Characterization of CD9-CXCL12-CXCR4 Expression in Biological Subtypes of Childhood ACUTE Lymphoblastic Leukemia: Preliminary Findings and Future Perspectives

Abstract Background. Although the prognosis of acute lymphoblastic leukemia (ALL) has improved considerably in recent years, some cases still exhibit therapy-resistance and late relapse. CD9-CXCL12-CXCR4 pathway has been implicated in hematopoietic and leukemic stem cell homing, promoting cell adhesion and survival in bone marrow stromal niches and mediating cell dissemination to secondary lymphoid organs. We characterized the CD9-CXCL12-CXCR4 expression in specific biological subgroups of childhood ALL, comparing, in some cases, diagnosis vs relapse, in order to investigate the role of these genes, in the process of leukemia relapse and to preliminarily evaluate their impact as prognostic markers, even for those subtypes with well-established good outcome. Materials and Methods. We analyzed bone marrow (BM) samples from 68 children with ALL, 3 cases with chronic myeloid leukemia, as control for t(9;22) positive leukemia, and 4 healthy donors (HDs). Patients were enrolled and treated at our Center from 2000 to 2010. We reverse-transcribed 500 ng of patients' diagnostic RNA and performed a Real-time PCR using the SYBRTM Green PCR Master Mix (Applied Biosystems®), calculating the median fold-changes (MFCs) among different reactions and comparing with HDs. GUS gene mRNA was used as an internal positive control, showing no significant variation in our experiments. Each PCR experiment was carried out in duplicate. Sequences of oligonucleotides used for experiments, together with RQ-PCR protocol were previously published (Gandemer V. et al Leukemia Research 2010) Results. We analyzed for CD9, CXCL12 and CXCR4 expression 39 children with t(12;21) positive ALL, 12 cases with t(1;19) positive ALL, 8 cases with t(9;22) positive ALL, and 9 cases resulted negative for chromosomal translocation screening. Among the t(12;21) positive ALL we found that CD9 was overexpressed in 13 out of 39 patients (33%), CXCL12 in 14 out of 39 (35%) and CXCR4 in 9 out of 39 (23%), respectively. We noted that in 2 cases who presented a late relapse, one (CT13) showed a normal expression of the analyzed genes, conversely the other case (CT24), who suffered from a isolated extramedullary relapse, showed a high overexpression of CD9. In the t(1;19) subgroup we found only one case with overexpression of both CD9 and CXCR4 genes, respectively. Moreover we noted that comparing diagnosis vs relapse in one case, we observed a dramatic increased expression of CXCL12 (CT70 diagnosis 3,33 FCs vs relapse 38 FCs). In the subgroup of children with t(9;22) positive ALL, we observed an extremely high expression of the CD9-CXCL12-CXCR4 genes. In particular, we found an overexpression of CD9 in 4 out of 8 (50%) cases with this subtype of ALL; interestingly, all these case showed a BM relapse. One of these (CT56) showed an overexpression of all the three pathway's components. As negative counterpart, we also analyzed three cases with a t(9;22) positive CML, finding a normal expression of all genes. In order to determine this pathway's expression in children presenting an ALL without known chromosomal aberration, we analyzed other 9 cases. In only one (CT44) we found an overexpression of both CD9 and CXCL12. This patient suffered from an early relapse (<24 months from diagnosis). Conclusions. Our preliminary findings demonstrated that the CD9-CXCL12-CXCR4 pathway is mainly altered in children with t(12;21) and t(9;22) positive ALL. In the latter subgroup we found a linear correlation between overexpression and impending relapse, confirming that this pathway can be potentially used as prognostic marker. Among t(1;19) positive ALL, we found few cases with single gene aberration. More importantly, we found an overexpression of one or two pathway's components in diagnostic samples of those cases, who presented a subsequent relapse. These data need to be confirmed in a larger population and will pave the way to identify new prognostic marker and/or therapeutic target, since CXCR4 has already antagonist drugs in clinical use, showing very recent promising results (Randhawa S et al. British Journal of Hematology 2016). Disclosures No relevant conflicts of interest to declare.

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Flow Cytometric Leukemic Blasts Detection in Cerebrospinal Fluid of Children with Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v124.21.3799.3799 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3799-3799

Author(s):

Mette Levinsen ◽

Hanne Vibeke Marquart ◽

Line Groth-Pedersen ◽

Thomas Leth Frandsen ◽

Birgitte Klug Albertsen ◽

...

Keyword(s):

Bone Marrow ◽

Cerebrospinal Fluid ◽

Acute Lymphoblastic Leukemia ◽

Conflicts Of Interest ◽

Leukocyte Count ◽

Lymphoblastic Leukemia ◽

Leukemic Cells ◽

Newly Diagnosed ◽

Childhood All ◽

Blast Count

Abstract Background: Central nervous system (CNS)-directed treatment has reduced risk of CNS relapse for childhood acute lymphoblastic leukemia (ALL), which accounts for 30-40% of initial relapses. Compared with CNS-negative patients, patients with CNS leukemia (>5 leukocytes/µL cerebrospinal fluid (CSF) and lymphoblasts) suffer from more CNS+ relapses. Conventional cytology (CC) is specific (>95%), but nonsensitive (<50%), since leukocytes in CSF decay within few hours after lumbar puncture. Method: We prospectively assessed centralised multi-parameter flow cytometry (FCM) of fixated CSF versus local CC in Nordic/Baltic childhood ALL. Diagnostic samples from 172 children aged 0-18 years with de novo and eight children with relapsed ALL were investigated in 297 CSF samples from 180 patients. Kinetics of disappearance of leukemic cells in the CSF was evaluated until day 15. Antibody-combinations reflected the immunophenotype of leukemic blasts in bone marrow. Result: Of 172 newly diagnosed patients, 51 (30%) had CSF involvement by FCM, while CC was positive in 16 patients (9%) (p<0.001). CSF involvement was detected by both FCM and CC in four of eight patients with relapse (50%). Among newly diagnosed patients, samples positive by FCM and CC had higher leukemic blast count compared to samples positive by FCM only (medians: 0.545 (range: 0.005-4.801) versus 0.016 (range: 0.003-1.38) leukemic blasts/µL; p<0.001). Among newly diagnosed patients with samples positive by FCM and CC, the CSF blast count was related to the CSF leukocyte count (rs=0.82; p=0.001). Compared to newly diagnosed patients who were FCM-negative, those with FCM-positivity had higher WBC (median: 37 versus 9 x 109/L; p<0.001), were younger (medians: 3 versus 5 years, p=0.04), and more often had T-cell ALL (12/51 (24%) versus (6/121 (5%), p<0.001). Five (16%) of 31 newly diagnosed patients with FCM detected blasts at diagnosis and available data at day 15 still had CSF leukemic blasts on day 15. So far the two patients who later developed CNS and/or bone marrow relapse were positive by flow (1.38 and 0.178 blasts/µL), but CNS negative by CC or had CSF leukocyte count <5/µL and lymphoblasts on CC, respectively. Conclusion: Leukemic blasts are present in spinal fluid of one third of newly diagnosed ALL. CSF involvement is associated with other higher risk characteristics. The prognostic value of these findings awaits prospective evaluation. Disclosures No relevant conflicts of interest to declare.

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Study of Some Biochemical Parameters in Iraqi Children with Acute Lymphoblastic Leukemia

Baghdad Science Journal ◽

10.21123/bsj.12.2.371-378 ◽

2015 ◽

Vol 12 (2) ◽

pp. 371-378

Author(s):

Baghdad Science Journal

Keyword(s):

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

Biochemical Parameters ◽

Serum Proteins ◽

Lymphoblastic Leukemia ◽

White Blood Cells ◽

Total Serum ◽

Childhood All ◽

Marrow Cavity ◽

Increased Risk

Leukemia or cancer of the blood is the most common childhood cancer, Acute lymphoblastic leukemia (ALL), is the most common form of leukemia that occurs in children. It is characterized by the presence of too many immature white blood cells in the child’s blood and bone marrow, Acute lymphoblastic leukemia can occur in adults too, treatment is different for children. Children with ALL develop symptoms related to infiltration of blasts in the bone marrow, lymphoid system, and extramedullary sites, such as the central nervous system (CNS). Common constitutional indications consist of fatigue (50%), pallor (25%), fever (60%), and weight loss (26%). Infiltration of blast cells in the marrow cavity and periosteum often lead to bone pain (23%) and disturbance of normal hematopoiesis. Thrombocytopenia with platelet counts less than 100,000 are seen in approximately 75% of patients. About 40% of patients with childhood ALL present with hemoglobin levels less than 7 g/dL. Although leukocyte counts greater than 50,000/mm3 occur in 20% of cases, neutropenia defined as an absolute neutrophil count less than 500 is common at presentation and is associated with an increased risk of infection. The aim of this study was to investigate the differentiations in some biochemical parameters (Hb, PCV, total serum proteins Aspartate amino transferase(AST), Alanin amino transferase (ALT), and Malondialdehyde (MDA) in blood which can be conceder as a marker of ALL. Samples were collected from 50 patients (between 1-16 years old) diagnosed with ALL after one month treatment with induction therapy, compared with 30 control samples taken from healthy persons at the same age . The ALT and MDA showed a significant increase p < 0.001 and p

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TEL-AML1 Fusion Transcript in Relapsed Childhood Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v91.5.1716 ◽

1998 ◽

Vol 91 (5) ◽

pp. 1716-1722 ◽

Cited By ~ 13

Author(s):

Karlheinz Seeger ◽

Hans-Peter Adams ◽

Dirk Buchwald ◽

Birgit Beyermann ◽

Bernhard Kremens ◽

...

Keyword(s):

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

Lymphoblastic Leukemia ◽

Prognostic Significance ◽

Philadelphia Chromosome ◽

Fusion Transcript ◽

Childhood Acute Lymphoblastic Leukemia ◽

Long Term Results ◽

Childhood All ◽

Cryptic Translocation

Abstract The cryptic translocation t(12;21)(p13;q22) has been recently recognized as the most common genetic rearrangement in B-lineage childhood acute lymphoblastic leukemia (ALL). The resulting fusion transcript, termed TEL-AML1, has been associated with an excellent prognosis at initial ALL diagnosis. Hence, we postulated that the incidence of TEL-AML1 fusion should be lower in patients with ALL relapse. To address this assumption and to investigate the prognostic significance of TEL-AML1 expression in relapsed childhood ALL, bone marrow samples of 146 children were analyzed by reverse-transcriptase (RT)-polymerase chain reaction (PCR). All children were treated according to Berlin-Frankfurt-Münster (BFM) ALL relapse trial protocols (ALL-REZ BFM 90-96). Their clinical features and outcome were compared with those of 262 patients who could not be tested due to lack of bone marrow samples. Thirty-two of 146 children with relapsed ALL were TEL-AML1–positive. Four of the negative patients had T-lineage and nine Philadelphia chromosome (Ph1)-positive leukemia. Thus, the incidence ofTEL-AML1 in relapsed Ph1-negative, B-cell precursor ALL is 32 of 133 (24%). The 32 TEL-AML1–positive and 101 negative patients differed significantly with respect to duration of last remission (42.5 v 27 months; P = .0001) and age at initial diagnosis (53.5 v 74 months;P = .0269). At a median follow-up time of 21.5 months, children positive for TEL-AML1 had a significantly (P = .0011) higher probability of event-free survival (EFS; 0.79 v 0.33). The predominant majority of patients had been treated for initial ALL according to German multicenter BFM (108 of 133) or Cooperative ALL study group (CoALL) (19 of 133) frontline protocols. The comparison of tested and not-tested (N = 262) patients showed no significant difference.TEL-AML1 positivity predicted a favorable short-term outcome; long-term results are unknown. Screening for TEL-AML1 should become routine at relapse diagnosis and might be used for therapy stratification in future trials.

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Cranial Irradiation Does Not Result in Pituitary-Gonadal Axis Dysfunction in Very Long-Term Male Survivors of Childhood Acute Lymphoblastic Leukemia.

Blood ◽

10.1182/blood.v114.22.989.989 ◽

2009 ◽

Vol 114 (22) ◽

pp. 989-989

Author(s):

Niels J. van Casteren ◽

Rob Pieters ◽

Gert Dohle ◽

Manita van Baalen ◽

Sebastian Neggers ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Cranial Irradiation ◽

Gonadal Function ◽

Free T4 ◽

Childhood All ◽

Male Survivors ◽

Igf I

Abstract Abstract 989 Poster Board I-11 Introduction: One of the risks of childhood cancer treatment is fertility impairment later in life. In the past a large proportion of children with acute lymphoblastic leukemia (ALL) has received cranial irradiation as part of their treatment. The aim of this study was to evaluate whether cranial irradiation negatively affects pituitary regulated gonadal function in male survivors of childhood ALL. Patients and Methods: We examined gonadal function, including Inhibin B, LH, FSH, testosterone, and pituitary axis function by measuring TSH, Free-T4 and IGF-I levels in 89 long-term male survivors of childhood ALL after a median follow-up time of 19 year (range 7-34 years). Results: Twenty-nine out of 89 male ALL survivors received cranial irradiation. Inhibin, FSH, LH, Testosterone, testicular volume as well as TSH and Free-T4 levels were not different in the cranial irradiated group as compared to the non-irradiated group (table 1). In contrast, IGF-I levels were significantly lower in the cranial irradiated group. Survivors treated with total body irradiation or testicular irradiation had significantly decreased gonadal function based on hormone levels. Conclusions: These data show that, in contrast to the negative influence on the growth hormone axis, cranial radiotherapy as part of ALL treatment does not have a deleterious long-term effect on the hypothalamic–pituitary-gonadal axis or pituitary-thyroid axis. Disclosures: No relevant conflicts of interest to declare.

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Implication of Genetic Variations of Ikzf1, ARIDB5, and CEBPE Genes In the Risk of Childhood Acute Lymphoblastic Leukemia In Korea

Blood ◽

10.1182/blood.v116.21.3235.3235 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3235-3235

Author(s):

Dong Kyun Han ◽

Hee Nam Kim ◽

Min Ho Shin ◽

Minenori Eguchi-Ishimae ◽

Mariko Eguchi ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Genetic Variations ◽

Childhood Acute Lymphoblastic Leukemia ◽

Asian Countries ◽

Childhood All ◽

Genomic Variations ◽

Genotype Frequencies ◽

The Impact

Abstract Abstract 3235 Background: Recent western studies have showed the implication of the germline genomic variations in IKZF1 gene at 7p12.2, ARIDB5 gene at 10q21.2, and CEBPE gene at 14q11.2 on the risk of childhood acute lymphoblastic leukemia (ALL); the most significant association was observed in the single nucleotide polymorphism (SNP) rs4132601 which located at 3' region of the IKZF1. IKZF1 plays important role in lymphocyte differentiation, proliferation and function, ARIDB5 in embryogenesis and growth regulation, and CEBPE in regulation of myelopoiesis. Genomic variants in these genes are therefore considered to be involved in transcriptional regulation and differentiation of B cell progenitors. However, there have been no reports on the role of germline variations in leukemogenesis of childhood ALL in Asian countries. The aim of this study is to show the impact of these genetic variants on childhood ALL in Korea. Patients and Methods: To examine the association between genetic variations (IKZF1 rs4132601, ARIDB5 rs7089424, and CEBPE rs2239633) and the risk of childhood ALL, we here analyzed 228 children with ALL and 508 healthy individuals in Korea. Results: In ARIDB5 rs7089424, TG and GG genotypes were significantly associated with a risk for ALL (odds ratio [OR], 1.63; 95% confidential interval [CI], 1.07–2.48; P=0.02 for TG genotype, OR, 2.69; 95% CI, 1.42–5.07; P=0.002 for GG genotype). The allele incidence of ARIDB5 rs7089424 was also significantly associated with a risk for ALL (OR, 1.66; 95% CI, 1.24–2.22; P=0.0006). CEBPE rs2239633 TT genotype showed a significant association with a decreased risk for ALL (OR, 0.54; 95% CI, 0.33–0.90; P=0.02 for TT genotype). The allele incidence of CEBPE rs2239633 was also associated with a decreased risk for ALL (OR, 0.77; 95% CI, 0.61–0.97; P=0.02). There was no significant association between IKZF1 rs4132601 polymorphism and a risk for ALL in this study. Conclusion: These results suggest that genomic variations of ARIDB5 and CEBPE may play an important role in the risk for childhood ALL in Korea, compared with findings from western countries showing a significant relation between IKZF1 and childhood ALL. Several factors should be considered to explain a discrepancy between our results and the previous studies, which include different genotype frequencies in polymorphisms and varied susceptibility to ALL in different ethnic groups. Further studies incorporating larger number of cases and analyzing other SNPs or other Asian countries are warranted in childhood ALL. Disclosures: No relevant conflicts of interest to declare.

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JAK2 mutations and CRLF2 rearrangements in Down Syndrome-Associated Acute Lymphoblastic Leukemia in Japan

Blood ◽

10.1182/blood.v118.21.1451.1451 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1451-1451

Author(s):

Isamu Hanada ◽

Kiminori Terui ◽

Tsutomu Toki ◽

Ko Kudo ◽

Tomohiko Sato ◽

...

Keyword(s):

Bone Marrow ◽

Down Syndrome ◽

Acute Lymphoblastic Leukemia ◽

Fusion Gene ◽

Lymphoblastic Leukemia ◽

The Other ◽

Pseudoautosomal Region ◽

Rt Pcr ◽

Childhood All ◽

Western Countries

Abstract Abstract 1451 Children with Down syndrome (DS) have a 10- to 20-fold increased risk of developing acute lymphoblastic leukemia (ALL). In DS-associated ALL (DS-ALL), the chromosome aberrations which are generally common in childhood ALL, such as hyperdiploidy and t(12;21), are less frequent. Recent studies have shown that activating JAK2 mutations and overexpression of cytokine receptor-like factor 2 (CRLF2) gene are identified in approximately 20% and 50–60% of DS-ALL in Western countries, respectively. Most of the patients with CRLF2 overexpression have been reported to be associated with interstitial deletions of the pseudoautosomal region 1 (PAR1) of the sex chromosomes and the P2RY8-CRLF2 fusion gene. In addition, one report showed that the activating CRLF2 F232C mutation was identified in about 10% of DS-ALL. However, there have been no studies to determine the incidence of these genetic aberrations in Asian patients with DS-ALL. In this study, 23 patients with DS-ALL in Japan were screened for mutations in the pseudokinase domain of the JAK2 gene, the P2RY8-CRLF2 fusion gene, and the CRLF2 F232C mutation by PCR/RT-PCR and direct sequencing. Fourteen patients, whose bone marrow RNAs were available, were also screened for CRLF2 overexpression by real-time quantitative RT-PCR. We identified the JAK2 R683G mutation in 2 patients (9%) and the P2RY8-CRLF2 fusion gene in 4 patients (17%). The CRLF2 F232C mutation was not detected in any patient. CRLF2 overexpression was observed in 2 of 14 patients examined (14%). Although bone marrow RNA was available in only 1 of 4 patients positive for P2RY8-CRLF2, high-level expression of CRLF2 was confirmed in this patient. The other patient with CRLF2 overexpression was negative for P2RY8-CRLF2, indicating the involvement of the other type of CRLF2 rearrangement, IGH@-CRLF2 in this patient. We also performed a preliminary study on JAK1, JAK3, and Interleukin-7 receptor-α (IL7R) mutations, and 14, 11, and 12 patients were screened for mutations in the pseudokinase domain of JAK1, JAK3, and exon 5 and 6 of IL7R, respectively. However, no mutations were identified in any patient. Our results show the lower incidence of CRLF2 rearrangements in DS-ALL patients in Japan than that in Western countries. Gene alterations other than CRLF2 rearrangements may contribute to leukemogenesis in Japanese patients with DS-ALL. To clarify if the incidences of the mutations in JAK1-3, CRLF2, and IL7R are also lower in DS-ALL patients in Japan than those in Western counties, more patients need to be studied. Disclosures: No relevant conflicts of interest to declare.

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Effects of G-CSF On Acute Lymphoblastic Leukemia.

Blood ◽

10.1182/blood.v120.21.2564.2564 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2564-2564

Author(s):

Jordan Basnett ◽

Adam Cisterne ◽

Kenneth F Bradstock ◽

Linda J Bendall

Keyword(s):

Bone Marrow ◽

Disease Progression ◽

Microarray Analysis ◽

Environmental Changes ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Hematopoietic Stem ◽

Childhood All ◽

Extracellular Matrix Components ◽

The Impact

Abstract Abstract 2564 G-CSF is commonly used to treat chemotherapy-induced neutropenia and for the mobilization of hematopoietic stem cells for transplantation in patients with leukemia. Administration of G-CSF has profound effects on the bone marrow microenvironment including the cleavage of molecules required for the maintenance of lymphopoiesis, including CXCL12 and VLA-4. We have recently reported that G-CSF results in the dramatic suppression of B-lymphopoiesis. This, together with previous reports by ourselves, and others, showing that disruption of CXCL12 or VLA-4 slow the progression of B-lineage ALL lead us to consider that G-CSF may similarly antagonize the progression of ALL. To explore this possibility, we examined the impact of G-CSF administration on six human ALL xenografts using a NOD/SCID mouse model. Mice were engrafted without radiation and G-CSF commenced when 1% of the bone marrow consisted of ALL cells. G-CSF was administered twice daily for 10 days, at which time all animals were culled and leukemia assessed in the blood, bone marrow and spleens. Surprisingly G-CSF was found to increase disease progression in two of xenografts investigated (1345 and 0398, referred to as G-CSF responsive xenografts hereafter), while the remainder demonstrated a small reduction in leukemia, with one showing a statistical significant decrease. No evidence for a direct mitogenic effect of G-CSF could be demonstrated in any of the xenografts using exogenous G-CSF in vitro cultures in the presence or absence of human or murine stromal support. Consistent with these findings, and previous reports, little to no G-CSF receptor was detected by flow cytometry or microarray analysis of xenografts. Microarray analysis of the xenografts revealed significant differences in gene expression between the G-CSF responsive xenografts and the remainder of the samples. A total of 83 genes were expressed at a higher level and 127 genes at a lower level in the G-CSF responsive xenografts. The more highly expressed genes included cell cycle regulators (eg cyclin A1), adhesion molecules (eg ALCAM), extracellular matrix components and surface receptors. Perhaps the most interesting was the exclusive expression of the acetylcholine receptor (cholinergic receptor, nicotinic, beta 4, nAChRb4) in the G-CSF responsive cases. Analysis of a large public dataset of childhood ALL samples revealed significantly higher expression of this gene in ALL samples with rearranged MLL (p<0.03). However, small numbers of cases in all ALL subgroups had greater than an 2 fold higher expression compared to normal B cell progenitors. The role of nAChR in the response of ALL cells to micro-environmental changes induced by G-CSF remains to be determined, however, nAChR has known roles in cell proliferation and inhibition of apoptosis. Furthermore G-CSF is known to induce acetylcholine production in other tissues. In summary, G-CSF inhibited leukemia progression in the majority of patient xenografts, however, in a subset of samples G-CSF accelerated disease progression. Clinically, G-CSF administration to ALL patients has not been associated with any major adverse outcomes. However our data suggest that a small subset of patients may experience accelerated disease. Identification of features associated with adverse responses to G-CSF will permit the identification of patients for whom G-CSF may present a risk for increased disease progression. Disclosures: No relevant conflicts of interest to declare.

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Successful Engrafment After HLA Identical Granulocyte Transfusion As Support Therapy in a Septic Shock Patient with Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v120.21.4507.4507 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4507-4507

Author(s):

Roberto Ovilla ◽

Claudia Barrera-Carmona ◽

Nicolas Guzman-Bouilloud ◽

Elizabeth Buganza-Torio ◽

Rosa Jimenez-Alvarado ◽

...

Keyword(s):

Bone Marrow ◽

Septic Shock ◽

Acute Lymphoblastic Leukemia ◽

Bone Marrow Transplant ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Immunosuppressive Treatment ◽

Marrow Transplant ◽

Granulocyte Transfusion ◽

Shock State

Abstract Abstract 4507 A 53 years old male started in February 2010 with ecchymosis, petechiae and spontaneous gum bleeding. He was diagnosed with acute lymphoblastic leukemia, and was started on HYPER-CVAD, in combination with anti-tumoral lysis syndrome measures and antimicrobial, antiviral y antifungal prophylaxis. After finishing HYPER-CVAD phase A, he developed severe myelosuppression even with the use of G-CSF, 72 hours later he presented with abdominal cramping, fever up to 38.2°C and hypotension, with a high clinical suspicion of neutropenic colitis; his mean arterial pressure average was 45 mmHg, he was started on intravenous colloids, dobutamine and norepinephrine drips. Because of severe myelosuppression, septic shock and myocardial depression, a granulocyte transfusion without previous mobilization was performed with an identical sibling donor with concomitant use of granulocytic colony stimulation factor. Severe myelosuppression was maintained during 7 days, however shock state was reversed some hours after performing granulocyte transfusion, without infectious signs, 36 hours later a mononuclear cell infusion was performed from the same donor, with previous 2 days G-CSF mobilization. A gradual increase in leukocyte number appeared, with 400, 900 and finally 1900. A new bone marrow aspiration was performed, where hematopoietic recovery was confirmed from his sister's cells with confirmation by karyotype and microsatellites. He was then considered bone marrow grafted HLA compatible. On April 2010 he presented with acute diarrheic syndrome secondary to a CMV infection, he was started on ganciclovir and intravenous immunoglobulin. On May he presented an Aspergillus pneumonia that was treated both clinically with antifungal therapy and surgically with thoracoscopy to remove the fungi lesion. On October 2010 he started with graft versus host manifestations on the skin and in the liver. He started immunosuppressive treatment with Prednisone and Sirolimus. On November he developed anogenital herpes zoster infection. After this complication, every GVHD manifestation ceded. Now, 28 months post-bone marrow transplant he presents full remission with no evidence of leukemia. Granulocyte transfusion is an uncommon technique. Preferably it should be done with and identical donor. In this case report, the justification of the procedure was to be able to achieve a remission from a potentially irreversible septic shock; this was successfully done, in an unexpected clinical scenario with severe life threatening, and in a casual manner, the patient achieved a successfully bone marrow transplant, and now 30 months post-granulocyte infusion the patient is free from any evidence of disease. Disclosures: No relevant conflicts of interest to declare.

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Chromothripsis-Mediated Structural Variations and Clonal Evolution In Recurrent Childhood High Hyperdiploid Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v122.21.233.233 ◽

2013 ◽

Vol 122 (21) ◽

pp. 233-233

Author(s):

Cai Chen ◽

Christoph Bartenhagen ◽

Michael Gombert ◽

Vera Okpanyi ◽

Vera Binder ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Copy Number ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Clonal Evolution ◽

Copy Number Variations ◽

Chromosome 3 ◽

Structural Variations ◽

Childhood All ◽

A Genome

Abstract High hyperdiploid acute lymphoblastic leukemia (HeH-ALL) is characterized by 51-67 chromosomes and nonrandom gains of specific chromosomes (X, 4, 6, 10, 14, 17, 18, and 21). It presents the most frequent numerical cytogenetic alteration in childhood pre B-cell ALL occurring in 25-30% of cases. Recurrent disease will affect 15-20%. Pre-leukemic HeH clones are generated in utero, but cooperating oncogenic lesions are necessary for overt leukemia and remain to be determined. Recently, a phenomenon termed chromothripsis has been described in which massive structural variations occur in a single aberrant mitosis. Whole or partial chromosomes are shattered and some fragments are lost in the process of rejoining. Thus, characteristically, chromosomal copy numbers oscillate between two copy number states. Chromothripsis has been suggested to be a tumor-driving alteration that may be present in 2-3% of all human cancers. Its role as a potential cooperating or initiating lesion in HeH-ALL has not been determined. We applied state-of-the-art whole-genome next-generation-sequencing to analyze structural variations in six pediatric patients with recurrent HeH-ALL. Matched sample sets taken at diagnosis, remission and/or relapse were compared. Paired end sequencing was carried out on a Genome Analyzer IIx or a HiSeq 2000 (Illumina), respectively. Reads were aligned against the human reference genome (GRCh37) using BWA. Translocations were detected by GASV. Copy number variations were analyzed by FREEC. Structural variations were validated by PCR/Sanger sequencing and FISH. Of the six patients analyzed, five harbored on average one interchromosomal translocation or intrachromosomal inversion, but one patient presented with massive genomic rearrangements (Figure). These affected chromosome 3, 11, 12 and 20. Ten copy number shifts on chromosome 3 oscillating between two copy number states (2 and 3) indicated that these rearrangements were caused by chromothripsis. Breakpoint sequencing revealed that one of the identified translocations (t(12;20)(p13.1;p12.3)), was indeed a three-loci-rearrangement composed of small fragments derived from chromosomes 3, 12 and 20. Characteristically for chromothripsis, the breakpoints clustered closely. Three breakpoints separated by 224 bp and 64 kb were located in the transducin (beta)-like X-linked receptor 1 (TBL1XR1) gene. Other genes repeatedly targeted included the MACRO domain-containing protein 2 (MACROD2) gene (a deacetylase involved in deacetylation of lysine residues in histones and other proteins), the KIAA1467 gene (a transmembrane protein of the integrin alpha FG-GAP repeat containing 3 (ITFG3) family), and a novel regulatory lincRNA (ENSG00000243276). MACROD2 was previously observed as a target of chromothripsis in a colorectal carcinoma. Thus, the characterized breakpoints may identify fragile genomic sites prone to chromothriptic rearrangement. DNA repair was effectuated by non-homologous-end-joining as typical addition of non-template nucleotides with microhomologies of two to four nucleotides at the breakpoints demonstrated. Copy number profiles of this patient showed that at least two distinct leukemic clones could be identified at diagnosis. One had acquired chromothriptic alterations and presented the dominant clone at relapse indicating chemotherapy resistance and tumor-driving potential. Prior whole-exome sequencing did not reveal mutations in known oncogenes or tumor suppressor genes. Therefore, loss of function or expression of genes affected by chromosomal rearrangements, such as TBL1XR1 that is recurrently mutated in childhood ALL with ETV6-RUNX1 translocation, may account for the tumor-driving effect. All leukemic cells at diagnosis showed conformity concerning number and pattern of whole chromosome gains demonstrating that chromothripsis was not an initiating oncogenic event, but occurred secondary to high hyperdiploidy. Further aberrations (t(4;7), loss of 4q) were gained by the chromothriptic clone and could be detected by FISH in minor subclones pointing at ongoing clonal evolution. Taken together, our study reveals chromothripsis as a novel assisting and tumor-driving lesion in HeH ALL. Chromothripsis in HeH-ALL. Copy number variations and translocations at diagnosis (left) and relapse (right). (magenta: chromothriptic translocations; green: other translocations) Disclosures: No relevant conflicts of interest to declare.

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CITED2 Facilitates Apoptosis Induced By Histone Deacetylase Inhibitor SAHA In Human Pediatric Pre-B Acute Lymphoblastic Leukemia Cells

Blood ◽

10.1182/blood.v122.21.2913.2913 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2913-2913

Author(s):

Jinwei Du ◽

Shigemi Matsuyama ◽

Yu-Chung Yang

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Side Effects ◽

Mononuclear Cells ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Dependent Manner ◽

Mouse Development ◽

Childhood All ◽

Normal Peripheral Blood

Abstract Acute lymphoblastic leukemia (ALL) is the most common malignancy in childhood, representing 31% of all tumors, and about 85% of children with ALL have B-cell ALL. Although the survival rate is approaching 90%, ALL remains the main cause of death from disease in children and young adults. The activity of histone deacetylases (HDAC) in childhood ALL is increased compared with that in normal peripheral blood mononuclear cells or bone marrow cells. Treatment of mice engrafted with T or B-ALL cells with HDAC inhibitor (HDACi) increases the acetylation of Histone 3 and Histone 4 and prolongs survival of these mice. <>Vorinostat (Suberoylamilide Hydroxamic Acid, SAHA) was the first HDACi approved by the FDA for the treatment of refractory cutaneous T-cell lymphoma. Currently, several clinical trials are being conducted to evaluate its effects on other cancers, including ALL. However, some patients are resistant to HDACi therapy, and concerns regarding toxic side effects of HDACi exist due to the roles of HDACs in multiple pathways. Therefore, identification of new therapeutic targets is required which could improve the efficacy of HDACi by reducing the dose of HDACi administered without compromising the treatment benefits but alleviating the side effects of HDACi. <>CBP/p300-interacting transactivator with glutamic acid (E) and aspartic acid (D)–rich tail 2 (CITED2) is a cytokine-inducible gene that plays various roles during mouse development and, in particular, is essential for normal hematopoiesis. While the role of CITED2 in the pathogenesis of leukemia is currently unclear, dysregulation of CITED2 has been implicated in various types of leukemia, including ALL, in which downregulation of CITED2 is frequently observed. In this study, we tested the hypothesis that CITED2 may enhance the sensitivity of human pediatric pre-B ALL cells to HDACi SAHA. <>SAHA treatment of NALM-6 and 697 cells (human pediatric pre-B ALL cell lines) significantly induced apoptosis and cell cycle arrest in a dose dependent manner. The protein level of CITED2 was not affected by SAHA treatment. Although overexpression of CITED2 alone only slightly increased apoptosis, it significantly enhanced apoptosis resulted from SAHA treatment in both NALM-6 (15.1% versus 39.2%) and 697 cells (9.4% versus 14.6%) as assessed by annexin V/Propidium Iodide double staining and flow cytometry analysis (Figure 1). Accordingly, compared with control (i.e. NALM-6 cells transduced with GFP), overexpression of CITED2 also greatly reduced mitochondrial membrane potential of NALM-6 cells caused by SAHA treatment. To explore the potential mechanisms underlying enhanced apoptosis by overexpression of CITED2 in NALM-6 cells treated with SAHA, we determined the levels of pro- and anti- apoptotic proteins by Western blot and real-time quantitative PCR. We found that SAHA treatment increased the levels of pro-apoptotic molecules Bak, Puma, and Noxa, and decreased the levels of anti-apoptotic molecule Bcl-xL and apoptosis inhibitors XIAP and survivin. Importantly, overexpression of CITED2 markedly increased the protein levels of pro-apoptotic molecules Bak and Bim. Furthermore, knockdown of Bim by shRNA significantly attenuated apoptosis in Cited2 overexpressing NALM-6 cells treated with SAHA. Taken together, these results suggest that modulation of the CITED2 activity may confer its cooperative effect with SAHA in pre-B ALL cells and warrant future evaluation of such a combination in inducing apoptosis of primary ALL cells. Disclosures: No relevant conflicts of interest to declare.

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