Limited Reliability of Ig/TCR Based MRD Monitoring in BCR/ABL-Positive Childhood ALL: Comparison to Quantitative Fusion Transcript Detection.

Abstract Leukemias with the t(9;22) translocation resulting in BCR/ABL fusion protein expression comprise 3–5% of childhood ALL. Despite modern therapeutic regimens, their prognosis is inferior. Minimal residual disease (MRD) based on leukemia-specific immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements has become a tool influencing clinical decisions in many therapeutic trials for childhood ALL. The presence of BCR/ABL fusion gene offers a possibility of the fusion transcript detection - a faster and cheaper alternative to Ig/TCR-based MRD monitoring. Up to now, no direct comparison based on a sufficient number of samples has been done. We analyzed 350 follow-up samples from 16 children (aged 4–17 years) with BCR/ABL-positive ALL by Ig/TCR-based real-time quantitative PCR (RQ-PCR) and by reverse-transcriptase (RT) RQ-PCR for BCR/ABL transcripts. Beta-2 microglobulin housekeeping gene was used for cDNA quality normalization. WBC, age, immunophenotype and blast proportion in the bone marrow (BM) and peripheral blood (PB) showed no relation to the initial BCR/ABL level. All children expressed m-BCR/ABL transcript at the time of diagnosis; 3 of 16 children expressed both m-BCR/ABL and M-BCR/ABL transcripts representing the p190 and p210 variant of BCR/ABL protein, respectively. The expression levels of m-BCR/ABL in diagnostic samples differed up to 3 logs, being the lowest in patients expressing both variants of the fusion gene. In 38 samples from those patients, M-BCR/ABL expression was generally higher than m-BCR/ABL expression, being negative by m-BCR/ABL and positive by M-BCR/ABL in 13 samples. For further analysis we used the higher value of m- and M-BCR/ABL as the BCR/ABL MRD level. For the comparison with Ig/TCR-based method, MRD levels in follow-up samples were related to the expression levels in diagnostic samples, which were set to 1. In total, 133 (38%) and 127 (36%) samples were negative and positive by both methods, respectively. The quantitative levels differed by more than 1 log in 46 (36%) double-positive samples, being underestimated by Ig/TCR method in 25 cases and by m-BCR/ABL quantification in 21 cases. With the same sensitivity of both methods we found significantly more false-negative samples by Ig/TCR approach (70 samples) compared to BCR/ABL quantification (20 samples). Altogether, we tested 219 bone marrow (BM), 130 peripheral blood (PB) and 1 cerebrospinal fluid samples. The PB samples showed significantly worse correlation between the two methods compared to BM (p=0.02). Interestingly, some patients had higher MRD levels in PB compared to BM as shown by corresponding BM and PB samples. Our data suggest that BCR/ABL-positive childhood ALL is a biologically heterogeneous group. We show that all diagnostic samples should be screened for the simultaneous m- and M- BCR/ABL expression to avoid false-negativity when using m-BCR/ABL quantification only. In our hands, the quantification of BCR/ABL transcripts appears to be a more reliable method than the generally accepted Ig/TCR-based MRD monitoring as the number of false-negative samples by BCR/ABL quantification is significantly lower. This contention is further supported by our pilot data on transplanted patients where BCR/ABL positivity preceding transplantation seems to be a better predictor of subsequent relapse than Ig/TCR approach. Support: MSM0021620813, MZ00064203 and 62/2004 GAUK CR. KK and KM contributed equally to this work.

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Juxtaposition of the BCL11B Gene to a Novel Region at 17q by a t(14;17)(q32;Q21) in Childhood T-Cell Lymphoblastic Lymphoma.

Blood ◽

10.1182/blood.v110.11.4130.4130 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4130-4130 ◽

Cited By ~ 1

Author(s):

Sabine Strehl ◽

Margit König ◽

Katharina Spath ◽

Markus Pisecker ◽

Georg Mann

Keyword(s):

Bone Marrow ◽

T Cell ◽

Peripheral Blood ◽

Fusion Gene ◽

Fusion Transcript ◽

Regulatory Elements ◽

Lymphoblastic Lymphoma ◽

Fish Analysis ◽

Bac Clone ◽

Expression Levels

Abstract T-cell acute lymphoblastic lymphoma/leukemia is frequently associated with recurrent genetic aberrations that result in the deregulation of transcription factors. In this respect, BCL11B plays a key role in the differentiation and survival during T-cell development. The 3′-located regulatory elements of BCL11B are juxtaposed to TLX3 by a cryptic t(5;14)(q35;q32) in approximately 20% of childhood T-ALL, which leads to inappropriate expression of TLX3. BCL11B can also fuse to TRDC through an inv(14)(q11.2q32.31) resulting in the expression of a BCL11B-TRDC fusion transcript in the absence of wild-type BCL11B. Moreover, a t(6;14) involving BCL11B and the 6q26 region has been described. We have identified a novel BCL11B rearrangement in a case of childhood T-cell lymphoblastic lymphoma. Cytogenetics detected a t(14;17)(q32;q21) and subsequent FISH analysis using BCL11B-spanning and BCL11B 3′-breakpoint-cluster-region flanking BAC clones revealed that BCL11B itself was not disrupted. However, a translocation breakpoint downstream of the BCL11B was observed suggesting the activation of a juxtaposed gene usually residing at 17q by the transcriptional regulatory elements of BCL11B. To narrow down the breakpoint at 17q a FISH-based chromosome-walking strategy using a set of chromosome 17q-specific BACs was employed. A BAC clone encompassing - from centromere to telomere - the genes RAB5C (a member of the RAS oncogene family), KCNH4 (potassium voltage-gated channel, subfamily H (eag-related), member 4), HCRT (hypocretin (orexin) neuropeptide precursor), GHDC (GH3 domain containing; LGP1), STAT5B (signal transducer and activator of transcription 5B), and the 5′-end of STAT5A showed a split signal indicating that one of these genes was juxataposed to the BCL11B enhancer. RAB5C, KCNH4, GHDC, and STAT5B are transcribed in a telomere-centromere orientation, whereas STAT5A shows the opposite transcriptional direction. Together with the FISH pattern observed these data suggested that STAT5A was the most likely candidate gene that might be inappropriately expressed via the regulatory elements of BCL11B. However, semi-quantitative expression analysis showed that neither STAT5A nor STAT5B were significantly upregulated in the affected lymph node as compared to normal bone marrow, peripheral blood, and thymus. In fact, compared to the expression levels in the other tissues STAT5A seemed to be expressed at lower levels. Thus, also the expression levels of RAB5C, KCNH4, and GHDC were analyzed. KCNH4 expression was almost undetectable in bone marrow, peripheral blood, and thymus and for all three genes no elevated expression was observed in the T-cell lymphoma. Owing to the unchanged expression of these genes also the transcription level of STAT3, which is localized further distal to the breakpoint determined by FISH was analyzed, and similar to STAT5A showed lower expression. However, depletion of STATs usually results in reduced cell viability and apoptosis. Together, our data suggest several scenarios: rearrangements of the region containing the remote enhancer of BCL11B are not necessarily accompanied by high expression of a gene juxtaposed into the close vicinity, expression levels of the juxtaposed gene may be just modulated rather than strongly enhanced, the presence of a more complex translocation undetectable by cytogenetics that results in the overexpression of a gene not obviously affected by the translocation or the generation of a fusion gene.

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MRD Monitoring Reveals a Specific Biology of BCR/ABL-Positive ALL

Blood ◽

10.1182/blood.v112.11.2529.2529 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2529-2529

Author(s):

Marketa Zaliova ◽

Leona Reznickova ◽

Eva Fronkova ◽

Katerina Krejcikova ◽

Katerina Muzikova ◽

...

Keyword(s):

Residual Disease ◽

Fusion Gene ◽

Fusion Transcript ◽

Cell Receptor ◽

Quantitative Detection ◽

Gene Transcript ◽

Equal Distribution ◽

Childhood All ◽

Negative Results

Abstract Minimal residual disease (MRD) monitoring is an essential tool for current leukaemia therapy. The only standard method for MRD monitoring in childhood ALL is the quantitative detection of clonal immunoglobulin (Ig) and T-cell receptor (TCR) genes rearrangements. The quantitative detection of fusion genes or transcripts provides an alternative option for MRD monitoring. We aimed to compare the significance of these MRD methods by parallel monitoring of fusion transcripts/genes and Ig/TCR targets during the follow-up of children from the three most common ALL genotype groups. We analysed 117, 109 and 191 bone marrow samples from 28 TEL/AML1-positive, 7 MLL fusion-positive and 16 BCR/ABL-positive patients, respectively. To keep the comparability of different MRD approaches, we used qPCR detection systems with similar sensitivity (at least 10−4), we adopted ESG-MRD-ALL principles for MRD quantification and we related the MRD level in follow-up samples to the diagnostic level for all MRD methods. We found a very good correlation of fusion transcript- and Ig/TCR-based approaches (R2=0.903) with only 7% of samples differing by more than 1 log in a cohort of TEL/AML1-positive patients. A good correlation was also found between fusion transcript- and Ig/TCR-based MRD in MLL fusion-positive patients (R2=0.8419). Only 10% of samples differed by more than 1 log, being underestimated by Ig/TCR in 4.5% and by MLL-fusion transcript in 5.5%. For the follow-up of MLL-fusion-positive patients we further employed the monitoring of MLL-fusions on genomic level. The MRD based on genomic MLLfusion genes showed a very good correlation with Ig/TCR -based method (R2=0.9124) with only 5% of samples differing by more than 1 log, and it also closely correlated with MLL-fusion transcript levels (R2=0.9195). Strikingly, in BCR/ABL-positive patients we found a limited correlation of fusion transcript-based and Ig/TCR-based MRD (R2=0.6880) with 1/3 (34%) of samples differing by more than 1 log. In contrast to the MLL cases, the underestimation of MRD by individual methods was “asymmetrical”: 8% of the discordant samples had higher MRD measured by Ig/TCR and 26% by BCR/ABL transcript. Despite identical sensitivity of both methods, in 19% of samples the MRD positivity was revealed only by BCR/ABL approach while Ig/TCR approach gave negative results. Detailed analysis showed clinical significance of the discordant BCR/ABL vs. Ig/TCR MRD information. Altogether, 13 relapses occurred during the follow-up of our cohort. We compared number of BCR/ABL and Ig/TCR -positive samples among all BM specimens taken 6 and 12 months before relapse. While the majority of samples preceding relapse were BCR/ABL-positive (14/18 and 22/36 six and twelve months before relapse) only a minority of samples showed Ig/TCR positivity (7/18 and 12/36, respectively). The non-equal distribution of the BCR/ABL and Ig/TCR-positive samples was statistically significant (p=0.04 and p=0.03 for the two time-points, respectively). Our study shows, that in TEL/AML1 and MLL fusion-positive patients, fusion gene/transcript-based MRD monitoring provides information highly concordant to the standard Ig/TCR approach and thus it is useful as a complementary method in patients with absent or inadequate Ig/TCR targets (particularly in MLL cases where clonal Ig/TCR rearrangements are rare). The situation is different in BCR/ABL patients, where the MRD information from both approaches is discordant in a high subset of samples. This result probably reflects the dissimilar biology of this ALL subtype and the fact, that BCR/ABL-positive (prae-)leukaemic stem cell is different and multilineage involvement more common. Thus, in some cases, the fusion transcript monitoring reveals the existing pool of cells that increase the risk of relapse despite the Ig/TCR negativity. We conclude that MRD in all BCR/ABL–positive patients should be monitored not only by the standard Ig/TCR approach but in parallel also by the quantitative fusion transcript-based detection. Support: MSM0021620813, MZO00064203.

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Prognostic and diagnostic value of Ewing family of tumors (EFTs)-associated fusion transcripts detected in peripheral blood specimens.

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.15_suppl.10538 ◽

2012 ◽

Vol 30 (15_suppl) ◽

pp. 10538-10538

Author(s):

Joanna Przybyl ◽

Hanna Kosela ◽

Katarzyna Wiater ◽

Konrad Ptaszynski ◽

Anna Szumera-Cieckiewicz ◽

...

Keyword(s):

Peripheral Blood ◽

Fusion Gene ◽

Systemic Disease ◽

Prognostic Significance ◽

Fusion Transcript ◽

Added Value ◽

Rt Pcr ◽

High Detection Rate ◽

Blood Specimens

10538 Background: EFTs are characterized by chromosomal translocations leading to formation of oncogenic EWSR1-FLI1 fusion gene in 85-90% of cases. The aim of the study was to detect circulating tumor cells (CTCs) carrying EWSR1-FLI1 fusion transcript in peripheral blood and assess their added value to standard diagnostic procedures and utility as a prognostic marker in EFTs. Methods: 10mL of whole blood was collected from 35 untreated adult EFTs patients at the diagnosis (period: 2008-2011, median age 27 years) and 13 healthy controls. 13 patients presented metastatic disease (M1) at the diagnosis. Nested RT-PCR was applied in triplicate for the detection of EWSR1-FLI1 transcript. Blood specimen was regarded CTC-positive when at least 2 out of 3 nested RT-PCR assays were positive and results were confirmed by sequencing. FISH assay for EWSR1 rearrangement was performed on FFPE tumor tissue in 28 available cases. Median follow-up was 16 months. Results: EWSR1-FLI1 transcript was detected in peripheral blood of 71.4% (n=25) of patients. FISH assay was positive for EWRS1 rearrangement in 58% of cases. In 10 patients, where FISH assay could not be performed due to insufficient quality or lack of material, nested RT-PCR provided confirmation of immunopathological diagnosis. Specificity of RT-PCR blood test in healthy control was 91.4% (12/13 negative; p=0.0001). Median overall survival (OS) was 18 months. CTC-positive patients showed the trend for longer OS than CTC-negative patients (1-year OS: 89% vs. 58%; p=0.07), but longer follow-up is needed. Conclusions: Our results show that the fusion transcript detection in peripheral blood specimens may be a useful additional test to the standard clinicopathological diagnosis of EFTs. High detection rate of EWSR1-FLI1 transcript in peripheral blood of EFTs patients at the diagnosis may imply EFTs as a systemic disease with clinically evident metastases or micrometastases at presentation. Prognostic significance of CTCs in EFTs warrants further studies.

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Prognostic value of CDH2 and CDT2 expression levels in the peripheral blood (PB) specimens of patients with Ewing family of tumors (EFTs).

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.15_suppl.e22046 ◽

2013 ◽

Vol 31 (15_suppl) ◽

pp. e22046-e22046

Author(s):

Joanna Przybyl ◽

Maria Debiec-Rychter ◽

Tomasz Switaj ◽

Hanna Melania Kosela ◽

Slawomir Falkowski ◽

...

Keyword(s):

Peripheral Blood ◽

Fusion Gene ◽

Prognostic Significance ◽

Expression Level ◽

Healthy Individuals ◽

Expression Levels ◽

Reverse Transcription Pcr ◽

Blood Specimens ◽

Aggressive Clinical Course

e22046 Background: EFTs are characterized by oncogenic EWSR1-FLI1 fusion gene, which can be detected in 85-90% of tumors and in 20-70% of peripheral blood specimens. It has been previously reported that downregulation of CDH2 (encoding N-cadherin) and overexpression of CDT2 [also known as DTL, encoding denticleless homolog (Drosophila)]in EFTs tumors are associated with poor prognosis. The aim of the study was to evaluate the expression levels of these markers in the circulating tumor cells and assess their utility as prognostic markers in EFTs. Methods: 10mL of PB was collected from 24 untreated adult EFTs patients at the diagnosis (period: 2009-2011, median age 30 years). 9 blood specimens from healthy individuals, and 5 untreated frozen EFTs tumor samples were used as controls. 8 EFTs patients presented metastatic disease (M1) at the diagnosis and 5 patients died during the follow-up period. Median follow-up was 22 months. Quantitative reverse transcription PCR (qRT-PCR) for CDH2 and CDT2 expression were performed in triplicate using the TaqMan Gene Expression Assays (Applied Biosystems). Mean expression level of 2 reference genes PSMC4 and EIF2B1 served as endogenous control. The fold change was calculated using ddCt method with pooled healthy individuals as calibrator. Results: At least 2-fold decrease (range 2.11-16.35) of CDH2 expression level in PB has been observed in 79% (n=19) of EFTs patients (p=0.04) as compared to healthy control. In general, CDH2 expression level in PB of EFTs patients was 3.08-fold lower compared with healthy individuals (p=0.07). All of the patients who had M1 at diagnosis (n=8) and who died during the follow-up (n=5) had CDH2 gene underexpression. CDT2 overexpression (FC range 3.28-6.97) has been observed in 17% (n=4) of EFTs patients (p=0.01). Three of these patients had M1 at diagnosis and two of these patients died during the follow-up. PB specimens presenting CDT2 overexpression clustered together with the tumor specimens. Conclusions: Abnormalities in the expression of CDH2 and CDT2 genes in PB specimens are associated with aggressive clinical course of EFTs. Diagnostic and prognostic significance of these biomarkers warrants further studies.

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Bone marrow-derived NGFR+ cells regulate arterial remodeling and those poor mobilizations in peripheral blood in acute coronary syndrome predicts plaque progression at the non-targeted lesion

European Heart Journal ◽

10.1093/ehjci/ehaa946.1679 ◽

2020 ◽

Vol 41 (Supplement_2) ◽

Author(s):

S Takashima ◽

S Usui ◽

S Matsuura ◽

C Goten ◽

O Inoue ◽

...

Keyword(s):

Bone Marrow ◽

Acute Coronary Syndrome ◽

Peripheral Blood ◽

Funding Source ◽

Arterial Remodeling ◽

Plaque Progression ◽

Wild Type ◽

Plaque Volume ◽

Coronary Syndrome

Abstract Background In our previous 5-year cohort study, we demonstrated that low gene expression of nerve growth factor receptor (NGFR) in peripheral leucocytes in acute coronary syndrome (ACS) predicted repetitive coronary interventions at the de novo lesions. An NGFR-positive cell has been demonstrated to reside in bone marrow (BM) stromal fraction and to be increased in peripheral blood mononuclear cell (MNCs) fraction in patients with ischemic heart disease. Purpose To investigate whether the BM-NGFR+ cell is associated with arterial remodeling and the relationship between the levels of peripheral NGFR+ cells after ACS and coronary plaque progression in an experimental and prospective clinical study. Methods and results In an experimental study, 8-week-old C57B6/J wild type male mice were subjected to irradiation with 9.6 Gy and transplantation with BM (BMT) isolated from GFP-transgenic NGFR wild type (WT) or knock-out (KO) mice at day 1. Four weeks after BMT, the right carotid artery was ligated for 4 weeks. Induced neointimal area was increased (p<0.05), where cells under apoptosis were decreased (p<0.05) in NGFR-KO-BMT group compared to WT-BMT group (n=4). NGFR+ cells were not detected in wild type sham-operated artery, whereas in the ligated artery in WT-BMT group NGFR+ cells assembled in the developed neointima and exclusively presented double positive with GFP, but absent in NGFR-KO-BMT group (p<0.05, n=4). In a clinical study, thirty patients with ACS who underwent primary percutaneous coronary intervention (PCI) were enrolled. The peripheral blood sample was collected on days 0, 3 and 7, and 9 months follow-up and the number of NGFR+MNCs were measured by flowcytometric analysis. The plaque volume at non-targeted coronary lesion (non-TL:>5 mm proximal or distal to the implanted stents) were quantitatively analysed using gray-scale intravascular ultrasound (IVUS) and Q-IVUS™ software at the acute phase and 9 months follow-up. The number of NGFR+MNCs in peripheral blood was 1.5-fold increased at day 3 (0.064±0.056%) compared to day 0 (0.042±0.030%) (p<0.05). The change in normalized total plaque volume (TAVN) at non-TL at 9 months was negatively correlated with the number of NGFR+MNCs at day 0 (r=−0.51), day 3 (r=−0.51) and 9 months (r=−0.59) after ACS (p<0.05). Multiple regression analysis showed that NGFR+MNCs at day 0 (β=−0.48, p=0.01) and CRP (β=−0.53, P<0.01) are independent factors associating with TAVN change at non-TL at 9 months, regardless of LDL-cholesterol control level. ROC analysis revealed that NGFR+MNCs <0.049 at day 0 predicted the increase of TAVN with AUC 0.78; sensitivity 0.82 and specificity 0.67. Conclusions Bone marrow-derived peripheral NGFR+ cells negatively regulate arterial remodeling through appropriate apoptosis of neointimal cells and the peripheral level of NGFR+ cells in ACS predicts plaque progression at the non-targeted lesion. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): KAKENHI

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Abstract 239: Stem Cell Therapy Improves Critical Limb Ischemia

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.32.suppl_1.a239 ◽

2012 ◽

Vol 32 (suppl_1) ◽

Author(s):

Alaa Marzouk

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Embryonic Stem ◽

Limb Ischemia ◽

Control Group ◽

Chronic Limb Ischemia

Introduction: The journey from single cell to complex being is attributable to stem cells role. Adult stem cells originate during ontogeny & persist in specialized niches within organs. Asymmetric division of each stem cell during differentiation produces : one daughter stem cell & one daughter transit amplifying/intermediate cell having migratory properties. Forced migration of hematopoietic stem/progenitor cells (HSPC) from bone marrow into peripheral blood is called mobilization. Accumulating evidence suggests that attenuation of the chemokine stromal derived factor-1(SDF-1)-CXCR4 axis that plays a pivotal role in retention of HSPC in bone marrow (BM) results in the release of these cells from the BM into peripheral blood. Recently, adult cells have been genetically reprogrammed to an embryonic stem cell like state. Induced pluripotent stem cells (IPSCs) were similar to human embryonic stem cells in morphology, proliferative capacity, expression of cell surface antigens, & gene expression. Treatment of ischemic vascular disease of lower limbs remains a significant challenge. Unfortunately, if medical & surgical salvage procedures fail, amputation is an unavoidable result for those patients. Aim of Work: (Hypothesis) To assess the application of implantation of autologous stem/progenitor cell in the treatment of chronic limb ischemia & to evaluate the safety, efficacy & feasibility of this novel therapeutic approach. Methods: A total of 24 patients with chronic limb ischemia not eligible for arterial reconstruction or endovascular procedures were enrolled & randomized (1:1) to either the implanted group or the control group. Control group: Conventional medical therapy in the form of anti platelet therapy & vasodilators. Implanted group: Subcutaneous injection of 300μ g/day of recombinant human granulocyte colony stimulating factor (G-CSF) for 5 days to mobilize stem/progenitor cells from BM. Total leucocytic count is measured daily to follow up successful mobilization of bone marrow mononuclear cells (BMMNCs). Stem cell Harvesting After 5 days peripheral blood mononuclear cells (PBMNCs) were harvested using a cell separator. Samples from apheresis products are subjected to TLC measurement & immunophenotypic characterization of CD34+ cells by flow cytometry. The collected PBMNCs were implanted by multiple intramuscular injections into ischemic limbs. Results: There was significant increase in pain free walking distance & ankle/brachial index (ABI) & significant decreased rest pain. Effectiveness was documented by : reduced number of amputation, increase ABI & improvement of the quality of life in therapeutic group compared to control group. Conclusion: The novel therapeutic approach of PBMNCs implantation in patients with chronic limb ischemia is safe, feasible & effective in decreasing co-morbidity & rate of amputation. Safety was manifested by absence of complications during G-CSF therapy or during harvesting & injection of the stem cells. Recommendations: 1- Future studies on larger number of patients & longer follow up. 2- Controlled studies using different methods & different cell population (PBMNCs, BMMNCs or MSCs) to compare the outcome of each. 3-Studing the role of endothelial progenitor cell dysfunction in different ischemic diseases to develop successful gene therapy.

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Improved Outcomes in Patients with Thalassemia Major Transplanted with Peripheral Blood and Marrow Grafts in Comparison with Cord Blood and Bone Marrow Grafts from Sibling Donors

Blood ◽

10.1182/blood.v128.22.847.847 ◽

2016 ◽

Vol 128 (22) ◽

pp. 847-847

Author(s):

Qiao chuan Li ◽

Jian ming Luo ◽

Zhong ming Zhang ◽

Lian jin Liu ◽

Ling ling Shi ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Cord Blood ◽

Graft Rejection ◽

Peripheral Blood ◽

Cumulative Incidence ◽

Conditioning Regimen ◽

Thalassemia Major ◽

Matched Sibling

Abstract Background: Thalassemia major (TM) is a fatal genetic disease currently only curable with allogeneic stem cell transplantation. This is limited by the lack of suitable donors and the quantity of collected stem cells, and is often complicated by graft rejection and graft versus host disease (GVHD). Methods: The aim of the study was to compare the outcomes of TM patients transplanted with matched sibling cord blood (CB) and bone marrow (BM) grafts vs. matched sibling peripheral blood (PB) stem cell and BM grafts. The trial was designed as a prospective, open-label, single-center clinical protocol, where 204 TM patients were enrolled between January 2007 and November 2015 and transplanted with either PB + BM (n=99) or CB+BM (n=105), from an HLA-identical sibling donor. This study was approved by the Medical Ethics Committee of the First Affiliated Hospital of Guangxi Medical University and was registered at the Chinese Bone Marrow Transplant Registry (CBMTR). The primary end point was 2-year thalassemia free survival(TFS). Secondary end points included 2-year overall survival (OS), the cumulative incidence of GVHD, transplant related mortality (TRM), graft rejection (GF).The conditioning regimen were:1) busulphan (BU) (1.25 mg/kg) given orally four times per day for 4 days or 1mg/kg given intravenously (IV) four times per day for 4 days (day -9 to day -6); 2) fludarabine (FLU) (50mg/m2/day) given IV for 3 days (day -12 to day -11); 3) cyclophosphamide (CTX) (50 mg/kg/day) given IV for 4 days (day -5 to day -4); 4) anti-thymocytes globulin (ATG, Genzyme ) (2.5 mg/kg/day) given IV for 4 days (days -4 and day -1). All patients were placed on 30 mg/kg hydroxyurea orally once daily for 2-3 months before transplantation.GVHD prophylaxis consisted of a combination of cyclosporin A, methotrexate and mycophenolate mofetil regimen. [BMT 2009; 43(1):61-67]. Results : Patient and donor characteristics, and transplantation outcomes are listed in Tables 1 and 2, respectively. Data cut off for survival follow-up was March 31, 2016. The median follow-up time was 26 months (range, 4 months -105 months). Both neutrophil as well as platelet engraftment occurred significantly faster in the PB+ BM group than the CB+BM group (11 days vs. 13 days, P=0.001 and 15 days vs. 25 days, P=0.001, respectively). The rate of GF was the same in both groups (1.0%). The cumulative incidence of grade II-IV acute (a) GVHD and extensive chronic (c)GVHD in the PB+ BM group was higher than the CB+BM group: aGVHD=15.5% vs 1.0%, P=0.001; cGVHD= 6.4% vs. 0%, P=0.013. The cumulative rates of TRM at 2 years remained significantly lower in the PB+BM group compared to the CB+BM group with 2.0% and 12.5%,(P=0.005), respectively . Both OS and TFS at 2 years favored the PB +BM group compared to the CB+BM group : OS=98% vs. 86.5%,P=0.003;TFS= 97% vs. 86.5%, P=0.008.(Fig 1) Conclusion: Our results demonstrate that grafts composed of PB + BM had superior overall outcomes compared to CB + BM grafts, as evidenced by faster engraftment and lower TRM of the former despite substantially lower aGVHD and cGVHD rates of the latter. The mixed stem cell populaitons and the high cell dose achieved with the use of 2 different graft sources, toghether with the conditioning regimen used likely contributed to the superior outcomes seen with this regiem. This strategy could be of great benefit for the treatment of patient with TM and other benign hematologic disease. Disclosures No relevant conflicts of interest to declare.

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BCOR Mutation and TLS-ERG Expression in Acute Myeloid Leukemia with Monoclonal Immunoglobulinemia

Blood ◽

10.1182/blood-2019-122366 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5180-5180

Author(s):

Jian Huang ◽

Jingxia Jin ◽

Shuna Luo ◽

Xingnong Ye

Keyword(s):

Bone Marrow ◽

Gene Mutation ◽

Peripheral Blood ◽

Myeloid Leukemia ◽

Poor Prognosis ◽

Plasma Cells ◽

Fusion Gene ◽

Next Generation Dna Sequencing ◽

School Of Medicine ◽

Immunofixation Electrophoresis

Acute myeloid leukemia(AML) originates from the abnormal clonal proliferation of myeloblast which often combined with clinical symptoms. Cytogenetic and molecular abnormalities are frequent in AML patience. To date, the driver genes for leukemia remain largely undiscovered. Monoclonal immunoglobulinemia is a group of diseases caused by excessive proliferation of plasma cells or immunoglobulin-producing lymphoid plasma cells and B lymphocytes. It can develop into malignant plasma cell disease. Herein, we report a AML patient was concomitant with monoclonal immunoglobulinemia, the patient was also accompanied by BCOR mutation and TLS-ERG fusion gene. A 55-year-old married female was admitted into our hospital due to repeated edema for 3 weeks. On admission, peripheral blood counts: PLT142×10^9/L, HB77g/L↓, WBC35.2×10^9/L.Bone marrow examination showed the mononuclear cell system proliferated actively, and the primitive infantile monocytes accounted for 86%. Cell morphology suggested M5b(Figure1A ). Fusion gene screening in bone marrow revealed that TLS-ERG expression. Immunophenotype of bone marrow cell:Abnormal myeloid primitive cells accounted for 96.39% of the nuclear cells,expressCD33, CD13, CD123, CD34, CD9, MPO(Figure 1D). Karyotype analysis of bone marrow cells showed in Figure 1B. Thus, AML was diagnosed. Next-generation DNA sequencing technology showed that BCOR (51.7%),PLCG1(49.9%),DIS3(48.4%),BRAF(51.6%), JAK2(45.1%) ,JAK3(49.0%) were mutated. Meanwhile, we found that Peripheral blood immunofixation electrophoresis showed that Gamma region is seen with a monoclonal light chain lambda component((Figure 1C.).Then, the patient underwent one cycle of IA(Idabisine hydrochloride 10mg d1-4, cytarabine 0.075g q12h d1-7). Twenty-five after chemotherapy onset, bone marrow examination showed that primitive and immature monocytes accounted for 3%. Chromosome become normal. Minimal residual disease(MRD):0.01%. The disease reached complete remission(CR). Peripheral blood immunofixation electrophoresis turned negative. Fusion gene detection showed that TLS-ERG turned negative. BCOR mutation was not detected by Next-generation DNA sequencing. Mutations of PLCG1,DIS3,BRAF,JAK2,JAK3 still exist. Monoclonal immunoglobulinemia and AML are both clonal diseases, but originated from different clones. This case has both malignant clones of granulocyte stem cell and malignant clones of B line, so it is worthy of discussion. By comparing CR before and after we found that while the patient's M protein turned negative, the TLS-ERG fusion gene and BCOR gene mutation also disappeared. The TLS-ERG fusion gene is formed by the rearrangement of TLS and ERG genes on chromosomes 16 and 21. The current study holds that the expression of this fusion gene indicates rapid disease progression and poor prognosis. BCOR mutations can be found in AML and often coincide with DNMT3 gene mutations, suggesting it may affect the occurrence of leukemia through epigenetics. BCOR is a newly discovered corepressor of BCL-6, which can play a supporting role when BCOR combines with DNA; when BCOR is overexpressed, it can enhance the inhibition of BCL-6. BCL-6 is highly expressed in tumor cells,it encodes transcriptional repressors which are required for the formation of germinal center and may affect apoptosis. We thinked that the monoclonal immunoglobulinemia of this patient may caused by the BCOR abnormal expression which increased the inhibitory effect of BCL-6 and affect the apoptosis of B cells, and B cells continue to secrete immunoglobulin. BCOR mutations are associated with poor prognosis. The patient with TLS-ERG fusion gene which is a poor prognosis gene.However, the BCOR gene mutation site is a non-hot spot mutation which has few clinical studies. Whether the BCOR gene mutation results in the combination of the two diseases requires further study. Acknowledgment:The research was supported by fundings of the public technology research projects of Yiwu,China (2016-S-05), the key medical discipline of Yiwu,China(Hematology,2018-2020),and the academician workstation of the Fourth Affiliated Hospital of Zhejiang University School of Medicine. Correspondence to: Dr Jian Huang, Department of Hematology, The Fourth Affiliated Hospital of Zhejiang University School of Medicine. N1 Shangcheng Road. Yiwu, Zhejiang, Peoples R China. Email: [email protected] Figure 1 Disclosures No relevant conflicts of interest to declare.

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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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