bluuebunny/crossref_metadata_2025 huggingface dataset:
Anti-CD20 Monoclonal Antibody in the Treatment of Refractory Autoimmune Cytopenias in Adults.
Authors: Narat Santhosh , Gandla Jagdish , Mehta Atul B.
Published: April 2020

Anti-CD20 monoclonal antibody (rituximab) has been used in autoimmune cytopenia with variable success.We report 13 patients with chronic refractory autoimmune hemolytic anemia (AIHA) and idiopathic thrombocytopenic purpura (ITP) who each received 4 cycles of rituximab 375mg/m2 weekly. All 8 AIHA patients (6 idiopathic,2 secondary to a lymphoproliferative disorder, 5 splenectomised) had warm antibody type. Response was seen in 4(50%) of 8 patients (3 CR,1PR) and 3 patients remain in CR at 5,7,14 months post-therapy. Median time to maximum response (TMR) in responders was 9 weeks(range 6 – 18 weeks). In 5 ITP patients (4 splenectomised), 4(80%) responded (3CR) and one continues in CR 50 weeks after completion of rituximab treatment Median time to maximum response was 4 weeks (range 4 – 12 weeks). No pre-treatment clinical or laboratory parameters that predict response could be identifird in the AIHA or ITP groups.Our data indicate that rituximab is a relatively safe and effective treatment option in patients with refractory autoimmune hemolytic anemia and thrombocytopenia.

Table 1

Number of patients Mean age (Years) Sex Overall response Complete response No response Time to maximal response AIHA 8 47.75 (26– 73) 3M:4F 4 (50%) 3 (37.5%) 4 (50%) 9 weeks ITP 5 58.6 (28–89) 4M:1F 5 (80%) 3 (60%) 2 (40%) 4 weeks
Strain Variation in the Immune Response to Human Factor VIII in Normal and Hemophilic Mice.
Authors: Jin Linda X. , Soukhareva Nadejda N. , Scott David W.
Published: April 2020

Approximately 25–30% of Hemophilia A patients make inhibitors after treatment with therapeutic Factor VIII (fVIII). The ability to predict which patients may respond with inhibitors would be important in order to identify suitable patients for tolerance therapies prior to exposure to fVIII and the development of high inhibitor titers. Advances in identifying the nature of the fVIII mutations (inversion, stop codons, etc), HLA linkage and other factors have suggested these as important predictors of inhibitor responsiveness. As a step toward identifying other genetic factors that may predispose for inhibitor formation, we immunized both fVIII−/ − (knockout; C57Bl background) and fVIII sufficient mice of different MHC backgrounds with human fVIII (i.e. therapeutic doses, intravenously in saline with no adjuvant). Our hypothesis was that fVIII sufficient mice would only respond to epitope differences between human and mouse fVIII and that this response would be more sensitive to genetic variation. Our results showed a clear hierarchy of responsiveness both in terms of T-cell proliferation and ELISA titers in the following order: CAST/Ei>BALB/c>B10.A; CBA>C57Bl/6. Despite being bred on a C57Bl (low responder) background, knockout mice made the strongest response to human fVIII, presumably due to the lack of tolerance to mouse epitopes. These results suggest that MHC plays a role in fVIII responsiveness, as it does in the response to fIX or fVIII delivered via an adenovirus vector (http://www.abstracts-on-line.com/abstracts/hemphiladelphia03; Lozier, 2003; Brown et alia, J. Thromb. Haemost.2:111–118, 2004). Thus, it may be possible to map additional loci responsible for inhibitor formation in future studies. Moreover, the fact that fVIII sufficient mice respond to intravenous fVIII points out a unique immunogenic quality of this molecule that appears to be unlike most other plasma proteins. This offers the additional possibility of developing mutated fVIII molecules that lack this inherent immunogenicity.
Dramatic Improvement in CR Rate and CR Duration with Imatinib in Adult and Elderly Ph+ ALL Patients: Results of the GIMEMA Prospective Study LAL0201.
Authors: Vignetti Marco , Fazi Paola , Meloni Giovanna , Chiarenza Annalisa , Malagola Michele , Fabbiano Francesco , Recchia Anna , Kropp Maria G. , Offidani Massimo , Palmieri Salvatore , Cilloni Daniela , Torelli Giuseppe , Cimino Giuseppe , Foà Roberto , Baccarani Michele , Mandelli Franco
Published: April 2020

In 2001, the GIMEMA Group started a phase II study to evaluate Imatinib both in adult (study A, 18–60 years) and elderly (Study B, >61 years) patients with Philadelphia positive (Ph+) acute lymphoblastic leukemia (ALL).

In study A, adult patients with Ph+ ALL received the standard GIMEMA induction (VCR, DNR, PDN, L-ASP) and consolidation (MTZ and high-dose Ara-C) treatments, without imatinib. All patients in hematological complete remission (HCR) after the consolidation, either with or withouth a donor, received imatinib p.o. at the dosage of 800 mg/day for 6 months. After completing the 6-months therapy, in the absence of safety concerns, if the investigator considered the patient eligible for treatment, the therapy with imatinib was continued. A total of 22 patients have been enrolled [7 in molecular complete remission (MCR) and 15 still BCR/ABL+]: 18 are still in HCR after a median follow up of 20 months (2 – 26). In particular, all the 7 patients PCR- when started imatinib are still in HCR (6 PCR- and 1 becoming PCR+), after a median follow-up of 19 months (7.6 – 24) and eleven of the 15 PCR+ patients are in CHR after a median follow-up of 19 months (1.2 – 26). Of the remaining 4 PCR+ patients, 1 died in CR after an alloSCT and 3 patients relapsed at 4, 9 and 11 months, respectively. The probability of disease-free survival at 2 years is 79% (C.I. 95%: 58,4 – 100,3).

Figure Figure

In study B, planned for elderly patients usually not eligible for intensive treatments, the induction therapy consisted of imatinib 800 mg/day p.o. associated to prednisone 40 mg/sqm/day for 30 days, withouth any other chemotherapy. Up to date only 12 patients have been enrolled. Median age was 67.5 yrs (61–78), 9 were females. Eleven (92%) obtained a CR with the prednisone-Imatinib association induction treatment, 1 patient discontinued the treatment for toxicity. The post-remissional treatment mainly consisted of imatinib alone: 8 patients are still in CR, after a median follow up of 7 months (4 – 15), 2 relapsed at 4 and 4 months and 1 died in CR due to a 2nd neoplasia. In conclusion, these studies, planned 3 years ago to verify the feasibility and toxicity of imatinib in adult and elderly Ph+ ALL patients, clearly suggest a strong activity of Imatinib also in this disease, not only giving the possibility of inducing a CR as monotherapy, but even of maintaining long-lasting hematological and molecular CR withouth allogeneic stem cell transplantation. Further studies are mandatory to finally identify the best way to integrate imatinib in the entire treatment strategy of this disease.
Treatment of Philadelphia-Positive Acute Lymphocytic Leukemia (Ph+ ALL) in the Elderly with Imatinib Mesylate (STI571) and Chemotherapy. an Interim Analysis of the GRAALL AFR09 Trial.
Authors: Delannoy Andre , Lhéritier Véronique , Thomas Xavier , Castaigne Sylvie , Farhat Hassan , Rigal-Huguet Françoise , Raffoux Emmanuel , Gressin Remy , Legrand Ollivier , Witz Francis , Dubruille Viviane , Turlure Pascal , Troussard Xavier , Delain Martine , Bilhou-Nabera Chrystele , Macintyre Elizabeth , Delabesse Eric , Vernant Jean-Paul , Dombret Hervé , Fière Denis
Published: April 2020

Ph+ ALL accounts for approximately one third of ALL cases in patients aged 55 years or older. The median survival of older Ph+ ALL patients is one year, with practically no long-term survivor (Blood, 98, Supp1 p319a, 2001). Imatinib has demonstrated remarkable, although transient, activity in relapsed and refractory Ph+ ALL, which prompted the GRAALL to implement a treatment protocol associating imatinib and chemotherapy in previously untreated elderly patients: ALL patients aged 55 years or older are treated with steroids during one week and Ph+ve cases are then offered a specific therapy including an induction treatment with steroids, cyclophosphamide, daunorubicin and vincristine, followed, irrespective of response to induction chemotherapy, by imatinib, 600 mg daily, combined with intermittent steroids during 2 months. Patients in complete response are then given 10 blocks of alternating chemotherapy, including 2 additional two-month blocks of imatinib, for a total treatment duration of 2 years. Therapy of occult central nervous system leukemia includes 5 intrathecal injections of methotrexate and cranial irradiation. The study is intended to include 30 patients and its main objective is to improve overall one-year survival to 70%. Results are compared with those obtained in 21 Ph+ ALL elderly patients treated according to our previous protocol. Since January 2003, 21 patients aged 58 to 78 years (median: 64.7 years) were included in the AFR09 protocol. Their median follow-up is 3 months. 15/19 patients are in complete response after induction chemotherapy vs 6/21 in the historical controls given similar induction regimen but with no steroids before chemotherapy (p=0.002). The projected overall survival is 95% at 9 months vs 62% in the control group (p=0.08, log-rank test). The 9-month projected event-free survival is 83% vs 10% (p<0.0001) and the projected 6-month relapse-free survival is 79% vs 22% (p=0.006, log-rank test). In conclusion, although preliminary, this interim analysis suggests that the use of imatinib in elderly patients with Ph+-ALL is very likely to dramatically improve prognosis. Of note, an unexpected high proportion of patients accrued in this study achieved a CR after induction chemotherapy possibly denoting a beneficial impact of steroids given before starting chemotherapy.
Pharmacokinetics and Pharmacodynamics of Three Asparaginases in Pediatric Patients with Higher Risk Acute Lymphoblastic Leukemia: A Report from CCG-1961.
Authors: Panosyan Eduard H. , Seibel Nita L. , Grigoryan Rita S. , Avramis Ioannis A. , Jaques Jon D. , Ettinger Lawrence J. , Gaynon Paul S. , Sather Harland N. , Franklin Janet , Cecilia Fu , Siegel Stuart E. , Fingert Howard J. , Avramis Vassilios I.
Published: April 2020

Deamination of asparagine (Asn) and glutamine (Gln) by asparaginase (ASNase) induces apoptosis of ALL lymphoblasts, which lack ability to synthesize Asn de-novo. Early treatment intensification after Induction, including PEG-ASNase instead of native E.Coli-ASNase, in addition to more vincristine and intravenous methotrexate, improves outcome in pediatric ALL patients presenting with unfavorable features, who show rapid early response to Induction chemotherapy (Seibel NL, et al., Blood, 2003;102:224a). Erwinase was used for patients with overt clinical allergy to E.Coli-ASNase. We evaluated the population pharmacokinetics and pharmacodynamics (PK-PD) of the three ASNase formulations employed on CCG-1961. The PK-PD parameters of native and PEG-ASNase in this group of HR ALL patients were similar to those reported from SR ALL patients from CCG-1962 (Avramis VI, et al., Blood, 2002;99:1986–1994). Population PK (NONMEM) showed the expected half-lives of native E.Coli-ASNase (6000 IU/m2): 1.3 days; PEG-ASNase (2500 IU/m2): 6.5 days; and Erwinase (6000 IU/m2): 0.8 days. Repeated doses resulted in an accumulation of activity with moderate prolongation of elimination half-lives for all formulations. PEG-ASNase provided > 0.3 IU/ml activity for > 21 days for most patients in Delayed Intensification. We measured simultaneous amino acid levels in 430 sera samples obtained from various phases of CCG-1961 from these 187 fully evaluable patients with detectable post-treatment ASNase activity. The averages of pre-treatment control serum levels for Asn and Gln were 44.8 ± 3.0 μM (mean ± SE) and 363.8 ± 23.9 μM, respectively. There were no statistical differences between the PD effects of ASNase formulations on serum Asn deamination, although the lowest Asn levels averaging 2.6 ± 0.4 μM were obtained on 11–14 days post-PEG-ASNase samples (n=14). Glutamine deamination enhanced Asn depletion, and deamination correlated with ASNase activity. At levels >0.4 IU/ml, PEG-ASNase and native ASNase provided 95 ± 8% and 85 ± 26% Gln deamination, respectively (p=0.04). Eleven to 14 days after PEG-ASNase, Gln levels averaged 13 ± 5 μM (n=14). In general, Erwinase provided greater Gln deamination (94 ± 5%, n=19) 2–3 days post administration, than either native or PEG-ASNases at comparable activity levels, which were 70–80% (p<0.001). Greater Gln deamination yielded lower Asn levels after Erwinase. These PD results provide insight into the benefit of PEG-ASNase intensification and possibly Erwinase substitution in the treatment of patients with high risk ALL treated with augmented BFM therapy and need to be further explored.
Non-Myeloablative Allogeneic Transplantation in Patients with Relapsed or Refractory Multiple Myeloma.
Authors: Gerull Sabine , Hegenbart Ute , Goerner Martin , Benner Axel , Moehler Thomas , Goldschmidt Hartmut , Ho Anthony D.
Published: April 2020

Patients with recurrent and refractory multiple myeloma have a very limited survival expectance. Allogeneic transplantation might offer an option for cure in myeloma and the recent development of non-myeloablative conditioning regimens has reduced transplant related morbidity and mortality and rendered this treatment feasible in elderly patients.

The role of non-myeloablative allogeneic transplantation for multiple myeloma however, has not yet been defined. We have analyzed the results of patients with relapsed or refractory multiple myeloma treated at our institution.

Between 08/1999 and 02/2004, 56 patients with relapsed (n=54) or refractory (n=2) myeloma were treated with non-myeloablative allogeneic transplantation. The median beta2microglobulin at the time of diagnosis was 2.75 mg/l, and median age at the time of transplant was 54.5 years (39.2–67.8). The median time from diagnosis to transplant was 3.6 years. Prior to allogeneic transplantation, patients received reinduction chemotherapy which included an autologous transplantation for 30 patients. The median number of previous cycles of conventional chemotherapy was 9. The conditioning regimen was 2 Gy TBI with (n=43) or without (n=3) fludarabin 3 x 30 mg/m² for 46 patients, the remaining 10 patients received a melphalan containing regimen.

Acute toxicity was low with a WBC < 500/μl and platelets < 50/μl for a median of 0 days. Engraftment was prompt with 90 % of patients having achieved > 90 % donor chimerism by day 56. Acute GvHD Grade II-IV occurred in 36 % of patients with 22 % Grade III-IV, and 61 % experienced chronic GvHD. Total transplant related mortality reached 20 %, with a day 100 TRM of 5 %. 32 patients experienced relapse or progressive disease, and 32 % of patients died due to relapse. The Kaplan-Meier estimate of overall survival and progression free survival at 18 months was 40 % and 25 %, respectively, with a median follow up of survivors of 21 months. Patients who experienced cGvHD had a significantly higher overall survival estimate (60 % vs. 20 % at 18 months, p=0.03). The number of cycles of pretreatment before allogeneic transplantation had a statistically significant negative influence on overall (p=0.02) and progression free survival (p=0.006).

We conclude that non-myeloablative allogeneic transplantation is feasible in patients with relapsed multiple myeloma. The significant poor prognostic factors we identified were absence of chronic GvHD and number of cycles of pretreatment. Allogeneic transplantion should therefore be considered as an option earlier in the course of the disease.
Successful Treatment of Advanced and Refractory AML with Sirolimus Based Non-Myeloablative Allogeneic Stem Cell Transplantation.
Authors: Claxton David F. , Ehmann Christopher , Rybka Witold
Published: April 2020

Non-myeloablative conditioning has extended the use of allogeneic hematopoietic transplant to many previously ineligible patients but has to date been disappointing in patients with advanced acute myelogenous leukemia (AML). We added the immunosuppressive and antitumor agent sirolimus (rapamycin) to an established transplant regimen of fludarabine 25 mg/M2 days -7 through -3 and cyclophosphamide 1000mg/M2 days −7 and −6, with tacrolimus and methotrexate immunoprophyllaxis. 23 patients with AML were treated, with median age 59 (range 28–72) at transplant. 6 patients received matched sibling, 11–unrelated donor, 1–5/6 matched and 5-haploidentical (“haplo” −3/6 or 4/6 matched) transplants. The haplo recipients also received antithymocyte globulin on days −1, 1, 3, and 5. Only 7 patients in total were in CR, but all of these were at high risk for relapse because of prior MDS, chromosome status, or other features. 9 patients were in chemotherapy refractory progression, and 7 were primarily refractory to induction therapy. All patients engrafted. In patients without graft versus host disease by day 30 (9 patients) tacrolimus was discontinued between days 45 and 100 while sirolimus was continued. 12/23 patients survive at 61–903 days of followup post transplant (median 525) and 11/12 survivors are currently free of detectable leukemia. 3 of these 12 relapsed at 83, 88, and 243 days, but the first 2 of these have received chemotherapy and DLI and are in CR 771 and 311 days from relapse. The figure below shows overall and progression free survival (PFS). All survivors but the single patient undergoing therapy for progression have performance status 0 or 1 and are free of need for transfusion. 9/12 survivors continue on sirolimus therapy. Only 2 patients, both recipients of haploidentical cells, have died from transplant related causes. Non-myeloablative hematopoietic cell transplantation with sirolimus (rapamycin) based immunosuppression effectively treats many patients with advanced and poor prognosis AML. Given the very limited anti-leukemic activity of the conditioning chemotherapy, this suggests that disease control has been via graft versus leukemic effects alone or combined with anti-leukemic activity of the sirolimus.

Figure Figure
Improved Long Term Survival with Minimal GVHD after Myeloablative Unrelated Donor Stem Cell Transplantation Using In Vitro and In Vivo T Cell Depletion with CAMPATH-1H.
Authors: von dem Borne Peter A. , Beaumont Floor , Starrenburg Ingrid , Oudshoorn Machteld A. , Hale Geoff , Falkenburg J.H. Frederik , Fibbe Willem E. , Willemze Roel , Barge Renee M.Y.
Published: April 2020

In allogeneic stem cell transplantation (SCT) T-cell depletion reduces transplant related mortality by diminishing GVHD. We have investigated a myeloablative regimen for matched unrelated donor SCT using both in vivo and in vitro CAMPATH-1H for effective T-cell depletion, utilising DLI at a later time point for graft versus tumor effect if necessary.

Thirty patients (median age 33 years, range 18–48) were transplanted from January 1997 to June 2002. Diagnoses were: CML CP (n=9), CML AP (n=2), AML/MDS (n=9), ALL (n=8), NHL (n=1) and Fanconi anemia (n=1). Six patients had one HLA mismatch, the others were identical for HLA A, B, C, DR and DQ. Conditioning consisted of CAMPATH-1H 5mg/d on days −8 to −4, TBI 6 Gy on days −8 and −7 and cyclofosfamide 60 mg/kg on days −6 and −5. T-cell depletion was performed by in vitro incubation of the graft with 20 mg CAMPATH-1H for 30 minutes (Campath “in the bag”). Post-transplant GVHD prophylaxis consisted of cyclosporine A and methotrexate. The stem cell source was bone marrow in 19 patients (63%) and peripheral blood in 11 patients.

One graft failure was observed, all other patients had sustained engraftment of donor cells. Acute GVHD was observed in 12 patients (40%), maximally grade I-II skin. No severe acute GVHD (grade III-IV) was experienced. Limited chronic GVHD developed in 2 patients, resolving after treatment. Only in one patient extensive chronic GVHD developed, which did not resolve. CMV reactivation occurred in 23% of patients, one patient developed CMV disease. No EBV disease was observed. Ten patients received donor lymphocyte infusion (DLI) at a median of 17.4 months after SCT (8 patients with relapsed CML, one patient with relapsed ALL, one patient with autoimmune hemolytic anemia). After DLI acute GVHD grade I-II developed in 4 patients, and GVHD grade III-IV in 3. Chronic GVHD developed in 5 patients, of which 2 extensive, resolving in all except one patient. With a median follow up of 37 (range 21–84) months 17 patients are alive (57%). One of the CML patients shows persistence of molecular disease not responding to increasing doses of DLI. All other patients are in CR with the CML patients in molecular remission. Five patients (17%) died because of relapsed disease (2 AML/MDS and 3 ALL). Treatment related mortality was 26% (1 rejection, 2 GVHD, 1 myocardial infarction, 4 infections).

In conclusion, matched unrelated donor SCT following myeloablative conditioning using T-cell depletion with CAMPATH-1H in vivo as well as in vitro results in good engraftment, minimal grade I-II GVHD and an overall survival of 57%. Relapse rate was not increased with this strategy. This regimen appears to be successful for young adults with high-risk malignancies.
The Transcription Factor Pbx1 Is Required for the Development of Double Positive Thymic T Cells.
Authors: Sanyal Mrinmoy , Tung James W. , Dejbakhsh-Jones Sussan , Strober Samuel , Herzenberg Leonore A. , Cleary Michael L.
Published: April 2020

It has become increasingly apparent that transcription factors originally discovered at the sites of chromosomal translocations in acute leukemias serve critical roles for hematopoietic cell development and function, which require fine orchestration of gene expression. Pbx1 is one such transcription factor, which forms a chimeric oncoprotein, E2a-Pbx1, resulting from t(1;19) chromosomal translocations between the E2a and Pbx1 loci in a subset of the pediatric pre-B cell acute lymphoblastic leukemias. Previously, our laboratory has shown that mice nullizygous for Pbx1 die in utero with substantial myelo-erythroid defects associated with profound fetal anemia. To determine whether Pbx1 is also required for normal lymphoid development, we performed Rag1-deficient blastocyst complementation assays using Pbx1 homozygous null embryonic stem (ES) cells. Interestingly, analysis of resulting chimeric mice displayed a partial rescue of the Rag1 phenotype as evidenced by T cell-restricted lymphocyte development. Pbx1 null ES cells produced circulating CD4 and CD8 single positive T cells, however in contrast to the typical naive T cell phenotype (CD62LhighCD44loCD45RBhigh) in control mice they exclusively displayed a characteristic memory T cell phenotype (CD62LlowCD44highCD45RBlow). Analysis of the thymi of the chimeric mice for T cell developmental precursors showed the complete absence of CD4/CD8 double-positive intermediates. Rather, T cells in the thymus, and all secondary lymphoid organs, were single positive CD4 or CD8 cells that exclusively displayed a similar memory T cell phenotype typical of T cells derived from bone marrow resident committed T cell progenitors (CTP). The striking absence of classical CD4/CD8 double-positive cells in the thymus indicates that Pbx1 deficiency interrupts the intrathymic T cell developmental pathway. Furthermore, the phenotypic resemblance of Pbx1 null T cells with T cells derived from bone marrow resident CTP strongly suggests that T cell development in the absence of Pbx1 may occur via an extrathymic pathway.
Differential Sensitivity of Normal and CML-Derived CD34+ Cells to Inhibition of SHP-2 Gene Expression by RNA Interference (RNAi).
Authors: Scherr Michaela , Battmer Karin , Chaturvedi Anuhar , Schultheis Beate , Ganser Arnold , Eder Matthias
Published: April 2020

RNA interference (RNAi) has rapidly evolved into an efficient tool for functional genomics in a variety of organisms. Stable expression of shRNA (short hairpin RNA) driven by pol III promoters upon retro- or lentiviral gene transfer can induce long-term gene silencing in mammalian cells, including human hematopoietic cells. We recently demonstrated that lentivirus mediated anti bcr-abl RNAi can specifically silence bcr-abl gene expression, inhibit oncogene driven cell proliferation, and eradicate leukemic cells depending on the dose of lentivirus-mediated shRNA expression (Scherr et al. Gene Therapy 2004). Since effective depletion requires a threshold of lentiviral integrations into target cell genomes, the risk of insertional mutagenesis may limit the therapeutic value of this approach. We therefore applied lentivirus-mediated RNAi for functional genomics in purified primary normal and CD34+ cells from chronic phase CML patients harvested at initial diagnosis. Several SHP-2 shRNAs were generated according to established rules and were functionally evaluated using a bicistronic reporter system as described earlier. Effective shRNA expression cassettes were subsequently cloned into lentiviral plasmids encoding RFP to track lentiviral transduction. Transduction of K562, U937, NB-4 and TF-1 cells with lentiviral supernatants results in a reduction of SHP-2 mRNA and protein by more than 90 %. Interestingly, anti-SHP-2 shRNA induced almost complete depletion of RFP+ cells in all four cell lines, demonstrating that SHP-2 expression is essential for proliferation and survival in these cells. We next transduced normal and CML-derived CD34+ cells with a puritiy of > 95% with control and anti-SHP-2 lentiviruses, and stimulated methylcellulose cultures of the cells with high (GM-CSF: 20 ng/ml; IL-3: 10 ng/ml) or low (GM-CSF: 0.2 ng/ml; IL-3: 0.1 ng/ml) cytokine concentrations. This assay relies on the fact that colony formation of CML-CFU is mediated by both cytokine receptor and bcr-abl signaling. Therefore differential numbers of transduced, i.e. RFP+ colonies under different cytokine stimulations reflect the role of the RNAi-target in normal or malignant CFU. Whereas anti-SHP-2 RNAi did not reduce the proliferation of normal transduced CFU (n=5), proliferation of CFU from CML patients was specifically reduced between 50 to 85 % under low cytokine concentration (n= 9). These data suggest that primary normal cells are more resistant to inhibition of SHP-2 gene expression than leukemic cell lines and CD34+ cells from CML patients and identify SHP-2 as a potential target for anti bcr-abl therapy.
Temporal Gene Expression after BCR-Crosslinking Based upon Zap70 Related Genomic Features.
Authors: Vallat Laurent D. , Park Yuhyun , Rassenti Laura , Gribben John G.
Published: April 2020

Ig mutation status discriminates aggressive from more indolent chronic lymphocytic leukemia (CLL). Several gene expression studies distinguished CLL from other leukemia but failed to discriminate these two types of patients. Nevertheless, differentially expressed genes including zap70 were identified and further validated as an important prognostic factor. Zap70 is a normal component from the T cell receptor pathway, and may have a critical role in the B cell receptor (BCR) signaling pathway in CLL. We questioned whether zap70 might alter response to BCR signaling in CLL cells and explain the difference in biologic characteristics of this disease. To examine this we crosslinked the BCR in CLL cells from 12 patients and B cells from 6 healthy donors. We examined the response to BCR stimulation on cell proliferation, apoptosis and gene expression profile. Here we analyzed the temporal gene expression after BCR crosslinking at different time points, in addition to the basal level of gene expression. Based on the results of a pilot study examining multiple different time points, four time points were analyzed in all cases ranging from 60 min to 390 min and gene expression of stimulated (S) and unstimulated (US) CLL and control cells analyzed for a total of 170 HU133 plus2.0 DNA-chips. We defined the expression data from the logarithmic ratio log(S/US) for each time point. In keeping with previous findings, the basal gene expression distinguished CLL samples from healthy B-cells, but did not distinguish mutated from unmutated patients. We defined the linear combination first for the initial time point and then for all four time points to score for expression over time. This temporal gene profile of BCR engagement was based on filtering of 2850 genes that now distinguish healthy B-cells from CLL-cells, but is now capable also of distinguishing a specific BCR signature of a group of patients. Of note, this distinguished patients based less upon Ig gene mutation status but on zap70 protein level, with a mean protein expression difference of 9 fold between these two groups. This more aggressive pattern reveals changes in gene expression in important genetic pathways associated with BCR crosslinking, including JUN and NFkB. Stimulating a crucial pathway for CLL biology identified temporal gene expression pathways linked with the aggressiveness of this leukemia. This suggests that a fuller understanding of the biologic differences in leukemia cells may be identified by a more dynamic approach to gene profiling by examination of response to specific stimuli, rather than examination at the basal level. Based upon these results, ongoing studies examine the biologic impact of gene silencing of zap70 using RNAi on BCR stimulation in CLL cells.
Decrease in Sensitivity of PIG-A-Mutant Cells to Natural Killer Cells Conferred by Missing of Stress-Inducible Membrane Proteins ULBPs.
Authors: Hanaoka Nobuyoshi , Kawaguchi Tatsuya , Horikawa Kentaro , Nagakura Shoichi , Ishihara Sonoko , Mitsuya Hiroaki , Nakakuma Hideki
Published: April 2020

The mechanism by which paroxysmal nocturnal hemoglobinuria (PNH) clones expand to manifest symptoms is still unknown. PNH cells with PIG-A mutations do not synthesize glycosylphosphatidylinositol (GPI), resulting in deficiency of a series of GPI-linked membrane proteins. Blood cells with PNH phenotype (GPI− cells) expand in patients with bone marrow (BM) failure syndromes including PNH, aplastic anemia, and myelodysplastic syndromes. The diseases share an immune-mediated BM injury possibly by cytotoxic lymphocytes such as natural killer (NK) cells and cytotoxic T lymphocytes (CTL). It is suggested that the BM injury allows PNH clones survive selectively. Indeed, we have shown that human leukemic cells (K562) decrease their sensitivity to NK cells in vitro when K562 cells acquire PIG-A mutations (Nagakura et al, Blood 2002). In the present study, we show that the decrease in NK sensitivity of PIG-A mutant cells is ascribable to the deficiency of GPI-linked membrane proteins, ULBPs. ULBPs bind cytomegalovirus protein UL-16, consist of ULBP1-3, and emerge in cell membrane when cells are infected or transformed. They trigger off a NK activation signal, which overrides an inhibitory signal from MHC class I (Cosman et al, Immunity 2001). ULBPs also activate CTL besides NK cells. As target cells, a pair of GPI+ control and GPI− mutant cell lines was prepared from a GPI− K562 cell line bearing a PIG-A mutation by transfection with a PIG-A cDNA and a vector alone, respectively. Flow cytometry detected ULBP1-3 on the surface of GPI+ but none of GPI− K562 cells. As effector cells, we used cultured human NK cells (KHYG-1) deficient in Fc γR type III (CD16). GPI− cells were more resistant than GPI+ cells to the killing by NK cells in the 51Cr-release assay. GPI+ cells decreased their sensitivity to NK cells to the level of GPI− cells in the presence of antibodies to both ULBP1 and ULBP2, while antibodies to ULBP3 exerted no effects. None of the antibodies to ULBPs showed any effects on the killing of GPI− cells, while antibodies to NKG2D, which is a NK receptor for both ULBPs and MICA/B, inhibited the killing of both GPI+ and GPI− cells. Thus, GPI− cells that lack membrane ULBPs show a survival advantage in the setting of immune attack in vitro. Of clinical interests, ULBPs were detected on the cell surface of GPI+ but none of GPI− granulocytes of patients with PNH. GPI+ granulocytes of healthy individuals were negative for ULBPs. There appears pressure to induce membrane ULBPs in patients with PNH, leading to BM injury. These findings suggest that the failure of membrane expression of ULBPs permits selective expansion of PNH clones in patients with PNH and other BM failure syndromes.
Nuclear Localization of Codanin-1, a Product of a Newly Identified Gene Mutated in Congenital Dyserythropoeitic Anemie Type I.
Authors: Tamary Hannah , Chernikovsk Naama , Lotan Sharon , Edlitz-Markus Tali , Shalev Hanna , Shaklai Sigal , Fibach Eithan , Yaniv Isaac , Resnitszky Peretz , Dgany Orly
Published: April 2020

Congenital dyserythropoietic anemia (CDA) type I is a rare autosomal recessive macrocytic moderate to severe anemia rarely associated with distal skeletal deformities. Erythroid bone marrow findings are pathognomonic with internuclear chromatin bridges, spongy heterochromatin, and invagination of the nuclear membrane. Our and others studied suggested erythroid S phase arrest. We recently identified the gene mutated in this disease, CDAN1 and named its ubiquitous encoded protein codanin-1. The function of this protein remains unknown. The aim of the present study was to determine the sub cellular localization of codanin-1. Human erythroid cultures as well as Hela, K562 and osteodarcoma cell line (U2OS) were grown. A polyclonal anti-codnain-1 antibody was generated by immunizaing rabbits with a synthetic 17 aa peptide. The anti-sera was affinity-purified against the immunizing peptide. Western blots, immunoflurescence stains and immunogold electromicroscopic studies were performed. Western blots of human erythroid cultures as well as HeLa, K562 and osteosarcoma cell lines identified one expected band of 130kD. Fluorescent and confocal microscopic studies of osteosarcoma and HeLa cells using anti-codanin-1 antibody showed that codanin-1 was an abundant nuclear protein. Immunogold electron microscopic studies with anti codanin-1 antibodies demonstrated that coodanin-1 is located within the heterochromatin. Future studies will focus on the role of codanin-1 within the heterochromatin.
Hematopoietic Cell Transplantation in Fanconi Anemia Patients with Biallelic BRCA2 Mutations.
Authors: MacMillan Margaret L. , Auerbach Arleen D. , Wagner John E.
Published: April 2020

Fanconi anemia patients with biallelic BRCA2 mutations have an exceptionally high risk of developing hematological malignancies, Wilms tumors and medulloblastoma at a very young age. We report on the hematopoietic cell transplant (HCT) results of 6 patients from 4 kindreds, all of whom have biallelic BRCA2 mutations. Two patients (800/2 and 984/2) were found to have a Wilms tumor at time of work-up for HCT despite normal previous renal ultrasounds. One was a stage 1 favorable histology, the second a stage II poorly differentiated tumor with diffuse anaplasia. In each case, the patient underwent nephrectomy and proceeded to HCT 3 weeks later. All unrelated or mismatched related donor marrow grafts were T cell depleted either by elutriation in patient 632/1, or CD34 selection with Isolex in the remaining cases. Five of 6 patients achieved neutrophil engraftment. Only one patient developed severe regimen related toxicity and one patient GVHD. Two of 6 patients are alive and well after HCT, both whom had T-cell ALL, which is uncommon in FA patients.

Patient and Transplant Characteristics

Kindred, IFAR No. Age/Gender Clinical Hx Donor Preparative Rx Day to ANC>500 Toxicity, GVHD Outcome 1, 632/1 3.7 yr/F AML at 36 mo, chemotherapy refractory 6/6 URD BM x 2 CY, TBI, ATG, CSA, MP; CY, ATG, MP, CSA graft failure; +31 None AML relapse after both HCT, died day +76 after 2nd HCT 1, 632/2 1.9 yr/F AML at 21 mo, chemotherapy refractory 6/6 MSD BM CY, TBI, FLU, ATG, CSA, MP Engrafted None AML relapse, died day +288 2, 800/1 1.8 yr/M AML at 11 mo, chemotherapy refractory 6/6 URD TCD BM CY, TBI, ATG, CSA, MP +11 Grade II aGVHD AML relapse day +110, died day +124 2, 800/2 0.8 yr/M Neutropenia at 5 mo, Wilms tumor at 9 mo 6/6 URD TCD BM CY, TBI, FLU, ATG, CSA, MP +9 Resp and renal failure Died day +60 from pulmonary hemorrhage 3, 900/1 4.9 yr/M T cell ALL at 5.2 yr, CR after chemotherapy 5/6 Mat TCD BM BU, CY, FLU, ATG, CSA, MP +10 None Alive and well 19 mo after HCT 4, 900/2 6.7 yr/F T cell ALL at 4.9 yr, remission with chemotherapy, relapse with AML 17 mo later, Wilms at 6.6 yr 6/6 URD UCB BU, CY, FLU, ATG, CSA, MP +13 None Alive and well 3 mo after HCT

In summary, HCT has limited effectiveness after the onset of leukemia in patients with Fanconi anemia. While these patients have tolerated chemoradiotherapy well in most cases, refractoriness of the leukemia is the principle obstacle to success. Therefore, patients with biallelic BRCA2 mutations should be screened for solid tumors and referred for HCT as soon as possible, prior to the onset of leukemia. In addition, these patients require routine screening for solid tumors after HCT, the incidence of which remains to be determined.
Isolation of Human CD4+CD25+ T Cells with Regulatory Capacity for Clinical Trials.
Authors: Hoffmann Petra , Eder Ruediger , Piseshka Biserka , Havasi Andrea , Orso Evelyn , Dada Ashraf , Andreesen Reinhard , Holler Ernst , Edinger Matthias
Published: April 2020

Donor derived CD4+CD25+ regulatory T (Treg) cells do not induce graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (BMT), but actively suppress GVHD induced by CD25− conventional T (Tconv) cells when co-transplanted at a 1:1 ratio in murine model systems. Purified human CD4+CD25+ Treg cells display similar phenotypic and functional characteristics as their murine counterparts, i.e. they constitutively express high levels of intracellular CTLA-4, preferentially express the forkhead/winged helix transcription factor Foxp3, they are anergic to T cell receptor-mediated stimulation, and suppress the proliferation of Tconv cells after polyclonal or allogeneic activation in vitro. Thus, their adoptive transfer is a promising strategy for the prevention of GVHD in patients after allogeneic BMT. For potential clinical applications, efficient protocols for the isolation of CD4+CD25+ Treg cells from peripheral blood are required. In laboratory-scale experiments we determined that depletion of B cells (that partially express CD25), followed by repetitive positive selection of the remaining CD25+ cells with paramagnetic beads, yields a more than 90% pure population of CD4+CD25+ T cells. We now applied this protocol for the large-scale isolation of human CD4+CD25+ T cells from leukapheresis products of healthy volunteers under GMP conditions using the CliniMACS® device. B cell depletion with CD19-beads, followed by three cycles of CD25-enrichment with CD25-coupled microbeads resulted in an average 90% purity of CD4+CD25+ T cells (range 86% to 94%). Positively selected cells constituted on average 1.56% of MNC (range 0.84% to 2.0%), resulting in absolute numbers of 63 to 261 x 106 cells, depending on the initial size of the leukapheresis product. Since T cells with regulatory capacity predominantly reside within the CD4+CD25high subpopulation, we further determined the relative proportion of CD25high to CD25intermediate CD4+ T cells within the cell product. CD4+CD25high T cells represented on average 42% of the purified CD4+CD25+ T cell population (range 26% to 60%) as compared to 10.6% (range 8.5% to 13%) of the CD4+CD25+ T cells within the starting population. CD4+CD25+ T cells isolated according to this GMP protocol expressed higher levels of Foxp3 mRNA and Foxp3 protein as well as higher levels of intracellular CTLA-4 as compared to CD4+CD25− T cells. In addition, they showed functional characteristics of Treg cells as they were hypoproliferative to polyclonal stimulation in vitro and inhibited the proliferation of co-cultured CD4+CD25− T cells in a standard suppression assay. The ability to isolate highly enriched Treg cell populations in sufficient numbers under GMP conditions will accelerate their clinical application in allogeneic BMT.
Isolation and Expansion of Functional Differentiating Mesenchymal Stem Cells with a Serum Deprived Medium.
Authors: Dominici Massimo , Mueller Ingo , Cafarelli Luigi , Kordowich Sandra , Sternieri Rita , Spano Carlotta , Chiodino Caterina , Mariotti Ilaria , Petak Istvan , Horwitz Edwin , Paolucci Paolo , Conte Pierfranco
Published: April 2020

The infusion of mesenchymal stem cells (MSC) is an increasingly utilized therapeutic approach for congenital and acquired diseases in regenerative medicine. Recently, MSC have been used for the treatment of graft-versus-host disease after allogeneic stem cell transplantation[1]. These strategies require ex vivo MSC expansion with media containing fetal bovine serum (FBS). Since the use of FBS may be associated with immunological responses against the cultured MSC[2] and may unknowingly transmit prions[4], the development of a serum free medium (SFM) for MSC isolation and expansion is essential for the widely applicable MSC therapy.

We evaluated a novel serum deprived medium (Quantum 333R) for the isolation and expansion of MSC as compared to the standard medium (SM), DMEM + 10% FBS. Fibroblastoid colony forming unit (CFU-F), as MSC progenitors, and MSC expansion potential were considered as measurable parameters. The number of CFU-F found at 14 days of culture in the SFM (average: 1 CFU-F/ 1.78 x 105 bone marrow mononuclear cells, BMMNC) and the SM (1 CFU-F/ 2.3 x 105 BMMNC) were similar (p=0.18 t-test), but the trend suggested that the SFM may be superior. Passage 5 MSC in both culture conditions showed similar percentages of proliferating, i. e. BrdU+ cells: 7,5% (+/− 1,3%) and 10% (+/− 2,1%) in MSF and in SM, respectively. Flow cytometric analysis of MSC for side and forward scatter properties showed that MSCSFM were smaller in the side scatter than MSC SM. Further immunophenotypical characterization showed, in the MSCSFM, the absence of CD45, CD34, CD31 and the presence of CD90, CD105, CD73 confirming the phenotype of the “classical” MSCSM. CD73 and CD105 were upregulated in MSCSFM in comparison to MSCSM. Still, MSCSFM retained the capacity to inhibit the cytokine-induced proliferation of PBMC as reported for MSCSM. In addition, to test the differentiation potential of MSCSFM, the cells were successfully driven into osteogenic, adipogenic, myogenic and chondrogenic phenotypes. Our data support that Quantum 333R is able to isolate and expand MSC clonogenic precursors without affecting their proliferation, immunophenotype, differentiation and immunomodulatory potentials, thus suggesting a superior technique to isolate clinical grade cell population for safer clinical applications.
Ex-Vivo Expansion of Megakaryocyte Progenitors.
Authors: Giulivi Antonio , Halpenny Mike , Birch Paul , Yang Lin , Martin Lisa
Published: April 2020

Following Bone Marrow Transplant, thrombocytopenia and neutropenia always occur and patients require additional post transplant support in the form of platelet transfusions.

Megakaryocytes (Mk), the precursors of platelets, are contained in hematopoietic progenitor cell products but their number is variable and relatively low. The infusion of ex vivo expanded Mk precursors could be beneficial by shortening the time to platelet engraftment and therefore reducing the amount of platelet transfusion support required by bone marrow transplant patients. The objective of this project was to investigate the expansion of Mk progenitors from peripheral blood stem cell (PBSC) harvests from patients with haematological malignancies. Briefly, CD34+ cells were isolated and cultured in serum free media supplemented with thrombopoietin (TPO) and Interleukin 1 (IL-1) then incubated at 37°C /5% CO2 for 8 – 12 days. Megakaryocyte progenitor analysis was accomplished using flow cytometry analysis (CD34+/41+, CD41+, CD61+) and Mk culture analysis (CFU-Mk) (Stem Cell Technologies). Mk progenitor expansion efficiency was determined as “fold expansion” of Mk progenitors produced over input levels.

After 8 days of culture, a mean expansion of 46 fold (range 1.2 – 327.0, n=10) in megakaryocytic cells (CD61+) and a 15 fold expansion (1.2 – 41.7, n=10) in megakaryocyte progenitor cells (CD34+/41+) was observed. After 12 days, a 116 fold expansion (1.5 – 286, n=7) in megakaryocytic cells (CD61+) and a 19 fold expansion (2.4 – 40, n=7) in Mk progenitors (CD34+/CD41+) was observed.

This study demonstrates that CD34+ cells can be used to effectively expand megakaryocytic cells using just two cytokines for an incubation period of 8 – 12 days. This data could be used to develop future protocols for use in clinical applications.
Genome-Wide Array-Based CGH for Mantle Cell Lymphoma: Precise Mapping of Genomic Aberrations and Identification of Novel Candidate Genes.
Authors: Tagawa Hiroyuki , Karnan Siversundram , Suzuki Ritsuro , Ota Akinobu , Tsuzuki Shinobu , Morishima Yasuo , Nakamura Shigeo , Seto Masao
Published: April 2020

Mantle cell lymphoma (MCL) is characterized by 11q13 chromosomal translocation and CCND1 overexpression, but the additional genomic changes are also important for lymphomagenesis. To identify the genomic aberrations of MCL in higher resolution, we analyzed 29 patient samples and seven cell lines (SP-53, Z-138C, Granta 519, REC-1, NCEB-1, Jeko-1 and JVM2) using array-based comparative genomic hybridization (array CGH) that consisted of 2,304 BAC/PAC artificial chromosome clones, covering the whole genome at a 1.3 mega base resolution. Tumor specimens obtained from 29 MCL patients, comprising 16 males and 13 females all from the Aichi Cancer Center, were included in the study. The median age of the patient was 67 years (49–92 years old). 67% of cases were leukemic, 93% were in an advanced stage III-IV, 30% had elevated LDH, 18% had a poor performance status and 78% had more than one extranodal site of involvement. Genomic profiles of MCL were significantly different from those of other B-cell lymphomas such as diffuse large B-cell lymphoma (n=66) (Cancer Res 2004: 64, in press) and follicular cell lymphoma (n=13). Frequent imbalances detected by array CGH in patients were gains of chromosomes 3q26, 7p21, 6p21, 8q13-8q24, 10p12 and 17q23 (frequencies: 21%–48%), and losses of chromosomes 1p36, 1p21–p32, 1q42, 2p11, 2q13, 5q21, 6q16–q24, 8p12–p23, 9p21–p24, 10p14, 9q22, 11q22, 13q21, 13q34, 17p11.2–p13, 19p13 and 22q12 (frequencies: 17%–83%). Among these gains and losses, patents with a gain of 8q13-q24, and losses of 1p22–p32, 6q16–q24, 8p12–p23 and 17p11.2–p13 showed prognostically inferior survival rate to those without such a gain or losses (P<0.001). The incidence of genomic aberrations identified by array CGH was generally higher than that of chromosomal CGH studies. For example, our data show more frequent losses of 1p21–p22 (52%) and 9p21 (41%, INK4/ARF locus) as well as of 11q22 (55%, ATM locus), than the corresponding losses detected by chromosomal CGH (e.g., loss of 1p21–p22: 24%; 9p21: 28%; 11q22: 24%). We found recurrent regions of high-level genomic gains (amplifications) at 10p12 (BMI-1 locus), 13q31–q32 and 18q21 (BCL2 locus), and homozygous losses (deletions) such as 2p11 (Igk locus), 2q13 and 9p21.3–p24.1 (INK4a/ARF locus) in either patient or cell line. Furthermore, we investigated genomic gain of 13q31–q32 and homozygous loss of 2q13 and identified the C13orf25 as a candidate gene at 13q31 gain (Cancer Res 2004: 64, p3087) and the BIM gene at 2q13 loss. Precise mapping of genomic aberrations by array CGH allowed identification of novel candidate genes that might be associated with the lymphomagenesis of MCL.