bluuebunny/crossref_metadata_2025 huggingface dataset:
MEL1 at 1p36.3 Is Fused to Several Partner Genes Including HOXA9 Located at Different Chromosomal Bands from 3q21.3 in t(1;3)(p36.3;q21.3)-Leukemia.
Authors: Xinh Phan Thi , Anh Hoang , Tanaka Hideo , Fujisawa Shin , Taketazu Fumitoshi , Rowley Janet D. , Tokunaga Katsushi , Sato Yuko
Published: October 2019

In MDS/AML with t(1;3)(p36;q21), overexpression of MEL1 lacking PR domain (MEL1S) driven by the RPN1 promoter at 3q21.3 is reported to be closely associated with the pathogenesis. Previously, we have defined the 1p36.3 breakpoint (BP) in 5 patients and the 3q21.3 BP in 4 patients with BAC/PAC probes. The BPs at 1p36 were clustered within 3 BP regions (BR), spanning an approximately 200-kb area: BR-1, a 70-kb region in the first intron of MEL1 covered by RP1-163G9; BR-2, a 29-kb region in 5′ region of MEL1 covered by RP5-907A6; BR-3, a 68.5-kb region between the BR-1 and BR-2. On the other hand, the BPs at 3q21.3 were clustered within only one BR, a 108.9-kb region belonging to RP11–475N22, centromeric to RPN1, in which GR6 gene and the promoter region of GATA2 gene are included. Among 5 patients, 2 had the 1p36.3 BPs within the first intron of MEL1, suggesting the existence of MEL1 fusion genes. As expected, using 3′- and 5′-RACE, we have identified 10 MEL1 fusion genes including MEL1/HOXA9 among the 4 patients, not only in patients with the 1p36.3 BP within MEL1 but also in patients with the 1p36.3 BP outside MEL1. None of reciprocal fusion transcripts were detected. Using two normal bone marrow samples and HNT-34 cell line as control, no MEL1 fusion transcripts were detected with TA cloning analysis of 50 clones obtained from each sample. Surprisingly, none of these partner genes were located at band 3q21.3, suggesting that different mechanisms from chromosome translocation might be involved in generating these fusion transcripts. This is the first report showing that MEL1 is involved in the (1;3)(p36;q21)-leukemia not only by inappropriate expression but also by generation of fusion transcripts. Taken together, our findings suggest that in addition to overexpression of MEL1S, MEL1 fusion transcripts may play important roles in the pathogenesis of t(1;3)-leukemia.
In Vivo Activated CD103+CD4+ Regulatory T Cells Ameliorate Ongoing Chronic GVHD.
Authors: Zhao Dongchang , Zhang Chunyan , Yi Tangsheng , Lin Chia-Lei , Todorov Ivan , Kandeel Fouad , Forman Stephen , Zeng Defu
Published: October 2019

The αEβ7 integrin CD103 is an excellent marker for identifying in vivo activated CD4+ regulatory T (Treg) cells. CD103− naive Treg cells from donors are effective in prevention but ineffective in treatment of graft versus host disease (GVHD). It is unknown whether in vivo activated donor CD103+ Treg cells can effectively treat ongoing GVHD. We have recently reported a new chronic GVHD model, in which donor DBA/2 (H-2d) spleen cells were transferred to the irradiated BALB/c (H-2d) recipients. The recipients showed chronic GVHD-like syndrome with high-levels of serum autoantibodies, proteinuria, and hair-loss, and the disease induction required both donor CD4+ T and B cells in a cell dose dependent manner (Blood, 2006). In the current studies, we observed that the percentage of CD103+ cells among donor CD4+ T cells in the recipients without disease was up to 45% and was more than two fold higher than that of the recipients with disease. The CD103+CD4+ T cells were more than 97% FoxP3+, which was similar to natural CD25hi Treg cells, but the former expressed markedly higher levels of CCR5 as compared to the latter. The CD103+ Treg cells showed markedly stronger suppression capacity as compared to freshly isolated as well as in vitro anti-CD3-activated CD25hi natural Treg cells. When injected into ongoing chronic GVHD recipients with severe proteinuria, CD103+ Treg cells (1x106) reversed proteinuria and tissue damages in 92% (11/12) of the recipients, and the recipients survived for more than 100 days. In contrast, the in vitro activated natural Treg cells reversed disease in only 25% (2/8) of the recipients, and the freshly isolated CD25hi natural Treg cells reversed none (0/12), and the treated recipients died within 45 days, which was similar to control PBS treated recipients. Furthermore, we found that infusion of the CD103+ Treg cells significantly reduced CD138+ antibody-secreting plasma cells in spleen and reduced serum levels of autoantibodies; and that infusion of the CD103+ Treg cells also significantly reduced CD44loCD62LhiSCA-1hi post-mitotic CD4+ T memory stem cells in spleen as well as pathogenic IFN-γ+CD4+ T cells in liver and nephritogenic IFN-γ+IL-10+CD4+ T cells in kidney tissues. Our results indicate that, different from CD103− naive natural Treg cells that prevent GVHD via suppressing donor T cell activation and expansion, CD103+ Treg cells ameliorate ongoing chronic GVHD via reducing activated pathogenic T and B cells.
Peripheral Blood Cytogenetic Studies in Hematological Neoplasms: Predictors of Obtaining Metaphases for Analysis.
Authors: Hussein Kebede , Ketterling Rhett P. , Dewald Gordon W. , Hulshizer Rachael L. , Kuffel Daniel G. , Wiktor Anne E. , Van Dyke Daniel L. , Tefferi Ayalew
Published: October 2019

Background: Peripheral blood (PB) is sometimes used in place of bone marrow (BM) for cytogenetic studies during the evaluation of hematologic malignancies. We looked for clinical or laboratory features that predict success in obtaining analyzable metaphases during PB chromosome studies.

Methods: The Mayo Clinic cytogenetics database was queried to identify adult cases (age > 18 years) with suspected or established hematologic neoplasm in whom PB cytogenetic studies were performed. Success defined as the acquisition of at least two metaphases, was correlated with clinical and laboratory information corresponding to the time of the PB cytogenetic study.

Results: A total of 242 PB cytogenetic studies were performed: clinical diagnosis was a myeloid neoplasm in 169 patients (70%), lymphoid neoplasm in 50 (21%), and unexplained cytopenia or leukocytosis in 23 (9%). The 169 myeloid cases included 59 patients with either primary (n=39) or post-polycythemia vera/essential thrombocythemia (post-PV/ET MF) myelofibrosis (n=20), 42 with acute myeloid leukemia (AML), 15 with chronic myeloid leukemia, 9 with myelodysplastic syndrome (MDS), 8 with ET, 6 with PV, and 30 with other MPDs. The 50 lymphoid cases included 19 with chronic lymphocytic leukemia, 12 with lymphoma, 11 with acute lymphocytic leukemia (ALL), and 8 with plasma cell proliferative disorders. PB cytogenetic studies resulted in at least two analyzable metaphases (median 20, range 2–31) in 142 of the 242 study cases (59%); in univariate analysis, this was predicted by the specific clinical diagnosis (p<0.0001), presence and degree of circulating myeloid progenitor cells (p<0.0001), higher leukocyte count (p<0.001), lower platelet count (p=0.003), lower hemoglobin level (p=0.002), and presence of palpable splenomegaly (p=0.002). In multivariable analysis, only the presence of circulating myeloid progenitor cells sustained its significance and this was consistent with the high yield rates seen in PMF (80%), post-PV/ET MF (85%), AML (76%), and ALL (80%) as opposed to the low rates seen in ET (0%) and PV (2%). In 104 cases, BM cytogenetic studies were performed within one month of the PB cytogenetic studies; an abnormal BM cytogenetic finding was another independent predictor of a successful PB study (p=0.002).

Conclusion: PB cytogenetic studies are most appropriate in diseases characterized by presence of circulating myeloid progenitors or blasts (e.g. PMF, AML, ALL); the yield otherwise is too small to be cost-effective. The current study also suggests a higher likelihood of a successful PB cytogenetic study in the presence of an abnormal bone marrow karyotype.
Regulation of Bim Expression by IGF-1 in the 5T33MM Murine Model for Multiple Myeloma.
Authors: De Bruyne Elke , Bos Tomas , Deleu Sarah , Atadja Peter , Menu Eline , Van Valckenborgh Els , Van Camp Ben , Vanderkerken Karin
Published: October 2019

Insulin-like growth factor-1 (IGF-1) is an important factor in proliferation, cell survival and migration of multiple myeloma (MM) cells. Bcl-2 like 11 (Bim) is a pro-apoptotic protein belonging to the BH3-only group of Bcl-2 family members. Three major forms of Bim are known: BimEL, BimL and BimS. In leukemia, beta1 integrin mediated adhesion has been shown to reduce Bim levels by enhanced proteasome activation. We used the 5T33MM syngeneic, immunocompetent murine model for MM. Both the exclusively in vivo growing 5T33MMvv cells and the stroma independent in vitro growing 5T33MMvt cells were used. The present study showed that Bim levels were reduced in MM cells after IGF-1 treatment. IGF1-mediated regulation of Bim expression in 5T33MMvv cells was demonstrated to be a result of reduced gene expression on the one hand and increased proteasomal-mediated degradation of Bim protein levels on the other hand. Reduced expression was related to activation of the Akt pathway and inactivation of the transcription factor FoxO3a. Increased degradation of Bim was related to activation of the mitogen-activated protein kinase pathway, as IGF-1 treatment resulted in phosphorylation of ERK1/2. Reducing Bim levels by transducing 5T33MMvt cells with a lentiviral vector with a short hairpin RNA cassette inhibited serum starved and the clinical relevant deacetylase inhibitor panobinostat (LBH589, Novartis) induced cell death. Together our data indicate that disrupting IGF-1-mediated regulation of Bim degradation may increase the efficacy of drugs. In the past few years, methylation of DNA and acetylation of histones have been proven to be important regulators of gene expression. Moreover, acetylation of non-histone proteins has emerged as a dynamic posttranslational modification regulating numerous cellular processes. Co-treatment of 5T33MMvv and 5T33MMvt cells for 24h with the demethylation agent 5-Aza-2′deoxycytidine and the deacetylase inhibitor panobinostat resulted in a strong induction of Bim expression at protein level. In the future, we will investigate the contribution of IGF-1 to the epigenetic silencing of Bim by i) comparing the Bim promoter methylation pattern of both human IGF-1 responsive cell lines and murine 5T33MMvv cells treated or not with IGF-1 using bisulfite PCR sequencing and ii) using the epigenetic modulating agents 5-Aza-2′deoxycytidine and panobinostat.
Host Indoleamine 2,3-Dioxygenase Is a Critical Regulator of Acute GVHD Lethality.
Authors: Jasperson Lisa K. , Panoskaltsis-Mortari Angela , Taylor Patricia A. , Bucher Christoph , Mellor Andrew L. , Munn David H. , Blazar Bruce R.
Published: October 2019

Graft versus host disease (GVHD), the major cause of morbidity and mortality after bone marrow transplant (BMT), is initiated following activation of donor T cells by host antigen presenting cells (APCs). The immunosuppressive enzyme indoleamine 2,3-dioxygenase (IDO) is expressed by APCs and parenchymal cells and is further inducible by inflammation. We investigated whether lethal conditioning and GVHD induce IDO and if IDO prevents tissue injury by suppressing immune responses at the induction site. Using a fully MHC-mismatched murine GVHD model, we determined that IDO is a critical regulator of GVHD, most strikingly in the colon, where epithelial cells dramatically upregulated IDO expression during GVHD. No IDO upregulation was detected in mice that received irradiation only or irradiation and T cell-depleted BM without added alloreactive T cells. When mice with a genetic ablation of the IDO gene (IDO−/−) were used as recipients of BM and T cells, they suffered accelerated GVHD lethality compared to wild-type recipients, displaying increased colonic inflammation and T cell infiltration. This was true using grafts of either CD4+ T cells or combined CD4+ and CD8+ T cells. In addition, the IDO inhibitor 1-methyltryptophan caused accelerated GVHD in wild-type recipients. Lethality was not due to increased radiation sensitivity, as sublethally irradiated IDO−/− recipients showed no morbidity, and syngeneic BM was capable of rescuing lethally irradiated IDO−/− recipients. GVHD protection was not mediated by early control of T cell proliferation or apoptosis in lymph nodes or spleen. Furthermore, IDO did not affect expression of T cell effector molecules in lymphoid organs. Though studies have suggested a link between IDO and T regulatory cell (Treg) function, Treg depletion of donor grafts did not abrogate the IDO effect, indicating donor Tregs are not required for the suppressive function of IDO. Conversely, cultured Tregs added to the graft delayed GVHD in IDO−/− recipients, proving Tregs do not require host IDO for their function in this model. Instead, colons from IDO−/− hosts contained more proliferating CD4+ cells than wild-type colons after transplant. Together, this evidence suggests that in GVHD IDO acts primarily at the site of its expression to decrease effector T cell proliferation, diminish colonic inflammation, and reduce disease severity. These studies are the first to identify a function for IDO in GVHD lethality and suggest that modulation of the IDO pathway may be an effective strategy for treatment of this disease.
Bortezomib Protects Osteoblasts from Glucocorticoid-Induced Damage, and Enhances Glucocorticoid-Induced Toxicity Against Osteoclasts and Myeloma Cells.
Authors: Soe Kent , Andersen Thomas L. , Kupisiewicz Katarzyna , Plesner Torben , Delaisse Jean-Marie
Published: October 2019

Introduction: Multiple myeloma is characterized by the accumulation of malignant plasma cells in the bone marrow, and leads most often to bone destruction by osteoclasts and prevention of bone repair by osteoblasts. Bortezomib and glucocorticoids are both powerful anti-myeloma drugs that are used for killing malignant plasma cells in the patients. Furthermore bortezomib has direct anti-osteoclastic and pro-osteoblastic properties that may contribute to bone protection in multiple myeloma, while glucocorticoids have more ambiguous effects on these bone cells and are clearly anti-osteoblastic. Recent clinical trials based on the combination of bortezomib and glucocorticoids drew the attention on the very promising anti-myeloma efficiency of this combination. However, the bone cell response of this combination has not been tested. In order to address this question, we performed an in vitro study, and importantly adapted our in vitro model to mimic the pharmacokinetics of bortezomib and glucocorticoid in the patients.

Methods: Myeloma cell lines, primary human osteoclasts and osteoblast-like cells (MC3T3) were pulse-treated or not with clinically relevant doses of bortezomib (12.5, 25 or 50 nM) for 3 hours. Subsequently, the cells were exposed during a 3-day culture to 1.6 μM prednisolone which approximately corresponds to a dose of 50 mg prednisolone in a patient. The impact of the treatment on the cells was determined by survival, activity and gene expression.

Results: Bortezomib as a single treatment was very efficient in killing sensitive myeloma cells (OPM2) whereas the more resistant cells (U266) were more efficiently killed in combination with prednisolone. The release of TRAP from primary human osteoclasts, a marker of osteoclastic activation, was strongly inhibited by bortezomib treatment alone, but only in combination with prednisolone did it result in killing of osteoclasts. Survival of osteoblast like cells was uninfluenced by treatment with bortezomib alone. In contrast, as shown previously, prednisolone strongly reduced osteoblast survival. Most importantly however, a 3 hr pre-treatment with bortezomib protected the osteoblasts against the detrimental effects of glucocorticoids. Ongoing investigations by Q-PCR indicate that important markers of osteoblast maturation remain high if the osteoblasts were pre-treated with bortezomib prior to prednisolone exposure.

Conclusion: Our study demonstrates in conditions relevant to treatment of myeloma patients, that combining bortezomib and glucocorticoids has a direct synergistic effect against myeloma cells and osteoclasts, and that bortezomib protects directly osteoblasts from the negative impact of glucocorticoids. Thus, the combination of bortezomib and glucocorticoids is not only a powerful treatment of multiple myeloma itself, but also shows promise for treating myeloma bone disease.
A Phase I Study of the Proteasome Inhibitor Bortezomib in Patients with Myelofibrosis.
Authors: Barosi Giovanni , Gattoni Elisabetta , Barbui Tiziano , Vannucchi Alessandro M. , Rambaldi Alessandro , Silverman Lewis , Goldberg Judith , Marchioli Roberto , Hoffman Ronald
Published: October 2019

Myelofibrosis (MF) is the most serious myeloproliferative disorder, characterized by clonal myeloproliferation associated with cytokine-mediated bone marrow stromal reaction including fibrosis and osteosclerosis. Because the NF-κB pathway is implicated in the abnormal release of cytokines in MF, the proteasome inhibitor bortezomib might be a potential therapy. We conducted a phase 1 trial of intravenously administered bortezomib (Velcade®) in 12 adults with advanced primary MF (11 patients) or post-PV/post-ET MF (1 patient). Eligibility was defined as refractory or not suitable to first line chemotherapy. Median age was 57 years (range, 22 to 69 years). Three cohorts of patients were treated with bortezomib at day 1, 4, 8, and 11 from 0.8 to 1.3 mg/m2, every 21 days x 6 cycles. Three patients received less than 5 cycles and are not considered evaluable for response (cutaneous vasculitis, respiratory distress syndrome and peripheral neuropathy). Dose-limiting toxicity (DLT) occurred in 1 patient treated with bortezomib in the 1.3 mg/m2 cohort, consisting on respiratory distress syndrome. The patient was hospitalised and improved with corticosteroid therapy. The maximum-tolerated dose was 1.3 mg/m2 for 4 days every 3 weeks. Other frequent non-DLTs were thrombocytopenia (2/12), fatigue (3/12), oedema (1/12), cutaneous rash (2/12). No complete, major or moderate responses according EUMNET response criteria were documented. One minor response was documented due to complete response in platelet count (platelet count 150–400 x 109/L at the end of the sixth cycle of therapy). Our results show that bortezomib in MF is clinically tolerable at the 1.3 mg/m2 every 21 days x 6 cycles. The efficacy of the drug is under examination in a phase II study.
A Single-Center Study To Evaluate the Safety and Efficacy of Intravenous/Oral Fludarabine in Untreated Gastric MALT Lymphoma. Impact of t(11;18)(q21;q21) in Molecular Response Rates.
Authors: Salar Antonio , Bellosillo Beatriz , Seoane Agustín , Martinez Luz , Ferrer Ana , Serrano Sergio , Besses Carlos
Published: October 2019

Background: The treatment of gastric mucosa-associated lymphoid tissue (MALT) lymphoma in the absence of H. pylori infection or when the lymphoma fails to regress after adequate antibiotic treatment remains controversial. Fludarabine (F) is an active agent for indolent lymphoma, however, its clinical activity in gastric MALT lymphoma has not been studied. The aim of the study is to assess the efficacy and safety of single-agent fludarabine in gastric MALT lymphoma and to analyze the molecular response (MR) after this treatment.

Methods: Treatment consisted of fludarabine (25 mg/m2 IV) given on days 1–5, every 4 weeks, for 6 cycles; after the first cycle, oral fludarabine was allowed to be given orally at 40 mg/m2 with the same schedule. Molecular response (MR) was assessed by RT-PCR analysis of t(11;18) or by PCR assays for analysis of IgH gene rearrangements analyzing FR1, FR2 and FR3 in endoscopic biopsies.

Results: Eight consecutive patients were included. Median age: 60 years (range: 45–77); 3 pts were in stage I, 2 stage II-1 and 3 stage IV according to Lugano system. Four out of 5 (80%) pts achieved a CR after three cycles and all eight cases (100%) achieved a CR after six cycles, for an overall response rate of 100%. After a median follow-up of 44 months (range 14.5–58 mo) no patient has shown clinical or endoscopic relapse. Hematological toxicity occurred in 75% of pts, mainly mild neutropenia and generally after the third cycle. Three cases received G-CSF (after the 2nd, 3rd and 6th cycle) and three patients required dose modification or delay (3–7 days) in the delivery of the following cycle. No blood transfusions were required. Only one patient had to be admitted because of non-neutropenic fever. None case of myelodisplasia has been detected at last follow-up. Four out of 8 pts (50%) achieved MR during the study-period (see figure). Four out of 5 (80%) pts without t(11;18) achieved MR. In contrast, no patient carrying t(11;18) achieved MR. Sequencing analysis of monoclonal PCR products will be presented.

Conclusions: Fludarabine, either intravenous or oral, is safe and achieve a high response rate when given in gastric MALT lymphoma, with many pts achieving MR. In those pts carrying t(11;18), residual disease can be detected by PCR but do not determine relapse at present follow-up.

Figure Figure
90Y-Ibritumomab Treatment for Relapsed and/or Refractory B Cell Type Non-Hodgkin’s Lymphoma. Multiinstitutional Argentinian Study.
Authors: Cacchione Roberto , Dupont Juan , Milone Jorge , Bordone Javier , Garay Guy , Ardaiz Maria , Castro-Rios Miguel , Prates Virginia , Basso Alfredo , Iabstrebner Marcelo , Avila Graciela , Diaz Alicia , Argentieri Daniel , Rudoy Silvia , Trouboul Alicia , Palmer Luis , Pombo Gonzalo , Rappaciotti Silvina , Negri Pedro , Lafalse Diana , Riera Leandro , de Tezanos-Pinto Miguel , Riveros Dardo , Arnold Mario Brown , Carrasco Antonio , Bezares Fernando
Published: October 2019

The radionucleid conjugate with monoclonal antibodies (antiCD20/90Y-ibritumomab tiuxetan) have been approved for the treatment of relapsed, refractory and transformed (high grade) follicular lymphomas. Between September 2005 and August 2007, 41 patients with refractory/relapsed lymphoma were enrolled. Median age was 62 yrs old (45–83). Twenty were women and 21 were men. Thirty three were follicular and 5 were mantle cell lymphoma and 3 transformed lymphoma. Eighteen patients had bulky disease, 8 had bone marrow involvement and 21 had stage III-IV disease. Median time from diagnosis was 5 years (0.5–29). Twenty one had received 1–2 previous treatments, and 20 pts had received 3–5 previous treatments including 5 pts with autologous bone marrow transplantation. All had previously received anti-CD20 monoclonal antibody therapy. Six pts received previous radiotherapy. 90Y-Ibritumomab (Zevamab™ Bayer-Schering Argentina) was administered at 0.3 or 0.4 mCi, based on initial platelet count. Seven days before, and on the same day of the immunoconjugate administration, pts received rituximab 250 mg/m2. Thirty eight were evaluable for response. Thirty four (89%) pts responded: 20 CR (53%) and 14 PR (36%). With a median follow-up of 10 months, 60% of pts were expected to continue in CR at 18 months. Overall survival at the same period for the entire group was 89%. Nineteen pts (46%) required filgrastim administration for neutropenia, Ten pts (24%) required platelet transfusions, 10 pts had neutropenia plus fever and they had to be admitted for complicated pancytopenia, 8 pts required red blood cells transfusion. Two patient died 30 and 40 days after treatment with hypoplastic bone marrow complicated with septicemia and in the post autologous bone marrow transplantation period, respectively. Four pts with previous bone marrow transplantation, required filgrastim, transfusions and 2/3 had febrile neutropenia. Our experience with 90Y-ibritumomab in relapsed and refractory folicullar lymphoma shows 53% CR. Even heavily treated pts, that had previous bone marrow transplantation were able to receive the radioimmunoconjugate, although they required extra support. Our experience favours the use of 90Y-Ibritumomab tiuxetan in relapsed and refractory lymphomas even if they had received previous autologous bone marrow transplantation.
Efficacy and Safety of Rituximab+CHOP in IR and Maintenance Therapy with Rituximab vs Observation in De Novo Aggressive Non-Hodgkin Lymphoma.
Authors: Tripp Francisco , Báez Enrique , Ávila Elsa , Baltazar Severiano , Garcés Oscar , Reyes Gustavo , Batista Benjamín , Zaragoza Alva , Vela-Ojeda Jorge , Villela Luis
Published: October 2019

Introduction: Patients with aggressive NHL may respond to treatment, but 4-year DFS is < 40%. Rituximab has shown activity as monotherapy in patients even in refractory or relapsing post-MOT patients. Besides, preliminary results from maintenance therapy studies are promising.

Objective: To determine if maintenance with Rituximab in first complete remission increases disease-free survival and overall survival compared to observation.

Methods: A clinical trial with post-remission randomization was carried out, including patients with Aggressive Non-Hodgkin Lymphoma, stages IIb- III- IV, with ECOG 0–2, and ≥15 years old. All patients received the Rituximab + CHOP/CHOP-Like regimen during induction to remission (IR), and those with Complete Remission (CR) were randomized to receive rituximab-based maintenance therapy biannually (2 x 375 mg/sc) vs observation.

Results: 217 patients have been enrolled to IR, age 52±14 years old, 114 (52.5%) masculine, 174 of them (80.2%) presented remission; 149 CR, 25 PR. Two (0.9%) patients showed stable disease, 25 (11.5%) developed disease progression, and 16 (7.4%) died. Out of the 174 patients with remission, 96 patients have received maintenance therapy for over 6 months, 45 in the rituximab group, and 51 in the observation group, with a median follow-up of 14.4 months (pt 25– 75, 7.74 – 21.88 months). To date only 1 patient per group had a progress and none has died. During IR -considering the maximum toxicity developed per patient -, acute toxicity was remarkable: fever I, II in 13%, III–IV 1%, and chills I, II 8.2%, III 1%. And not acute: anemia I,II 54%, III 1.9, IV 1.4%, neutropenia I,II 46.1%, III 18.4, IV 2.8% and infection I,II 16.4%, III 1.0%, and IV 1.4%.

Discussion: Overall response to Rituximab + CHOP regimen is very good (80.2%), and to date 96 patients included in maintenance therapy with a median follow-up of 14.4 months, the difference between the groups is not evident yet; therefore, surveillance should be continued.
Detection and Follow Up of BCR/ABL Translocation by Cromogen In Situ Hibridization with ABL Centromeric and Telomeric (T/C) Probe Pair. Molecular, Accurate and Inexpensive Assay.
Authors: Barragan Carlos Sergio , Hansson Silvia B. , Cicchini Silvina A. , Tamashiro Monica , Dinardo Pablo G.
Published: October 2019

Objective: To Identify and to standardize a reliable t(9,22) BCR/ABL Molecular diagnosis method for developing countries.

Patients, Materials and method: Nineteen patients: Fourteen newly diagnosed Chronic Myeloid Leukemia confirmed by PCR and Cytogenetic study, one Acute Myeloid Leukemia (AML), one Acute Lymphoblastic leukemia (ALL) Phi positive, two myeloproliferatives Phi negative and a Chronic Lymphocyte Leukemia were studied. Mononuclear cell was obtained by Histopaque separation cell medium (Sigma Aldrich) from Bone Marrow and Blood samples. A translocation probe pair ABL (Chromosome 9 band 9q34) T/C digoxigenin and biotin labeled (Zymed - Invitrogen) were used for hybridization both cellular; blood and bone marrow smear. Immunoassay detection system was applied to localize BCR/ABL under bright field microscope by counting between 200 and 500 cells. We performed fifty assays with these samples.

Result: No differences between cell, blood or bone marrow smear were found. We improved the method fixing the samples with acetone. The assay was performed in fresh and frozen samples after six months without relevant differences. We were able to identify translocation, deletion and trisomy. The assay is semi quantitative and we can see a diminished number of BCR/ABL after five months with Tyrosin Kinase inhibitor, Imatinib (Glivec, Novartis) in six patients. (Table 1 and graphic 1). We detected a BCR/ABL LLA Phi positive and AML. All the cases were tested with RT-PCR, and five of them with QRT- PCR. We established an accuracy cutoff higher than 5 to 8% of positive cells for accepting BCR/ABL presence in the sample.

Conclusion: This low cost method is useful, accurate, easy and fast to diagnose and follow up CML patients treated with Imatinib. Molecular diagnosis is hardly perform in developing countries or in far away towns or cities; training personal with this method in laboratories with basic equipment would solve this critical problem for many people throughout the world.
FLT3 Is Up-Regulated in Blast Crisis of Chronic Myeloid Leukemia and Combination of Small Interference RNA of FLT3 and Gleevec (STI571) Synergistically Induces the Apoptosis in K562 Cells.
Authors: Lee Young Y. , Ahn Kwang-Sung , Yoon Sung-Soo , Choi Jung H. , Park Byoung B. , Kim Byoung K.
Published: October 2019

To identify a gene signature for prognostic markers at transition from chronic phase to blast crisis of chronic myeloid leukemia (CML), we have applied Affymetrix Genechips of 22,000 transcripts to analyze total RNA of CML cells from 12 patients with chronic phase and 12 patients with blast crisis. Data analysis using GeneSpring 6.0 generated a list of 143 differentially expressed genes. A total of 89 genes were up-regulated and 54 genes were down-regulated in blast crisis of CML, and vice versa in chronic phase of CML. Array data for 32 genes was validated using quantitative realtime PCR analysis. The expression levels of HSA6591, FLT3, NTE5, RSG1, LAF4, CPA3, ATF, FCGR3A, MYD88, IFIT1, TP73L, DTNA, MDA, and IL18R1 showed statistically significant difference (p < 0.05) between chronic phase and blast crisis. Since CML cells of blast crisis were generally unresponsive to STI571, we further analyzed roles of FLT3 which is known to be a poor prognositic marker in acute myeloid leukemia. For this experiment, K562 cells (CML blast cells) were transfected with small hairpin RNAs (shRNAs), also referred to as small interfering RNAs, to target human FLT3, resulting in the significant inhibition of FLT3 expression at mRNA and protein levels. MTT assay demonstrated that FLT3 knockdown K562 cells by shRNAs were more sensitive to STI571 compared to wild type of K562, although there was no difference at high concentration of STI571 (320 nM) between FLT3 knockdown K562 cells and wild type of K562 cells. The higher expression levels of apoptosis related genes (PARP, caspase-3, Bax) were observed in FLT3 knockdown K562 cells compared to wild type of K562 cells. Thus, RNA interference-directed targeting of FLT3 might be a novel treatment modality in STI571 refractory CML patients.
Allogeneic Hematopoietic Stem Cell Transplantation for Patients with Chronic Myeloid Leukemia in Second Chronic Phase Attained by Imatinib after Onset of Blast Crisis.
Authors: Wang Ying , Wu De Pei , Sun Aing-Ning , Jin Zheng-ming , Miao Miao
Published: October 2019

The prognosis for patients with chronic myeloid leukemia (CML) in blast crisis (BC) remains dismal even with the availability of the BCR-ABL tyrosine kinase inhibitor imatinib, since it only offers short-term benefit in most cases. Allogeneic hematopoietic stem cell transplantation (HSCT) seems to be a viable option for BC-CML patients who attained remission. We treated 10 patients (9 males, 1 female) with ablative allogeneic HSCT, who achieved second chronic phase (CP) by the use of imatinib after onset of BC between October 2003 and August 2006. Median patient age was 32 years (range, 17–46). Imatinib was given orally at daily doses ranging from 600 to 800mg according to patients tolerance for at least 2 months (range, 2–5) prior to HSCT Among them, 4 patients received HSCT from human leukocyte antigen mismatched haplo-identical family donors, the others underwent a transplant from HLA matched related (n=5) or unrelated (n=1) donors. At the time of transplantation, 5 patients were in complete hematologic response with 3 patients achieved a cytogenetic response, 5 patients were in partial hematologic response. After a median follow-up of 26 months (range, 10–44), 6 (60%) out of the 10 patients were alive with mean Karnofsky score reaching 80. Among them, 5 patients achieved a molecular remission. 1 patient died in relapse 4 months after transplantation, the others died of severe acute graft-versus-host disease and associated infections. No unusual organ toxicities and engraftment difficulties were observed. Extensive chronic GVHD developed in 3 of 6 patients who could be evaluated. Patients transplanted with haplo-identical donors had a high treatment-related modality, 3 out of 4 patients died. These results suggest that allogeneic HSCT may represent a feasible treatment for patients with CML in second CP attained by imatinib after onset of BC especially when a suitable donor is available.
PHA-680626 Exhibits Anti-Proliferative and Pro-Apoptotic Activity on Imatinib-Resistant Chronic Myeloid Leukemia Cell Lines and Primary CD34+ Cells by Inhibition of Both Bcr-Abl Tyrosine Kinase and Aurora Kinases.
Authors: Gontarewicz Artur , Balabanov Stefan , Keller Gunhild , Panse Jens , Bernhardt Mirja , Bokemeyer Carsten , Fiedler Walter , Moll Jurgen , Brummendorf Tim
Published: October 2019

Emergence of resistance to Imatinib complicates the treatment of chronic myeloid leukemia (CML). Second-generation Bcr-Abl inhibitors are capable to overcome resistance mediated by most mutations except T315I. As this mutation is causative for ∼20% of clinically observed resistances, the need for novel treatment strategies becomes obvious. Here, we report on a novel kinase inhibitor PHA-680626 exhibiting strong inhibitory activity on both Bcr-Abl and Aurora kinases. Significant anti-proliferative and pro-apoptotic effects were observed in human BCR-ABL positive cell lines and murine BaF3 cells ectopically expressing wt BCR-ABL or the Imatinib resistant BCR-ABL mutants M351T, E255K and T315I. Treatment with PHA-680626 decreased phosphorylation of CrkL and histone H3. As CrkL represents a typical down-stream target of Bcr-Abl while histone H3 phosphorylation is an indicator for Aurora kinase B activity, these findings indicate that effects of PHA-680626 are mediated via inhibition of both pathways. Moreover, high antiproliferative activity of PHA-680626 was observed in primary CD34+ cells derived from CML patients at diagnosis or in blast crisis as well as from an individual harbouring the T315I mutation. Thus, both Bcr-Abl and Aurora kinase inhibition contribute to the efficacy of PHA-680626 against Imatinib-resistant BCR-ABL positive leukemias, particularly those harbouring the T315I mutation.
RARS-T without Thrombocytosis?.
Authors: Ravoet Christophe , Azerad Marie-Agnès , Delrieux Vanessa , Meuleman Nathalie , Bron Dominique
Published: October 2019

Background: Recently, a provisional entity called “refractory anemia with ringed sideroblasts with thrombocytosis” (RARS-T) has been characterized in the MDS/MPD overlap category of the WHO classification. Diagnostic criteria include RARS features in association with sustained platelet counts > 600 (500) x 109/l and exclusion of cytogenetic abnormalities such as 5q−, t(3,3) and inv(3). Reticulin fibrosis in the bone marrow has been significantly associated with RARS-T. Splenomegaly also seems more frequent than in other RARS subtypes. Among these patients, approximately two thirds present with a JAK2 V617F mutation. On the contrary, JAK2 V617F mutation appears extremely rare in patients with typical RARS (Szpurka H. Blood 2006, Cabello AI. Leuk Research 2005, Malcovati L. ASH 2006, Remacha AF. Haematologica 2006).

Case report: In 2001, we observed a 67 years old patient with RARS (22 to 45% ringed sideroblasts). Conventional cytogenetics revealed a trisomy 8 and deletion of chromosome Y. A mild to moderate fibrosis was observed in the bone marrow. At diagnosis, the patient was anaemic but had normal platelet counts which remained strictly normal during the next 6 years. After failure of various treatments, the transfusion requirements increased due to progressive alloimmunization and probably a moderate splenomegaly which had appeared 3 years after the diagnosis (splenic volume evaluated by ultrasonography was completely normal until 2004). In July 2006, the haemoglobin level was 6.1 g/dl despite the transfusion of 11 RBC units in the previous month. In the aim to overcome the suspected hypersplenism, a splenectomy was performed. In the subsequent days following splenectomy, we observed a rapid and persistent increase in platelet count with a peak of 2550 x 109/l. A JAK2 V617F mutation was demonstrated. The normalization and maintenance of platelets was obtained with small doses of anagrelide (0.5mg four doses/week) with RBC transfusion requirements decreasing by 50%.

Conclusion: This patient presented most of the main characteristics of RARS-T except thrombocytosis (note that trisomy 8 has been described in association with RARS-T). Our observation suggests that thrombocytosis could be absent in some patients presenting with a very similar pattern of features as described in RARS-T. We postulate that hypersplenism (and possibly other particular biological characteristics) could, in some cases, mask the complete picture of RARS-T.
ETV6: Its Role in Mouse Development and Leukaemogenesis Using Knock-Out and Knock-In Models.
Authors: Hart Stephen , Duke Veronique , Pannell Richard , Rabbitts Terence , Naresh Kikkeri , Foroni Letizia
Published: October 2019

Disruption of the transcription factor ETV6 (TEL) is believed to play a pivotal role in the development of many types of leukaemia. As well as there being more than 15 ETV6 fusion gene partners described to date, there are other genetic alterations of ETV6 including deletions, point mutations and disruption in the ETV6 promoter region that may contribute to the malignant phenotype. We generated two Etv6 knock-out mouse models (Met1 and Met43) to investigate the role of Etv6 in mouse embryonic development and two knock-in models carrying the t(12;21) and t(9;21) fusion genes to investigate the role of the Etv6-RUNX1 and Etv6-ABL fusion oncoproteins in leukaemogenesis. Etv6-Met1−/− was compatible with full embryonic development. However, from a total of 382 animals generated from the Etv6-Met1+/− cross only 39 were identified as homozygous suggesting some embryonic lethality. A second knock-out construct (Etv6-Met43) was generated in which the second transcription start site at AA position 43 was removed. Of the 235 offspring genotyped from the Etv6-Met43+/− cross, no homozygous animals were identified. Further investigation revealed that Etv6-Met43−/− foetuses lacked definitive haematopoiesis and died between dE10.5–11.5. These data indicate that an Etv6 transcript generated from the methionine at AA position 43 can partially rescue the Etv6-Met1−/− phenotype. No mice homozygous for the Etv6-RUNX1 were identified from the 628 offspring genotyped from the Etv6-RUNX1+/− cross. Further investigation revealed that Etv6-RUNX1−/− foetuses displayed a similar phenotype to the Etv6-Met43−/− animals and died in-utero at a similar time. Etv6-RUNX1+/− animals were fertile and remained well for the first year of life. However, after this time in a group of 91 animals it was noticed that among 41 Etv6-RUNX1+/− animals, the majority were dying from a blastic haematological tumour formation best identified in spleens, at a higher incidence than in the 51 wild-type littermates. Chimeric mice targeted with the Etv6-ABL construct remained well for a short period after birth but all subsequently sickened and died within the first year of life. Upon examination peripheral blood as well as post-mortem organs showed a homogeneous infiltration of granulocytic cells resembling a chronic myeloid proliferation. RT-PCR on RNA isolated from the peripheral blood, bone marrow and spleen of three chimeric animals showed the presence of the fusion gene. Our study demonstrates that Etv6 is essential for mouse embryonic development. The role of Etv6 in malignant transformation is complex and would appear to be dependent on the function of the partner fusion gene rather than on the common Etv6 part of the protein.
Prediction of Survival in Patients with Myelodysplastic Syndrome According to WHO Classification-Based Prognostic Scoring System (WPSS).
Authors: Jang Jun Ho , Kim Kihyun , Jung Chul W. , Park Keon Woo
Published: October 2019

Background Based upon the classification of FAB criteria, International Prognostic Scoring System(IPSS) has been a standard prognostic model to predict survival and progression in MDS. In 2000, the WHO has formulated a new classification of myelodysplastic syndrome(MDS). The aim of this study was to evaluate the prognostic value of WHO classification-based prognostic scoring system(WPSS) in MDS.

Patients and methods One hundred forty-nine patients who were diagnosed as having de novo MDS at the Division of Hematology-Oncology, Samsung medical center, Seoul, Korea, between Dec. 1994 and Feb. 2007, were evaluated retrospectively for clinical and hamatologic features at diagnosis, transfusion dependence, overall survival(OS), and progression to leukemia(LFS). Risk group stratifications in MDS patients were done according to IPSS and WPSS.

Results 18 patients(12.1%), 93 patients (62.4%), 29 patients(29%) and 9 patients(6%) had IPSS risk scores of low, intermediate-1(Int-1), intermediate-2(Int-2) and high, respectively. According to WPSS risk scores, 8 patients(5.4%), 30 patients(20.1%), 41 patients(27.5%), 57 patients(38.3%) and 13 patients(8.7%) were classified to very low, low, intermediate, high and very high risk group, respectively. In IPSS, median OSs of low, Int-1, Int-2 and high subgroup were 65.2, 32.9, 14.3 and 9.1 months respectively (p<0.001). According to WPSS, median OSs of very low, low, intermediate, high and very high risk subgroup were not reached, 55.4, 27.4, 19.0 and 6.2 months respectively (p<0.001). Between subgroups classified according to WPSS, significant differences in OS were noted in low vs. intermediate risk group (p=0.047), in intermediate vs. high risk group (p=0.046) and in high vs. very high risk group(p=0.003) but statistically not significant difference in OS was observed between very low and low risk group (p=0.08). The mean and median OS of the lowest risk group(low risk) in IPSS are 65.33 and 55.43 months, respectively. The mean and median OS of the lowest risk group(very low risk) in WPSS are 102.8 months and not reached, respectively.

Conclusion These data show that WPSS with five risk groups might provide more refined prognostic stratifications of MDS than IPSS with four risk groups. Especially, new prognostic system appears to discriminate a subset of patients with very low risk, who could have long term survival.
Allelic Expression Imbalance of JAK2 V617F Mutant Contributes to Phenotypic Variation in BCR-ABL Negative Chronic Myeloproliferative Disorders.
Authors: Shin Myung-Geun , Kim Hye-Ran , Kim Hyeoung-Joon , Kim Mi-Ji , Kim Hee-Nam , Kim Yeo-Kyeoung , Kim Soo-Hyun , Shin Jong-Hee , Suh Soon-Pal , Ryang Dong-Wook
Published: October 2019

Background: An acquired somatic mutation in the JAK2 gene (V617F) could present the primary causative lesion in BCR-ABL-negative chronic myeloproliferative disorders (CMPD). However, some of the clinical and genetic data implied that the pathophysiological role of JAK2 V617F mutation in the CMPD would be more complex. Quantitative assessment of JAK2 V617F mutation has shown substantial heterogeneity in the genomic DNA. Recently, it has been reported that allelic variation in gene expression is common in the human genome. To test the hypothesis that JAK2 V617F mutant allele could be increased in cDNA, we examined JAK2 V617F mutation status in the genomic DNA and cDNA from a total of 78 patients with BCR-ABL-negative CMPD.

Patients and Methods: Enrolled patients with BCR-ABL-negative CMPD in this study comprised 42 cases of essential thrombocythemia (ET), 26 polycythemia vera (PV), 7 idiopathic myelofibrosis (MF) and 3 unclassifiable (UC) CMPD. A 364-bp PCR product containing JAK2 V617F mutation was bidirectionally sequenced from total BM cells. A quantitative real time PCR-based allelic discrimination assay and pyrosequencing (Pyrosequencer PSQ96) were developed for quantitative analysis of JAK2 V617F mutation status. Homozygous JAK2 V617F mutation was defined if the mutant peak was more than 50% of total peak area.

Results: The proportion of mutant alleles ranged from 36% to 100% in real-time PCR and pyrosequencing analysis. Patients with MF had higher percentages of JAK2 mutant alleles than patients with ET (MF > PV > ET). The prevalence of homozygous V617F mutations was significantly higher in PV patients (73%) than in patients with ET (17%). Allelic expression imbalance of heterozygous JAK2 mutation was common in patients with PV and ET. Interestingly, allelic expression imbalance in patients with MF was not remarkably impaired, but preferential expression of JAK2 mutant allele showed threefold increase from the cDNA compared with the genomic DNA from patients with PV and ET.

Conclusion: Allelic imbalance in the gene expression of JAK2 V617F mutant could provide the underlying mechanisms to elucidate phenotypic variation in BCR-ABL negative CMPD.
Familial Essential Thrombocythemia Showing Spontaneous Megakaryocyte Colony Formation and Acquired JAK2 Mutations without a Germline Mutation in C-MPL or TPO.
Authors: Higgs Jillian R. , Sadek Irene , Neuman Paul E. , Ing Vincent , Berman Jason N. , Greer Wenda L.
Published: October 2019

Essential thrombocythemia (ET) is a chronic myeloproliferative disorder, characterized by increased proliferation of megakaryocytes and elevated platelet counts that usually occurs sporadically. We report a family with 7 affected individuals in three generations, including one individual with a phenotype resembling polycythemia vera, a related disorder. Megakaryocyte (CFU-MK) colony formation occurred in the absence of added cytokines in cultures of peripheral blood from affected family members. Some reports of familial ET have identified mutations in the genes coding for thrombopoietin (TPO), the cytokine that primarily regulates platelet production and its receptor, c-MPL. In this family, the MPL gene was excluded by linkage analysis. Although TPO levels were elevated in most affected family members and evidence for linkage was found between the disease and TPO (𝛉 = 0.0, Zmax = 3.0), a TPO mutation was not identified by DNA sequencing. The JAK2 V617F mutation that has been associated with 50% of sporadic cases of ET was identified as a somatic mutation in the peripheral blood of the two most clinically affected family members. These patients also had the most elevated TPO levels, raising the possibility of an association between JAK2 and TPO production. Further study of familial myeloproliferative diseases will help elucidate the initiating genetic events underlying ET.