bluuebunny/crossref_metadata_2025 huggingface dataset:
Minimal Residual Disease (MRD) Evaluation by Flow Cytometry in Acute Leukemia Patients Undergoing Allogeneic Stem Cell Transplantation.
Authors: Vidriales Maria-Belen , Pérez Jose J. , López-Berges Consuelo , Gutierrez Norma C. , Ciudad Juana , Vazquez Lourdes , del Cañizo Consuelo , Díaz-Mediavilla Joaquín , Fernández-Calvo Javier , Ramos Fernando , Rodriguez María Jesús , Calmuntia María José , Orfao Alberto , Miguel Jesús San
Published: April 2020

Investigation of minimal residual disease (MRD) in acute leukemias by immunophenotyping is increasingly used for disease monitoring. Most MRD studies using flow cytometry techniques have focused on patients treated with conventional chemotherapy, while information on its value in patients undergoing allogeneic transplantation is scanty. The aim of the present study is to evaluate whether or not immunophenotypical assessment of MRD could also be a valuable tool in patients undergoing allogeneic transplantation for acute leukaemia. For that purpose we have analysed the level of MRD before and after (month +3) transplantation, by multiparameter flow cytometry, in a series of 38 acute leukaemia patients (26 ALL cases -20 cases in 1st CR and 6 in 2nd CR- and 12 AML cases -all of them in 1st CR), that showed an aberrant immunophenotype at diagnosis. Although the level of MRD in the BM obtained before transplantation showed a tendency to predict RFS, differences did not reached statistical significance. By contrast, the evaluation of the BM obtained 3 months after allo-transplantation had significant prognostic value. Thus, patients with low MRD levels (<0.05% for ALL and <0.2% for AML) (n=17) had a significantly longer RFS than patients with MRD positive (median RFS: 40 vs 16 months) (p=0.001). Multivariate analysis showed that the immunological evaluation of MRD after transplantation had independent prognostic value for RFS in acute leukaemia, together with age, WBC count, and cytogenetics. Our results suggest that immunological analysis of MRD should be included in the follow-up evaluation of acute leukaemia patients undergoing allogeneic stem cell transplantation.
Long Distance Cyclin D1 Activation in B Cell Malignancies: Role of RNA Polymerase II.
Authors: Liu Hui , Wang Jin , Maziarz Richard T. , Epner Elliot M.
Published: April 2020

Translocations involving the immunoglobulin heavy chain gene locus (IgH) are common in B cell malignancies. One common target gene is cyclin D1, which is deregulated in most patients with mantle cell lymphoma (MCL) and approximately 15–20% of patients with multiple myeloma (MM). Cyclin D1 is not expressed in normal lymphocytes; cyclin D1 expression in B cell malignancies is deregulated by IgH translocations and insertions. We have shown that the cyclin D1 locus, including the promoter and upstream regions, are acetylated and DNA hypomethylated in both malignant and nonmalignant B cells (Liu et al, Blood in press). Using chromatin immunoprecipitation (ChIP) assays, we have found that RNA polymerase II (Pol II) is a) present at the cyclin D1 promoter b) located at regions far upstream of the cyclin D1 gene and c) present at 3′ Cα IgH regulatory elements only in cyclin D1 expressing malignant B cells. Pol II is present at the translocation breakpoint in a MCL cell line, but we do not find Pol II present continuously from the IgH regulatory regions to the cyclin D1 promoter. Confirmatory RT-PCR analyses also do not demonstrate continous evidence of RNA transcripts extending from the IgH regulatory elements to the cyclin D1 promoter. Mutants derived from gene targeting experiments that have lost the translocated chromosome show Pol II binding only at the Eu intronic enhancer, consistent with known promoter acivity and sterile transcripts originating at this location. Our data suggest that Pol II may play a important role in initiating long distance cyclin D1 deregulation by translocated IgH sequences. Analogous to the mouse β-globin gene locus, a polymerase transfer (LPT) model may be invoked, where other proteins such as CTCF effect Pol II transfer from 3′ Cα IgH regulatory sequences to the cyclin D1 promoter and initiate cyclin D1 transcription.
Constitutive FLT3 Activation Results in Specific Changes in Gene Expression in Myeloid Leukemic Cells.
Authors: Kim Kyu-Tae , Baird Kristin , Davis Sean , Levis Mark , Piloto Obdulio , Li Li , Chen Peili , Meltzer Paul , Small Donald
Published: April 2020

Constitutively activating internal tandem duplication (ITD) mutations of the receptor tyrosine kinase FLT3 play an important role in leukemogenesis. They are the most common genetic alteration in AML and their presence is associated with poor prognosis. To better understand FLT3 signaling in leukemogenesis, we have examined the changes in gene expression induced by FLT3/ITD or constitutively activated wild type FLT3 signaling by cDNA microarray analysis. In order to minimize gene expression changes that might be drug specific and not related to FLT3 inhibition or might be cell-type specific, we used three different FLT3 inhibitors, CEP-701, CEP-5214, and AG1296, and three different constitutively activated FLT3-expressing leukemia-derived cell lines, EOL-1, MOLM-14 and MV4-11. We considered to be FLT3 responsive only those genes whose expression consistently changed in response to FLT3 inhibition by each of the three FLT3 inhibitors in all of the cell lines. RNA for hybridization to the microarrays was harvested from cells both before and after increasing times of FLT3 inhibition to determine genes which decreased or increased in response to FLT3 signaling. In addition, because the inhibitors are all reversible, RNA was also harvested from the cells at increasing times after release from FLT3 inhibition. This enabled us to confirm that, for example, genes whose expression appeared FLT3 dependent and were thus down-regulated by FLT3 inhibition, returned towards normal levels after FLT3 signaling was allowed to resume. Statistical analysis of the microarray results indicated a limited set of genes are highly and consistently affected by FLT3 inhibition and return toward pretreatment levels after release from inhibitor. We confirmed the cDNA microarray data using quantitative real-time PCR. Several of the most significantly affected genes are involved in the Ras/MAPK pathway including DUSP6, DUSP7, MAPK6, TNF, and cMyc. Other sets of genes are involved in JAK/STAT or Wnt signaling pathways including Pim-1, cMyc, Cyclin D3, IL4 receptor, and CISH. These genes are all consistently down-regulated after FLT3 inhibition. These data further confirm the role of constitutively activating FLT3 in mediating multiple signal transduction pathways. We also found several transcriptional factors (RUNX1/AML1, MAFG, XBP1, TGFBI4, and BRD8), several genes involved in receptor-mediated signaling (IL1RAP, CDC42EP3, PLAUR, LY64), and several genes involved in cell proliferation, metabolism, or structure (BCL7A, APTX, GHRH, SET7, ATAD2, CLIC1, CRIP2, MRPL12, VIM) to be consistently differentially expressed. In summary, we have found by cDNA microarray analysis and confirmed by QPCR, a consistent pattern of FLT3 dependent gene expression. The alteration of the gene expression profile in these cells is likely the mechanism of FLT3-mediated leukemogenisis.
Gene Expression Profiling Analysis in Splenic Marginal Zone Lymphoma Allows To Predict Survival and Histological Transformation.
Authors: Thieblemont C. , Ballester B. , Nasser V. , Gazzo S. , Doucet G. , Felman P. , Callet-Bauchu E. , Berger F. , Loi L. , Salles G. , Birnbaum D. , Coiffier B. , Houlgatte R.
Published: April 2020

Splenic Marginal Zone Lymphoma (MZL) is described as an indolent lymphoma with a long term survival. However classical prognostic factors can not distinguish between patients who are likely to have good or poor outcomes. Moreover histological progression occurs in 10–20% of cases at the time of recurrence, but may be present at diagnosis, inducing a shorter survival with a median around 2 years. New model based on molecular understanding are needed to better discriminate these patients for their prognosis and disease evolution.

Biopsy samples of splenic MZL from 43 patients treated in one institution and 8 with transformed splenic MZL were examined for gene expression using a nylon cDNA microarray consisting of 7, 000 human genes. Two of them came from a group of matched pair of splenic MZL and the transformed counterpart. An additional group comprising 4 transformed follicular lymphomas and 1 transformed small lymphocytic lymphoma were also analysed. Hierarchical clustering realized with all samples allowed to visualized patterns of gene expression already described in our previous work1 and corresponding to precise functions (proliferation, early response...), cell or tissue subtype (T cells, stroma …) or lymphoma subtype (MZL, MCL, SLL). The ability of individual genes to distinguish 1/ MZL and transformed-MZL subtypes, and 2/ alive patients and dead patients was calculated using a supervised method (discriminating score and bootstrap resampling). This allowed us to construct molecular predictors of survival and histological transformation.

Transformation discriminating genes regrouped 73 genes related to cell proliferation such as CDC10, CDK4, PRKDC, PLCG2, LDHA, MCL2, and NPM1. P53 was down-expressed in all the transformed samples. Survival discriminating genes regrouped 26 genes from a unique cluster related to cell proliferation such as LDHA, NME1, MYC, ENO1. Although these genes seem to participate to the same function, only one gene was found discriminating the 2 parameters (LDHA). Gene ontology analysis using GOminer showed that the histological transformation was more related to cyclin-dependent protein kinase and that the survival was more related to metabolism. We used 26 genes to construct a predictor for survival in MZL patients. This gene-based predictor allowed predicting survival (p <0.00001) in MZL patients better than with classical prognostic factors (age, performance status, stage, LDH, monoclonal component, IPI…), but also in transformed MZL patients. In conclusion, genes involved in histological transformation and survival in MZL patients are implicated in independent but complementary functions leading to cell proliferation.
Hypermethylation of Retinoic Acid Receptor Alpha 2 (RARα2) Promoter in Human AML Cells Correlates with Poor RARα2 Expression and Response to Differentiation Inducers.
Authors: Glasow Annegret , von Lindern Marieke , Waxman Samuel , Arthur Zelent
Published: April 2020

All-trans retinoic acid (ATRA) signaling pathway is of critical importance for optimal myelomonocytic differentiation and its disruption by translocations of the RARα gene leads to acute promyelocytic leukemia (APL). APL associated fusion oncoproteins, such as PML-RARα and PLZF-RARα, function through recruitment of histone deacetylases (HDACs) and DNA methyltransferases (DNMTs), thus promoting an inactive chromatin state and leading to repression of RARα target genes. Recently, we demonstrated that up-regulation of RARα2 expression by ATRA directly correlates with differentiation in non-APL AML cells. Treatment of ATRA non-responsive cells with the demethylating agent 5-Aza-2′-deoxycytidine (decitabine) restores RARα2 expression and differentiation response to ATRA in different AML subtypes, suggesting abnormal epigenetic changes as a possible mechanism underlying silencing of RARα2 and contributing to a differentiation deficit in these cells. Here we compared the methylation status of ATRA-inducible RARα2 and constitutive RARα1 promoters in 5 human AML cell lines and in normal human CD11b+/CD33+ leukocytes (NLs) by sequencing their respective bisulfite modified CpG islands. Whereas the CpG-island of the RARα2 promoter was completely methylated in all AML samples, it was not methylated in NLs nor in a control preB cell line, NALM-6. The methylation status of the RARα2 promoter directly correlates with the RARα2 expression level, which was shown by real-time RT-PCR to be 100–1000 fold higher in NLs or NALM-6 cells than in AML cell lines. Expression of RARα2 in human primary AML cells was found to be as low as in AML cell lines. In contrast, RARα1 promoter was not methylated in any of the samples and RARα1 expression levels were similar in AML cells and NLs. We have previously shown that stimulation of AML cell differentiation by ATRA and G/GM-CSF (GFs) is associated with a 10 fold induction of RARα2 expression through direct effects on receptor activity. We now show that pre- or co-treatment with decitabine results in up to 100 fold further increase in RARα2 expression levels, rendering the RARα2 mRNA levels similar to these observed in NLs. This synergism in RARα2 induction is paralleled by marked enhancement of leukemic cell differentiation. These results highlight the potential therapeutic use of epigenetic modifiers like decitabine, which may relieve the repression of aberrantly silenced genes required for cellular maturation and amplify the effects of differentiation inducers like ATRA and GFs in AML.
HDAC2 Plays a Role in Protecting t(14;18) Lymphoma Cells from Apoptosis by Up-Regulation of Bcl-2.
Authors: Duan Hong , Heckman Caroline A. , Boxer Linda M.
Published: April 2020

Histone deacetylases (HDACs) usually cause repression of gene expression through deacetylation of histones H3 and H4. Inappropriate transcriptional repression by HDACs has been observed in some malignancies, and several HDAC inhibitors are in clinical trials to treat solid tumors and hematologic malignancies. While the transcriptional activation of genes by HDAC inhibitors has been consistently associated with increased histone acetylation, transcriptional repression by HDAC inhibitors is less well understood. Previously, we showed that two structurally unrelated HDAC inhibitors, Trichostatin A (TSA) and Sodium Butyric Acid (NaB), induced apoptosis and down-regulated bcl-2 expression in follicular lymphoma cells, in which the t(14;18) translocation results in increased bcl-2 expression and resistance to apoptosis. In the current study we further explored the mechanisms underlying the HDAC inhibitor-induced bcl-2 transcriptional repression. Transient transfection of expression vectors encoding three Class I HDACs and real-time reverse transcriptase PCR analysis showed that while overexpression of HDAC2 consistently increased bcl-2 mRNA transcription, overexpression of HDAC1 and HDAC3 did not have a significant effect on bcl-2 levels. These results indicate that HDAC2 has a functional role in the regulation of bcl-2 expression. Furthermore, immunoprecipitation and Western blot analysis showed that TSA treatment dramatically increased the acetylation of two bcl-2 promoter sequence-specific transcription factors, Sp1 and C/EBPα, which are required for bcl-2 P1 and P2 promoter transcriptional activity as well as for TSA-induced bcl-2 transcriptional repression. Increased acetylation of Sp1 and C/EBPα was accompanied by decreased association of HDAC2 but not HDAC1 with these two transcription factors. More importantly, quantitative chromatin immunoprecipitation assays revealed decreased binding of HDAC2 but not HDAC1 to the bcl-2 promoter regions in vivo. These results suggest that posttranslational acetylation of Sp1 and C/EBPα by TSA disrupts the association of HDAC2 with them and leads to decreased binding to the bcl-2 promoters. The current study along with our previous findings suggest that HDAC2 may be an important target of HDAC inhibitor-induced bcl-2 transcriptional repression and provide a mechanistic rationale for the use of HDAC inhibitors in the treatment of human follicular lymphoma.
CA-125 as a Marker for the Early Diagnosis of Hepatic Veno-Occlusive Disease Following Hematopoietic Stem Cell Transplantation.
Authors: Zuckerman Tsila , Ben Harush Shani , Avivi Irit , Gavish Israel , Haddad Nuhad , Fineman Riva , Dann Eldad , Hoffman Ron , Brenner Benjamin , Rowe Jacob M. , Zuckerman Eli
Published: April 2020

Hepatic veno-occlusive disease (VOD), a common complication of hematopoietic stem cell transplantation (HSCT), is associated with significant morbidity and mortality. The diagnosis is mainly based on clinical criteria that are neither sensitive nor specific enough to establish early diagnosis. CA-125 is a tumor marker, which may also increase in chronic liver disease, especially with ascites formation. Since VOD is known to result from liver injury and is associated with ascites, we examined whether CA-125 can be used as a serum marker for early diagnosis of VOD.

Methods: 74 consecutive patients undergoing HSCT were prospectively followed for clinical signs and symptoms of VOD. Serum samples for CA-125 were taken before conditioning, on day 0 and then every 7 days until patient’s discharge from hospital. CA-125 serum levels were measured by ELISA assay.

Results: 19 of 74 patients undergoing HSCT developed VOD according to Jones criteria. 89% of these patients underwent allogeneic stem cell transplantation, 47% received the regimen containing busulfan/cytoxan and 37% were treated with total body irradiation (TBI) and cytoxan. 26% of the patients had severe VOD. Diagnosis of VOD was established on average on day 7 (range 2–20). No difference in CA-125 was observed pre-transplant between the two groups (VOD versus non-VOD), but CA-125 levels increased in 68% of the patients with VOD as opposed to only 15% of those without VOD (p<0.0001) on average on day 9 post transplant. In addition, the peak CA-125 levels were 149.2u/ml versus 45.6u/ml (p<0.0001), respectively. On day 7 post transplant a cut off level of 43u/ml can detect VOD with specificity and sensitivity of 79% and 72%, respectively. The specificity rose to 94% in patients with normal CA-125 levels pre-transplant.

In conclusion: CA-125 levels rise early in patients developing VOD with a good sensitivity and specificity and can therefore be used as a marker in the diagnosis of VOD. Whether CA-125 is a true predictor for the development of VOD or is a surrogate marker needs to be evaluated prospectively by measuring day serum CA-125 levels starting on day 0. This is currently being evaluated in our center.
Prevention of Veno-Occlusive Disease in Hematopoietic Stem Cell Transplantation Due to Defibrotide Prophylaxis.
Authors: Hasenkamp Justin , Conradi Imme , Wulf Gerald , Jung Wolfram , Truemper Lorenz , Glass Bertram
Published: April 2020

Veno-occlusive disease (VOD) is a serious complication of intensive chemotherapy regimens used in hematopoietic stem cell transplantation (HSCT). Incidence ranges from 10–60% (Chopra et al., 2000). Despite numerous therapeutic attempts described such as rtPA, TIPS and defibrotide mortality remains 80% (Fried et al., 1996). Risk factors for developing VOD are previous high-dose chemotherapy, unrelated donor transplants and busulfan-based conditioning regimens. In face of the frequency and high mortality due to absence of effective therapeutic options there is need for prophylaxis. We incorporated defibrotide into the supportive regimen for high risk patients undergoing a HSCT with busulfan containing conditioning regimens. From 01/2003 to 08/2004 44 patients (median age 52y, 24 male) receiving a busulfan containing regimen for HSCT (2 autologous, 42 allogeneic, 23 unrelated donors) were treated prophylactically with defibrotide as 800mg/24h continuous infusion from start of conditioning chemotherapy up to day +21 (group 1). Underlying diseases were: 10 AML, 24 NHL, 3 HD, 6 MM, 1 CML. Median number of previous regimens were 3. 27 patients had high-dose chemotherapies followed by autologous HSCT as prior therapy. From 07/2001 to 12/2002 16 patients (median age 45y, 11 male) receiving busulfan-based conditioning for allogeneic HSCT (7 unrelated donors) received heparin (300IE/h) for prophylaxis of VOD and catheter related thrombosis (group 2). Disease entities were 7 AML, 7 NHL, 1 HD, 1 ALL. Median number of previous regimen was 3. 11 patients had high-dose therapies followed by autologous HSCT as prior therapy. While in the latter group 1/16 patients developed and finally died of VOD, 3/44 in the defibrotide group did so. Date of VOD diagnosis in the patient not prophylatically treated with defibrotide was day +13 after HSCT compared to day +21, +25 and +27 in patients who received defibrotide prophylaxis. There were no serious adverse events (e.g. bleeding) attributable to defibrotide. Overall incidence of VOD in both groups is low (6,8% group 1; 6,3% group 2). Although patients in group 1 were at high risk of VOD, no VOD occurred during the period of defibrotide prophylaxis. We conclude that defibrotide is save and efficient in prevention of VOD. Defibrotide delayed the onset of VOD beyond its application. However, with defibrotide prophylaxis late onset of VOD has to be encountered. The optimal application period has to be determined.
Venocclusive Disease of the Liver Is Associated with the Early Development of Post-Transplant Microangiopathy in Allogeneic Stem Cell Transplantation.
Authors: Zeigler Zella Rose , Shadduck Richard K. , Lister John
Published: April 2020

Thirty-three patients were followed prospectively for the development of venocclusive disease (VOD) of the liver and Post-Transplant Microangiopathy (PTM) on days 0,7,14,21,30,60,90 following allogeneic stem cell transplantation. The grading system of Zeigler, ZR et al (Bone Marrow Transplant, 1995) was used to categorize patients with the clinical syndrome of PTM. The diagnosis of venocclusive disease was made using the criteria of Jones, RJ, (Transplantation, 1987). Patients with and without VOD had similar peak grades of PTM, that ranged for Grade 2 – 4. However, the development of PTM occurred earlier in those with VOD. Data regarding the development of PTM is shown in the Table below as medians and 10th and 90th percentiles. Patients following allogeneic SCT without evidence of PTM have a median % fragmented cells of 1.4% (10th and 90th percentiles of 0.2 and 3.6%). Median LDH elevations expressed as x ULN in patients following allogeneic SCT (without evidence of PTM) were 0.5 ( 10th and 90th percentiles of 0.5–1.0).

When compared with patients without VOD, patients with VOD experienced earlier an earlier onset and a higher grade of PTM. The sequence of events is an increase in bilirubin assoicated with an elevation of the LDH level at day 7 followed by the recognition of grade 2 or higher PTM by day 14. There is also an increase in fragmented cells differentiating the two groups by day 21. By day 28, those patients with VOD who have survived have similar LDH and fragmented cell levels as those that did not have VOD. We conclude that PTM that occurs less than or equal to day 21 post-SCT represents regimen toxicity and often accompanies the diagnosis of VOD.

Parameter With VOD N=15 Without VOD N=18 P Day to Development of PTM 14 (7–31) 33 (21–58) <0.001 Day to peak PTM 18 (14–32) 43 (7–112) 0.02 LDH x ULN Day 7 1.0 (0.5–2.1) 0.6 (0.5–0.8) 0.02 LDH x ULN Day 14 1.5 (0.7–3.7) 0.7 (0.5–2.5) <0.01 LDH x ULN Day 21 2.3 (0.7–12.2) 0.9 (0.7–1.6) 0.01 % FC Day 21 6.7 (1.8–17) 2.0 (0.2–3.8) <0.01 Bilirubin (mg/dl) Day 7 3.3 (1.2–6.5) 1.0 (0.5–2.7) <0.001
Rasburicase Improves Renal Function and Uric Acid Nephropathy in Patients Developing Acute Renal Failure after Conditioning for Hematopoetic Stem Cell Transplant (HSCT).
Authors: Strader Donovan , Oholendt Michael S. , Popat Uday , Carrum George , Heslop Helen E.
Published: April 2020

Significant reduction in renal function secondary to protein deposition in the renal tubules is a common occurrence among patients with plasma cell dyscrasias such as Multiple Myeloma (MM) and Amyloidosis undergoing HSCT. Conditioning chemotherapy and uric acid release following cellular lysis may worsen already compromised renal function, leading to acute renal failure. Rasburicase, a recombinant urate oxidase enzyme, has been approved by the U.S. Food and Drug Administration for the treatment and prevention of hyperuricemia associated with tumor lysis syndrome in pediatric patients with leukemia, lymphoma, and solid tumor malignancies undergoing chemotherapy. We hypothesized that prompt treatment of hyperuricemia with rasburicase may prevent uric acid nephropathy and improve renal function, obviating the need for dialysis and improving the eventual outcome. Six adults, four with MM and two with amyloidosis, with rising uric acid levels after high dose Melphalan (200mg/m2) were given a single dose of rasburicase (0.15 mg/kg). At baseline, all patients had rising serum creatinine [mean: 2.1 mg/dL (range: 0.9–4.5 mg/dL)], rising BUN [mean: 27.6 mg/dL (range: 1.3–70 mg/dL)], and declining creatinine clearance (CrCL) [mean: 50.5 ml/min (range: 16–75 ml/min)]. After one dose of rasburicase, renal function significantly improved according to CrCL at 48 hours [mean: 55.2 ml/min (range: 17–82 ml/min)] and continued to improve at five days [mean: 68.2 ml/min (range: 18–109 mg/dL)]. Baseline uric acid was elevated [mean: 8.61 mg/dL (range: 7–9.9 mg/dL)] and improved 24 hours after treatment with rasburicase [mean: 0.2 mg/dL (range: 0–0.6 mg/dL)]. None of the patients required renal replacement therapy. There were no serious adverse events associated with rasburicase therapy. We therefore conclude that a single dose of rasburicase is promising in improving deteriorating renal function post high dose chemotherapy in patients with compromised renal function; further controlled studies are warranted.
Predicitve Value of Pretransplant Testing.
Authors: Copelan. Edward A.
Published: April 2020

Routine evaluation prior to stem cell transplantation generally includes assessment of left ventricular ejection fraction (EF), pulmonary function, renal function, liver function, and functional status. Patients in whom compromised organ function is detected, are often deemed ineligible for myeloablative transplantation. However, the predictive value of specific pretransplant tests is not well documented, and has not been critically analyzed in patients receiving busulfan-based preparative regimens. We analyzed the prognostic value of the pretransplant evaluation performed in 1035 consecutive patients who underwent autologous (n=444) or allogeneic (n=591) transplantation with busulfan-based regimens between 2/84 and 12/03 for acute (250) or chronic leukemia (198), myelodysplasia (50), multiple myeloma (88), or Hodgkin’s (139) or non-Hodgkin’s lymphoma (303). Ages ranged from 4–75 (median 41). BuCy was given to 477 and BuCyVP16 to 558 individuals.

Ninety three patients had EF < 50%, 61 had DLCO < 65%, 103 had elevated creatine, 130 had elevated tranaminases or bilirubin, and 116 had Karnofsky scores < 80%. For the entire group, multivariable analysis demonstrated only Karnofsky score < 80% (P < .001) and elevated LFTs (P=.04) to have significant adverse prognostic influence on survival. Karnofsky score was significant in patients undergoing autologous (P <.001) or allogenic (P <.001) transplantation. Neither EF nor DLCO were predictive of poor outcome for the overall group (P >.2), nor for the autologous or allogeneic transplant groups. However in patients undergoing transplants from unrelated donors (n=119) DLCO < 65% was a significant adverse prognostic factor (P=.004). There was no association of compromised cardiac or pulmonary function with cardiac or pulmonary failure as a primary cause of death. This study suggests that moderately compromised organ function, particularly cardiopulmonary limitations which might not be clinically apparent, are of limited value in prediction of transplant outcome. Direct clinical assessment, including functional status are of much greater value in prediction of outcome. It is hoped that this investigation will stimulate larger studies of pretransplant predictors of outcome with attention to their relevance to specific preparative regimens, sources of stem cells, and other treatment variables.
Endothelial Cell Injury of Carmustine (BCNU) in Hepatic Sinusoids: Contribution of Oxidation and Immune Reaction.
Authors: Ozdemir Oner , Ravindranath Yaddanapudi , Savasan Sureyya
Published: April 2020

Background: Carmustine (BCNU) is used for the treatment of brain tumors and is included in conditioning regimens of stem cell transplantation. It has been long believed that BCNU causes hepatotoxicity by inducing pericholangitis and intrahepatic cholestasis leading to biliary cirrhosis. Either hepatocytes or biliary ductal cells have been thought to be the target in BCNU-induced liver toxicity. However, the mechanisms operational at cell level have not been well elucidated. Possible explanations include glutathione depletion in cells, DNA damage accompanied by cellular oxidative stress and/or acceleration of apoptosis. It has been shown that BCNU treatment inhibits hepatic glutathione reductase activity in rats. Recently, a two-step process involving free radical formation and immune reaction has been proposed as a mechanism of BCNU-induced alveolar epithelial cell damage by our group (Savasan S, Ozdemir O, Buck S, et al., 95th annual meeting of the AACR, Orlando, March 27–31, 2004. Abstract#: 1133, 2004). In this study, we have investigated BCNU effect on human liver sinusoid endothelial cells (HLS-EC) in vitro.

Material and Methods: Cytotoxicity in HLS-EC was measured by flow cytometric detection of annexin V/PI-positive cells, DiOC6(3) /PI staining, further quantification of fluorosphere-adjusted events and trypan blue exclusion test in repeated experiments. The generation of reactive oxygen species (ROS) was measured by flow cytometric DCF staining. Lymphokine activated killer (LAK) cell-mediated cytotoxicity against BCNU-treated and untreated HLS-EC was measured by flow cytometric cell mediated cytotoxicity assay. The influence ROS-scavenging compound butylated hydroxyanisole (BHA) and anti-inflammatory dexamethasone (DXM) on BCNU treatment was also studied.

Results: Severe HLS-EC damage occurred following BCNU treatment shown by four different cytotoxicity methods. This effect was associated with oxidative stress demonstrated by significant increase in DCF staining. Although pre-treatment of cells with DXM did not affect BCNU cytotoxicity, BHA suppressed BCNU cytotoxicity by 25% (p < 0.05). BCNU treatment also resulted in increased LAK cell-mediated elimination of HLS-EC (65%) compared to untreated cells (16%). Similar to its effect on direct BCNU cytotoxicity, BHA prevented BCNU treated HLS-EC killing by LAK cells by 25%. However, DXM did not influence LAK cell-mediated killing of BCNU-treated HLS-EC cells. These results suggest that both ROS generation and immune reaction against perturbed endothelial cells play a central role in BCNU-associated HLS-EC injury. In contrary to our findings on human alveolar epithelial cells, BHA ameliorated BCNU-induced direct and immune-mediated cytotoxicity on HLS-EC, but not DXM.

Conclusion: Reactive oxygen species scavenging may be useful in prevention of liver toxicity of BCNU.
Phase I Trial of CG53135-05 to Prevent Mucositis in Patients Undergoing High-Dose Chemotherapy (HDCT) and Autologous Peripheral Blood Stem Cell Transplantation (PBSCT).
Authors: Schuster Michael W. , Shore Tsiporah B. , Greenberg June , Jalilizeinali Bita , Possley Scott , Annino Vincent I. , Hahne William
Published: April 2020

Mucositis is a painful side effect of many transplant conditioning regimens used in HDC and PBSCT. This complication often requires treatment with potent narcotic analgesics and may even require intravenous patient-controlled analgesia (PCA) for adequate pain relief as well as total parenteral nutrition (TPN). Infections in this setting may also result from disruption of the mucosal barrier with subsequent migration of intestinal bacteria into the blood stream. Previous treatments with oral rinses and topical applications of soothing gels have largely been ineffective. Recently, a breakthrough class of drugs in the fibroblast growth factor (FGF) family holds great promise in more effectively ameliorating or even preventing oral mucositis (OM). We report on the results of a Phase I trial with CG53135-05, a novel investigational protein therapeutic (FGF-20) that promotes epithelial and mesenchymal cell proliferation in vitro and has demonstrated activity in animal models. 14 patients (ages 25–75) undergoing HDCT with PBSCT were treated with escalating doses of study drug, including 0.1 mg/kg, 0.2 mg/kg and 0.33 mg/kg (concentrations are determined by the UV method which are equivalent to 0.3, 0.6, and 1 mg/kg by the Bradford method previously used). Conditioning regimens used included melphalan (Mel 200), cyclophosphamide, carmustine and etoposide (CBV), carboplatin and thiotepa (CT), cyclophosphamide, etoposide and carmustine (CEC) and busulfan/cyclophosphamide (targeted BuCy). The primary objective of this phase I trial was to evaluate safety, tolerability and pharmacokinetics of CG53135-05. Patients (pts.) were also scored daily for presence of OM using both the WHO and OMAS (oral mucositis assessment scale) grading scales. 7/14 pts. in this study experienced no OM (including 2 Mel 200 patients), 5 pts. experienced only grade 1 OM. while 2 pts. (both treated with Mel 200) experienced grade 3 OM, and no pts. experienced grade 4 OM. 1 pt. experiencing grade 3 OM required TPN. Only 4 pts. experienced diarrhea that lasted more than 4 days and only 1 pt. had gut mucositis-associated (E. coli) bacteremia. The median day of engraftment (ANC>500/uL) occurred on day 14 (range: day 11–19). Patients tolerated the study drug well with no significant side effects up to a dose of 0.33 mg/kg. At that dose, 2 pts. experienced an infusional reaction consisting of fevers, nausea, and mild hypotension. Pharmacokinetics were measured at all dose levels and will be presented. CG53135-05 is a member of a breakthtrough drug class (FGF family) that was well tolerated in autologous stem cell transplant patients at doses up to 0.33 mg/kg with apparent clinical effects in ameliorating or preventing OM - 12/14 pts, thus, avoided severe (grades 3–4) mucositis following HDCT. A larger Phase II clinical trial is planned.
t(8;21)-Positive AML with Mutant Kit Is a Novel Therapeutic Target of Imatinib Mesylate.
Authors: Asou Hiroya , Kyo Taiichi , Inaba Toshiya
Published: April 2020

Point mutations in the kinase domain of c-Kit are frequently associated with t(8;21)-acute myeloid leukemia (AML). In our study, eleven (26 %) of 43 patients had mutations: six Asp816Val, two Asp816Tyr, one Asp816Ala, one Asp816His, and one Asn822Lys. Here, we provide evidences that proliferation of leukemia cells expressing both the AML1-ETO chimera and a c-Kit mutation heavily depends on signals originating from mutated c-Kit, and this mutation is a possible therapeutic target for imatinib mesylate. We initially investigated effects of imatinib on the growth of the Kasumi-1 cell line, which harbors both t(8;21) and a c-Kit kinase domain mutation (Asn822Lys). Imatinib inhibited autophosphorylation of c-Kit at the standard concentration (0.1 μM), and induced cell cycle arrest and apoptosis in an even faster time course than Ph1-positive cell lines treated with this drug. By contrast, growth of SKNO-1, another t(8;21)-positive leukemia cells without c-Kit mutation was not affected by imatinib. To test whether imatinib is effective for Asp816 c-Kit mutants, we isolated t(8;21)-positive fresh leukemia cells from untreated patients. Numbers of cells with an Asp816 mutant from four patients after short-term cultures in the presence of imatinib (0.1 μM, 4 days) were 20–30 % of those in the absence of imatinib, while no significant difference was observed for cells isolated from four patients without c-Kit mutation. (Viability of fresh leukemia cells without imatinib was maintained over 80% during this short-term culture.) Moreover, autophosphorylation of mutated c-Kit in leukemia cells from one patient with an Asp816 mutant was inhibited by imatinib. Our results disagree with those of previous studies, which indicated that cells with c-Kit mutations in the kinase domain are resistant to imatinib in murine IL-3-dependent cells and human mast cell leukemia cells. This discrepancy could be explained by high expression levels of c-Kit mutants in IL-3-dependent cells by powerful ectopic promoters, since overexpression of Bcr-Abl kinase is one of the major causes of resistance to imatinib in the treatment of CML patients. In addition, drug metabolism may be different between human t(8;21)-positive leukemia cells and in murine IL-3-dependent cells or mast cell leukemia cells. Although t(8;21) in AML represents a favorable prognostic indicator for achievement of cure, a substantial number of these patients relapse and eventually die of their disease. Indeed, of five patients harboring both t(8;21) and c-Kit mutations who we identified and followed up for more than five years, four relapsed. Therefore, our results suggest that imatinib would be useful for eliminating minimal residual disease in these patients after achievement of complete remission.
Bone Marrow-Derived Stem Cells Reduce Radiation-Induced Damage to Salivary Glands.
Authors: Wierenga Pieter K. , Lombaert Isabelle , Visser Willy , Kampinga Harm H. , de Haan Gerald , Coppes Rob P.
Published: April 2020

The salivary glands are often included in the field of irradiation during radiotherapy of head and neck cancer. This can result in severe side-effects that reduces the quality of life of the patient and may even limit the treatment dose. Late damage to the salivary glands is mainly caused by exhaustion of the tissue specific stem cells. Post-irradiation replacement of salivary gland stem cells with donor stem cells may ameliorate radiation-induced complications. Bone marrow-derived stem cells (BMSC) have been shown to be multipotent and thereby able to engraft in many tissues after injury. In this study, we assessed the potential of BMSC to reduce irradiation-induced salivary gland damage. C57BL/6 mice were transplanted with bone marrow from eGFP transgenic animals. After two months the salivary glands of these chimeric mice were locally irradiated with 15 Gy. BMSC were mobilized 10, 30 and/or 60 days after irradiation by s.c. injection of rHu-PEG-G-CSF. Saliva secretion (μl/15 minutes) was measured up to 90 days after irradiation by pilocarpine induction. Hereafter, the glands were extirpated and examined for eGFP-expression. From every individual animal one parotid and one submandibular gland was used to prepare single cell suspensions in order to detect eGFP-positve cells by flow cytometry. The other parotid and submandibular glands were analyzed using confocal laser fluorescence scanning microscopy and light microscopy. G-CSF treatment yielded in an increase of saliva flow for all time points. The optimal time-point for mobilization, however, was 30 days after irradiation as is demonstrated by an improvement of salivary flow from 5 to 30% when compared to radiation alone. FACS analysis showed that up to 10% of the isolated cells were eGFP-positive. Microscopic analysis revealed a similar amount of positive cells and an improved morphology. Immuno-histochemistry using anti-SM-actin antibodies showed the close vicinity of actin and eGFP within the cells, demonstrating the occurence of BMSC derived myoepithelial cells in irradiated salivary glands. Furthermore, using cell-type specific antibodies, the meyoepethilial nature of the eGFP positive was revealed. In conclusion, the results show that BMSC home to severely damaged salivary glands after mobilization. Hence, BMSC mobilization could become a promising modality to ameliorate radiation-induced complications in salivary glands after radiotherapy.
Juxtaposition of the HPFH2 Enhancer Is Not Sufficient To Reactivate the g-Globin Gene in Adult Erythropoiesis in bYAC Transgenic Mice.
Authors: Xiang Ping , Ye Xin , Han Hemei , Stafford Mary , George Stamatoyannopoulos , Li Qiliang
Published: April 2020

Hereditary persistence of fetal hemoglobin (HPFH) is characterized by high-level expression of the g globin gene in adult patients. HPFH2 contains a genetic deletion of approximately 83.5 kb from the middle of intron 2 of the yb gene to the region 66 kb 3′ to the b gene. The deletion juxtaposes an enhancer located downstream to the 3′ breakpoint to the vicinity of the g gene. To understand the reactivation mechanism of this fetal stage specific globin, we introduced the deletion into a 213 kb b-YAC. Four transgenic lines bearing 1–3 intact copies of the YAC construct were established. Globin gene expression at different developmental stages was measured by RNase protection assays. In the HPFH2 transgenic mice the e and g genes were expressed at high levels similar to the wild type YAC mice in embryonic and fetal stages. Unexpectedly, the g gene was completely silenced in the adult mice. The failure of g gene reactivation by juxtaposition of the HPFH2 enhancer contradicts the results reported by the Forget lab (Mol. Cell. Biol.17:2076–2089, 1997) and the Strouboulis lab (Blood102:3412–3419, 2003). The discrepancy could be due to the differences between the constructs used in the different labs. The construct used in the Forget lab was a cosmid containing the mLCR linked a 13 kb Gg and Ag fragment. The cosmid used by the Strouboulis lab contained the 22 kb LCR and a 5 kb Ag fragment. The bYAC construct we used in this study contains the whole b globin locus and spanned from about 40 kb 5′ to the e gene to 100 kb 3′ to the b gene. The failure of g gene reactivation in the HPFH2 YAC mice suggests that additional elements, which are missing in the 213 kb bYAC construct, are involved in reactivation of the g gene in the HPFH patients. Based on data from literature (Bender et al., Mol. Cell5:387–393, 2000; Forrester et al., Genes & Dev.4:1637–1649, 1990) and our own studies (Ping et al., this meeting) we speculate that the 200 kb upstream region is the candidate for harboring this activity.
A Contigs Strategy of Southern Blot Hybridization: Screening for DNase I Hypersensitive Sites in the 200 Kb Region 5′ to the B-Globin Locus LCR.
Authors: Xiang Ping , Han Hemei , Fang Xiangdong , Stamatoyannopoulos George , Li Qiliang
Published: April 2020

Formation of DNase I hypersensitive sites is an indication of local disruption of chromatin conformation. It has been documented that HS sites are frequently associated with functional DNA sequences, such as, promoters, enhancers, and insulators. While Southern blot hybridization is the standard method to detect HS sites, this procedure is time-consuming and labor intensive. To improve the efficiency of HS detection through Southern blot hybridization, we designed a contigs strategy of Southern blot hybridization and test it in the 200 kb region 5′ to the LCR in the b-globin locus. Based on the human genome sequence we made physical maps of seven 6-bp-cut restriction enzymes in the 200 kb region. From the map we selected continuous contigs of 10 to 15 kb fragment; and designed hybridization probes for the 5′ and 3′ ends of each fragment (some probes can be used in two neighboring fragments). The screening was performed on erythroid (K562) and non-erythroid (Jurkat) cell lines. We found about 40 HS sites within the region. The major sites were either erythroid specific (for instance, HSs at −66 kb, −142 kb, and −236 kb, the cap site of the e-globin gene is +1), or non-erythroid specific (for instances, HSs at −111 kb, −164 kb, and −205 kb). These HS sites will be investigated for enhancer, promoter, and insulator function using transient and stable transfection studies. Due to the limited number of enzyme required and the fact that each blot could be used several times, this strategy can greatly expedite the screening process for presence of DNase I hypersensitive sites. Estimated efficiency of this screening approach is about 0.5 to1 Mb per person per year.
Association of Skin and Blood Dendritic Cells with Acute Graft Versus Host Disease after Human Haematopoetic Cell Transplantation.
Authors: Auffermann-Gretzinger Susanne , Eger Lars , Bornhaeuser Martin , Thiede Christian , Schaekel Knuth , Oelschlaegel Uta , Illmer Thomas , Schaich Markus , Ehninger Gerhard
Published: April 2020

Both number and origin of dendritic cells (DC) in the blood have been associated with acute graft-versus-host disease (aGvHD), graft-verus-leukemia effect, relapse and graft failure after human allogeneic haematopoietic cell transplantation (aHCT). Aim of this study was the systematic simultaneous investigation of skin and blood DC subtypes, the analysis of their host/recipient origin after HCT and the correlation of DC kinetics with treatment, outcome and incidence of aGVD and its treatment in 31 recipients of allogeneic HCT. Epidermal shave biopsies and peripheral blood samples were collected from patients pre and at defined time points following aHCT, DC from skin and blood isolated and examined by FACS and quantitative PCR for Short Tandem Repeat markers. A significant reduction in number of skin and blood DC subtypes was observed already before start of conditioning chemotherapy in patients compared to healthy volunteers. A majority of donor derived epidermal Langerhans Cells (median 95%, range 53–100%) and also dermal DC (median 94%, range 39–100%) was found in all examined patients even early after transplant independent from chemotherapy regimen, graft, occurrence of skin GvHD or time point after transplantation. Numbers of both skin DC subsets remained low for months post HCT. Reconstitution kinetics of CD 11c+ preDC1 and CD 123+ preDC2 blood DC were similar to each other and pre transplant numbers were reached again around day +112 post transplant. Recipients of grafts containing higher T cell numbers had lower preDC1 counts on day +28. PreDC2 reconstitution was negatively affected by steroid treatment. Patients with aGvHD tended to have more preDC1 on day +28 and lower numbers of both preDC subsets on day 56 post transplant. Residual host blood DC were rare at all time points (median <3% at all investigated timepoints range 2–97%). The kinetics of DC reconstitution at different sites might become an important diagnostic tool to predict the occurrence of acute and chronic GvHD and to monitor the efficacy of prophylactic and therapeutic interventions.