male was diagnosed in 2018 with MF/MPN based upon a hypercellular bone marrow biopsy with diffuse reticulin fibrosis. He was negative for JAK2, but was not tested for CALR or MPL mutations. Treatment was ruxolitinib and hydroxyurea. Two years later, the patient was diagnosed with CML after being admitted for recurrent fevers, headache, and shortness of breath. At that time, a type 2 CALR mutation (CALR K385Nfs*47) was noted. He started dasatinib and decitabine, which resulted in a drop from 28.31% BCR-ABL to 0.0196% (Table 1A). However, hyperuricemia and renal failure led to a change in regimen and the patient was started on asciminib and ropeginterferon alfa-2b-njft in January 2022. By December, the patient's BCR-ABL PCR dropped below quantification, but he received a diagnosis of AML based on a bone marrow biopsy which showed 43% blasts. He received cladribine and cytarabine, but was found to have leukemic infiltration of the spinal cord five months later. The patient was referred to hospice and passed away in June 2023. Discussion To our knowledge, this is the first MPN-CML patient reported to have been treated with asciminib. This case illustrates its effectiveness in treating CML, but highlights the need for improved treatments for CALR+ MPN. Moreover, this case illustrates the importance of testing for CALR, MPL, and BCR-ABL in addition to JAK2 immediately when a patient presents with an MPN. It is unknown whether this patient's CALR mutation preceded the BCR-ABL translocation, and there is debate in the literature with respect to mutation chronology. The patient's BCR-ABL burden dropped from 28.31% to 0.0196% while the CALR burden remained around 94% over the same period (Table 1B). Thus, it is unlikely that the BCR-ABL mutation preceded the CALR mutation within the same clone. The patient's >90% CALR allele burden is higher than reported in the other reported cases, where the burden ranged from 10% to 68%. Furthermore, none of the other cases were reported to progress to AML, though follow up data is lacking. The heavy allelic burden in our patient may have been a contributing factor to the development of AML, as has been reported elsewhere. The patient's overall mutational profile is of interest. Type 2 CALR mutations have been reported to have less favorable prognoses than type 1 mutations. In addition to the CALR and BCR-ABL mutations, the patient had ASXL1 A861Hfs*6, EZH2 D730*, and NRAS G12V. All three mutations are associated with unfavorable outcomes in MDS, AML, and/or CML. Genomic instability has been discussed in relation to both CML and JAK2-positive MPN. Our patient's heavy mutational burden suggests CALR-positive neoplasms may also present against a background of inherent genomic instability. Conclusion We reported a case of MPN, followed by CML, and then AML. The patient was positive for CALR and BCR-ABL. He achieved a complete response in CML after targeted treatment, with asciminib consolidating the gains the patient made on earlier therapies. However, he always maintained a high CALR burden. This patient's mutational profile and disease course argue for the importance of early testing for CALR and BCR-ABL in addition to JAK2 and MPL in the setting of an MPN and for further research into the underlying causes of these overlap conditions.

Pediatric chronic myeloid leukemia (CML) presenting with ocular manifestations: A case report

A 14-year-old male child presented to the ophthalmologist with complaints of floaters and headache. Ophthalmic examination revealed dilated and tortuous retinal veins, perivascular infiltrates scattered throughout the fundus, and multiple white centered hemorrhages (leukemic infiltrates). A provisional diagnosis of leukemia or lymphoma was made. The diagnosis was confirmed by further systemic workup and complete blood count. A final diagnosis of CML in chronic phase was made after bone marrow aspiration, biopsy, and presence of BCR-ABL translocation. This case report highlights the role of an ophthalmologist in diagnosing and prognosticating a rare entity of pediatric CML with ocular manifestation as its presenting symptoms.

Philadelphia Chromosome Positive and Philadelphia-Like Acute Lymphoblastic Leukemia in Children and Adolescents: Current Management, Controversies and Emerging Concepts.

Philadelphia chromosome positive (Ph+) acute lymphoblastic lymphoma (ALL) is an uncommon subtype of ALL in children, seen in 2-5% cases. Diagnostic evaluation includes conventional karyotyping and detection of BCR-ABL1 translocation by fluorescence in-situ hybridization (FISH) or reverse transcriptase polymerase chain reaction (RT-PCR). For children, the frontline management includes combination of intensive chemotherapy along with imatinib (300-340mg/m2/d) or dasatinib (60-80mg/m2/d). Imatinib/dasatinib should be introduced in induction as soon as results for BCR-ABL are available. Minimal residual disease (MRD) monitoring is essential; multi-parametric flowcytometry and immunoglobulin/T-cell receptor rearrangement PCR are the preferred methods. Intrathecal therapy with at least 12 doses of methotrexate is adequate for central nervous system (CNS) prophylaxis, but cranial radiation is necessary for CNS3 involvement. Allogeneic hematopoietic stem cell transplantation (HSCT) in first remission may be considered in high-risk cases (persistent MRD positivity/induction failure). Maintenance therapy with tyrosine kinase inhibitors (TKI) in children is debatable, with potential concerns for long term adverse effects. At relapse, the choice of TKI is guided by the presence of BCR-ABL tyrosine kinase domain resistance mutations, although the frequency of resistance mutations in children are lower. Allogeneic HSCT is essential for consolidation in second remission, if not done.Ph-like ALL is a newly recognized molecular entity, with gene expression profile similar to Ph+ALL and poor survival outcomes. In resource-constrained settings, a stepwise cost-effective diagnostic evaluation should be considered among high-risk patients without recurrent genetic abnormalities. Current treatment strategies remain similar to Ph-negative ALL. Enrolment in clinical trials is encouraged for such children to evaluate potential targeted agents in this subtype.

Clonal evolution analyses of a chronic myeloid leukemia patient with hematopoietic stem cell transplantation based on deep sequencing

BackgroundThe Philadelphia (Ph) chromosome is the hallmark chromosome aberration in chronic myeloid leukemia (CML), which confers the cancer phenotype of the disease. However, how the Ph chromosome forms and the genetic clonal evolution structure after targeted Ph treatment are still unclear.MethodsIn this study, we performed genome sequencing and clonal evolution analyses in a series of bone marrow specimens and skin biopsy from a CML patient who had received hematopoietic stem cell transplantation from her sister, then relapsed (lymphoid blast crisis), and received Ph-targeted therapy.ResultsThe Ph chromosome was the “driver” clonal change in the original CML and the relapse. Both the patient and her sister had micro-deletions in the BCR gene region; however, the patient had a frameshift BRIP1 mutation that may account for the malfunctioning homologous recombination DNA repair of the BCR gene region and formation of the Ph chromosome.ConclusionWe found that the BCR-ABL1 translocation was the driving force of the patient’s CML and relapse. The malfunctioning double-strand DNA break repair caused by the BRIP1 mutation could be the cause of Ph chromosome formation in the patient.

IL-18 and VEGF-A trigger type 2 innate lymphoid cell accumulation and pro-tumoral function in chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is a hematologic malignancy associated to an unregulated growth of myeloid cells in bone marrow (BM) and peripheral blood (PB), characterized by the BCR-ABL1 translocation. Given the known cytokine impairment in the leukemic niche of CML, we investigated the impact of this microenvironmental dysregulation on innate lymphoid cells (ILC), whose role in cancer has recently emerged. Three ILC subsets are identified based on transcriptional profiles and cytokine secretion. We observed that interleukin 18 (IL-18) and vascular endothelial growth factor A (VEGF-A) are increased in CML patients' sera and that ILC2 are enriched in CML PB and BM. We found that IL-18 drives ILC2 proliferation and that CML ILC2 highly express CXCR4 and CXCR7 BM-homing receptors, potentially explaining their enrichment in PB and BM, respectively. Next, we showed that ILC2 are hyper-activated through a tumor-derived VEGF-Adependent mechanism, which leads to higher IL-13 secretion. In response to IL-13, leukemic cells increase their clonogenic capacity. Finally, we discovered that the pro-tumoral axis involving VEGF-A, IL-18 and ILC2 was disrupted upon tyrosine kinase inhibitor treatment, normalizing the levels of all these players in CML patients responding to therapy. Overall, our study uncovers the involvement of ILC2 in CML progression, mediated by VEGF-A and IL-18.

GENETIC CHIMERISM IN MYELOPROLIFERATIVE NEOPLASMS: CLINICAL CASE OF COEXISTENCE OF JAK2V617F MUTATION AND BCR-ABL TRANSLOCATION

Essentialy, the diagnostics feature of myeloproliferative neoplasms (MPNs) is a genetic mutations. While bcr-abl translocation belongs to CML, Jak2V617F mutation is commonly associated with other MPNs. The recent decade has been clarified a data when a human blood presents bcr-abl translocation and Jak2V617F mutation both. This clinical case reports of bcr-abl translocation and Jak2V617F mutation when they were detected simultaneously by polymerase chain reaction (PCR) in a patient with myeloproliferative neoplasm, unclassifiable (MPN-U).

Outcomes of Patients with B-ALL with Concomitant BCR::ABL1 and CRLF2 Rearrangements

Background: Outcomes of patients with Philadelphia chromosome positive (Ph+) ALL have improved significantly with the introduction of tyrosine kinase inhibitors (TKI). Ph-like ALL is a high-risk subtype of B-ALL with gene expression profile similar to Ph+ ALL, but without the BCR-ABL translocation. About 50% of the patients with Ph-like ALL have CRLF2 rearrangement (CRLF2-R) and are associated with poor outcomes with conventional chemoimmunotherapy. The co-occurrence of both Ph+ and CRLF2-R is rare (Jain, Haematologica 2017) and long-term outcomes unknown. Methods: We retrospectively analyzed the Leukemia and Pathology databases for patients who presented to the MD Anderson Cancer Center (MDACC) and were noted to have concomitant Ph+ and CRLF2-R. Ph+ was assessed by conventional cytogenetics / FISH / PCR for BCR-ABL1. CRLF2-R was assessed by flow cytometry / FISH. Outcomes assessed were best response (complete remission (CR), relapse, minimal residual disease (MRD), event-free survival (EFS) and overall survival (OS). Results: We identified 8 patients (5 newly diagnosed and 3 R/R B-ALL) who had concurrent Ph+ and CRLF2-R rearrangement (Table 1). The median age was 29 (range, 14-57), 6 (75%) were of Hispanic ethnicity, and 6 (75%) were men. The median clone size of CRLF2-R and Ph+ were 73% (56-92%) and 52% (4-96%), respectively. The most frequent mutated gene was JAK2 in 3/8 (38%) patients. All patients were treated with chemoimmunotherapy or immunotherapy + TKI (ponatinib, n=7; dasatinib, n=1). The median follow-up was 23 months. The best response achieved was CR in 6/8 (75%). The median EFS was 8.6 months; 12-month OS was 75% (Figure 1). A total of 3/6 (50%) responders had a subsequent relapse with 2/3 relapses occurring in the extramedullary compartment (one in central nervous system and one intraperitoneal); both with CRLF2-R clone without Ph+. Of the 5 patients who presented to MDACC as newly diagnosed B-ALL, two were treated with frontline with Hyper-CVAD + ponatinib/dasatinib, two with blinatumomab + ponatinib and the one with a pediatric regimen + dasatinib. Best response was CR with negative MRD in 5/5 (100%). All 5 patients cleared both rearrangements (Ph+ and CRLF2) at the time of remission; 2/5 patients showed CRLF2-R during relapse (without the Ph+). The 12-month OS and EFS were 80% and 60%, respectively. Three patients presented to MDACC with R/R B-ALL. They received HCVAD + ponatinib/dasatinib +/-ruxolitinib. Best response was CR in 1/3 (33%) patients. All 3 patients cleared Ph+, but CRLF2-R persisted in 2 patients. Conclusions: Patients who harbor concomitant Ph+ and CRLF2-R represent a rare but high-risk subgroup of B-ALL. High rate of relapse was noted despite treatment with chemotherapy + TKI +/- ruxolitinib; several patients cleared the Ph+ clone, but CRLF2-R clone persisted suggesting novel interventions against CRLF2-R B-ALL are warranted. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

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.

Abstract 12981: Non-Conventional Causes of ST-Elevation Myocardial Infarction: Essential Thrombocytosis

Case: A 61-year-old female with no significant past medical history, except hospitalization for COVID-19 pneumonia complicated by myocarditis ten months prior, presented to the emergency department with worsening chest pain over the last two weeks. The admission electrocardiogram (ECG) was without ischemic changes. The initial impression was that the patient's symptoms were attributed to her prior myocarditis. However, a repeat ECG obtained six hours later, due to worsening chest pain, demonstrated ST-segment elevations in leads V2, I and AVL. Emergent coronary angiography was performed, showing a 90% occlusion of the proximal LAD, which was managed with a drug-eluting stent. Admission platelet count was significantly elevated at 1,049,000mm 3 , which resulted in a hematology consultation. Her further work up included a positive JAK2-V617F mutation and negative BCR-ABL1 translocation, which confirmed the diagnosis of essential thrombocythemia. During the prior hospitalization for her COVID pneumonia, she underwent invasive coronary angiogram for suspected myocardial infarction in the setting of elevated troponin, which showed a mild, non-obstructive lesion in the proximal LAD. She underwent a cardiac MRI that demonstrated both sub-epicardial and mid-wall late gadolinium enhancement consistent with myocarditis. Discussion: Essential thrombocythemia is a clonal myeloproliferative disease caused by a pathological expansion of megakaryocytes, which results in excess platelet production. Essential thrombocythemia leads to a hematologic hyper-viscosity and subsequent increased risk of vasomotor, thrombotic and hemorrhagic manifestations. Myocardial infarction as the primary clinical presentation of essential thrombocythemia is rare but an important risk factor that should be considered in patients presenting with acute coronary syndromes and elevated platelet counts, both with and without traditional cardiovascular risk factors.

MPN-166 Myeloproliferative Neoplasm (MPN) Post-Renal Transplant (RT)-A Single-Center Review.

MPN post-RT is a rare disease. There is little data on the clinicopathological features and outcomes of patients with post-RT MPN. The main objective of this study was to analyze the clinical outcome of MPN post-RT. This was a single-center retrospective study Settings: Patients who had post-RT MPN at Memorial Herman Hospital, Texas Medical Center. We reviewed 1998 patients who underwent RT between January 1, 1999, and December 31, 2019. Three cases of post-RT MPN were identified. None Main Outcome Measures: We analyzed the incidence, the interval between RT and MPN diagnosis, MPN treatment, and clinical outcome of patients with post-RT MPN. Among 1998 RT recipients, 3 cases (0.1%) of MPN were identified. All occurred in women with a mean age of 53 years. Races were African American (n=2) and white (n=1). Causes of end-stage renal disease were diabetes, hypertensive, and IgA nephropathy. Two patients received RT, and one patient simultaneous kidney and pancreas transplant. All three patients were on tacrolimus, mycophenolate, and prednisone for post-transplant immunosuppression. Two patients developed chronic myeloid leukemia (CML), and one had polycythemia vera (PV). The mean interval between RT and diagnosis of MPN was 9 years. Cytogenetics demonstrated JAK-2 mutation in the patient with PV and BCR-ABL translocation in the two CML patients. The CML patients received BCR-ABL tyrosine kinase inhibitors (TKIs; imatinib/dasatinib) and continue to have a major molecular response. The patient with PV remains on hydroxyurea (HU) and aspirin with excellent hematocrit control. Mycophenolate was discontinued in all three patients after the diagnosis of MPN. TKI and HU were well tolerated. No thrombotic events or leukemic transformation were reported. One CML patient developed cytomegalovirus colitis. No other infectious complications were noted. She also developed immune-related hemolytic anemia and thrombocytopenia that were successfully treated with glucocorticoids. All three patients still have preserved allograft function and are alive at a median follow-up of 42 months. MPN post-RT is rare. Standard-of-care therapies for MPN are well tolerated. Further studies are required to explore the pathophysiology and outcomes of MPN in RT recipients.

PARP INHIBITION INDUCES PHILADEPHIA P190 POSITIVE ACUTE LYMPHOID LEUKEMIA CELL DEATH IN LEVELS SIMILAR TO IMATINIB TREATMENT

Acute Lymphoid Leukemia (ALL) is the most common leukemic subtype among pediatric populations. The prognosis is worse among patients which are Philadelphia chromosome positive (Ph+), detected with BCR-ABL1 p190 translocation, and, although treatments strategies utilizing tyrosine-kinase inhibitors improved Ph+ patient's outcomes, the development of new therapeutic approaches is of utmost importance in the oncologic practice when facing the inevitable cases of resistance emergence. This study aims to evaluate the cytotoxic effect of inhibiting poly-ADP-ribose 1 (PARP1), a signaling pathway for DNA repair machinery, in an ALL Ph+ cell model. Cytotoxicity analyses of the compounds AZD2461, Imatinib and Doxorubicin were performed by Alamar Blue assay in three cell lines: SUP-B15 – B-lymphoblast Ph p190+ ALL –, Raji and Namalwa – both B-lymphocyte Burkitt's lymphoma. Drug cytotoxicity was determined through serial dilutions resulting in final concentrations of 10 μM, 2 μM, 0.4 μM, 0.08 μM, 0.016 μM and 0.0032 μM for every drug. All concentrations were analyzed in triplicates for each cell line after a period of 72 hours of treatment, except for SUP-B15, which was also analyzed with all the drugs in periods of 24 and 48 hours. After the treatment period, Alamar Blue was added to each well, plates were incubated and then read for fluorescence. Viability results were analyzed through non-linear regression using GraphPad Prism (v. 5.01). AZD2461 showed cytotoxic activity comparable to that of Imatinib in Ph+ SUP-B15, represented by an IC 50 at 72 hours of treatment of 344.3 nM and 329.2 nM, respectively. The use of PARP inhibitors showed little to no efficacy when treating either of the remaining cell lines, with an IC 50 that was not reached in treatment of Raji and a high IC 50 of 20.9 μM in the treatment of Namalwa. Doxorubicin showed high cytotoxic activity against all cell lines, with IC 50 values at 72 hours of low nanomolar levels; 20.32 nM, 85 nM and 75.93 nM for SUP-B15, Raji and Namalwa, respectively. Due to non-responsiveness of Raji and Namalwa to the targeted therapies, only SUP-B15 was evaluated at 24 and 48 hours and treatment. AZD2461 and Imatinib's IC 50 remained comparable at 1421 nM and 1230 nM at 48 hours, respectively, and 3295 nM and 3966 nM at 24 hours, respectively. The use of PARP inhibitors (PARPi) shows great clinical efficiency in the treatment of BRCA1/ 2-mutated tumors and other malignancies that harbor defects in double-strand-breaks repair through homologous recombination. Although Raji and Namalwa have been previously shown to considerably overexpress PARP1 in relation to SUP-B15, the presence of BCR-ABL1 translocation is highly associated with increased chromosomal instability through intracellular accumulation of reactive oxygen species and promotion of non-conservative repair pathways. The major cytotoxic effect of AZD2461 over SUP-B15, in levels comparable to those of Imatinib, shows that ALL Ph+ models seem to be over reliant on pathways that regulate genomic damage and minimize the instability brought by BCR-ABL1 , specifically PARP1. The use of PARPis shows great effect over the viability of ALL Ph+ models and PARP1 represents a potential new target for investigation in BCR-ABL1 positive tumors. However, studies are being conducted to confirm cell death through on-target effects of AZD2461 and the possible effects of PARPi in the expression levels of BCR-ABL1.

HYPERDIPLOID KARYOTYPE ASSOCIATED WITH PHILADELPHIA CHROMOSOME IN A PEDIATRIC PATIENT WITH ACUTE LYMPHOBLASTIC LEUKEMIA

An exacerbated proliferation of B-cell progenitors in bone marrow can evolve to a Precursor B-cell Acute Lymphoblastic Leukemia (B-ALL). Molecular and chromosomal abnormalities affect differentiation and proliferation of lymphoid precursors. Chromosomal alterations (hypodiploidy and hyperdiploidy), rearrangements involving KMT2A gene and the translocations BCR-ABL (Ph+), TCF3-PBX1 and ETV6-RUNX1 can be used as biomarkers to predict prognostic. The association of hyperdiploidy with chromosomal translocations is observed in 1%‒4% of hyperdiploidy patients in B-ALL. The aim of this study was to describe a rare case of B-ALL in an adolescent with BCR-ABL and hyperdiploidy. Immunophenotyping was used to characterize the leukemia type. Molecular tests were performed to investigate the presence of recurrent chromosomal translocations in B-ALL. G-banding and Fluorescence in situ hybridization (FISH) were carried out to characterize the karyotype. A 16-year-old girl was admitted in Hospital Sarina Rolim, São Paulo in February 2019 after experiencing 3 months of recurrent limb pain along with loss of weight, skin pallor and muscle weakness. The peripheral blood count showed 8.800 white blood cells/mm 3 , platelets count 90.000, hemoglobin 10 g/dL and hematocrit 30%. The immunophenotyping showed: CD19++/+++, CD20−/+/++, CD10+/++/+++, CD66C+/++/+++, CD123−/+/++, CD9+/++/+++, CD127−/+, CD44+++. The RT-PCR was negative for TCF3-PBX1, ETV6-RUNX rearrangements. It was detected the presence of Ph+ (P190). The results were consistent with B-ALL. The karyotype was: 58≍61,XX,+2,+4,+6,+8,t(9;22)(q34;q22),+10,+11,+12,+13,+19,+20,+21,+22,+22,+der(22)[cp16]/46,XX[1]. The patient was treated according to ALL BFM 2009 protocol associated with imatinib 400 mg/day. During the follow-up of minimal residual disease (D33), the patient presented hematological and infectious toxicity stage IV, requiring hospitalization due to febrile neutropenia and sepsis. Also, exhibited aseptic necrosis of the femoral head and underwent orthopedic surgery. The treatment has been completed in February 2021. In July 2022, the patient had no evidence of disease recidive but she acquired modest motor sequelae due to the orthopedic surgery. Hyperdiploidy occurs in 30% of pediatric B-ALL. The most frequent chromosome gains are X, 4, 6, 10, 14, 17, 18 and 21. However, BCR-ABL translocation comprises 2%‒5% of childhood ALL. There are only a few case reports of patients presenting BCR-ABL associated to hyperdiploidy. The outcome of patients can change according to these variations in genetic factors. Although BCR-ABL is associated with poor prognosis, the outcome has improved with implementation of tyrosine kinase inhibitors in Ph+ patients'therapy. We described an unusual case of pediatric B-ALL with hyperdiploid and Ph+ karyotype. This study and the literature review demonstrate the importance of cytogenetic and molecular tests during the diagnosis aiding to choose the treatment and improving the survival for ALL pediatric patients.

Evaluation of the Effect of Curcumin and Imatinib on BCR-ABL Expression Gene in Chronic Human k562 Cells

Background and Aims: Detection of overexpression in tumor-inhibiting genes provides valuable information for leukemia diagnosis and prognosis. Chronic myeloid leukemia (CML) is a stem cell disorder determined by a well-defined genetic anomaly involving BCR-ABL translocation in the Philadelphia chromosome. Curcumin is a chemo-preventive agent for the primary cancer targets, such as the breast, prostate, lung, stomach, duodenum, colon cancers, and leukemias. Imatinib (Gleevec®, Glivec®) is a synthetic tyrosine kinase inhibitor that treats CML. This study aimed to investigate Curcumin and Imatinib's effect on K562, a human CML cell line that expresses p210 BCR-ABL.
 Materials and Methods: In this study, Curcumin nanomicelles and Imatinib's apoptotic effects on the K562 cells and the expression of BCR-ABL were studied. BCR-ABL gene expression was evaluated using real-time polymerase chain reaction.
 Results: The findings indicated a decrease in the desired gene expression, but the BCR-ABL gene expression of the samples treated with Curcumin nanomicelles did not differ significantly from Imatinib (group control). The amount fold change of the BCR-ABL gene for Imatinib and Curcumin nanomicelle was 0.497 and 0.540, respectively.
 Conclusions: The present study showed that treating cellular category k562 with Curcumin nanomicelle and Imatinib reduces the BCR-ABL gene expression. Also, data showed that the Curcumin nanomicelle and Imatinib induced the apoptotic process.

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.

Frequency of BCR-ABL Gene Translocation in B-ALL Patients Associated with Clinicopathological Parameters

Background: B-Acute Lymphoblastic Leukemia (B-ALL) accounts for 25% of childhood malignancies. Chromosomal abnormalities like translocations lead to the formation of oncogenes, some of which are strong predictors of prognosis and response to anti-leukemic therapy. This study aimed to find out the frequency of BCR-ABL gene translocation in B-ALL patients by fluorescence in situ hybridization (FISH) and its association with their clinicopathological parameters. Methods: Patients (n=150) aged 1-17 years with a confirmed diagnosis of B-ALL were selected. Peripheral blood and/or bone marrow aspirate samples were obtained and Breakpoint cluster region-Abelson murine leukemia viral oncogene (BCR-ABL) translocation by FISH was observed. The patient’s demographics, hemoglobin levels, total leucocytes count, platelet count, FISH results, CNS status and risk stratification were recorded. Data was stratified and the Chi-square test was applied, p-value < 0.05 was considered statistically significant. Results: There were 100 (66.67%) males and 50 (33.33%) females. The average age of the patients was 7.03±4.51 years. The frequency of BCR-ABL translocation in B-ALL was 16(10%). A significant association was found between age and BCR-ABL translocation (p-value <0.05), however, an insignificant association was recorded among gender, Hb levels, TLC, platelet count, CNS status, proposed risk stratification system of B-ALL and BCR-ABL translocation (p-value >0.05). Conclusion: The frequency of BCR-ABL translocation in B-ALL was significantly high in the targeted population. FISH improves detection of the BCR-ABL translocation in either metaphase or interphase cells. Therefore, BCR-ABL expression can be considered as a prognostic approach and assist in effective treatment planning and better management of these children. Keyword: Leukemia; In Situ Hybridization; Fluorescence; Pathology.

Correlation of Sokal Scoring System with Molecular Response in pts of CML Taking CML Specific Tyrosine Kinase Inhibitors

Background: Chronic myeloid leukemia (CML) is a clonal myelo-proliferative disorder of a pluripotent stem cell. Aim: To find the frequency of low, intermediate and high risk category in patients of chronic myeloid leukemia and to compare frequency of complete molecular response. Study design: Descriptive case series. Methodology: Present study was conducted at Hematology Department, Sheikh Zayed Hospital Lahore. 260 patients fulfilled the selection criteria were selected. Sokal score was calculated. Low risk, intermediate and high risk category was assessed. Patients were followed up for 3 months. BCR-ABL translocation detection was done after 3months of treatment and complete molecular response was recorded. All this information was recorded on proforma. Data was analyzed using SPSS version 20. The chi-square test was used to compare the groups. A p-value of 0.05 was considered significant. Results: The mean age of patients was 49.18±20.33years. There were 144(55.4%) males and 116(44.6%) females. The mean duration of CML diagnosis was 3.52±1.69months. At baseline, 172 (66.2%) had low risk, 76(29.2%) had intermediate risk and 12 (4.6%) had high risk. In the study, complete molecular response was observed in 0% patients. Conclusion: It was concluded that there is regression in grades of Sokal score in patients of Chronic myeloid leukemia after 3 months treatment with TKIs but there is no complete molecular response observed in any patient after 3 months of treatment with TKIs. Keywords: Chronic Myeloid Leukemia, Low, Intermediate, High Risk and Sokal Score.

Patient-Derived In Vitro and In Vivo Models of Cancer.

Over the last two decades, cancer researchers have taken the promise offered by the Human Genome Project and have expanded its capacity to use sequencing to identify the genomic alterations that give rise to and sustain individual tumors. This expansion has allowed researchers to identify and target highly recurrent alterations in specific cancer contexts, such as EGFR mutations in non-small cell lung cancer (Lynch et al, N Engl J Med 350:2129-2139, 2004; Sharifnia et al., Proc Natl Acad Sci U S A 111:18661-18666, 2014), BCR-ABL translocations in chronic myeloid leukemia (Deininger, Pharmacol Rev 55:401-423. https://doi.org/10.1124/pr.55.3.4 , 2003; Druker et al, N Engl J Med 344. 1038-1042, 2001; Druker et al, N Engl J Med 344:1031-1037. https://doi.org/10.1056/NEJM200104053441401 , 2001), or HER2 amplifications in breast cancer (Slamon et al, N Engl J Med 344:783-792. https://doi.org/10.1056/NEJM200103153441101 , 2001; Solca et al, Beyond trastuzumab: second-generation targeted therapies for HER-2-positive breast cancer. In: Sibilia M, Zielinski CC, Bartsch R, Grunt TW (eds) Drugs for HER-2-positive breast cancer. Springer, Basel, pp 91-107, 2011). Despite these advances in our capacity to identify the genetic alterations that drive tumor initiation, survival, and proliferation, our ability to target these alterations to provide effective treatment options for patients in need, particularly those with rare or advanced cancers, remains limited (Gould et al, Nat Med 21:431-439. https://doi.org/10.1038/nm.3853 , 2015). Patient-derived models of cancer offer one potential mechanism to overcome this barrier between the bench and bedside. Through the development and testing of patient-derived models of cancer, functional genomics efforts can identify tumor-specific drug sensitivities and thereby provide a connection between tumor genetics and effective therapeutics for patients in need of treatment options.Recognizing that cancer is a multifaceted set of disease states, the development of personalized models of cancer that can be used to compare treatment options, identify tumor-specific vulnerabilities, and guide clinical decision-making has tremendous potential for improving patient outcomes. This chapter will describe a representative set of patient-derived models of cancer, reviewing each of their strengths and weaknesses and highlighting how selecting a model to suit a specific question or context is critical. Each model comes with a unique set of pros and cons, making them more or less appropriate for each specific research or clinical question. As each model can be leveraged to gain new insights into cancer biology, the key to their deployment is to identify the most appropriate model for a specific context, while carefully considering the strengths and limitations of the selected model. When used appropriately, patient-derived models may prove to be the missing link needed to bring the promise of personalized oncology to fruition in the clinic.