bluuebunny/crossref_metadata_2025 huggingface dataset:
Economic Analysis of Prophylactic Pegfilgrastim in Cancer Patients Receiving Chemotherapy.
Authors: Eldar-Lissai Adi , Cosler Leon E. , Lyman Gary H.
Published: October 2019

Introduction: Neutropenia and its complications are major dose-limiting toxicities of systemic cancer chemotherapy. Febrile neutropenia (FN) generally prompts immediate hospitalization for evaluation and empiric broad-spectrum antibiotics adding to the cost of cancer care. Randomized controlled trials (RCTs) have demonstrated that prophylactic recombinant granulocyte colony-stimulating factor (rG-CSF; filgrastim) is capable of reducing the risk of FN, infection related mortality (IRM) and duration of hospitalization associated with FN. In the absence of compelling clinical indications, current guidelines support the use of prophylactic G-CSF only when using regimens associated with FN risk of 40% or higher. While recent RCTs have demonstrated that a long acting, pegylated form (pegfilgrastim) is at least as effective and safe as filgrastim, its economic impact has received little study.

Methods: An economic analysis was conducted comparing prophylactic pegfilgrastim to no prophylactic growth factor in patients receiving systemic chemotherapy. Direct medical cost estimates (US$ 2002) were obtained from the University HealthSystem Consortium and Marketscan databases. Pegfilgrastim costs were based on the average wholesale Red Book© prices. Risk and efficacy estimates were based on recent RCTs and meta-analysis of prophylactic rG-CSF. Sensitivity analyses and estimation of cost neutral thresholds for the range of feasible values of all variables were conducted based on cost-minimization.

Results: Under baseline conditions (FN= 17%; RRR=0.941), an incremental cost savings with pegfilgrastim of $428 was estimated. At baseline, a cost neutral threshold for FN risk of 14.6% was estimated with an expected cost for either strategy of $2721. Sensitivity analysis demonstrates a baseline threshold for RRR of 0.74 with lower rates of RRR supported by increasing hospital cost or length of stay, increasing infection-related mortality or incorporation of indirect costs. Threshold estimates for FN risk were robust over all possible values assumed for RRR of infection related mortality. The FN risk threshold falls below 10% when hospitalization cost per day exceeds $3000. Further results, including a Monte-Carlo simulation will be presented.

Conclusions: Incorporation of recent RCTs and cost data into clinically relevant models demonstrates that pegfilgrastim is cost saving at levels of FN risk associated with common chemotherapy regimens (< 20%). Primary prophylaxis with pegfilgrastim should be considered in patients being treated for curative intent with moderately myelosuppressive regimens.
Impact of Immunosuppressive Drugs on Adaptive and Innate Immune Responses to Aspergillus fumigatus Antigens.
Authors: Hebart Holger , Mickan Andreas , Haddad Ziad , Loeffler Juergen , Latge Jean-Paul , Einsele Hermann
Published: October 2019

Appropriate activation of the innate and adaptive immune system is crucial for the successful control of invasive aspergillosis (IA). Acute and chronic graft-versus-host disease as well as corticosteroids were described as major risk factors for the development of IA. In this study, we assessed the impact of immunosuppressive agents (dexamethasone, rapamycin, Cyclosporin A, FK506) on the A. fumigatus induced activation of monocyte-derived immature dendritic cells (iDC) and A. fumigatus-specific T-cell responses in well established cell culture models.

Immature DCs were found to be activated and to differentiate into mature DCs in response to A. fumigatus antigens. The upregulation of CD86 was inhibited by dexamethasone (D) in 3 out of 3 experiments, and of CD40 and CD80 in 2/3. CSA and FK506 had a variable impact on the upregulation of CD86, but not on CD40 and CD80, whereas the expression of co-stimulatory molecules was found unchanged upon incubation with rapamycin.

Autologous DCs were found to restore A. fumigatus-specific T-cell responses. T-cell proliferation to A. fumigatus hyphae and a cellular extract of the culture filtrate were found to be strongly inhibited by rapamycin and dexamethasone (n=3), whereas the effect of CSA and FK506 (n=3) at the concentrations analysed was variable. The release of IFN-g in culture supernatants upon stimulation with A. fumigatus antigens was strongly reduced in the presence of rapamycin (n=3), whereas the release of IL-4 was found to be increased in the majority of experiments (n=3). Comparable results were observed upon stimulation with tetanus toxoid and a CMV lysate (n=3). These data indicate, that A. fumigatus-spec. T-cell responses may be directed towards a TH2 phenotype in the presence of immunosuppressive agents.

In summary, immunosuppressive agents were found to exert differential effects on adaptive and innate immune responses directed against A. fumigatus. Whereas dexamethasone was found to modulate the expression of co-stimulatory molecules on A. fumigatus activated iDCs and to suppress A. fumigatus-specific lymphoproliferation, rapamycin exerted only minor effects on DC-activation but had a strong impact on A. fumigatus-induced T-cell responses. These results may help to tailor immunosuppressive regimens in patients at high risk for invasive aspergillosis.
Pharmacokinetics of Ganciclovir Following Oral Valganciclovir Versus Intravenous Ganciclovir in Allogeneic Hematopoietic Stem-Cell Transplant Patients with Stable Graft-Versus-Host Disease of the Gastrointestinal Tract.
Authors: Winston Drew J. , Ratanatharathorn Voravit , Baden Lindsey , Emmanouilides Christos , Gabriel Don , Li Sherry , Lange W. Robert
Published: October 2019

Cytomegalovirus (CMV) disease can be effectively prevented in allogeneic hematopoietic stem cell transplant (HSCT) patients by ganciclovir (GCV) given as prophylaxis or preemptive therapy. Due to the low bioavailability of oral GCV capsules, GCV is usually administered intravenously (IV) to HSCT patients. Valganciclovir (VGCV) is the valine ester prodrug of ganciclovir. In healthy subjects, HIV-infected patients, and solid-organ transplants, the oral bioavailability of VGCV is about 60%, or 10-fold higher than oral GCV capsules. The bioavailability and total GCV exposure provided by oral VGCV relative to IV GCV in HSCT patients with gastrointestinal (GI) GHVD has not been established.

METHODS: HSCT patients were eligible for the study if the following criteria were satisfied: 1) ≥16 years of age; 2) biopsy-proven GHVD of GI tract with nausea and/or diarrhea (300–1500 ml/day) or proven GVHD of skin or liver plus diarrhea with no other explanation; 3) no active CMV infection or disease; 4) neutrophil count ≥1000/μL; 5)creatinine clearance >60 ml/min. Following a standardized breakfast, eligible patients were randomized to receive a single dose of open-label study drug (900 mg of oral VGCV or 5 mg/kg of IV GCV). After a minimum 48 hr. washout period, patients were crossovered to alternate study drug. Blood for levels of GCV and VGCV were obtained predose and then over the 24 hours after dosing. Pharmacokinetic (PK) parameters were derived by noncompartmental methods.

RESULTS: Data from 16 patients are currently available. Patient demographics include mean age 45 yrs (range 23 to 58 yrs); males 13, females 3; mean weight 80kg (range 52 to 107 kg); mean creatinine clearance 96 ml/min (range 62 to 184 ml/min). Median time after transplant for study was 303 days (range 102 to 988 days). Mean GCV PK parameters are summarized in the following table.

Mean GCV Value (Coefficient of variation in %)

Oral VGCV-900 mg IV GCV-5mg/kg Parameter N = 16 N = 16 AUC o -τ (μg•hr/mL) 43.58 (37) 46.74 (40) AUC o-∞ (μg•hr/mL) 46.03 (41) 48.89 (43) C(max) μg/mL) ( 6.45 (30) 12.53 (30) T max (hr) 3.13 (22) 0.97 (8) T½ (hr) 4.97 (31) 5.09 (29)

GCV AUC values were similar, although maximum GCV concentrations were higher and acheived earlier with IV GCV. Terminal elimination half-life of GCV with oral VGCV and IV GCV were similar. After 900 mg of oral VGCV, mean plasma Cmax for VGCV was low (0.22 μg/mL), which is consistent with rapid and almost complete metabolism of VGCV to GCV.

CONCLUSION: These preliminary results suggest that systemic exposure to GCV after 900 mg of oral VGCV is comparable to that achieved with IV GCV in HSCT patients with stable GI GVHD. Oral VGCV could be a useful alternative to IV GCV in certain HSCT patients requiring prophylaxis or preemptive therapy for CMV.
Cidofovir Is Safe and Effective in the Treatment of Adenoviral Infection in Pediatric Hematopoietic Stem Cell Transplantation.
Authors: Yusuf Usman , Hale Gregory , Woodard Paul , Benaim Ely , Kasow Kimberly , Horwitz Edwin , Leung Wing , Srivastava Deo , Handgretinger Rupert , Haden Randy
Published: October 2019

Adenovirus (ADV) infections are increasingly recognized as significant cause of morbidity and mortality after hematopoietic stem cell transplantation (HSCT). Early diagnosis and prompt initiation of effective treatment are important in preventing often fatal disseminated ADV disease.

We report our experience with using cidofovir (CDV) for the treatment of adenovirus infection in 57 HSCT patients, median age 8 years (range 0.5–26). Fifty-four patients received allogeneic HSCT, 35 of whom were T-cell depleted with 3 patients receiving autologous marrow. Blood was tested weekly for ADV by quantitative real-time PCR, with viral culture performed on urine, stool and throat swabs. Antiviral therapy was initiated immediately upon detection of ADV by PCR, culture or tissue histopathology. Cidofovir was given at 5mg/kg once weekly for 2 weeks, then every 2 weeks untill 3 negative PCR or cultures were documented. ADV was first detected at a median of 53 days (range 6–319 days) after HSCT. The most common clinical manifestations were diarrhea (53%), fever (21%), hemorrhagic cystitis (12%), and pneumonitis (11%). Fourteen (25%) patients were asymptomatic and four (7%) patients had exacerbation of an ongoing GVHD.

The virus was isolated from stool 53%, blood 50%, urine 16%, respiratory specimens 16% and the cerebrospinal fluid in 1 patient. Twenty (35%) patients had the virus isolated from more than 2 sites. Of the 30 patients who initially had ADV only in stool, 77% of them became PCR-positive in the blood while on CDV therapy, at a median of 10 days after ADV was first detected from the stool. The median duration of therapy was 60 days (range 1–270), with a median of 5 doses given (range 1–22). CDV treatment was associated with resolution of diarrhea, hemorrhagic cystitis, fever and pneumonitis in 56 patients in whom the virus became undetectable by both cultures and quantitative PCR. There was one adenovirus-related death due to pneumonitis and ARDS. No cases of dose-limiting nephrotoxicity were observed. Cidofovir was demonstrated to be safe and effective for the treatment of ADV infection in this predominantly pediatric HSCT patient population. Vigilant surveillance for ADV and early treatment with CDV can prevent the poor outcomes associated with ADV disease. A larger prospective study will be needed to further determine the role of CDV in the treatment of ADV in patients after HSCT.
Pharmakokinetics of Valganciclovir after AlloSCT: A Fixed Oral Dose Can Be Used for Preemptive Therapy in Patients with Normal Body Weight - Even with Intestinal GVHD.
Authors: Einsele Hermann , Reusser Pierre , Hebart Holger , Hertenstein Bernd , Bornhaeuser Martin , Ehninger Gerhard , Kroeger Nikolaus , Kahls Peter , Chalandon Yves , Rohde Frank
Published: October 2019

Background: After alloSCT, oral valganciclovir (VALGCV) is a promising alternative to IV GCV against CMV but its pharmacokinetics (PK) have not been studied.

Methods: We investigated the PK of GCV after VALGCV in a randomized, crossover phase II-study of 48 patients (pts) after alloSCT. Pts who had >400 copies/ml of CMV DNA in plasma measured by quantitative PCR were randomized to receive a fixed dose of VALGCV 900 mg BID (adjusted for renal function) for 7 days, followed by IV GCV as 1-h-infusion at 5mg/kg every 12 h for another 7 days, or to receive the inverse sequence of study drug administration. The PK of GCV were assessed on days 4 and 11. Safety monitoring was done until day 84 after alloSCT.

Results: 28 pts were fully assessable for PK analyses. Among the 22 pts without intestinal graft-versus-host disease (GVHD), the mean±SD AUC 0–12 (mg/L*h) was 53.8±18.0 on VALGCV vs 39.5±13.9 on IV GCV (mean difference 14.3 [95% CI 7.6 to 20.9]); Cmax (mg/L) was 8.8±2.4 vs 10.3±2.1; tmax was 2.7±0.8 vs 1.1±0.6; and t1/2 was 4.2±1.1 vs 3.4±0.8. Among the 6 pts with intestinal GVHD, the mean±SD AUC 0–12 (mg/L*h) was 46.6±24.9 on VALGCV vs 35.3±12.8 on IV GCV (mean difference 11.3 [95% CI −13.4 to 35.9]); Cmax (mg/L) was 7.1±3.6 vs 11.1±3.1; tmax (h) was 2.7±0.8 vs 1.2±0.4; and t1/2 (h) was 5.6±2.0 vs 3.3±0.7. The bioavailability of GCV after VALGCV was 53%. Using a VALGCV fixed oral dose (1.800mg) for preemptive therapy in pts with low body weight led to a sharp increase of the VALGCV / IV GCV ratio (i.e. 50kg = 1.9). With a limited number of pts with low body weight included in this study no severe GCV associated toxicity was seen in these pts. Non-fatal CMV pneumonia developed in 2 pts during follow-up after antiviral therapy, and servere neutropenia < 500/μl occurred in 3 pts. Clearance of CMV DNA was equally effective in both arms.

Conclusions: In alloSCT, exposure to GCV after preemptive therapy using VALGCV is higher compared to that with IV GCV in patients with and without intestinal GVHD. In addition to patients with renal dysfunction patients with low body weight < 60kg and normal renal function should be treated very carefully to avoid over-exposure to GCV. Although no severe toxicity was demonstrated in this pk-study a further study to address the safety and efficacy of VALGCV in alloSCT is needed.
B Cell Development in the Absence of PU.1.
Authors: Ye Min , Ermaermakova-Cirilli Olga , Graf Thomas
Published: October 2019

Mice deficient of the ETS-family transcription factor PU.1 lack B cells as well as macrophages. While most macrophage specific genes are known to be regulated by high levels of PU.1, the reason for the defect in B cell formation is not known. Here we analyzed a mouse strain in which a floxed version of the PU.1 gene, surrounding exon 4 and 5, which encode the DNA, binding and PEST domains (developed by C. Somoza and D. Tenen), was excised by Cre mediated recombination. As expected, this strain lacks both B cells and macrophages and die at birth. Surprisingly, however, we were able to establish lymphoid cell lines from fetal livers of these mice (day 14 to day 18), which proliferated on S17 stromal cells supplemented with IL-7 and stem cell factor. These cells expressed the B lineage cell surface markers CD19, CD43, BP-1 and CD24, but not B220. They also expressed B cell transcription factors, EBF, E47, Pax5, and their target genes, Rag1, IL7R, λ5 and v-preB, as detected by RT-PCR, exhibited DJ and VDJ immunoglobulin heavy chain rearrangements, and expressed IgM after IL-7 withdrawal. We then tested the effect of PU.1 deletion in B cells in adult animals by crossing the floxed PU.1 strain with a CD19 Cre mouse line. The spleen and peripheral blood (but not bone marrow) of these mice contained B cells that were CD19+ IgMlow, IgDhigh but B220 negative and instead expressed CD43. Thus PU.1 is not essential for immunoglobulin production and late B cell development. Although PU.1−/− fetal liver cells can give rise to cells, resembling Pre-B in vitro, the process of B cell formation was delayed by almost 12 days, compared with wt fetal liver, and the efficiency was reduced approximately 25-fold. In addition, PU.1 deficient B cells demonstrated an impaired ability to engraft into the bone marrow, when injected into irradiated SCID mice. We have found that PU.1 deficient B progenitors showed reduced or undetectable levels of the SDF1 receptor CXCR4, a receptor that has been implicated in B cell homing. Taken together, our observations suggest that PU.1 plays two different roles during B cell development: for early B cell formation and for proper migration and engraftment, which might be mediated through regulation of CXCR4 expression.
Tissue Iron Overload Is Common Post Transplantation (Allo BMT) and Is Associated with Red Cell Transfusion Load and HFE Genotype.
Authors: Carradice Duncan , Shuttleworth Peter , Szer Jeffrey , Roberts Andrew , Grigg Andrew
Published: October 2019

In order to analyse the incidence of iron overload after allo BMT and assess the role of venesection in preventing complications, we retrospectively analysed 168 consecutive patients undergoing allo BMT at our institution from 1998–2003 surviving at least one year. Iron studies were performed routinely pre-BMT, at D100, one & two years post BMT. Iron overload was defined by at least one of the following criteria i)liver biopsy (n=24), one of : a) dry weight iron concentration >80μmol/g; b) iron index >1.9; c) Perl’s stain grade 3 or 4, ii) CT liver iron >1.0mg/ml (n=13) iii) raised ferritin >1000 μg/L and transferrin saturation >55% on 2 occasions, persisting >6/12 post BMT (n=11), iv) venesection >5g iron (n=1). Using these criteria, iron overload occurred in 49/168 (29%) pts. 12/119 in the non-overloaded group had further investigation but did not meet the criteria; liver biopsy (n=10) or CT (n=2). Elevated ferritin, particularly early post-transplant, did not reliably predict for iron overload, with 55/91 evaluable patients having values >1000μg/L at D100 not fulfilling the criteria for iron overload. There was no difference between overloaded and non-overloaded patients with respect to age or sex. Acute (15/49 vs. 26/113) or chronic liver GVHD (25/46 vs. 47/105) was not different between the two groups (both p>0.05). Only 3 patients were hepatitis B sAg+ or hepatitis C Ab+. The iron overloaded group was more likely to i) have been transplanted for acute leukaemia (29/49 vs. 33/119; p 0.0002) ii) be C282Y heterozygotes (11/46 vs. 10/110, p 0.02) (iii) been transfused more units of red cells (mean 42 vs. 19; p<0.0001) and iv) have persistently abnormal liver function post-transplant, ALT (IU/ml; normal <55) at 1 year 77 vs. 52 and at 2 years 67 vs. 37 (all p<0.05). There was no effect of hetero- or homozygosity for H63D. 63 patients were analysed for the S65C, V59M and Q283P mutations. One patient was heterozygous for the S65C mutation (non-overloaded group). A mean of 12.3 units were venesected in 22 patients (range 2–46), all of whom had received >25 units of red cells. ALT fell significantly (mean pre venesection 189 IU/ml, post 36, p<0.05), as did transferrin saturation (mean pre venesection 68%, post 29%, p<0.05). We conclude that tissue iron overload is common after BMT, that biochemical measures of iron stores (ferritin and transferrin saturation) may be unreliable in this context, particularly in the early post BMT period and that radiological or histological assessment to distinguish hyperferritinaemia due to inflammation from true tissue iron overload may be required. Patients at risk of iron overload (transfusions >25 units, C282Y heterozygotes) should be closely monitored and early venesection therapy instituted to minimise organ damage.
Primary Central Nervous System Lymphomas (PCNSL) Are Distinct from Systemic DLBCL for Expression Pattern of Germinal Center and Activation B-Cell Markers. A GOELAMS Study.
Authors: Camilleri-Broet Sophie , Delwail Vincent , Moreau Anne , Gardembas Martine , Raphael Martine , Lamy Thierry , Sautes-Fridman Catherine , Colombat Philippe , Martin Antoine
Published: October 2019

Most of the PCNSL of immunocompetent patients are diffuse large B-cell lymphomas (DLBCL). They differ from systemic DLBCL in their outcome, a poor survival in most cases contrasting with a lack of systemic dissemination. Concerning their histogenic derivation, a germinal center B (GCB) cell origin has been hypothesized, based on BCL-6 expression and ongoing mutational activity. Using cDNA microarrays, two main prognostic subgroups have been described in systemic DLBCL: the favorable GCB-cell like subgroup and the activated-B cell like, characterized by a non-favorable outcome. These prognostic subgroups are defined accurately by immunohistochemistry, using a panel of GC B-cell (CD10, Bcl-6) and activation (MUM1/IRF4, CD138) markers. The goal of the present study was to determined the immunoprofile of PCNSLs and its potential prognostic significance by analyzing retrospectively 28 cases included in two clinical trials from the GOELAMS (Groupe Ouest Est des Leucémies et Autres Maladies du Sang). According to the pathological panel review, all cases were classified as DLBCL, not associated with Epstein-Barr virus as shown by in situ hybridization. Immunohistochemistry was performed for CD10, BCL-6 and MUM1/IRF4 and CD138 on paraffin-embedded sections. Labelings were interpreted independently by two pathologists without knowledge of clinical data with 10% increments. Cases were considered positive when positive tumor cells accounted for more than 30%. CD10 was expressed in 1 out of the 28 cases. BCL-6 was expressed in more than 30% of tumor cells in 15/28 cases (54%). Strong nuclear expression of IRF-4 was found in almost all cases (26/28; 93%) and none of the tested cases expressed CD138. Thus, only one case was subclassified in the favorable GCB-cell like subgroup (CD10+/BCL-6+/IRF4−/CD138−). The other cases, classified in the non favorable activated-B cell like prognostic subgroup. The latter may be divided in two patterns: an activated or “late” GC B-cell pattern (CD10−/BCL-6+/IRF4+/CD138-) and an activated non-GC pattern (CD10−/BCL-6−/IRF4+/CD138−) observed in 54% and in 42 % of the cases, respectively. This study shows that PCNSL display an relatively homogeneous immunoprofile, corresponding to an activated late GC or early post-GC stage of B cell maturation, different from that published in systemic DLBCL. This result may partly explain the usual poor prognosis of PCNSL and raises the question of considering PCNSL as a separate biological entity, distinct from systemic DLBCL.
Follicular Lymphoma: Design of a Protein-Based Survival Predictor Using Tissue-Microarrays (TMA).
Authors: Camacho Francisca I. , Arranz-Saez Reyes , Caleo Alessia , Cannata Jimena , Cereceda Laura , Gonzalez Leocricia , Perez-Martin Mª Elena , Sanchez-Verde Lydia , Montalban Carlos , Garcia Juan F. , Piris Miguel A. , Bellas Carmen
Published: October 2019

Follicular lymphoma (FL) is the most common type of low-grade non-Hodgkin’s lymphoma. A subset of FL patients shows a favorable treatment response, and remains in complete remission with long-term follow-up, while another subset of patients has a clinical course characterized by frequent relapses and a shorter survival. Survival predictors for FL are mainly based on clinical data, and they seem to lack accuracy enough predicting survival among patients with advanced stage disease. The aim of this study is to build a survival predictor based on a set of biological markers using Tissue Micro Arrays (TMA). To this purpose, we have retrospectively analysed the expression of a group of 40 selected genes - related with apoptosis control, cell cycle, B-cell differentiation and signaling- in a series of 192 FLs using TMA. The association of these molecules with survival, and their usefulness to discriminate among FLIPI groups was evaluated. Results were quantified using different tools; singularly nuclear markers were scanned using the Bliss system and the quantitative expression was measured using the TMAscore v.1.0 image analysis software (Bacus Laboratories, Inc.). The mean overall survival (OS) was 74 months, and 38 months for progression-free survival (PFS). Statistically significant differences in OS were found with the Follicular Lymphoma International Prognostic Index (FLIPI) score (p < 0.01). No significant OS or PFS differences were observed between FL grades 1–3, between grades 3a and 3b, or using Ki67 expression. Univariate analysis revealed several TMA markers with capacity to predict OS and PFS. After multivariate analysis, a set of 4 apoptosis and cell-cycle markers was integrated into a FLIPI-independent clinical predictor, with the capacity to recognize two groups of FL patients with statistically significant differences in OS (83% versus 43% of OS at 120 months; p< 0.001). Then, patients were classified into low-risk (FLIPI: 0–2) and high-risk groups (FLIPI: 3–5), and the protein-based predictor model was used in both groups. High-risk FLIPI patients were stratified by the protein-based predictor into two groups with OS probability of 79,3% versus 14,2% at 120 months, p < 0.001, and low-risk FLIPI patients were also separated into two groups with OS probability of 100% versus 60,4% at 120 months, p < 0.01. The model is now being validated in a blind set. These results suggest that an integrated use of the FLIPI and the protein-based model could reach a higher accuracy predicting survival in FL.
High Resolution Screening of Genomic Aberrations in Follicular Lymphoma Using Microarray Based Comparative Genomic Hybridization (MATRIX-CGH).
Authors: Schwaenen Carsten , Wessendorf Swen , Viardot Andreas , Ruf Sandra , Enz Martina , Kohlhammer Holger , Kestler Hans A. , Barth Thomas F.E. , Moeller Peter , Doehner Hartmut , Lichter Peter , Bentz Martin
Published: October 2019

Follicular Lymphoma (FL), one of the most frequent lymphoma entities in the western world, is characterized by a highly variable clinical course reaching from rapid progression with fatal outcome to cases with long term survival. In a recent study applying chromosomal comparative hybridization (CGH) to FL, in 70% of the cases genomic aberrations were detectable and a loss of genomic material on chromosomal bands 6q25-q27 was the strongest predictor for short overall survival. However, limitations of CGH as a screening method are a restricted genomic resolution to 3–10 Mbp and demanding non-automated evaluation procedures. Thus, high throughput analysis of genomic alterations for risk adapted patient stratification and monitoring within treatment trials should rely on efficient and automated diagnostic techniques. In this study, we used array CGH to a novel generation of DNA Chips containing 2800 genomic DNA probes. Target clones comprised i) contigs mapping to genomic regions of possible pathogenetic relevance in lymphoma (n=610 target clones mapping to e.g. 1p, 2p, 3q, 7q, 9p, 11q, 12q, 13q, 17p, 18q, X); ii) selected oncogenes and tumor suppressor genes (n=686) potentially relevant in hematologic neoplasms; and iii) a large genome-wide cluster of 1502 target DNA clones covering the genome at a distance of app. 2 Mbp (part of the golden path clone set). This chip represents a median genomic resolution of app. 1.5 Mbp.

In total, DNAs from 70 FL samples were analyzed and results were compared to data from chromosomal CGH experiments and clinical data sets. The sensitivity of array CGH was considerably higher compared to chromosomal CGH (aberrations in 95% of cases vs 70% of cases). Most frequent aberrations were gain mapping to chromosome arms 2p (21%), 7p (24%), 7q (30%), 12p (17%), 12q (21%), 18p (21%) and 18q (34%) as well as losses mapping to chromosome arms 1p (19%), 6q (23%), 7p (20%), 11q (26%) and 17p (20%). In addition, several genomic aberrations were identified which have not been described before in FL. Currently, these aberrations are characterized in more detail and results will be correlated with the clinical data set. Moreover, three recurrent sites of genomic polymorphisms in human beings affecting chromosomes 5q, 14q and 15q were identified. In conclusion, these data underline the potential of array CGH for the sensitive detection of genomic imbalances in FL.
Quantitative Analysis of Protein Expression Changes Induced by Geldanamycin in NPM-ALK Positive Lymphoma Cells by Functional Proteomics.
Authors: Schumacher Jonathan A. , Crockett David K. , Lin Zhaoshang , Elenitoba-Johnson Kojo S.J. , Lim Megan S.
Published: October 2019

A subset of anaplastic large cell lymphomas are characterized by the t(2;5)(p23;q35) chromosomal translocation. Fusion of the nucleophosmin (NPM) gene on chromosome 5q35 with the anaplastic lymphoma kinase (ALK) gene on chromosome 2p23 results in the constitutive expression of the NPM-ALK oncoprotein. Heat shock protein 90 (HSP90) is a molecular chaperone involved in the folding of a number of substrate proteins implicated in cancer. The list of known HSP90 client proteins is expanding and includes oncogenes, cell cycle-associated proteins, and kinases including NPM-ALK. The ansamycin-based anti-cancer drug, geldanamycin (GA) has been shown to inhibit HSP90 by interfering with its function as a molecular chaperone, resulting in ubiquitin-mediated proteasomal degradation of its client proteins. To date, the cellular effects of inhibition of HSP90 by GA remain largely uncharacterized. Here, we describe a functional proteomics approach to identify the differentially expressed proteins in NPM-ALK positive lymphoma cells exposed to GA. GA induced a dose- and time-dependent decrease in cell viability with an IC50 of 20μM, which was associated with a G2/M cell cycle arrest followed by caspase-3 mediated apoptosis. Equal amounts of total cell lysates from DMSO-control and GA-treated SUDHL-1 cells were labeled with either the light or heavy cleavable ICAT™ reagent. Samples were combined, digested with trypsin, and separated by strong cation exchange chromatography and avidin affinity chromatography. Peptides were subjected to liquid chromatography (LC), electrospray ionization (ESI) and tandem mass spectrometry (MS/MS). The resulting peptides were searched against the NCBI and Swiss-Prot databases using SEQUEST™ for protein identification and relative quantitation determined by XPRESS™. A total of 117 proteins were differentially expressed by greater than 1.5-fold of which 48 were overexpressed and 69 were underexpressed in GA-treated cells relative to control. The 48 overexpressed proteins included those that are involved in apoptosis, protein degradation, cell communication, and cell cycle regulation. Of the 69 underexpressed proteins, many included proteins involved in ubiquitin-proteasome function (proteasome beta 2 subunit, HSP90-organizing protein, CBL-C), cytokines (IL-14 precursor), and DNA repair. Notably, a number of previously reported targets of HSP90/70 (male germ cell-associated kinase and IL-26 precursor) were identified. Our results provide insights into the role of HSP90 in the pathogenesis of NPM-ALK positive lymphomas and demonstrate the diverse cellular functions that are deregulated as a consequence of HSP90 inhibition.
Expression of Activation-Induced Cytidine Deaminase and Transcripts of Immunoglobulin Variable Heavy Chains in Mature B-Cell Lines Lacking Immunoglobulin Protein Expression.
Authors: Matsuo Yoshinobu , Drexler Hans G. , Harashima Akira , Okochi Ayumi , Daibata Masanori , Takakuwa Tetsuya , Orita Kunzo
Published: October 2019

Expression of immunoglobulin (Ig) chains is a definitive marker of B-cell origin. Ig chains are generated during B-cell maturation by somatic rearrangement of Ig gene sequences. This rearrangement generally follows an orderly progression beginning with the heavy (H) chain genes and proceeding to the light (L) chain genes. The fact that Ig expression in B-cells generally reveals single L and H chains implies monoclonal cell expansion. Nevertheless, several exceptions have been reported, including double light chain positive cases. However, only little is known about mature B-cell malignancies lacking any Ig protein expression at all. In order to clarify the mechanisms of lacking any Ig protein expression in mature B-cell malignancies, we have analyzed the expression of activation-induced cytidine deaminase (AID), terminal deoxynucleotidyl transferase (TdT), recombination acting gene (RAG)-1 and RAG-2, transcription factors (TFs) and mRNA transcripts of Ig variable heavy (IgVH) chains using five both surface membrane (Sm) and cytoplasmic (Cy) Ig negative B-cell lines derived from acute lymphoblastic leukemia L3 type (ALL-L3), Burkitt’s lymphoma (BL) and pyothorax-associated lymphoma (PAL). BALM-9N and BALM-16 are ALL-L3, KATATA is BL, OPL-1 and OPL-3 are PAL-derived cell lines (EBV+). In cytogenetic analysis, t(8;14)(q24;q32) in BALM-9N and KATATA and t(8;22)(q24;q11) in BALM-16 were detected, respectively. Band 14q32 was not involved in the PAL cell lines. All cell lines showed rearrangement of IgH chain genes. Expression of RAG-1 and RAG-2 was variable, TdT was negative for all, AID was expressed in all cell lines except for PAL cell line OPL-3. The seven TFs BOB1/OBF1, E2A, IKAROS, OCT1, OCT2, PAX5 and PU1 were universally expressed at the mRNA level in all cell lines. Reverse transcriptase polymerase chain reaction elucidated the IgVH gene usage. BALM-9N = VH4; BALM-16 = VH2; KATATA = VH3; OPL-1 = VH3; OPL-3 = VH3. In summary, the present study demonstrates that Ig gene recombination, class switch recombination and somatic hypermutation machineries are not the cause for the lacking expression of both Sm/Cy Ig chains in mature B-cell malignancies. The lack of any Sm/Cy Ig expression in such mature B-cells may indicate rather a germinal center origin.
Acquirement of Rituximab Resistance Is Associated with the Development of Chemotherapy Resistance in B-Cell Lymphoma Cells: Evidence of Shared Pathways of Resistance between Chemotherapeutic Agents and Biological Therapies.
Authors: Olejniczak Scott H. , Hernandez Francisco J. , Czuczman Myron S.
Published: October 2019

Pre-clinical studies have demonstrated that rituximab triggers apoptotic signals in B-cell lymphoma cells upon biding to CD20 antigen. Downstream signaling events observed in lymphoma cells following in vitro exposure of rituximab or chemotherapy include: 1) activation of the intrinsic apoptotic pathway and 2) increased mitogen activated protein kinase (MAPK) activity. In addition, pre-clinical and clinical studies strongly suggest that rituximab may sensitize lymphoma cells to apoptotic effects of various drugs used to treat NHL. Despite its anti-tumor activity, many patients relapse after initial response to rituximab-based therapy and demonstrate variable degrees of rituximab resistance. To further study the impact of rituximab resistance in cellular responses to chemotherapy we developed several rituximab resistant cell lines (RRCL) derived from Raji, SU-DHL-4 and RL cells by exposing cells to escalating doses of rituximab with (4RH cells) or without (2R cells) human serum. The rituximab resistance of each RRCL was confirmed by immunological assays. Subsequently, lymphoma cells (parental and RRCL) were exposed to various chemotherapeutic agents (cisplatin, doxorubicin, paclitaxel, or vincristine) for up to 48 hours. Detection of cell death was determined by trypan blue and/or propidium iodine staining. Caspase-3 activity was measured by PhiPhi Lux G1D2 enzymatic cleavage. Bcl-2 expression was determined by Western blotting. Chemotherapy resistance to all agents tested was observed in RRCL when compared to parental Raji, SU-DHL-4 and RL cell lines. In addition, caspase-3 activity was lower in RRCL following chemotherapy exposure than in parental cell lines. A significantly lower percent of RRCL cells within sub-G0/G1 peaks in cell cycle histograms confirmed that RRCL were less sensitive to chemotherapy-induced apoptosis. Additionally, an increase in Bcl-2 expression was observed in RRCL when compared parental cell lines. Our data strongly suggest that chemotherapy resistance emerges concomitantly with the acquirement of rituximab resistance in lymphoma cells. Chronic exposure to rituximab appears to cause overexpression of Bcl-2, which likely renders RRCL less susceptible to the apoptotic effects of chemotherapy agents. Ongoing studies are aimed at identifying and overcoming rituximab/chemotherapy shared cellular pathways of resistance.
Allogeneic Stem Cell Transplantation for Children with Advanced Primary MDS: Results from the EWOG-MDS Study Group Employing a Pre-Transplant Preparative Regimen with Busulfan, Cyclophosphamide and Melphalan.
Authors: Niemeyer Charlotte Marie , Zecca Marco , Korthoff Elisabeth , Duffner Ulrich , Zintl Felix , Ebell Wolfram , Starý Jan , Dilloo Dagmar , Peters Christina , Schmugge Markus , Sedlacek Petr , Messina Chiara , van Heuvel Marry , Bergstraesser Eva , Trebo Monika , Noellke Peter , Locatelli Franco
Published: October 2019

MDS in children is a rare disorder characterized by dysplasia and defined genetic abnormalities. In most patients (pts) MDS arises without known predisposing conditions (primary MDS). Here, we report the results of 55 males and 30 females with advanced primary MDS enrolled in the prospective EWOG-MDS trial 97. Data were analysed according to the most advanced FAB-type prior to SCT: 32 pts were classified as RAEB, 40 as RAEB-t and 13 as myelodysplasia-related AML (MDR-AML). Median age at diagnosis was 9.5 yrs (0.1–17.6) and median time from diagnosis of advanced MDS to SCT 4 mo (0.5–31). Cytogenetics revealed monosomy 7 in 32 pts, trisomy 8 in 7, a complex karyotype in 9 and other abnormalities in 9; karyotype was normal in 26 pts and unknown in 2. 31 pts had received AML-like therapy prior to SCT. All pts were given an unmanipulated graft after condititioning with busulfan 16 mg/kg, cyclophosphamide 120 mg/kg and melphalan 140 mg/m. Source of stem cells was bone marrow in 56 pts, peripheral blood in 25, cord blood in 2 and unknown in 2. 36 pts were transplanted from an HLA-identical relative (MFD), 49 pts from an HLA-identical or 1-antigen disparate unrelated donor (UD). GVHD prophylaxis consisted of CSA alone for MFD, whereas recipients of a UD graft generally received CSA, methotrexate and anti-lymphocyte globulin. Two pts suffered graft failure. The cumulative incidence of grade II-IV acute GVHD and chronic GVHD was 40% (SE 5%) and 25% (SE 5%), respectively. 18 pts suffered transplant-related mortality (TRM), the cumulative incidence of TRM in pts grafted from a MFD or UD being 14 and 25%, respectively (p=n.s.). Presence of acute GVHD II-IV (p<0.01), spleen size at SCT ≥ 1 cm below the costal margin (p=0.03) and age ≥ 12 years (p=0.04) predicted an increased risk of TRM. 17 patients relapsed at a median of 12 mo after SCT (1–107). The 5-year probability of leukemia recurrence was 29%, with no difference between MFD and UD transplants. While the highest FAB type prior to SCT predicted relapse with a cumulative incidence rate increasing from RAEB (13%, SE 10%) to RAEB-t (24%, SE 11%) and MDR-AML (69%, SE 20%) (p=0.03), the use of intensive chemotherapy prior to SCT or blast percentage at SCT did not. With a median observation time after SCT of 29 months (6–107) the EFS at 5-years was 62% (SE 12%) and 39% (SE 11%) for pts given SCT from a MFD or an UD, respectively (p=n.s). These results indicate that a large proportion of pts with advanced MDS can be rescued by SCT. Disease recurrence remains the main cause of treatment failure. Intensive chemotherapy prior to SCT should not be routinely employed.
Improved Outcomes for Peripheral Blood Stem Cell Transplantation in Advanced Myelodysplastic Syndrome.
Authors: Solomon Scott R. , Childs Richard , Montero Aldemar , Sloand Elaine , Wisch Laura , Wehrlen Leslie , Barrett John
Published: October 2019

Allogeneic marrow or peripheral blood stem cell transplantation (PBSCT) is the only curative treatment for myelodysplastic syndrome (MDS). Historically, transplantation for MDS has produced long-term disease-free survival rates of 30–40%, partially due to high procedural mortality (~40%) in this patient population. Although transplant outcomes in younger patients with low-risk disease have been favorable, inferior results are seen in older patients and those with more advanced disease. Evidence suggests that the lower transplant-related mortality (TRM) and improved graft-versus-leukemia seen with PBSCT may translate into improved clinical outcomes for MDS patients. Forty-four patients, aged 12–73 years (median 50) received a PBSCT from a matched related sibling donor (MRD). Patients aged <55 years, without prohibitive comorbidity, received myeloablative conditioning consisting of total body irradiation and cyclophosphamide, followed by a T cell depleted allograft and scheduled post-transplant donor lymphocyte infusions (MST, n=23). Patients ineligible for an ablative transplant due to age or poor health received reduced intensity conditioning (fludarabine and cyclophosphamide, melphalan, or busulfan) followed by a T cell replete allograft (n=21). Six patients had low-risk MDS (RA/RARS), while the majority of patients (86%) had advanced disease (RAEB [9], RAEBT [6], AML [13], therapy-related MDS [10]). Median follow-up is 15.3 (range 2–82) months. Patients with therapy-related MDS had a significantly lower survival rate due to a very high risk of relapse (figure). The actuarial probabilities of overall survival (OS), disease-free survival (DFS), relapse, and TRM were 64%, 59%, 26%, and 23% for primary MDS patients, and 51%, 47%, 40%, and 25% for the whole cohort. Transplant-related mortality in patients under 50 years of age was 11% vs. 45% in patients ≥50 years (p=0.03). OS and DFS were significantly better in recipients of MST (64%, 57%) than in patients receiving reduced-intensity PBSCT (33%, 34%), due to a higher risk of relapse in the latter group (55% vs. 29%, p=0.10). In nineteen patients <50 years receiving MST, actuarial probability of OS, DFS, relapse, and TRM were 81%, 72%, 23%, and 7%, respectively. In summary, PBSCT yields superior outcomes for patients with primary MDS, even in patients in transformation to AML. The inferior outcomes seen in therapy-related MDS suggest alternative therapies are required for this patient population. Reduced intensity transplantation permits curative therapy for MDS patients not amenable to MST, but at the price of increased TRM and relapse in this older cohort.

Figure Figure
Gene Expression Analysis of Peripheral T-Cell Lymphoma Not Otherwise Specified Reveals the Existance of Two Subgroups Related to Different Cellular Counterparts and Recurrent PDGFRA Deregulation.
Authors: Piccaluga Pier-Paolo , Agostinelli Claudio , Zupo Simona , Califano Andrea , Went Philip , Ascani Stefano , Basso Katia , Facchetti Fabio , Falini Brunangelo , Ferrarini Manlio , Gallamini Andrea , Klein Ulf , Novero Domenico , Paulli Marco , Tura Sante , Zinzani Pier Luigi , Baccarani Michele , Dalla-Favera Riccardo , Pileri Stefano A.
Published: October 2019

Peripheral T-cell lymphoma not otherwise specified (PTCL/NOS) is a rare non Hodgkin lymphoma characterized by heterogeneous morphologic and phenotypic features, aggressive clinical behavior, poor response to conventional treatments and dismal prognosis. Its molecular basis and relationships to normal T-cells are still unclear. We performed gene expression profiling of 17 PTCL/NOS cases, collected at diagnosis before any treatment, and 20 samples of normal T-lymphocytes. The comparison of the gene expression profiles of PTCL/NOS and purified normal T-cell subpopulations, including CD4+, CD8+, resting (HLA-DR-), and activated (HLA-DR+) elements, shows that PTCLs/NOS are more related to activated peripheral T-lymphocytes either CD4+ or CD8+, suggesting derivation from these compartments. Notably, immunohistochemistry on routine sections by using anti-CD4 and CD8 antibodies does not vicariate molecular analysis, as there is no correspondence between the global gene expression profile and CD4/CD8 antigen positivity. When compared with normal T-cells, PTCLs/NOS displayed a remarkable deregulation of functional programs often involved in tumorigenesis, such as apoptosis, proliferation, cell adhesion, and matrix remodeling. In addition, our analyses identified several genes that are specifically expressed in PTCLs/NOS. Their expression was confirmed at the protein level by immunohistochemical analysis of tissue micro-arrays including 160 primary PTCL/NOS cases. We applied several different markers that provided significant information as concerns the ectopic, paraphysiologic or stromal expression of the proteins corresponding to the deregulated genes. Among others, PTCLs/NOS aberrantly express CYR61, a molecule involved in drug resistance, and PDGFRA, a tyrosine kinase receptor whose deregulation is often related to a malignant phenotype. Notably, we demonstrated the constitutive activation of PDGFRA in the majority of cases - as indicated by its phosphorylation. Finally, we showed that imatinib mesylate significantly reduces PTCL/NOS primary cells viability after in vitro exposure at 1 mM concentration, independently from their sensitivity to conventional cytotoxic agents. These results are provided with biological implications relevant to the pathogenesis of the tumor, as well as for its clinical management.
Androgen Modulates Thymopoiesis by Regulating Thymocyte Precursor Immigration.
Authors: Williams Kirsten M. , Chu Yu-Waye , Gress Ronald E.
Published: October 2019

Impaired thymopoiesis contributes to immune deficiency in aging, AIDS, and following allogeneic bone marrow transplantation. However, little is known of the mechanisms of thymic regulation and the determinants of thymic function. Exogenous administration of testosterone and estrogen has been shown to accelerate thymic involution. Conversely, studies have demonstrated that castration of male mice resulted in increased thymic size and thymocyte number. We present data that thymic enlargement in castrated male mice is due to enhanced thymopoiesis. Comparing castrated post-pubertal male mice with age-matched control littermates, we found a statistically significant doubling of thymic weight, thymocyte number, and double-positive CD4+CD8+ thymocytes as early as eight days post-orchiectomy. Major thymocyte subsets defined as CD4− CD8−, CD4+CD8−, CD8+CD4− were also increased post castration and the ratio of subsets was unchanged suggesting that castration augmented overall thymic activity. This was further corroborated by a concomitant 4–6 fold increase in thymic T cell-receptor excision circles (TREC). Significantly, at one month post-castration, early thymic progenitors (Lin- CKIT hi CD44 hi) were increased threefold in the castrated cohort. The increase in thymic productivity led to a subsequent increase in peripheral cell populations, with a significant increase in splenic CD4+ and CD8+ T cells. The greatest proportion of this increase was due to naïve splenic CD4 and CD8 T cells total numbers as defined by CD44lo expression. Memory splenocytes, CD44hi CD4+ and CD8+ cells, were slightly increased as well. Additionally, there was an increase in the number of splenic recent thymic emigrants (RTE) as enumerated by TREC. Our data suggest that androgen withdrawal leads to an increase in thymopoiesis, as enumerated by a significant influx of ETPs, increased total thymic TREC, subsequent increases in single positive thymocytes, and increased peripheral recent thymic emigrants. Furthermore, the data suggest that a mechanism by which this occurs is through increased immigration of thymocyte precursors from the bone marrow into the thymus identifying this as a central point in thymic regulation.