bluuebunny/crossref_metadata_2025 huggingface dataset:
Clinical Presentation, Treatment Response and Survival in Adult Acute Biphenotypic Leukemia: Single Centre Experience.
Authors: Abdulwahab Amal S. , AlJurf Mahmoud D. , Elkum Nasser M. , Almohareb Fahad I. , Owaidah Tarek M.
Published: October 2019

Background: Acute leukemia is classified as lymphoid or myeloid depends on antigen expression. Knowledge about acute biphenotopic leukemia (BAL) is limited; both in terms of clinical and biological presentation and also in regard to treatment outcome. Hereby we are completing the work done by our group which looked at the laboratory features of BAL by reviewing the clinical presentation and response to different chemotherapy protocols used at our institute for treatment of patients with BAL to estimate over all survival.

Methods: Out of 478 adult patients with acute leukemia, we analyzed 15(3.1%) cases classified as BAL according to EGIL score system. Clinical data were collected retrospectively and analyzed. Five different chemotherapy protocols had been used. Six(40%) patients had received VCEPM (Vincristine 1.4mg/m2 x 4 doses, Cyclophosphamide 600mg/ m2 , Etoposide 100mg m2 daily for 5 doses, Prednisone 60mg/m2 for28 days). Four (27%)cases had received 1423 KFSH protocol (Cyclophosphamide 1200mg/m 2 x 1 dose, Daunorubicin 45mg/m2 for 3 doses, Vincristine 1.4mg/m2 once every week x 4 doses, Prednisone 60mg/m2 for 21 days, L-Asparaginase 6000 iu/m 2 every 3 days x 6 doses). Standard ICE protocol was used for induction in 3 cases and in 2 patients modified St Jude protocol (Vincristine 1.5mg/m 2 once every week x 4 doses, Daunomycin 25mg/m2 once every week x 2 doses, Prednisone 40mg/m 2 daily for 25 dose, L-Asparaginase 10000u/m 2 every 3 days for 6 doses, Etoposide 300mg/m 2 every 4 days x 3 doses and Cytarapin 300mg/m 2 every 4 days x 3 doses).

Results: We had 11 males and 4 females with mean WBC count on presentation of 74.7 x 109/L.Chromosomal karyotype was done for all except one, 4(29%) were normal and 8(53%) had complex karyotype Of all patients with BAL leukemia, 4(27%), 8(53%) and 10(67%) had hepatomegaly,splenomegaly and lymphadenopathy respectively. Initial evaluation done on D14 post chemotherapy revealed evidence of residual leukemia (> 5% blasts) in 1 out of 6 patients treated with VCEPM and 7 out of the other 9 patients received different other protocols. Out of the eight patients with residual leukemia, 4 had received salvage chemotherapy with FA (Fludarabine 30mg/ m2, Cytarabine2mg/ m2 x 5doses) and 2 had received MEC (Mitoxantrone 6mg/ m2 , Etopside 80mg/ m2, Cytarabine1mg/ m 2 x 6 doses) and one received modified St Jude while one underwent for SCT. ALL except one had achieved CR. Total of 10(66%) underwent SCT in CR. Although there were 9 (60%) died during the follow up, the OS at two years was (50%).

Conclusion: Adult BAL is an aggressive disease that is associated with complex karyotype with poor response to single lineage treatment. The use of hybrid drugs (VCEPM like) that are effective for lymphoid and myeloid leukemia improve the respond.

Figure Figure
Hypersensitivity and Allergic Reations to L-Asparaginase in Childhood ALL.
Authors: Santo Shelly , Kapelushnik Joseph , Moser Asher M.
Published: October 2019

Asparaginase (ASP) is an effective antileukemic agent. However, hypersensitivity and/or allergic reactions (HA), estimated to occur in 2.5%–45% of patients are major limitations for its clinical use. The risk of developing hypersensitivity and/or an allergic reaction to ASP increases with higher doses, IV (vs. IM), continuous infusions and repeat treatment after prolonged abstinence. We surveyed the rate of HA in pediatric ALL in Israel, and identified additional factors of clinical significance.

Methods: Data was gathered from the medical records of 245 children treated for ALL during the years 2000–2006 from 4 Pediatric Hematology-Oncology wards. All children were enrolled on the Israel National Leukemia Study 1998 or 2003, modified from BFM protocols. In Brief, patient received 8 doses of ASP in the induction and 4 additional doses in each reinduction, starting after a two-month period free of ASP. The uniform treatment used an E coli L-ASP preparation and was replaced with a pegylatedE coli ASP preparation in case of HA. The definition and severity of the HA was defined by the professional opinion of the treating medical team.

Results: Forty-Eight of the 245 patients (19.6%) developed an HA reaction. 25 of the 48 were defined as severe. A wide range of HA was noticed between the treating centers (6.4%–29.4%). 16/48 of the children with HA were treated on a high-risk protocol (33% vs. 14% of the entire group, p<0.05), including 10/25 (40%) with severe HA. A higher incidence of HA was noticed at the start of the reinduction, with 23 (47.8%) of the patients developing a HA to ASP on the reinduction cycle’s first or second dose. Pegylated ASP had significantly more HA vs. E. coli L-ASP treatments (8/145; 5.5% vs. 37/2610; 1.4%, p <0.01). PEG was also associated with allergies of higher severity, (8/9; 88.9% vs. 43.6%). A careful analysis revealed no differences between the allergenic and non-allergenic groups of children in relation to sex, age, mortality and/or chromosomal abnormality.

Conclusions: The overall HA rate (with the noticed wide variations) and the high incidence at the beginning of the reinduction protocol are within the described literature, We believe that the lack of steroid treatment that precedes ASP in the reinduction contributes to the high rate of HA at this point. The findings regarding the PEG were unexpected. We found a relationship between the higher risk group and the development of HA as well as the severity. We believe that the differences observed between treating wards are due to unclear definitions of reaction and of severity .We have noticed a wide variability in executing the supporting treatment. We recommend a revision of the reinduction protocol to initiate steroids at the beginning of all treatment cycles and to consider the use of a different than E. coli source after a hypersensitivity or allergic reaction.
Asparagine Depletion, Safety and Antibody Production after E Coli Asparaginase Treatment in Children with Newly Diagnosed Acute Lymphoblastic Leukaemia Treated with EORTC 58951 Protocol: A Single Center Report.
Authors: Bertrand Yves , Goutagny Marie Pierre , Poulat Anne Lise , Girard Sandrine , Acquaviva Cecile , Saban Christine Vianey , Vermot-Desroches Claudine , Bourdin Laurence , Alex Cedric , Rougemond Eric , Kebaili Kamila
Published: October 2019

Randomised trials in pediatric ALL show that multiple doses of E Coli Asparaginase (ASP) are associated with better outcome. In the EORTC 58951 protocol, all the patients except those with high risk features (HR or VHR) had 8 IV doses of ASP during induction Ia and were randomized during induction Ib to receive or not ASP (8 IV doses vs 0) and protocol II (8 IV doses vs 4), respectively long arm and short arm. The aim of this study was to report on safety, asparagine (ASN) depletion and antibody production in 28 consecutive patients with newly diagnosed previously untreated ALL.

Methods. Twenty-eight patients included in the EORTC 58951 protocol were analyzed: one had very low risk features, 22 were average risk patients and 6 had HR features. Thirteen patients were randomized in the short ASP arm and 9 in the long ASP arm. ASN levels were assessed on days 35 of Ia, 27of Ib and 22 of IIa. The amino acid assays employed the HPLC method. Serum samples were taken for antibody titers against ASP analyzed by ELISA on days 12 and 35 of Ia, 17 and 27 of Ib (long arm only) and 8 and 22 of IIa, and before the start of maintenance therapy.

Results. Twenty-eight patients aged 1year 3 months to 14 years 6 months (median 6 years) with newly diagnosed ALL (precursor B-cell: 25 ; T-cell: 3) were assessable. All patients achieved CR after induction phase Ia. Four patients had allogeneic bone marrow transplantation in first CR because of VHR features. Two other patients with HR features had intensified chemotherapy. Preliminary data showed that ASN depletion was obtained in all patients at the end of phase Ia, in all the patients “long arm” at the end of Ib and in one patient ” short arm” at the end of Ib All patients had depletion of ASN after protocol IIa We observed very few severe clinical allergic reactions in this series of patients (10%). Two patients were switched to Erwinia asparaginase because of clinical hypersensitivity. Investigation on correlation between the antibody production against ASP, clinical allergic reaction and depletion of ASN are in progress.

Conclusions. Multiple doses of ASP given intravenously were well tolerated in this series of 28 consecutive patients with previously untreated ALL. Preliminary results reveal that the rate of antibodies production was low and depletion of ASN was obtained in near all the patients. Some patients presented with clinical allergic reactions but had low levels of ASN and no appearance of anti ASP antibodies. Other had antibodies without clinical allergy and with depletion of ASN.
Fungal and Bacterial Infections in Acute Myeloid Leukemia Patients Treated with Induction Regimens Including Fludarabine: A Retrospective Analysis of 224 Cases.
Authors: Malagola Michele , Peli Annalisa , Damiani Daniela , Candoni Anna , Tiribelli Mario , Martinelli Giovanni , Piccaluga Pier Paolo , Paolini Stefania , Lauria Francesco , Bocchia Monica , Gobbi Marco , Pierri Ivana , Zaccaria Alfonso , Zuffa Eliana , Mazza Patrizio , Priccolo Giancarla , Gugliotta Luigi , Bonini Alessandro , Visani Giuseppe , Cristina Skert , Cesare Bergonzi , Aldo Roccaro , Carla Fili , Fanin Renato , Baccarani Michele , Russo Domenico
Published: October 2019

Infections are the major cause of morbidity and mortality of acute myeloid leukemia (AML). Invasive Fungal Infections (IFIs) occur in at least 10 to 20% of the patients submitted to induction and consolidation treatments, are responsible for death during induction in up to the 5% of the cases and may cause a delay in consolidation and intensification therapy. Among the risk factors for IFIs it has been included the use of Fludarabine (Fluda), which can induce severe and prolonged immunosuppression. In this study we retrospectively analyzed the infections occurred in 224 newly diagnosed AML patients, aged at least 65 years, consecutively treated with an induction regimen including Fluda, Ara-C and idarubicine with or without etoposide (FLAI/FLAIE). During induction phase, 181/224 (81%) patients experienced a fever of undetermined origin (FUO), the incidence of Gram negative and positive sepsis was 16% (37/224) and 29% (65/224) respectively and 7/224 (3%) patients developed a possible/probable IFI. In 6/224 patients (3%) a proven IFI was found (4 aspergillosis and 2 candidiasis). We then collected the data of the incidence of infections during the first consolidation course (FLAI: n=70; high-dose Ara-C [HD-AC]: n=65; idarubicine and HD-AC: n=89). The overall incidence of FUO was 34% (76/224), the number of Gram negative and positive sepsis was 52/224 (23%) and 49/224 (22%), respectively and 2/224 (1%) patients developed a proven IFI (3 aspergillosis and 1 candidiasis). We subsequently evaluated the incidence of infections in the three different consolidation groups. No significant differences were observed in terms of FUO, Gram positive and negative bacteraemia/sepsis and possible, probable and proven IFIs, during consolidation with Fluda-based regimen and with HD-AC-based regimens. Interestingly, the overall incidence of IFIs during consolidation with FLAI was significantly lower than during consolidation with HD-AC-based treatment program (0% vs 9%; p=0.02). These data, even though retrospectively collected, suggest that Fluda-based chemotherapy is not followed by increased incidence of infections, in particular IFIs, in comparison with conventional non-Fluda based regimens commonly used for AML induction. In our series, Fluda-based consolidation chemotherapy caused a significantly lower incidence of IFIs compared to HD-AC-based consolidation. This may be related to the lower duration of neutropoenia in patients treated with FLAI with respect to those treated with HD-AC/HD-AC + idarubicine.
Prognostic Index for Older Adult Patients with Newly Diagnosed Acute Myeloid Leukemia: The Edouard Herriot Hospital Experience.
Authors: Plesa Claudiu , Le Quoc-Hung , Chelghoum Youcef , Elhamri Mohamed , Tigaud Isabelle , Anglaret Bruno , Ducastelle Sophie , Perrin Marie-Claire , Nicolini Frank , Belhabri Amine , Troncy Jacques , Thiebaut Anne , Revesz Daniela , Wattel Eric , Dumontet Charles , Michallet Mauricette , Thomas Xavier
Published: October 2019

The treatment of older adults with acute myeloid leukemia (AML) is associated with unsatisfactory rates of complete responses and long-term overall survival. Therefore, a clinically usefull prognostic index can facilitate therapeutic decision making and evaluation of investigational treatment strategies in this patient population. Overall, 243 of the 432 patients (56%, 95% CI: 51–60%) achieved CR (229 of them after the first induction course and 14 after salvage therapy). The median disease-free survival (DFS) and the median overall survival (OS) of the entire cohort were 8.4 months (95% CI: 7.2–10.1 months) and 8.3 months (95% CI: 7.2–10 months) respectively. A prognostic score is presented based on the multivariate analysis of 432 newly diagnosed non-M3 AML patients aged more than 60 years, selected on the base of their initial performance status and the absence of severe co-morbidity factors, for entering onto five successive clinical trials combining an anthracycline and cytarabine. Four clinically relevant parameters are included in this index: cytogenetics at diagnosis, history of previous hematologic disorder, hematologic features at diagnosis, and LDH level at diagnosis. Using this stratification system, three risk groups were defined: a favorable-risk group A (OS of 39% at 2 years and 21% at 5 years), an intermediate-risk group B (OS of 19% at 2 years and 8% at 5 years), and a poor-risk group (OS of 5% at 2 years and 0% at 5 years). The prognostic index estimates the outcome of elderly AML patients usually selected for intensive chemotherapy trials using four easily determined parameters and might identify patients who are really candidates for this treatment strategy from those for whom investigational therapy or palliation may be most appropriate.
Modified Topotecan Combination with Cyclophosphamide and Ara-C (CAT) for Patients with Refractory/Relapsed Leukemia.
Authors: Wu Sui-jing , Du Xin , Lin Wei , Huang Zi-lun , Zhou Xiao-li , Weng Jian-yu , Zhong Li-ye , Lu Ze-sheng
Published: October 2019

The prognosis of patients with leukemia has improved significantly. With combination chemotherapy, 60%–80% of adult patients with acute myeloid leukemia (AML) and acute lymphoblastic leukemia(ALL) achieve complete remission(CR). But most patients eventually relapse and do not response to current induction regimens based on cytarabine and anthracyclines any more. This emphasize the need to discover new agents for the treatment of leukemia to improve long-term prognosis. Topotecan is a semisynthetic analogue of the alkaloid camptothecin which acts as a specific inhibitor of topoisomerase I,by stabilization of the topoisomerase I DNA complex which leads to cell death.It also appears to be active against leukemias which have acquired the multi-drug resistant phenotype.[ Ulukan et al. Drug, 2000; Weihrauch et al. Leuk Lymphoma, 2004].It also could go through blood brain barrier[Kollmannsberger et al. Oncology, 1999].Here we observe the effect and side-effects of cyclophosphamide, cytanthetic analogue of rabine and topotecan (Modified CAT) combination regimen for treatment of refractory or relapsed leukemia. The study population comprise 26 patients with relapsed/refractory leukemia,including 12 with AML, 9 with ALL, 2 with myeloid blast phase of CML, 3 with lymphoma. Median age was 35 years.Patients received cyclophosphamide 300mg/m2 every 12 hours for 6 doses on day1 to 3.On day 2, topotecan was given at a dose of 1.5mg/m2/day for 5 days on days 2 to 6, and cytarabine 1.0 g/m2 /day for 5 days on days 2 to 6.The regimen (cytarabine reduced to 0.5g/m2 /day) could be repeated one or two cycles after remission. The total response rate of one course was 57%, eleven patients (42%) achived complete remission(CR). Responses occurred in 7of 12 AML (57%), including 5 CR(41%); in 6 of 9 patients with ALL (67%), including 5 CR(56%); and in 1 of 2 myeloid blast phase of CML(CR 50%). The median overall survival time after treatment was 2+ months, and the median overall survival time after CR was 4+ months. Severe myelosuppression was universal (100% Grade 4 neutropenia and thrombocytopenia), the median time to recovery of neutrophils to ≥0.5×109/L was 18 days,and the median time to platelet recovery to ≥ 20×109/L was 26 days.Infection developed in 96% cases, the most frequent events were oral mucositis(77%, 20/26), sepedogenesis(46%, 12/26),anusitis(38%,10/26),pneumonia(31%, 8/26), two patients died of infection complications(8%). Non-hematologic toxicity was seen frequently in gastrointestinal but it was mild. Nausea and/or vomiting occurred in 13 patients(50%)with gradeI-II in all of them.Two patients(8%) had diarrhea and grade 1–2 in them. In summary, Modified CAT regimen is well tolerated and has significant anti-leukemia activity as salvage therapy for relapsed/refractory leukemia.
The Ontogeny of Definitive Hematopoiesis in the Zebrafish.
Authors: Traver David , Bertrand Julien Y. , Kim Albert D. , Stachura David L. , Cisson Jennifer L.
Published: October 2019

Shifting sites of blood cell production during development is common across widely divergent phyla. In zebrafish, like other vertebrates, hematopoietic development has been roughly divided into two waves, termed primitive and definitive. Primitive hematopoiesis is characterized by the generation of embryonic erythrocytes in the intermediate cell mass and a distinct population of macrophages that arises from cephalic mesoderm. The generation of definitive, or multilineage, hematopoietic precursors during embryogenesis remains less well understood. Here we show, using a combination of gene expression analyses, prospective isolation approaches, hematopoietic progenitor cultures, transplantation, and in vivo lineage tracing experiments, that definitive hematopoiesis initiates through committed erythromyeloid progenitors (EMPs) in the posterior blood island (PBI) that arise independently of hematopoietic stem cells (HSCs). EMPs isolated by coexpression of fluorescent transgenes driven by the lmo2 and gata1 promoters exhibit an immature, blastic morphology and express only erythroid and myeloid genes. Transplanted EMPs home to the PBI, show limited proliferative potential, and do not seed subsequent hematopoietic sites such as the thymus or pronephros. In vivo fate mapping studies similarly demonstrate that EMPs possess only transient proliferative potential, with differentiated progeny remaining largely within caudal hematopoietic tissue. By contrast, fate mapping studies of CD41:eGFP+ cells residing in the aorta/gonads/mesonephros (AGM) region demonstrate robust colonization of the pronephros and thymus. Using timelapse microscopy, we show that these sites of adult hematopoiesis are seeded by CD41+ cells that migrate along the pronephric ducts from the AGM. These studies provide phenotypic and functional analyses of the first hematopoietic stem and progenitor cells in the zebrafish embryo and demonstrate that definitive hematopoiesis proceeds through two distinct waves during embryonic development.
Efficacy and Tolerability of Lenalidomide/Dexamethasone in Intensively Pretreated Myeloma Patients: Experiences from the German Named Patient Program.
Authors: Klein Ulrike , Blau Igor W. , Engelhardt Monika , Simon Jan P. , Goldschmidt Hartmut , Einsele Hermann , Knop Stefan
Published: October 2019

Lenalidomide (Len), an immunomodulatory drug, in combination with dexamethasone (Dex) is a new treatment option for patients (pts) with relapsed or refractory multiple myeloma (MM). Reimbursement and drug laws in Germany allow treatment before marketing authorization of a given drug, if no alternatives are available for life-threatening diseases. AIM: To evaluate efficacy and tolerability of MM pts treated with Len/Dex in the German named patient (pt) program.

PATIENT AND METHODS: A comprehensive, previously tested, questionnaire was used to document data from consecutive pt series in five large and specialized university treatment centres. Response was assessed using the EBMT criteria.

RESULTS: 55 pts were documented so far (median age, 63 yrs; 92% stage III Durie & Salmon). Pts were heavily pretreated: 30% had up to 2 previous therapy lines, 35% had three, 13% four and 21% more than four; the median no. was 3 (interquartile range, IQR 2–4). Pts had received a median of 16 (IQR 10–22) previous courses of chemotherapy. Forty-nine patients (89%) had previously received a stem cell transplant (SCT): 41 an autologous and an additional eight allogeneic SCT. That correlates with a 2-fold higher proportion of pts with prior SCT compared to the Len registration trial. Furthermore, the proportion of pts treated with the new drugs thalidomide or bortezomib was even 7–10-fold higher than in the registration trial. Best response could be assessed in 49 pts.: 4% CR, 32% PR, 19% MR, thereby showing an overall reponse rate of 55%. Four patients (8%) progressed during therapy and two (4%) died before response evaluation. The table shows response in different subgroups according to previous therapies. Response (CR/PR/MR) ranged from 88% (7/8) with only 1 previous treatment line shown in the table to 52% (16/31) with 2–3 and 43% (6/14) with 4–5. The most common adverse event III°/IV° was infection occurring in 21/55 (38%) patients. Thrombo-embolic events were observed in three patients (5%). Haematological toxicity grade IV° was seen in 11/46 (24%) patients, most common in course 1 of therapy. Five patients (9%) died during treatment, four due to infection (one in neutropenia) and one patient due to cerebral bleeding. 70% of patients were alive at last follow-up.

CONCLUSION: In this pt cohort Len/Dex induced considerable antineoplastic activity despite extensive pretreatment. Toxicity was moderate and as expected for this risk-cohort. Nevertheless pts with more than 3 prior treatment lines should receive close clinical monitoring in order to prevent life-threatening infection.

Response in different subgroups Over all response Prev. alloSCT Prev. ASCT Prev. thalidomide Prev. bortezomib 3 prev. treatment lines>> n 49 8 40 30 39 19 CR/PR/MR 55% 50% 53% 50% 54% 42%
Alteration in Melphalan Conditioning Regimen for Multiple Myeloma: Reflection on Therapeutic Benefit and Length of Hospital Stay.
Authors: Gopaluni Srivalli , See Heidi , Coyle Thomas , Gentile Teresa C.
Published: October 2019

BACKGROUND: Melphalan is the most common conditioning regimen prior to autologous stem cell transplant in patients with Multiple Myeloma. Conventionally Melphalan was administered at a dose of 200mg/m2 for 2 days[100 mg/m2 each day] prior to transplantation on days -4 and -3 followed by autologous stem cell infusion on day 0. Recently we changed the scheduling of Melphalan to 200mg/m2 given on day-2 and stem cell infusion on day 0.

OBJECTIVE: A retrospective analysis was done to assess the effect of this change in Melphalan regimen on length of stay in hospital and gastrointestinal toxicity.

METHODS: We performed a retrospective analysis of 20 patients receiving melphalan as conditioning regimen prior to autologous stem cell transplant. All patients received similar induction therapy and were transplanted as part of their initial therapy. Assessment including maximum oral score per day of transplant (using the oral assessment guide developed by the University of Nebraska Medical Center) and number of days with nausea/vomiting/diarrhea were analyzed to compare gastrointestinal toxicity.

PATIENTS: Records on twenty patients with multiple myeloma undergoing autologous stem cell transplant from January 2003 until August 2006 were reviewed. Study population included10 females and 10 males. Average age was 60 yrs in melphalan 200mg/m2 over 2 days group and 55 in melphalan 200mg/m2 over one day group.

RESULTS: The average length of hospital stay was 17.4 days in the Melphalan 200mg/m2 over 2 days group and 15.4 days in the Melphalan 200 mg/m2 over 1 day group. The average number of days of reported nausea/vomiting and diarrhea was 7.1 in the Mel 200 mg/m2 over1 day and 7.6 days in the Mel 200mg/m2 over 2 day group. The average peak daily oral score in the Mel 200mg/m2 over 2 days was slightly higher (14 vs 12) and the duration of reported abnormal oral scores was slightly longer for the patients in the Mel 200mg/m2 over 1 day group (13 days vs. 10 days).

CONCLUSION: The major advantage of delivering Melphalan over one day as opposed to two days is decreased length of hospital stay and thereby the costs. However this does not appear to be at the expense of increased gastrointestinal toxicity. Although the duration of mucositis appears to be longer in the Melphalan 200mg/m2-1 day group, this did not lead to a prolonged length of stay in our patients. Reported nausea/vomiting/diarrhea was not significantly different in both groups.

Figure Figure Figure Figure
The Effects of Rapamune on NK Cell Post Bone Marrow Transplantation in Mice.
Authors: Chen Guanghua , Wu De Pei , Yang Ming Zhen , Tang Xiao Wen , Sun Ai-ning
Published: October 2019

Natural killer(NK) cells are innate effector cells of the immune system, believed to limit viremia and tumor burden before the onset of adaptive T and B cell immunity. NK cells are potent effector cells in allogeneic bone marrow transplantation. NK cell activity is partially controlled through interactions between killer Ig-like receptors on NK cells and their respective HLA class I ligands. Immunosuppressive agents including cyclosporin, FK506 and Rapamune are utilized in clinical hematopoietic stem cell transplantation. Little is known about the effects of immunosuppressive agents on NK cells post bone marrow transplantation. The in vivo effects of Rapamune on NK cells were determined in an allogeneic bone marrow transplantation model. Splenic NK cell levels in recipients treated with Rapamune decrease markedly. NK cell proliferation and function are significantly decreased in the presence of Rapamune. Studying the differential effects of immunosuppressive drugs on NK cell function is critical in clinical hematopoietic stem cell transplantation.
Uncovering the Epigenetic Pathomechanism in 13q14.
Authors: Mertens Daniel , Ruppel Melanie , Philippen Angela , Fleig Verena , Brakel Bianca , Stilgenbauer Stephan , Lichter Peter , Doehner Hartmut
Published: October 2019

INTRODUCTION: Deletions in chromosomal band 13q14.3 distal to RB1 occur in a variety of human neoplasms like B-cell chronic lymphocytic leukaemia (CLL), indicating a tumor suppressor mechanism in this region. Intriguingly, several characteristics of the region of interest point to an epigenetic pathomechanism:

candidate genes lack point mutations, yet these genes are downregulated in tumors, the presence of large non-coding RNA genes in 13q14.3 is reminiscent of imprinted regions where only one gene copy is active.

The data we show here led us to propose a novel oncogenic mechanism where already in healthy tissue only one gene copy is active while one gene copy is randomly chosen for silencing. Loss of the single active copy is then sufficient for complete loss of gene function in tumor cells. Currently we are trying to identify the (epi-)genetic element that controls the whole locus.

AIM: Identification of the epigenetic regulatory mechanism localized in 13q14.3.

METHODS and RESULTS: We performed FISH analyses of hematopoietic and non-hematopoietic cell lines to assess replication timing and chromatin packaging of the critical region. In line with an imprinting mechanism, we find that the two copies of the critical region replicate asynchronously and/or show delayed chromatid segregation, suggesting differential chromatin packaging of the two copies of 13q14.3. Next, we found by sequencing of SNPs that 13q14.3 candidate genes are expressed from one copy only in healthy probands. However, expression originated from either the maternal or paternal copy, excluding an imprinting mechanism. We could also show a functional interconnection of DNA methylation and gene expression, as demethylating agents and histone hyperacetylation induced biallelic expression. However, replication timing was not affected. Currently we are employing array- and capillary electrophoresis-based analysis of DNA-methylation (aPRIMES and bioCOBRA) and chromatin-immunoprecipitation on arrayed CpG-libraries (chIP on chip) with antibodies specific for histone modifications in order to identify the epigenetic element regulating the critical region.

CONCLUSIONS: We propose that differential replication timing represents an early epigenetic mark that distinguishes the two copies of 13q14.3, resulting in differential chromatin packaging and monoallelic expression. This has profound effects for the tumor suppressor mechanism localized in 13q14.3: Deletion of the single active copy of the region at 13q14.3, which is detected in more than 50% of CLL tumors, will suffice for complete loss of tumor suppressor function, as the remaining gene copies are epigentically silenced. In addition, we are currently identifying the locus control region that orchestrates gene expression in the critical region. Thus, we provide a model for the pathomechanism of 13q14.3 in CLL by the interaction of genetic lesions and epigenetic silencing.
Embryonic Stem Cells Transplanted In Utero Survive and Produce Teratocarcinomas in a Murine Model.
Authors: Nedelcu Elena , Srivastava Anand , Assatourian Gustavo , Varki Nissi , Carrier Ewa
Published: October 2019

Introduction: Embryonic stem (ES) cells have the capacity to differentiate into any cell type. We have previously established an in vivo murine model for the study of the in vivo differentiation of ES cells. The aim of this study was to monitor tumor formation in this model. Materials and

Methods: Murine ESC genetically engineered to express yellow fluorescent protein (YFP-ES cells) were cultured on mitomycin-treated feeder layers for four days prior to in utero transplantation in medium containing leukemia inhibitory factor. A cell dose of 5x104 YFP-ES cells (H-2b) was injected in utero intraperitoneally in the fetuses of Balb/c (H-2d) and C57BL/6 (H-2b) pregnant mice at E12- E14. The presence of YFP-ES cell-derived cells was analyzed in various tissues of the liveborn offsprings of different ages by fluorescence microscopy and quantified by digitalized fluorescence microscopy of frozen section from various tissues and organs and analysis of tissue supernatants for the presence of the YFP signal. Tumor formation was monitored in liveborn offsprings for up to one year and tissue sections were examined by routine and fluorescent microscopy.

Results: The YFP-fluorescent cells derived from YFP-ES cells were detected in liver and thymus without any evidence of migration in other organs. Extensive peritoneal teratocarcinomas with supradiaphragmatic involvement were generated in two out of five allogeneic in utero transplantation of YFP-ES cells. Tumor formation was not observed in the groups transplanted in utero with fetal liver or adult bone marrow hematopoietic cells, or in the control group. Clinical and histopathological pictures suggestive of graft-versus -host disease were also observed in two out of five mice transplanted in utero.

Conclusion: The promise and the potential risks of the ESC transplantation have to be carefully considered due to the high risk of teratoma/teratocarcinoma occurrence. Protocols using pre-differentiated cells from ES or adult stem cells may constitute a better approach for cellular therapies.
Cytotoxic Effects of CML28 Specific T Cells on Acute Leukemia Cells In Vitro.
Authors: Mao Hanwen , Liu Wenli , Gen Zhe , Huang Wei , Zhang Yicheng , Zhou Jianfeng , Sun Hanying
Published: October 2019

The antigen-specific cytotoxic T lymphocyte activated by antigen presenting cell is widely used in cell immunotherapy recently. CML28, which was screened from chronic myelogenous leukemia (CML) patients, was reported to be a specific tumour antigen and over-expressed on CML cells and acute leukemia cells. Therefore, CML28 could be a potential target for leukemia treatment. Dendritic cells (DC) are the most important antigen present cells, but it is hard to isolate and culture DCs for clinical use, which hampers the specific cell immunotherapy. Our investigation aimed to study the cytotoxic effects of CML28 specific T cells activated by artificial antigen presenting cells, on acute leukemia cells in vitro. Artificial antigen presenting cells were prepared by connecting CML28 to magnetic superbead that containing HLA-A2-Ig and B7-1 molecule. Mononuclear cells were isolated from the bone marrow or peripheral blood of healthy donors with positive HLA-A2. The artificial antigen-presenting cells were co-cultured with isolated mononuclear cells for four weeks. The activation and proliferation of CML28-specific T cells were measured by dimmer binding technique using flow cytometry. The cytotoxic effects of CML28-specific T cells on leukemia cells, which were isolated from leukemia patient, were evaluated by lactate dehydrogenase (LDH) releasing assay. Increased proportion of CML28-specific T cells was observed in artificial antigen-presenting group than in control group (29.27±3.54% vs 2.95±0.66%, p<0.05). For cytotoxic effects assay, significant higher killing efficiency was seen in artificial antigen-presenting group (41.47±4.23%vs3.56±0.71%, when the effector: target ration is 40:1, p<0.01). Therefore, we concluded that the artificial antigen presenting cells could mimic antigen presenting cells to induce specific T cell activation and proliferation, and cytotoxic effects on target cells, indicating that artificial antigen presenting cell-induced cytotocix T cells could be an option for leukemia treatment.
Recombinant Chimeric IgA1 and IgA2 Antibodies for Tumor Immunotherapy.
Authors: Dechant Michael , Beyer Thomas , Lohse Stefan , Schneider-Merck Tanja , Weisner Wencke , Berger Sven , Peipp Matthias , van de Winkel Jan G.J. , Valerius Thomas
Published: October 2019

The therapeutic potential of human IgA antibodies has hardly been explored, although IgA is the most abundantly produced antibody isotype in humans. Here, we describe the generation and functional characterization of human/mouse chimeric IgA antibodies against the epidermal growth factor receptor (EGF-R), which is a validated target antigen for tumor immunotherapy. Human IgG1, IgA1 and IgA2 variants of the C225 antibody were produced in CHO-K1 transfectomas using serum-free, non-adherent culture conditions. Productivity rates of selected IgA producing clones were approximately 5 pg/cell/day. A two-step purification procedure consisting of thiophilic agarose columns followed by gel electrophoresis on Superdex 200 yielded highly purified monomeric IgA antibodies, which were stable at 4° C. Silver staining and Western blots with human kappa-light- and human α-heavy chain antibodies confirmed the purity and correct assemby of IgA1 and IgA2 antibodies. Co-transfection of a His-tagged joining (J) chain significantly increased the amount of dimeric IgA antibodies, which were detected by anti-His antibodies. IgG1 and monomeric IgA antibodies demonstrated similar binding to EGF-R expressing A431 tumor cells (EC50 approx. 10 μg/ml) and to EGF-R transfectants. Furthermore, IgA1 and IgA2, but not IgG1 demonstrated binding to FcαRI (CD89)- transfected cells. All three isotypes were similarly effective in recruiting F(ab’)- mediated killing mechanisms such as blockade of EGF binding, inhibiting EGF-R phosphorylation, and mediating growth inhibition of EGF-R expressing cells. All three isotypes did not activate complement- mediated killing. Human IgG1 effectively triggered MNC- mediated ADCC, but activated PMN only weakly. Importantly, both human IgA isotypes were significantly more effective than IgG1 in recruiting neutrophils for ADCC (EC50 approx. 0.25 μg/ml), and triggered ADCC at effector to target cell ratios as low as 5:1. Since neutrophils constitute the most abundant effector cell population in human blood, IgA antibodies triggered higher levels of tumor cell killing in whole blood assays than human IgG1 antibodies - particularly when blood from G-CSF- primed donors was compared to healthy donor blood. Together, these data suggest that recombinant human IgA antibodies can be produced in reasonable amounts, and that they may constitute promising reagents for immunotherapy.
Immunostimulatory Properties of the Human Ortholog of the GMCSF/IL2 Fusion Protein for Cell Based Therapy.
Authors: Graduate Claudia Penafuerte , Galipeau Jacques
Published: October 2019

NK cells constitute a potential candidate for cancer cell therapy because they express a diverse array of inhibitory and activating receptors, which recognize and kill infected or tumor cells without prior immune sensitization. However, autologous NK cell mediated adoptive immunotherapy is restricted due to insufficient cytolytic activity of NK cells from patient with aggressive malignancies. In contrast, the infusion of alloreactive NK cells has shown more successful outcomes in the treatment of cancer, but this approach also presents difficulties such as the high doses of cytokines required to induce NK cell expansion ex vivo, which may also sensitize NK cells to apoptosis. Therefore, a critical issue for NK cell based therapy is the use of appropriate growth factors or cytokines that promote NK cell expansion and activation. We have previously shown that a murine GM-CSF/IL-2 fusion protein (aka GIFT2) displays novel antitumor properties in vivo compared to both cytokines in combination regarding tumor site recruitment of macrophages and significant functional NK cell infiltration [Stagg et al., Cancer Research (December 2004)]. In the present work, we found that human GIFT2 will lead to a substantial two fold proliferation of human blood-derived NK cells which is significantly (p<0.05) superior to either IL2 or GMCSF single cytokine treatment or both cytokines combined at equimolar concentration. In addition, we observed that GIFT2 leads to robust expression of NK-cell activation markers CD69 and CD107a. In conclusion, the human GIFT2 fusokine is a novel and potent tool for ex vivo expansion of activated NK cells which may be of use in cell-based immunotherapy of cancer.
KIR Incompatible NK Cells Effectively Lyse Osteosarcoma Cells.
Authors: Delgado David C. , Hardin Heather , Webster Dan , DeSantes Ken B. , Shaaban Aimen F.
Published: October 2019

Recurrent or metastatic pediatric solid malignancies, including osteosarcoma, carry a dismal prognosis despite modern multi-modality treatment approaches. Although the success of KIR (Killer Immunoglobulin-Like Receptor) incompatible, haploidentical stem cell transplantation has been documented in hematological malignancies in adults and children, this approach has not been thoroughly examined in pediatric solid tumors. In this study, we evaluated the potential for KIR-incompatible lysis of osteosarcoma cells, in vitro. We hypothesized that the killing of osteosarcoma cell targets could be predicted by the degree of inhibitory KIR receptor/ligand mismatch with NK cell effectors. To test this hypothesis, healthy donor NK cells were isolated by magnetic bead sorting and their KIR phenotype determined by flow cytometry. Consistent with previous studies, donor NK cells exhibited a high prevalence of all three relevant inhibitory KIRs (KIR2DL1, KIR2DL2/KIR2DL3, KIR3DL1). Conversely, examination of three established osteosarcoma cell lines (HOS, SaOS, and U2OS) demonstrated significant variability in cell surface HLA class I expression, by flow cytometry. QRTPCR analysis of these cell lines revealed variable expression of the three known inhibitory KIR ligands (HLA-C groups 1 and 2, HLA-Bw4). This variable KIR-ligand expression allowed evaluation of NK-mediated lysis of targets with varying KIR receptor-ligand incompatibility. Following a 12-hour incubation of donor NK cells in IL-2, lysis of osteosarcoma targets was measured in an annexin V flow cytometric assay. Osteosarcoma cell lines that expressed fewer KIR ligands consistently showed greater susceptibility to NK-mediated cytotoxicity. These findings were consistent using NK effectors from different donors. Additionally, we observed that at high passage number (>20), SaOS cells demonstrated down-regulation of KIR ligand expression. These changes correlated with increased lysis by the same donor NK cells. Our findings suggest:

Variable expression of KIR ligands in osteosarcoma cell lines allows potential susceptibility to KIR-incompatible, NK cell-mediated lysis; The killing of osteosarcoma cells by NK cells can be predicted by the degree of receptor/ligand mismatch; and During expansion, osteosarcoma cells may alter expression of KIR ligands, resulting in increased or decreased susceptibility to lysis by KIR-incompatible NK cells.

Further studies are needed to explore the utility of KIR-incompatible, haploidentical stem cell transplantation for patients with high-risk osteosarcoma.
Methylenetetrahydrofolate Reductase (MTHFR) Polymorphisms of Both Donor and Recipient Can Modify the Rate of Recurrence in Allogeneic Hematopoietic Stem Cell Transplantation.
Authors: Oh So Yeon , Kim Hye Jin , Yi Hyeon Gyu , Kim Jin Won , Piao Cheng Zhe , Chang Chongwon , Shin Eunsoon , Kim Inho , Yoon Sung-Soo , Park Seonyang , Kim Byoung Kook
Published: October 2019

Introduction Clinical significance of polymorphism in MTHFR gene is widely described in various disease entities. Polymorphism is associated with decreased metabolism of methotrexate. Because methotrexate is commonly used to prevent GVHD in HSCT, we studied transplantation outcomes according to genotypes of donor and recipient MTHFR; about the most studied two polymorphism sites (677C/T and 1298A/C). Materials and Methods Patients and donors who underwent allogeneic HSCT due to hematologic malignancy between 1998 and 2006 were retrieved. Patients had received MTX and cyclosporine for GVHD prophylaxis were included. The genotyping of MTHFR polymorphism was screened using SNaPShot Multiplex kit. Clinical data were collected retrospectively. Results We found 99 patients consisted of 60 males (60%) and 39 females (39%) in this homogeneous population. Median age was 34 years (range17–59). Diagnosis were acute leukemia (n=67), chronic leukemia (n=20) and other diseases (n=12). Donor was related(n=72) or unrelated(n=27). Conditioning was myeloablative in 95 patients and nonmyeloablative in 4 patients. Median PFS and OS were 6 (range 0–101) and 15 (range 0–108) months. Clinical factors and genotype are analyzed for risk of hepatic toxicity, recurrence, TRM and death. Peak AST and ALT levels are higher in the 677TT group than in the CC or TT group. In univariate analysis, recipient or donor 1298AA, AC, and CC were likely to relapse in ascending orders (figure1). This was reproduced in multivariate analysis (p=0.008 and 0.011, for each). About TRM and survival, there were no significant correlations with MTHFR genotypes in both univariate and multivariate analysis. Conclusion Simultaneously decreased activity of MTHFR due to 1298AC polymorphism of both donor and recipient might be translated into increased recurrence rate. Validation with randomized trial is required to compare methotrexate to other drug for GVHD prophylaxis.

Figure Figure
Results of a Randomized Phase II Trial of Pegfilgrastim Versus Filgrastim To Treat Neutropenia Post-Autologous Peripheral Blood Stem Cell Transplant (PBSCT) in Patients with Non-Hodgkin’s Lymphoma (NHL).
Authors: Rifkin Robert , Beveridge Roy , Spitzer Gary , Orloff Gregory , Mandanas Romeo , McGaughey Dean , Brunvand Mark , Zhan Feng , Boehm Kristi A. , Asmar Lina
Published: October 2019

Granulocyte colony-stimulating factor has been shown to decrease the time to neutrophil recovery following autologous PBSCT. Therefore, it was hypothesized that a single injection of pegfilgrastim (P) would mimic the role of filgrastim (F), resulting in at least an equivalent shortening of post-PBSCT neutropenia. PBSCT eligible NHL patients over the age of 18 years with preserved end-organ function were identified prior to the administration of high-dose chemotherapy, and following adequate stem cell harvest and cryopreservation (ie, >2.5 x 106 CD34+ cells/kg). All patients received either standard BEAM or BEAC high-dose chemotherapy (HDC). Prior to HDC, patients were randomly assigned to receive either P at a fixed-dose of 6 mg on Day +1(Arm A), or weight-based, dose-adjusted F rounded to the nearest prefilled syringe beginning on Day +1(Arm B) following transplantation. Between July 2003 and April 2007, 101 eligible patients were enrolled within our transplant network. Three patients were deemed ineligible (CHF death, patient withdrew consent, other). The analyses and results presented below outline the remaining 98 patients. The demographic characteristics of both arms were well-balanced with regards to stage at diagnosis, stage at treatment, ECOG Performance Status, histology, and prior therapy. The comparison of P vs F is summarized in the table below. Incidence of Grade 3–4 adverse events were comparable in both arms as was transplant-related mortality. In conclusion, administration of pegfilgrastim post-auto PBSCT appears to be equivalent to multiple daily dosing of filgrastim, and might be considered in lieu of filgrastim obviating the need for multiple injections.

Results - Comparison of Pegfilgrastim vs Filgrastim Variable Arm A (P) Arm B (F) No. of Patients Treated 50 48 Doses Received (mean +/− SD 1.0 +/− 0 12.7 +/− 2.6 Time to ANC Recovery (days) (mean +/− SD) 8.3 +/− 1.1 8.9 +/− 1.5 No. of RBC Transfusions (mean +/− SD) 1.7 +/− 0.9 1.9 +/− 1.2 No. Platelet Transfusions (mean +/− SD) 3.0 +/− 1.9 2.8 +/− 1.8 Positive Blood Culture Rate (%) 18.0% 29.0% Febrile Neutropenia (FN) Rate (%) 18.0% 16.7% Duration of FN (days) (mean +/− SD) 5.1 +/− 3.4 5.5 +/− 4.9