bluuebunny/crossref_metadata_2025 huggingface dataset:
Profound Hyperbilirubinemia during Induction of Adult Acute Lymphoblastic Leukemia: Capping L-Asparaginase Dose to 10,000 Units Attenuates It without Compromising Remission Rate.
Authors: Vora Amish D. , Pai Vasnat R. , Kumar Prabhash , Banavali Shripad , Nair Reena , Kurkure Purna , Parikh Purvish M.
Published: October 2019

Introduction: Hyperbilirubinemia is a well known side-effect of L-asparaginase. Grade 4 hyperbilirubinemia (bilirubin > 3) results in delayed chemotherapy; compromising dose intensity. Hence a strategy to prevent this is important.

Materials & Methods: At our center, we see around 125 cases of newly diagnosed adult acute lymphoblastic leukemia (age>24 years) every year. Since 2005, we have started treating these adult patients on modified BFM 90 protocol. This protocol includes 8 doses of L-asparaginase at 10,000 units/m2 during induction. Hyperbilirubinemia of grade 4 (median bilirubin upto 20) was encountered in the first few patients. To avoid delay in chemotherapy, we modified the protocol to cap L-asparaginase dose at 10,000 u for all patients.

Results: A total of 64 patients with ALL was treated on this modified protocol in 18 months. We compared the degree of hyperbilirubinemia in this group of patients with the first 20 patients who were treated with the standard dose of L-asparaginase. Median age of the full cohort (All 84 patients) was 34 years (24–60). Fifty four patients had precursor B-cell ALL while 30 patients had T-cell ALL. Male: Female ratio was 4.1:1. All the analysed patients have completed induction. In first group of 20 patients who had received standard dose of L-asparaginase, 6 patients developed grade 4 hyperbilirubinemia (median bilirubin 23). SGPT, SGOT and SAP were either normal or borderline elevated. Viral markers at diagnosis and at the onset of hyperbilirubinemia were negative. No significant hepatic abnormality was seen on USG abdomen. In four out of 6 patients’ bilirubin returned to normal after conservative management, over a period of 3 weeks. The two remaining patients developed sepsis, had deranged coagulation parameters and succumbed to sepsis. Of the second group of 64 patients ( L-asparaginase at 10,000 fixed dose), 5 patients developed hyperbilirubinemia (highest value of bilirubin 4). All other investigations (listed above) were normal.All patients’ bilirubin came back to normal within 1 week of interruption of chemotherapy. Complete remission rates were 90% in the first group of 20 patients and 92% in the second group of 64 patients.

Conclusion: In the cohort of 64 patients (second group), capping of L-asparaginase resulted in significant reduction in intensity, incidence and duration of hyperbilirubinemia. This modification did not affect CR rate, though we are currently evaluating long term survival to confirm sustained efficacy.
Remission Induction with Cladribine, Cytarabine and Idarubicin (CAI) in Elderly Patients with Relapsed AML - A Pilot Study.
Authors: von Lilienfeld-Toal Marie , Hahn-Ast Corinna , Orlopp Katjana , Ingo Schmidt-Wolf G.H. , Axel Glasmacher
Published: October 2019

Elderly (>55 years) patients with relapsed acute myeloid leukemia (AML) generally have a poor prognosis mainly due to a low rate of 2nd complete remission (CR). Commonly, salvage therapy consists of regimens including fludarabine. However, results have been disappointing and the efficacy of fludarabine in AML is questionable. In contrast, there is evidence that 2CdA, another purine analogue, has single drug activity in AML as well as enhancing the effect of other cytostatic drugs such as ARA-C. Here we present data from a pilot study evaluating CAI: Cladribine (2CdA) in addition to cytarabine (ARA-C) with idarubicin (Ida) as a salvage therapy in relapsed AML.

Patients and Methods: Two treatment protocols were tested from May 2005 to May 2006 at our institution:2CdA 5 mg/m2/12 h, d 1–5, ARA-C 1000 mg/m2/12 h, d1-5 and Ida 12 mg/m2/d, d1-32CdA 5 mg/m2/12 h, d 1–3, ARA-C 1000 mg/m2/12 h, d1–3 and Ida 8 mg/m2/d, d1–3.Protocol A was designed for patients < 60 years and considered fit for intensive therapy.

Results: Nine patients were included in the study after written informed consent. Eight of them are evaluable to date. Three patients received protocol A (median age 57 years), whereas 5 received protocol B (median age 65 years). Although one patient died during neutropenia and one patient had refractory disease, response after one cycle of CAI was favourable in 6 patients: 4 achieved CR and 2 CR with incomplete regeneration (response rate 75%). Of those, 3 patients went on to receive further consolidation therapy with a second cycle of CAI, one with ARA-C/Ida and one with 2CdA/ARA-C. Toxicity was predominantly neutropenia with subsequent infectious complications: median duration of neutropenia was 28 days (IQR 22–34) with all patients experiencing febrile neutropenia (median duration 8 days IQR 3–9) and 4 episodes of septicemia. The second course of CAI resulted in an even longer duration of neutropenia: 37 days (IQR 16–50) with all patients having septicaemia. Of the 6 responding patients 2 died subsequently, one of a second relapse and the other of neutropenic sepsis after the 2nd course of CAI. The other 4 patients are still alive (follow-up 4 months to 1 year).

Next steps: Because of these promising results we decided to pursue the study but change the protocol as follows:

Patients will only be treated according to protocol B, application of growth factors from day 15 onwards will be mandatory, antibiotic prophylaxis with a chinolone and twice weekly Cotrimoxazole will be mandatory and a second cycle will omit idarubicin.

Conclusion: Combination therapy with cladribine, cytarabine and idarubicin appears to be feasible and successful in elderly patients with relapsed AML. However, infections are a serious complication warranting intensive supportive care.
Abnormal Expression of TRAF Adapter Proteins in Waldenstrom’s Macroglobulinemia.
Authors: Leleu Xavier , Xu Lian , Hunter Zachary R. , Moreau Anne-Sophie , Jia Xiaoying , O’Sullivan Garrett , Bakthavachalam Vinod , Ho Allen W. , Hatjiharissi Evdoxia , N’Go Hai T. , Santos Daniel D. , O’Connor Kelly , Manning Robert , Soumerai Jacob , Patterson Christopher , Ghobrial Irene M. , Treon Steven P.
Published: October 2019

Background: Waldenström’s Macroglobulinemia (WM) is an incurable low-grade lymphoplasmacytic lymphoma with as yet unknown genetic basis for its pathogenesis. Several TNF family members (CD40L, APRIL and BAFF/BLYS) are known to regulate WM growth and survival. TRAFs are a novel family of adapter proteins that facilitate pro-apoptotic (TACI) or pro-survival/differentiation (CD40, BAFFR, BCMA) receptor signaling mediated by TNF family ligands. Therefore, understanding the TRAF system in WM may yield important clues about WM growth and survival.

Methods: WM cell lines (BCWM.1 and WSU-WM), IgM secreting low-grade lymphoma cell lines (MEK1, RL, Namalwa), and primary bone marrow CD19+ selected lymphoplasmacytic cells (LPC) from 20 WM patients and 6 healthy donors were evaluated for TRAF (TRAF 2, 3, 5, 6) expression using semi quantitative RT-PCR and/or western blot analysis.

Results: The TNF familiy receptors CD40, BAFFR, BCMA, and TACI were expressed in all cell lines tested as well as in CD19+ selected LPC from WM patients and healthy donors. Moreover, TRAF 2, 3, 5, 6 were expressed in all cell lines by both RT-PCR and western blot analysis. In contrast, we observed loss or abnormally low expression of both TRAF 2 and 5 in 6/20 (30%) patients, whilst TRAF 3 was absent or abnormally low in 3/30 (15%) patients. TRAF 6 was expressed in all patients. Among healthy donors, we observed expression of all TRAF adapter proteins.

Conclusion: Up to one third of WM patients demonstrate loss of TRAF 2 and 5 adapter proteins which facilitate signaling through the pro-apoptotic receptor TACI. Ongoing studies including gene sequencing and siRNA knockdown models are delineating a role for TRAF loss in the pathogenesis of WM.
Immunohistochemical Expression Patterns of Germinal Center and Activation B-Cell Markers in Diffuse Large B-Cell Lymphoma. Clinicopathologic Study of 62 Cases from Peru.
Authors: Quintana Shirley , Barrionuevo Carlos , Gomez Henry , Neciosup Silvia , Grados Oswaldo , Casavilca Sandro , Flores Claudio , Casanova Luis
Published: October 2019

Background: Recent studies have shown that diffuse large B-cell lymphoma (DLBCL) cases with gene expression profiles similar to germinal center (GC) B cells had a much better prognosis than DLBCL cases with gene expression profiles resembling activated B cells. DLBCL can be classified into three immunohistochemical expression patterns: GC B-cell pattern (pattern A) expressing CD10 and/or Bcl-6 but not MUM1; activated GC B-cell pattern (pattern B) expressing at least one of GC B-cell markers and MUM1; and activated non-GC B-cell pattern (pattern C) expressing MUM1 but not GC B-cell markers.

Purpose: To determine the frequency of these patterns, the immunohistochemical expression intensity of MUM1, BCL6 and BCL2, as well as International Prognostic Index (IPI) and therapeutical response.

Methods: Immunohistochemical analysis on paraffin-embedded tissues was performed using the streptavidin-avidin-biotin method in 62 cases of DLBCL. The intensity of immunohistochemical expression was performed as follows: positive (more than 50% expression), positive/negative (expression 20 to 50%), and negative (less than 20%expression).

Results: The average age was 60 years. Females were slightly more frequent than males. 84% cases were primary nodal. According to IPI, 60% of cases were low (L) or intermediate-low (IL) risk, and 40% were intermediate-high (IH) or high risk (H). The corresponding A, B and C patterns, by immunohistochemistry, were 10%, 19% and 65% respectively. The majority of patients were treated with CHOP. Complete response (CR) was achieved in 54%, partial response (PR) in 11%, and no response (NR) in 23%. 9 % were not assessed. 4/6, 8/10, 17/36 of the cases had CR in the patterns A, B, C respectively. 5/6, 8/12, 22/40 of the cases were L-IL in the same patterns, without significant difference between these groups. The immunohistochemical expression intensity for either marker was not statistically significant.

Conclusions: The most frecuent pattern was C. The activated patterns were most common. The correlation between different pattern expression, IPI and therapeutical response didn’t show statistical differences, probably due to the small size of the sample.
Impact of STI571 (Imatinib) on the Pharmacokinetics and In Vivo Efficacy of Etoposide and/or Ifosfamide.
Authors: Rezai Keyvan , Lokiec François , Grandjean Isabelle , Weill Sophie , de Cremoux Patricia , Arnaud Philippe , Bordier Vincent , Ekue Richard , Garcia Mickael , Lidereau Rosette , Poupon Marie-France , Decaudin Didier
Published: October 2019

Using a human small cell lung cancer (SCLC) xenografted in nude mice, we previously reported an increase of tumor growth inhibition following chemotherapy in combination with STI571 (imatinib)(Int J Cancer.2005;113:849–856). This led us to study the in vivo impact of STI571 on the pharmacokinetics of chemotherapy. Plasma, urine, and fecal concentrations of etoposide were determined by a validated high performance liquid chromatography method, and plasma concentrations of ifosfamide were determined by a validated GAS chromatography assay with nitrogen-phosphorus detection. C-kit and MDR1/ABCB1 transcripts were quantified using real-time quantitative reverse transcription-PCR assays. We first observed an increase of the Cmax of both etoposide and ifosfamide on days 1 and 3 when mice were treated by chemotherapy combined with STI571 compared to chemotherapy alone. We then observed a high significant increase of the AUC 0–3h (Area Under the concentration-time Curve) of etoposide when mice were treated with etoposide + STI571, due to a decrease of its fecal excretion, without impact of concomitant administration of the CYT3A4 inhibitor fluconazole. As previously shown, this effect was observed despite the absence of c-kit receptor mRNA expression. Finally, we showed that STI571 enhanced the etoposide-induced tumor growth inhibition of a human xenografted lymphoma, but not that of an in vivo human SCLC tumor, and that this antitumor effect was correlated with the mRNA level of the MDR1/ABCB1 gene. This last observation suggests that imatinib-induced pharmacokinetic changes could be due to inhibition mediated via the MDR modulator of the P-glycoprotein transporter resulting in decreased elimination of etoposide. In conclusion, STI571 can reverse MDR1 drug resistance and potentiate the effects of etoposide in MDR1-expressing cancer cells such as lymphoma cell lines. These results show that further prospective phase I and II clinical trials with combinations of etoposide-based chemotherapy and imatinib are therefore warranted in some cancer patients, such as malignant lymphoma patients, with careful toxicologic monitoring.
Hypophosphatemia Observed in Chronic Myeloid Leukemia Patients Treated with Imatinib Mesylate (Gleevec®) Is Related to Digestive Side-Effects.
Authors: Legros Laurence , Breuil Veronique , Ferrari Patricia , Testa Jean , Cassuto Jill-Patrice
Published: October 2019

The therapeutic strategy in chronic myeloid leukemia (CML) has been totally modified with the development of Imatinib Mesylate (Gleevec®), a specific inhibitor of Bcr/Abl tyrosine kinase activity. However, many side-effects are reported with Gleevec, generally low grade. We report a new side-effect in patients treated with Glevec® for CML: hypophosphatemia. Between September 2001 and February 2005, 38 patients with CML were treated with Gleevec®. Thirty-four patients (18 males, 16 females) had a complete clinical and biological follow-up including into bone metabolism. Fifteen patients presenting a low level of serum phosphorus. Hyphosphatemia appeared within a median of 15 months (range: 1–47). Comparison of normal and hypophosphatemic patients did not show any statistically significant difference regarding age, treatment duration and dose of Gleevec®. Moreover, in 17 patients, we investigated the plasma level of Gleevec® as described by Mahon FX et coll. And did not find a correlation between the phosphorus level and concentration of Gleevec® (1). We next investigated phosphorus metabolism starting with digestive excretion because Gleevec® is known to induce gastro-intestinal side-effects. A multivariate analysis showed a correlation between hypophosphatemia and diarrhea (p<0.05). It suggests that the hypophosphatemia is related to digestive dysfunction. Moreover, we gave oral phosphorus supplementation for 3 months to 3 patients and failed to rescue normal phosphorus levels but exacerbated in all patients the digestive side-effects. This finding does not support interest in phosphorus supplementation.

Interestingly, we observed a low cross-laps serum level in all patients, independently of their phosphatemia status. A similar observation was recently reported by Berman E and coll in 16 patients treated for gastrointestinal stromal tumors and CML (2). They suggested inhibition of the platelet-derived growth factor (PDGF) receptor b. Parathyroid hormone levels were similar in both groups and the low levels in the phosphorus group tended to correlate with an increased level of serum 1,25-dihydroxyvitamin D, but did not reach significance. This is could be in favour of an inhibition of bone remodelling by Gleevec® independently of hypophosphatemia. This suggests a possible implication of FGF23, a major regulator of phosphorus metabolism, that need to be further investigated.
Dlk1 Up Regulated Might Be an Important Event in Human MDS.
Authors: Xiao Q.F. , Chen Zi X. , Liu Dan D. , Cen Jian N. , He Jun , Gu Ming
Published: October 2019

The diagnosis of myelodysplastic syndrome (MDS) is made largely on the dysplastic morphology of BM cells from aspiration or biopsies. Prognosis scored by IPSS is depending on the percentage of marrow myeloblasts and the clonal cytogenetic abnormalities. To expand the understanding of genetic defects in hematopoietic cells of MDS in hope of finding novel genes correlated to pathogenesis and provide possible diagnostic marker for MDS, we have applied microarray to analyze the clinical samples from MDS patients.

Total RNAs of CD34+ cells from 8 patients ( 2 RAEBt,2 RAEB,2 RA,1 RAS,1 CAA ) and one healthy people were extracted followed by a double in vitro transcription to circumvent the limited number of CD34+ cells. Following a modified Affymetrix target amplification protocol. Biotinylated cRNA was synthesized from 50 ng total RNA by double-round amplification and hybridized to an Human Genome U133 Plus 2.0 Array (Affymetrix). From the expression profile of 18404 different genes, we revealed that DNTT,MLL3,IL1R2,MAPK1,IGLL1 were down regulated while EGR-1, Rap1GAP or MAF were up regulated compared with normal controls. Most notably, Dlk1 was up regulated in MDS, while down regulated in AML and normal. By real-time RT-PCR we confirmed that in BMNCs the median levels of Dlk1 transcript in patients with RA and RAS were 2.55 (range, 0.00–23.7), RAEB and RAEBt were 8.24(range, 2.01–18.44), AML were 1.88 (range, 0.12–5.13), and other patients were 0.37(range, 0.00–1.79), respectively. The abundance of Dlk1 mRNA in MNCs from most MDS patients was markedly greater than that in the MNCs from others (P <0.05 ). Dlk1 expression in RAEB and RAEBt is markedly higher than AML (P <0.05 ) Forced expression of Dlk1 in transfected K562 cells resulted in faster growth than control cells, affected apoptosis induced by As2O3. and reduced the G2 arrested cells induced by TPA. By using the same experimental system we found that forced expression of Dlk1 can increase the mRNA levels of HES1 and p21WAF1 transcript variant 1. To elucidate the mechanisms we analyzed the levels of phosphorylated-p38 and p38 in Dlk1 transfected K562 cells treated with TPA. Dlk1 inhibited p38 phosphorylation while expression of p38 kept no change. These results support further investigation on the role of Dlk1 in abnormal hematopoiesis in MDSheterogeneous cell component. Diagnosis is currently depending on the dysplastic morphology of.
Erythropoetin (EPO) and Granulocyte Colony Stimulating Factor (GCS-F) Based Therapy in Patients with Low Risk MDS: A Single Centre Experience from India.
Authors: Nair Velu , Mishra Deepak K. , Sharma Ajay , Das Satya N. , Kotwal J. , Mukerjee B. , Agarwal M.B. , Mehta S.R.
Published: October 2019

MDS is a group of clonal disorders characterized by ineffective erythropoiesis and peripheral cytopenias. We report our single centre experience of treatment of low risk MDS. A total of 68 patients (M:F 49:19) were treated with either EPO or EPO with G-CSF combination and followed up for a period of 6 years. MDS diagnosis was based on the WHO criteria and patients with MDS-RAEB were excluded. The mean age was 48 years(range:24–72yrs). This treatment was offered as an upfront therapy in view of non feasibility of other modalities of treatment(e.g., non- availability of HLA matched donors, poor clinical status and co-morbid conditions). Patients were randomly divided into 2 Arms. Arm ‘A’ (n = 55) received EPO(10,000 units) once a week and G-CSF(150μg) alternate day for 12 weeks. 13 Patients in Arm ‘B’ received EPO alone in same dose for 12 weeks. If a patient did not show an erythroid response in Arm ‘B’, he / she was shifted to the combination arm. Once adequate erythroid response was achieved, patients were put on lowest effective EPO dose. Patients who interrupted their treatment in between for whatever reasons were excluded (n =10) from study.14 Patients who showed a decline in response were given an escalated dose of EPO (20,000 units per week).

The overall erythroid response rate was 58%. It was partial (100% reduction in red cell transfusion requirement) in 30% of patients and complete (maintaining hemoglobin at >11.5 G/dL without red cell transfusion) in 28%. The median response duration was 28 months (range:6–72 months). Erythroid response was better in the first Arm (65%) vis-à-vis Arm B (33%). Patients, who started therapy before they became transfusion dependent, achieved an earlier response within 8 weeks(range:4–24 weeks). The complete responders also showed a longer duration of response than the partial responders.35% of patients progressed to AML after a median duration of 28 months(range:3–60 months). Eleven patients who stopped therapy (5:AML progression, 6:unrelated causes) were excluded from the analysis. Patients with IPSS Low group showed durable response than the patients with Int-1 group. Though there was no definite survival benefit, the responders became transfusion free or less dependent on transfusion without any adverse effect on the outcome, thereby improving the quality of life.

The erythroid response in our patients was better than in most reported series in the west. However, the progression to AML was 35% in our patients during the 60 months follow-up duration, which is marginally higher than the western series and it is important to be further studied. In conclusion, patients with IPSS low / Int-1 score should be offered EPO+ G-CSF combination therapy to achieve a durable response in contrast to the patients with high risk IPSS patients, in developing countries.
Interphase FISH in Multiple Myeloma.
Authors: Haaber Jacob , Sørensen Anne G. , Nibe Anne , Nielsen Ole , Abildgaard Niels , Plesner Torben , Kerndrup Gitte B.
Published: October 2019

192 patients with multiple myeloma (MM) and benign monoclonal gammopathy of undetermined significance (MGUS, n=10) were investigated by interphase fluorescence in-situ hybridisation (iFISH) without (n= 132) and with positive plasma cell identification (PC-ID+) (n= 50).

134 were investigated at diagnosis 32 at time of progression, 7 at time of relapse and 9 were investigated with partial remission or no response. 10 of the MM cases were investigated twice. The patients were investigated with FISH probes detecting 11q23 (n=61), 13q13-14 (n=181), 14q32 ((n=121), 17p13.1 (n=181), t(4;14) (n=76) and t(11;14) (n=73). 61/132 (46%) of patients investigated without PC-ID+ showed abnormalities as opposed to 45/49 of evaluable cases (92%) with PC-ID+. The increase in abnormal cases was mainly due to the detection of more cases with loss of 13q and 17p and der(14)(q32): For patients investigated at diagnosis without and with PC-ID+, respectively: 13q-: 17% and 28%, 17p-: 3% and 15%, and 14q split signals (excluding the specific translocations): 8% and 24%. Based on the relatively small number investigated, the t(4;14) and the t(11;14) were not detected more frequently. G-band cytogenetics was carried out in 72 patients (25 without PC-ID+ and 47 with PC-ID+). 19 cases were abnormal (26%). Concordance for 1 or more aberration was found in 14 patients. t(11;14) was detected by both methods in 4 of 5 patients. Out of 7 cases with either near-tetraploidy/triploidy or hypoploidy in the G-band karyotypes, the modal number in the G-banded karyotypes could not be elucidated with certainty in 4 by iFISH with the applied probes. 7/10 patients investigated twice by iFISH showed new abnormalities on reinvestigation, 5 of these had a normal 1st analysis. 3 of 10 MGUS patients showed abnormalities.

In conclusion, PC-ID+ is important for the detection of numerical aberrations and disclosing translocations involving 14q32, as translocations involving the IgH locus are frequent occurring in 64 % (n = 32) at diagnosis. Of these the t(4;14) and the t(11;14) constituted 8% and 20%, respectively. Re-examination of patients with a normal analysis should be considered in non-responders and progressing patients. Lastly, by applying the set of probes we chose in accordance with the proposed recommendations from the European Myeloma Network FISH Workshop, Royal Marsden Hospital, London UK, March 2005, failure to accurately the exsistence of detect near-tetraploid/near-triploid-, and hypoploid clones, is not insignificant (26%) and, based on the small number of cytogenetically abnormal cases it is recommended to include extra probes to classify the patients according to modal number by iFISH.
Baicalein Combined with Dexamethasone Can Induce the Marked PPARß-Mediated Suppression of Growth and Survival in Human Myeloma Cells.
Authors: Otsuyama Ken-ichiro , Amin Jakia , Uchida Emi , Abroun Saeid , Shamsasenjan Karim , Islam Khademul , Asaoku Hideki , Kawano Michio M.
Published: October 2019

PPAR (Peroxisome proliferator-activated receptor) ß is ubiquitously expressed in all cells and considered to be involved in the lipid metabolism and regulating the inflammatory response and cell proliferation. However, it remain to be clarified the role of PPARß in human myeloma cells. In order to identify the possible ligands for PPARß, we constructed the PPARß response element (PPREß)-lusiferase reporter gene and performed the reporter assay in Hela cell. We found that adrenal cortex hormones (DHEA and DHEA-S etc), dexamethasone (Dex) and baicalein augmented the expression of reporter gene. As already reported by us, baicalein, one of the major flavonoids in Scutellaria baicalensis (Chinese Skullcap), has the suppressive effects on cell proliferation and angiogenesis. Furthermore, baicalein as well as Dex upregulated the expression of PPARß target genes such as ILK and PPARg in myeloma cell lines. Moreover the combination of baicalein with Dex showed cooperative effect on the growth suppression in primary myeloma cells as well as myeloma cell lines. The expression of NF-kB target genes such as IL-6 was also markedly suppressed with the treatment with baiacalein and Dex in U266 cells. Since U266 cells constitutively express RelA(p65), RelB and p50 in the nucleus, we examined whether ligand (baicalein or Dex)-stimulated PPAR ß interacted with RelA in the nucleus of U266 cells or not. We detected physical interaction of PPARß with RelA in the nuclear fraction of U266 cells after stimulation with baicalein or Dex, and also confirmed that baicalein and Dex suppressed the DNA binding of RelA to kB site in the p65 ELISA. Therefore, these data suggest that baicalein combined with Dex can stimulate PPARß-mediated growth suppression in myeloma cells possibly through nuclear interaction between PPARß and RelA (NF-kB) more effectively.
Quantitative Assessment of Hematopoietic Chimerism after Allogeneic Nonmyeloablative Stem Cell Transplantation.
Authors: Chen Bao-An , Xiong Hui-Xia , Ding Jia-Hua , Gao Chong , Sun Yun-Yu , Wang Jun , Cheng Jian , Zhao Gang , Chen Ning-Na , Fei Fei
Published: October 2019

Objective:

To establish a method for quantitative analysis of hematopoietic chimerism by polymerase chain reaction (PCR) based on short tandem repeat (STR) locies. To investigate the correlation between the kinetics of chimerism and hematologic engraftment, graft rejection, disease relapse and graft versus host disease (GVHD) after allogeneic nonmyeloablative peripheral blood stem cell transplantation. To guide implementation of therapy at an early stage and to improve patients life quality.

Method:

Cell dilution experiments were performed by mixing mononuclear cells (MNCs) obtained from peripheral blood samples of unrelated individuals to test the sensitivity, accuracy and linearity of the assay. Quantitative assessment of hematopoietic chimerism was performed by short tandem repeat-polymerase chain reaction(STR-PCR), polyacrylamide gels, silver staining and analyzed by Image Analysis System. 28 patients received nonmyeloablative stem cell transplantation were evaluated. The conditioning regimen included fludarabine 30mg/(m2·d)×6d, busulphan 4mg/(kg·d)×2d, CTX 600mg·d−1×2d, ±Ara-C.

Peripheral blood were collected before and after transplantation in different period and the chimerism were analysed by this method.

Results:

Sensitivity varied from 1.25% to 5% depending on the STR locies being tested. vWA and D16S539 appeared better sensitivity from 1.25% to 2.5% than other locies. The quantitative results showed a linear correlation between the percent chimerism calculated and the DC proportion mixed(R2 =0.97~0.98, P<0.001). As an average of all informative markers, concordance between the calculated and mixed DC percent showed minimal deviation from 1(R2=0.996). On day 30, one patient failed to engraft, 22 cases formed complete chimerism (CC) and 5 cases were of mixed chimerism (MC).

The time of molecular engraftment preceded the recovery time of neutropils and platelets. One patient with MC rejected grafts. The incidence of aGVHD in group CC was significantly higher than that in group MC(P<0.05). There was no significant difference in the incidence of cGVHD and disease relapse between group CC and group MC(P>0.05). One patient relapsed in CC status lacking a transitional MC interveral. Patients received an early implementation of therapy transferred from MC to CC or got a longer disease-free time.

Conclusions:

STR-PCR is a sensitive, precise and reliable method for quantitative analysis of hematopoietic chimerism. Selecting sensitive markers to analyze is an economic approach for monitoring of chimerism after transplantation. Sequential and quantitative detection of chimerism may be of great value to predict engraftment, graft rejection, disease relapse and high risk of GVHD.

Furthermore it can provide a basis for early intervention of clinical treatment posttransplantation.
Treosulfan-Based Reduced Toxicity Regimen Prior to Allogeneic Hematopoietic Cell Transplantation in Non-Malignant Disorders.
Authors: Wojnar Jerzy , Giebel Sebastian , Markiewicz Miroslaw , Krawczyk-Kulis Malgorzata , Wylezol Iwona , Stella-Holowiecka Beata , Kruzel Tomasz , Holowiecki Jerzy
Published: October 2019

Graft rejection is a major cause of failure after alloHCT in non-malignant hematopoietic disorders including severe aplastic anemia (SAA) and paroxysmal nocturnal hemaoglobinuria (PNH). For patients with high risk of this complication we introduced a novel conditioning regimens, based on treosulfan - an alkylating agent possesing both immuno- and myeloablative properties. Between 2003–2006, eleven patients (age: 23(14–35) years) with SAA (n=6) or PNH (n=5) were treated in a single institution with alloHSCT from either HLA-identical sibling (n=3) or an unrelated volunteer (n=8). For patients with SAA conditioning regimen consisted of treosulfan 10 g/m2/d on days -7, -6, cyclophosphamide 40 mg/kg/d on d. -5, -4, -3, -2, and anti-thymocyte globulin (ATG) 2 mg/kg/d on d. -3, -2, -1. PNH patients received treosulfan 14 g/m2/d on days -6, -5, -4, fludarabine 30 g/m2 on d. -6, -5, -4, -3, -2, and (ATG) 2 mg/kg/d on d. -3, -2, -1. Bone marrow was used as a source of stem cells in 5 patients and peripheral blood - in 6 cases. Graft-vs.-host disease (GVHD) prophylaxis consisted of Cyclosporin A and short-course Methotrexate.

All patients engrafted with the median time to neutrophil >0.5×10e9/L and platelet >50×10e9/L recovery of 16 (14–22) days and 21.5 (12–29) days, respectively. Complete donor chimerism was achieved on day +30 in all cases. None of the patients developed grade III-IV acute GVHD, two patients experienced grade II acute GVHD. At one year the cumulative incidence of extensive chronic GVHD equaled 22%. No severe organ toxicity was observed. With the median follow-up of 19 months, the disease-free survival at 2.5 years was 91%. A single PNH patient died of haemorrhagic cystitis.

We conclude that treosulfan-based preparative regimens are well-tolerated and allow stable engraftment in SAA and PNH patients. The use of treosulfan allows intensification of the conditioning without providing an additional non-hematological toxicity.
Selective Targeting of the p53-hDM2 Interaction Using Hydrocarbon-Stapled p53 Peptides.
Authors: Bernal Federico , Walensky Loren D. , Tyler Andrew F. , Korsmeyer Stanley J. , Verdine Gregory L.
Published: October 2019

p53 is a transcription factor that induces cell cycle arrest and apoptosis in response to DNA damage and cellular stress, and thereby plays a critical role in protecting cells from malignant transformation. The E3 ubiquitin ligase hDM2 controls p53 levels through a direct binding interaction that neutralizes p53 transactivation activity, exports nuclear p53, and targets it for degradation via the ubiquitination-proteasomal pathway. Loss of p53 activity, either by deletion, mutation, or hDM2 overexpression, is the most common defect in human cancer. Tumors with preserved expression of wild type p53 are rendered vulnerable to pharmacologic approaches that stabilize and upregulate p53. In this context, hDM2 inhibition has emerged as a validated approach to restore p53 activity and resensitize cancer cells to apoptosis in vitro and in vivo. The inhibition of the p53-hDM2 interaction has been studied intensively with the goal of developing novel therapeutics for cancer. Here we describe the synthesis and evaluation of hydrocarbon-stapled α-helical peptides based on the transactivation domain of the p53 tumor suppressor protein. Select p53 stapled peptides exhibit subnanomolar binding to hDM2, displaying the highest affinity hDM2 binders reported to date. We find that these structurally-stabilized peptides are resistant to proteolysis and exhibit cell permeability as documented by flow cytometry and confocal microscopy analyses. In vivo binding of p53 stapled peptides to nuclear hDM2 is highly specific as demonstrated by co-immunoprecipitation experiments. In response to stapled peptide treatment, we observe upregulation of p53 and its transcriptional targets, including p21 and Bax. As a consequence, the peptides are capable of activating apoptosis in hDM2-overexpressing tumor cells through the p53 tumor suppressor signaling pathway. Thus, we find that hydrocarbon-stapled p53 peptides can trigger apoptosis in cancer cells by effectively modulating the p53-hDM2 protein interaction.
Routine Surveillance for Bloodstream Infections in a Pediatric Hematopoietic Stem Cell Transplant (HSCT) Cohort: Do Patients Benefit?.
Authors: Fernandez Conrad , Rigby Heather , Langley Joanne , Mailman Timothy , Crooks Bruce , Higgins Ann
Published: October 2019

Background: HSCT recipients are at high risk of late bloodstream infection (BSI). Controversy exists regarding the benefit of surveillance blood cultures in this immunosuppressed population. Despite the common use of this practice, its practical value is not well established in non-neutropenic children following HSCT.

Methods: At the IWK Health Centre, a tertiary health care center serving 2.5 million in the three Maritime provinces of Canada, weekly surveillance blood cultures from central venous access lines (CVLs) are drawn from children following HSCT until CVL removal. We reviewed data from a prospectively collected CVL database in association with a health record review to determine the utility and cost of this practice. Eligible participants were non-neutropenic pediatric HSCT recipients with CVLs followed at the IWK between 1999 and 2005. Patients routinely received PCP and penicillin prophylaxis. Cultures were considered to be for surveillance if the child was afebrile without focus of infection. The cost of laboratory investigations, nursing time, hospital stay, and interventions for positive surveillance cultures was calculated.

Results: 43 HSCTs were performed in 41 patients of whom 21 were male (49%). The median age was 7 years (range 1–18). Donors were related in 15 cases (35%), unrelated in 18 (42%) and autologous in 10 cases (23%). The most common underlying illness leading to HSCT was leukemia (19/41; 46%) followed by solid tumour (7; 17%), inborn errors of metabolism and Fanconi Anemia (4 each; 10%) and other (4;10%). The median length of study eligibility, defined as the point at which the patient returned to the IWK from the transplant centre until the time of central line removal, was 70 days (range 7–176). There were 316 patient contacts for surveillance cultures (mean 7 per transplant) and 577 central line lumens sampled. Three patients had potentially clinically significant surveillance blood cultures that were positive (3/43; 7%). The bacteria isolated were Klebsiella pneumoniae (n=2) and Corynebacterium jeikium (n=1). Repeat cultures were done prior to initiation of antimicrobial therapy and all were sterile. Patients were admitted for antimicrobial therapy if they were not already hospitalized for HSCT care. Median hospitalization for treatment of the positive culture was 5 days. All three patients had an uncomplicated course. The estimated total cost of BSI surveillance and treatment of asymptomatic infection over 6 years was $27,989.

Conclusion: This study suggests that BSI surveillance in children post engraftment has a very low yield and significant cost. It is unclear that it contributes to improved patient outcomes.
Monitoring JAK3 mRNA Levels in Patients with GVHD after Allogeneic Hematopoietic Stem Cell Transplantation by Real-Time Quantitative PCR.
Authors: Li Qihui , Du Xin , Geng Suxia , Weng Jianyu , Guo Rong , Lu Zesheng , Xing Haizhou
Published: October 2019

Background: Graft-versus-host disease (GVHD) represents a major hurdle impeding the efficacy of allogeneic hematopoietic stem cell transplantation (allo-HSCT). JAK3 plays pivotal roles in the initiation of cytokine-triggered signaling transduction. Recent studies about the murine GVHD model show that targeting JAK3 in donor lymphocytes with a chemical inhibitor such as WHI-P131 may be useful in the prevention of severe GVHD. However, the level of JAK3 expression in patients with GVHD has not been reported. The current study is to develop a reliable and rapid real-time quantitative PCR (Q-PCR) method using Taqman Probe for detecting JAK3 in allo-HSCT recipients.

Methods: 20 patients who received myeloablative-conditioning regimen for hematological malignancies and cyclosporine for GVHD prophylaxis have been investigated in this still ongoing study clinically. One of these patients developed acute GVHD (aGVHD), 9 patients developed chronic GVHD (cGVHD), and 10 patients showed no evidence of GVHD during the first 365 days post-transplant. RNA was extracted from white blood cells and cDNA templates were synthesized using 1μg RNA. The standard curve method was used for relative quantitation of JAK3.

Results: There is a clear trend for a increasing expression of JAK3 in 9 patients with GVHD. Interestingly, a significant decrease of JAK3 gene expression when the severity of GVHD was remission (P=0.04). One of the patient with cGVHD, the level of JAK3 gene expression has no changed before and after treatment. However, these patients post HSCT without GVHD have a comparable level of JAK3 expression before myeloablative conditioning regimen(P>0.05).

Conclusion: Based on our preliminary results, JAK3 mRNA level was obviously correlated with the development of GVHD. Analysis JAK3 mRNA expression may be useful in evaluating GVHD patients with allogeneic stem cell transplantation.
Double Syngeneic Transplantation in Plasma Cell Leukaemia.
Authors: Barrenetxea Cristina , Callis Manuel , Bueno Javier , Julia Antonio , Diaz Jose L.
Published: October 2019

INTRODUCTION Plasma cell leukemia (PCL) is a rare disorder, characterized by circulating clonal plasma cell.

It accounts for less than 1% of all plasma cell dyscrasias and has a fatal prognosis.

It can be primary or secondary, when there was a previously diagnosed plasma cell dyscrasia.

The median survival is 7–12 moths in the first case and 2 moths in the second.

CASE We present a 54 years old man, diagnosed in November 2003, with multiple myeloma IgA kappa, Bence Jones +, that presented weight loss, retinal hemorrhages, respiratory distress, hepatomegaly, splenomegaly, osteolytic lesions, the cariotype showed hyperploid, chromosome 13 monosomy, translocations t (1,12) and t (4,14).

He was treated upfront with tree cycles of a drug’s combination with melphalan, carmustine, vincristine and dexametasone with no response, therefore, was changed to cyclophosphamide, adriamycin, vincristine, methotrexate and citarabine.

After two cycles, the patient got complete remission.

The patient had a twin brother, and we decided, to do a double transplantation to consolidate the response. The first transplantation was condicionated with carmustine, etoposide, citarabine and melphalan (BEAM), and the second with cyclophosphamide and total body irradiation; the patient remained in complete remission.

Eight months after second transplant was admitted to the hospital with disorientation, bradypsiquia, headache, and sensoriomotor loss of lower extremity.

Laboratory examination showed differential count of leukocytes, haemoglobin and platelets were normal, LDH increase, absence of monoclonal gammophaty in blood and urine by immunofixation, and the brain’s computerized tomography showed multiple intraparenchymatous lesions, with peripheral edema, in both cerebral hemispheres, confirmed by magnetic nuclear resonance, all of that suggested a neoplastic disease.

These lesions were biopsied with the result of multiple myeloma lambda.

The patient died one day after biopsy because intracranial hypertension.

CONCLUSION Plasma cell disease has poor prognosis, and transplantation could be a good option for some patients, but in our case we only achieve to extend life a few months.
Regulation of Alpha Hemoglobin Stabilizing Protein (AHSP) mRNA Stability by a 3′ UTR Iron Response Element.
Authors: dos Santos Camila O. , Dore Louis C. , Ollala Sara T. , Costa Fernando F. , Weiss Mitchell J.
Published: October 2019

AHSP is an erythroid protein that binds the alpha globin subunit of hemoglobin (Hb) to maintain its structure and limit its oxidative activity. Biochemical and genetic studies demonstrate that AHSP is essential for normal Hb homeostasis and may act via at least two mechanisms: First, as a molecular chaperone to promote the folding and stability of alpha globin prior to its incorporation into HbA (alpha2beta2). Second, to bind and detoxify excess alpha globin that accumulates in normal erythroid precursors, and to a larger extent, in beta thalassemic ones. The existence of a potential iron responsive element (IRE) in the 3′-UTR of the AHSP mRNA raises the interesting possibility that iron homeostasis impacts on alpha globin stability via AHSP. IREs form stem-loop structures that bind cytosolic iron-sensing proteins (IRPs). Typically, IRP binding to the 3′ UTR stabilizes mRNA. Association with iron releases the IRP, enhancing mRNA degradation. Computational algorithms identified IRE-like stem-loop structures in AHSP mRNA of multiple species, yet the primary sequences deviate significantly from canonical IRE consensus sequences determined by studies of classical IREs, such as Transferrin receptor and Ferritin. Several lines of evidence now show that the AHSP IRE binds IRPs to regulate mRNA stability in an iron-dependent fashion:

1) in vitro gelshift competition assays demonstrate that human and mouse AHSP IREs bind tightly to IRPs. 2) AHSP mRNA co-immunoprecipitates with IRPs in erythroid cells in a fashion that depends on an intact IRE. 3) AHSP mRNA is destabilized by iron in both erythroid and heterologous cells; disruption of the IRE renders the mRNA constitutively unstable.

To study how iron regulates AHSP expression in vivo, we treated mice with iron dextran for 10 days and then examined AHSP mRNA in Ter119+ erythroid progenitors by RT-PCR. We found that short-term iron overload reduced AHSP mRNA levels by about 50% (p > 0.05). Our findings indicate that AHSP mRNA stability is regulated by iron via an atypical 3′-UTR IRE. These findings extend the potential repertoire for functional IREs that do not conform to previously defined canonical consensus sequences. In addition, they provide a potential mechanism by which erythroid cells can regulate globin stability according to iron status. For example, induction of AHSP during iron deficiency might stabilize apo- alpha globin that could accumulate from lack of heme. Conversely, iron overload could destabilize alpha globin by reducing AHSP levels. As such, iron overload, which occurs in patients with beta thalassemia, might aggravate the disease by further elevating the levels of toxic free alpha globin.
Unrelated Donor Hematopoietic Stem Cell Transplantation for High-Risk Leukemia.
Authors: Wu De Pei , Tang Xiaowen , Sun Aining , Fu Zheng-zheng , Qiu Huiying
Published: October 2019

objective: To investigate the efficacy of unrelated donor hematopoietic stem cell transplantation(URD-HSCT) for high-risk leukemia.

Methods: From 2001 to March, 2006, 24 patients of high-risk leukemia underwent HSCT with unrelated donor. Patients between 6 and 49 years of age(median age 21.5 years) There were 16 unrelated donor’s grafts came from Buddhist Tzu Chi Stem Cells center in Taiwan, and 8 unrelated grafts came from Red Cross Society of China Hematopoietic Stem Cell Marrow Donor Program Administration Center. Fifteen patients received bone marrow transplantation and another 9 patients received peripheral blood stem cell transplantation. Eighteen patients were fully matched with their donors for HLA loci A and B as well as for HLA-DRB1. Four of 24 patients had 1 molecular loci mismatch and 2 of 24 patients had 2 molecular locus mismatch. The conditioning regimen consisted of modified BU/CY or modified total body irradiation(TBI) plus CY. Eleven patients received cyclosporine (CSA), short-term methotrexate(MTX) and mycophenolate mofetil (MMF) as the regimen of prophylaxis of GVHD. Ten patients received CSA, MTX and MMF plus ATG/ALG for GVHD prophylaxis. The combination of CSA, MTX, MMF,ATG/ALG and CD25 monoclonal antibody for preventing GVHD in 3 patients.

Results: The incidence and severity of regimen-related toxicity were mild. The median number of MNC and CD34 positive cell was 4.18×108/kg and 7.75×106/kg respectively. The median time of the engraftment of neutrophil and platelet was 13 days and 19 days posttransplant respectively. The incidence of infection posttransplant was 70%(17/24). The complicated pneumonitis occurred frequently (13/17, 76%), The incidence of grade I-II and grade III-IV acute GVHD was 33% and 41% respectively. Limited chronic GVHD occurred in 25% patients and extensive cGVHD presented in 31% patients. Median follow up was 16 (range 1~57) months after transplantation. 75% patients achieved full donor chimerism(FDC) detected by STR-PCR and FISH. While 25% patients presented mixed chimerism(MC) and 2 of them converted to unstable MC. Decreasing values of donor chimerism were detected prior to the relapse of disease. Furthermore the treatment related mortality(TRM) was 33%(8/24), The incidence of relapse was 8%. Until now 14 patients are still alive. The median overall survival time were 26 months and 5-years expected survival was 47.7%.

Conclusion: URD-HSCT can be an effective and curable approach for leukemia with higher incidence of GVHD and infection. The treatment for the severe and deadly GVHD and pulmonary infection are still major problems need to be resolved in the future.