bluuebunny/crossref_metadata_2025 huggingface dataset:
Pococurante (Poc): An ENU-Induced Missense Mutation of MyD88 with Selective Effects on TLR Signal Transduction.
Authors: Jiang Zhengfan , Li Chenglong , Shamel Louis , Olson Arthur , Beutler Bruce
Published: October 2019

Toll-like receptors (TLRs) are key sensors of the innate immune system, and individual TLRs respond to specific molecules derived from microbes. MyD88 is a Toll/Interleukin-1/Resistance (TIR) domain-containing adaptor protein required for signaling by all TLRs except TLR3. While the structural basis of association between MyD88 and TIR-domain receptors is obscure, MyD88-deficient mice show no responses to bacterial flagellin, peptidoglycan (PGN), lipoteichoic acid (LTA), bacterial lipopeptides such as PAM2CSK4, PAM3CSK4 and R- or S-MALP-2, DNA bearing unmethylated CpG dinucleotides (CpG DNA), or Resiquimod (RSQ). Using germline ENU mutagenesis, we have produced a large number of phenotypic variants that have abnormal TLR signaling. We now report the identification of a new mutation called Pococurante (Poc), originally detected in screening because macrophages from this mouse showed no response to the tri-acylated lipopeptide PAM3CSK4, the di-acylated lipopeptide S-MALP-2, LTA, CpG DNA, RSQ, and a markedly reduced response to LPS: the ligands for TLRs 2/1, 2/6, 9, 7 and 4 respectively. They also had no response to interleukin-1, a cytokine that signals by way of a MyD88-dependent TIR domain receptor. However, Poc mice showed a normal response to PGN, as well as R-MALP-2 and PAM2CSK4 lipopeptides. The latter three ligands are sensed in a TLR2-dependent, MyD88-dependent fashion. The Poc phenotype was ascribed to a point mutation of MyD88 affecting a surface residue (I179N). Because the mutation is discriminatory, permitting MyD88 to carry a signal from some TIR domain receptors but not others, we infer that it resides at the receptor:adaptor signaling interface. A new model of TIR receptor:adaptor interaction is proposed on the basis of docking studies that take account of the Poc phenotype, made using the protein-protein docking program SURFDOCK. We note that S-MALP-2 is dependent upon TLR2/6 heterodimers, while PAM3CSK4 sensing depends upon TLR2/1 heterodimers. Since the Poc mutation forbids detection of both these ligands while it allows detection of PAM2CSK4 and R-MALP-2, it may be inferred that TLR2 signal transduction entails greater structural diversity than was previously supposed. The involvement of TLR2 homodimers, or the incorporation of subunits yet unknown into the receptor complex, cannot be excluded.
Imatinib Mesylate (GLEEVEC®) Is Effective in the Treatment of Polycythemia Vera: A Multi-Institutional Clinical Trial.
Authors: Silver Richard T. , Fruchtman Steven M. , Feldman Eric J. , Spivak Jerry L. , Salvado August J.
Published: October 2019

We previously reported that the erythrocytosis of polycythemia vera was controlled in a small group of patients with imatinib mesylate. This effect may involve inhibition of tyrosine phosphorylation of c-Kit, the stem cell factor receptor essential for erythroid progenitor cell proliferation. We now report treating 27 polycythemia vera patients with imatinib in a multi-institutional study. There were 13 men, 14 women, median age 52 years, range 29 to 83 yrs. All had an increased 51Cr RBC mass determination at diagnosis and fulfilled stipulated criteria for the diagnosis of polycythemia vera. Sixteen of 27 had platelets counts greater than 400,000/?l. Prior to imatinib, disease duration ranged from 1 month to 15 years (median 31 months, mean 52.1 months). Eleven patients had been treated only by phlebotomy; 16 with hydroxyurea, interferon, or anagrelide and supplemental phlebotomy. Men were initially phlebotomized to a hematocrit of 45% or less and women to a hematocrit of 42% or less. Prior to imatinib, the average number of phlebotomies per year ranged from 0 to 14 (median 6); after 1 yr of imatinib, phlebotomy number ranged from 0 to 6 (median 1). Imatinib was started at 400 mg qd escalating every 2 weeks by 100 mg to a maximum of 800 mg for persistent phlebotomy requirements or for platelet counts above 400,000/?l. All patients received 81 mg of aspirin daily. There were 5 complete remissions (CR) (phlebotomy-free, normal platelets, no splenomegaly) and 8 partial remissions (PR) (phlebotomy-free but platelet counts greater than 400,000?/l and/or splenomegaly). No patient with initial thrombocytosis achieved a platelet count less than 400,000/?l with imatinib. Four patients are too early to evaluate. Of the remaining 10, there were 2 failures (one developed myelofibrosis, one increasing thrombocytosis and a TIA but no neurologic sequelae), one protocol violation and 2 patients were removed for unrelated medical conditions. Five of these 10 patients developed drug toxicity: grade 3 dermatologic (n=2); grade 3 diarrhea (n=2); grade 3 bone pain (n=1); all recovered with drug withdrawal. The mean time to CR/PR was 4 months. The median duration on treatment was 11 months. Thus, of 23 evaluable patients to date, 13 (57%) have had a CR or PR. The difference in response of thrombocytosis in CML patients to imatinib compared to polycythemia vera is notable and requires further evaluation. We conclude that imatinib is useful for treating erythrocytosis and controlling splenomegaly in some patients with polycythemia vera, but not for controlling thrombocytosis or splenomegaly in others. These data suggest heterogeneity of the mechanisms involved in hematopoietic stem cell proliferation in PV or patient variability with respect to the metabolism of imatinib.
Phenotypic Description of Familial Chronic Myeloproliferative Disorders.
Authors: Najman Albert , Chaumarel Isabelle , Bellann-Chantelot Christine , Barbu Veronique , Labopin Myriam , Rain Jean D. , Casadevall Nicole , Varet Bruno , Bauters Francis , Thomas Gilles
Published: October 2019

Chronic myeloproliferative disorders (CMPD) are rare diseases of the bone marrow characterised by a clonal proliferation of one or several myeloid lineages. Familial clustering are regularly reported suggesting a role for inherited factors. To adress this issue, we collected with the help of a large network of haematologists clinical data and blood samples of 78 families defined by the presence of at least two affected subjects with one of the four CMPD: polycythemia vera, essential thombocythemia, chronic myeloid leukemia and agnogenic myeloid metaplasia. A total of 176 affected subjects including 83 polycythemia vera, 70 essential thrombocythemia, 12 chronic myeloid leukemia and 11 agnogenic myeloid metaplasia were recruited. 449 asymptomatic relatives, mainly first-degrees, were collected and among them 11%were carriers of endogenous erythroid colonies with normal blood counts. Phenotypic spectrum within families was either homogenous (46/78) or heterogeneous (32/78) and the main association (25/32) was polycythemia vera and essential thrombocythemia. A few cases of changes in disease phenotype was also observed during the course of CMPD. Clinical and haematological data of affected subjects at diagnosis and during the course of the disease were similar to sporadic CMPD. However, a marked anticipation of approximatively 20 years/generation at onset age was observed. No excess of carcinomas was noted either in affected subjects or relatives. An excess of acute leukemias, however, was noted in relatives. We did not show evidence of known environmental factors such as exposition to ionic radiations or chemical solvents. Familial cases were restricted to a single generation in 33 families. Occurence was vertical in 45 families involving 2 generations in 36 and 3 generations in 9. In those familial cases consistent with a dominant inheritance, we exluded molecular abnormalities of VHL and EpoR genes involved in particular forms of familial and congenital polycythemia. In conclusion, the analysis of these 78 familial CMPD cases highlights that (i) the observation of mixed phenotypes in a large proportion is consistent with the theory that MPD arise from clonal expansion of a pluripotent hematopoietic precursor cell (ii) no environmental factor is clearly involved in the onset of CMPD and (iii) the observation of familial aggregations and the low incidence of the pathology suggest the implication of genetic predisposition factors in the occurence of myeloproliferative disorders. This large collection of multiplex families enables us to initiate genome-wide linkage analysis.
Role of Cell Adhesion in the Maintenance of Hematopoietic Stem Cells in the Bone Marrow Niche.
Authors: Arai Fumio , Hirao Atsushi , Hosokawa Kentaroh , Suda Toshio
Published: October 2019

Interaction of HSCs with their particular microenvironments, known as the stem cell niches, is critical for adult hematopoiesis in the bone marrow (BM). HSCs are keeping the balance of the quiescence and the self-renewal in the stem cell niche, and are maintaining long-term hematopoiesis. We have demonstrated that HSCs expressing the receptor tyrosine kinase Tie2 are quiescent and anti-apoptotic, transplantable and comprise a side-population (SP) of HSCs, which contact closely to angiopoietin-1, a ligand for Tie2, expressing osteoblasts (OBs) in the BM niche. Tie2 and Ang-1 are part of a key signaling interaction between HSCs and niche cells. This signaling pathway regulates the feature of HSCs in the BM niche. The interaction of Tie2 with Ang-1 in vitro is sufficient to maintain the long-term blood-repopulating (LTR) activity of HSCs in vivo by preventing cell division. In addition, Ang-1 enhanced the ability of HSCs to become quiescent and also induced their adhesion to bone surface, resulting protection of HSC compartment from stresses which suppress hematopoiesis (Arai et al., Cell, 2004). In this study, we focused on the role of cell adhesion molecule on the maintenance of HSCs in BM niche. A previous paper reported that both quiescent HSCs and some of osteoblasts express N-cadherin. We found that Tie2+ HSC expressed N- and VE-cadherin, and OBs expressed N-, P-, and OB-cadherin. This suggests that an adherens junction between HSCs and OBs created via N-cadherin may contribute to HSC maintenance. Furthermore, N-cadherin overexpressing OP9 stromal cells (OP9/N-cadherin) maintained LTC-IC more than control OP9 cells did. Overexpression of N-cadherin in HSCs also maintained colony formation. In addition, in the presence of Ang-1 in coculture of OP9/N-cadherin and HSCs facilitated the maintenance of CFU-C and HPP-CFC formation after long-term culture. Furthermore, Ang-1 treatment upregulated the expression of N-cadherin in Tie2+ HSCs as well as β1-integrin. It suggests that Tie2/Ang-1 signaling enhanced cell-cell adhesion between HSCs and OBs.

Altogether, these observations led us to the novel model that the localization of HSCs on bone surface is regulated by stem cell specific adhesion molecules such as N-cadherin. Once the HSCs adjacently localize to OBs, Ang-1 produced by OBs may activate Tie2 on the HSCs and promote tight adhesion of HSCs in the niche, resulting in quiescence and enhanced survival of HSCs.
Phenotypic and Functional Characterization of Smooth Muscle-Like Cells Derived from Multipotent Adult Progenitor Cells (MAPCs).
Authors: Ross Jeffrey J. , Keirstead Susan A. , Felton Mike , Tranquillo Robert T. , Verfaillie Catherine M.
Published: October 2019

Smooth muscle cells (SMC) play a key role in vascular development and morphogenesis of blood vessels defined by a noncontractile, or synthetic SMC phenotype. Mature SMCs respond to vascular injury/repair by phenotypic modulation to a synthetic state, and hence are also involved in various clinical disorders including atherosclerosis and cancer. We have induced relatively homogenous differentiation of SMC-like cells from multipotent adult progenitor cells (MAPC).

MAPCs have been isolated from murine, rat and human bone marrow. MAPCs can differentiate into mesodermal, neuroectodermal, and endodermal cell types. Here we report that serum free medium supplemented with PDGF-BB and TGF-b1 allows differentiation of rat MAPCs to a population of cells expressing smooth muscle specific markers, as determined by Q-RT-PCR (a-SMC actin, calponin, SM-22a, myocardin, and smooth muscle heavy chain myosin),and immunohistochemistry (a-SMC actin staining in >90% cells and calponin staining in ~50% of cells, co-localization localized with defined stress fibers). Such SMCs can subsequently be passed for at least 15 passages (26 doublings) in the presence of FCS and PDGF-BB and TGF-b1, without loss of phenotypic characteristics. RT-PCR analysis for genes of other mesodermal lineages, including cardiac muscle and endothelium expressed genes (Nkx, Mef-2D, PCAM, and vWF) was negative.

To characterize the function of the SMC-like cell population, calcium handling and in-vitro remodeling properties were studied. Passage 4 SMC-like cells responded to endothelin-1, norepinepherine, oxytocin, arg-vasopressin, and bradykinin with an increase in intracellular calcium ion concentration, consistent with a smooth muscle phenotype. To assess the remodeling potential, passage 4 cells were entrapped into fibrin matrix hemispheres as previously described for neonatal rat SMC. After 5 weeks in culture, hydroxyproline assays quantified total collagen as (86.8 +/− 9.3 ug) approximately 50% the amount reported using neonatal SMCs. In addition, the remodeled hemispheres demonstrated defined mechanical properties (tensile strength= 0.20 MPa, modulus = 0.65 MPa), similar to what we have previously observed when neonatal rat SMCs are entrapped in fibrin disks. Masson Trichrome staining demonstrates areas of tissue organization characterized by fibrils and collagen production. While further characterization of this population is needed to determine their developmental plasticity, the data is suggestive of a vascular SMC-like cell population derived from MAPCs that demonstrates functional smooth muscle characteristics and could be used to tissue engineer blood vessels.
Early Consolidation with Myeloablative Radiochemotherapy Followed by Autologous Stem Cell Transplantation in First Remission Significantly Prolongs Progression-Free Survival in Mantle Cell Lymphoma - Long Term Follow up of a Prospective Randomized Trial of the European MCL Network.
Authors: Dreyling Martin H. , Lenz Georg , Schiegnitz Eva , van Hoof Achiel , Gisselbrecht Christian , Pfreundschuh Michael , Metzner Bernd , Truemper Lorenz H. , Reiser Marcel , Steinhauer Hjalmar B. , Boiron Jean-Michel , Boogaerts Marc A. , Aldaoud Ali , Silingardi Vittorio , Kluin-Nelemans Hanneke C. , Hasford Joerg , Parwaresch Reza , Michael Unterhalt , Wolfgang Hiddemann
Published: October 2019

Mantle cell lymphoma (MCL) is a distinct subtype of malignant lymphoma with an especially poor prognosis and a median survival of only 3–4 years. In 1996, the European MCL Network initiated a randomized trial to improve the otherwise dismal outcome of MCL, comparing early consolidation with myeloablative radiochemotherapy (TBI 12 gray, cyclophosphamide 120 mg/kg bw) followed by autologous stem cell transplantation (ASCT) to a conventional a -interferon maintenance (6x106 IE IFNa 3x weekly) in first remission after a CHOP-like induction regimen. Until March 2004, a total of 269 previously untreated patients (up to 65 years) were randomized upfront. 189 (82%) of 230 patients with advanced stage MCL completed initial induction chemotherapy and 142 (75%) achieved either a complete (45 pts., 24%) or partial response (97 pts, 51%). 122 pts proceeded to consolidation therapy, 62 pts received ASCT consolidation and 60 patients were assigned to IFN maintenance.

Patients in the ASCT study arm experienced a significantly longer progression-free survival (PFS) as compared to patients in the IFNa arm. The PFS at 2 years was 73% after ASCT as compared to only 43% in the IFNa arm (p = 0.0108). Similar results were achieved in the intent to treat analysis of all initially randomised MCL patients (p=0.0001). Accordingly, this advantage resulted in a trend towards an improved overall survival (OS) in the ASCT arm. After a follow-up of up to nearly 7 years (median follow-up of patients: 2.8 years), 3 year survival after ASCT was 83% in comparison to 77% under IFNa maintenance (p = 0.18). As expected, acute toxicity was higher in the ASCT group with an early mortality of 4.8%, whereas long-term effects were more frequently encountered under IFNa maintenance.

Conclusion : In first line treatment of advanced stage MCL, dose-intensified consolidation with myeloablative radiochemotherapy followed by ASCT after CHOP-like induction results in a significant prolongation of PFS. Current study concepts evaluate the benefits of combined immuno-chemotherapy to further improve the overall survival.
Risk Factors of Myelodysplastic Syndromes (MDS): A Case-Control Study.
Authors: Strom Sara S. , Gu Yun , Gruschkus Stephen K. , Estey Elihu H.
Published: October 2019

Little is known about the etiology of MDS. A hospital-based case control study was conducted to investigate the association between lifestyle characteristics and MDS risk. Cases included 354 newly diagnosed adult de novo MDS cases registered at M. D. Anderson Cancer Center (MDACC) between 1996 and 2003. Four hundred and fifty two age-, sex- and race-matched healthy controls were recruited among visitors to MDACC who accompanied patients to different clinics excluding the Leukemia clinic. A validated questionnaire was used to collect demographic and epidemiological information including lifetime history of smoking, alcohol drinking and occupational history. Multivariate logistic regression analysis adjusting for all variables simultaneously was used. The distribution by FAB type was 102 (29%) RA/RARS, 43 (12%) CMML and 209 (59%) RAEB/RAEB-T. The mean age of patients was 64 years (range: 24 to 89), the male to female ratio was 2.72, and 94% of cases were whites. Abnormal cytogenetics was found in 49% of the patients. Multivariate logistic regression analysis was performed among all MDS cases and among each FAB type. For all MDS combined, having a first degree relative diagnosed with a hematopoietic cancer (Odds ratio (OR)=1.92, 95% CI: 1.11–3.33), having ever smoked (OR=1.65, 95% CI: 1.19–2.29), having been exposed to agricultural chemicals (OR=4.57, 95% CI: 1.78–11.7) and solvents (OR=2.08, 95% CI: 1.22–3.54) were associated with MDS risk. Wine drinkers had a reduced risk of MDS as compared to those who never drank alcohol (OR=0.54, 95%: 0.37–0.79). For the RA/RARS type, smoking (OR=2.16, 95% CI: 1.3–3.6) and having been exposed to agricultural chemicals (OR=4.64, 95% CI: 1.27–16.9) were the only two risk factors identified. Wine drinkers were at reduced risk (OR=0.51, 95% CI: 0.29–0.91). For the RAEB/RAEBT type, a positive family history of hematopoietic cancer (OR=2.16, 95% CI: 1.17–3.98), smoking (OR=1.55, 95% CI: 1.05–2.30) and exposure to agricultural chemicals (OR=4.12, 95% CI: 1.51–11.3) or solvents (OR=2.83, 95% CI: 1.59–5.05) were independent risk factors. Also drinking wine seemed to reduce the risk by almost 50% (OR=0.51, 95% CI: 0.32–0.81). We also found a joint effect between smoking and chemical exposures with the largest OR found among smokers who were also exposed to solvents or agricultural chemicals as compared to never smokers without chemical exposures (OR=3.22, 95% CI: 2.11–4.92, Trend test: p<0.0001). Results from this large study suggest that several factors play a role in predisposing to MDS with possible joint effects. Risk profiles seem to differ by FAB type. Further population-based studies are needed to confirm these findings. Supported by NCI grants CA91340 and CA 100632.
Coordinated Chromatin-Association of Fanconi Anemia Network Proteins Requires Replication-Coupled DNA Damage Recognition.
Authors: Sobeck Alexandra , Stone Stacie , deGraaf Bendert , Costanzo Vincenzo , deWinter Johan , Wang Weidong , Joenje Hans , Gautier Jean , Hoatlin Maureen E.
Published: October 2019

Fanconi anemia (FA) is a genetic disorder characterized by hypersensitivity to DNA crosslinking agents and diverse clinical symptoms, including developmental anomalies, progressive bone marrow failure, and predisposition to leukemias and other cancers. FA is genetically heterogeneous, resulting from mutations in any of at least eleven different genes. The FA proteins function together in a pathway composed of a mulitprotein core complex that is required to trigger the DNA-damage dependent activation of the downstream FA protein, FANCD2. This activation is thought to be the key step in a DNA damage response that functionally links FA proteins to major breast cancer susceptibility proteins BRCA1 and BRCA2 (BRCA2 is FA gene FANCD1). The essential function of the FA proteins is unknown, but current models suggest that FA proteins function at the interface between cell cycle checkpoints, DNA repair and DNA replication, and are likely to play roles in the DNA damage response during S phase. To provide a platform for dissecting the key functional events during S-phase, we developed cell-free assays for FA proteins based on replicating extracts from Xenopus eggs. We identified the Xenopus homologs of human FANCD2 (xFANCD2) and several of the FA core complex proteins (xCCPs), and biochemically characterized these proteins in replicating cell-free extracts. We found that xCCPs and a modified isoform of xFANCD2 become associated with chromatin during normal and disrupted DNA replication. Blocking initiation of replication with geminin demonstrated that association of xCCPs and xFANCD2 with chromatin occurs in a strictly replication-dependent manner that is enhanced following DNA damage by crosslinking agents or by addition of aphidicolin, an inhibitor of replicative DNA polymerases. In addition, chromatin binding of xFANCD2, but not xBRCA2, is abrogated when xFANCA is quantitatively depleted from replicating extracts suggesting that xFANCA promotes the loading of xFANCD2 on chromatin. The chromatin-association of xFANCD2 and xCCPs is diminished in the presence of caffeine, an inhibitor of checkpoint kinases. Taken together, our data suggest a model in which the ordered loading of FA proteins on chromatin is required for processing a subset of DNA replication-blocking lesions that are resolved during late stages of replication.
TNF-α Hypersensitivity of Fanconi Anemia Type C Deficient Cells Is Dependent on the Apoptosis Signal-Regulating Kinase 1 Signaling Pathway.
Authors: Haneline Laura S. , Bijangi-Vishehsaraei Khadijeh R. , Werne Adam , McKenzie Kristina Wilson , Davis Susanna , Ichijo Hidenori , Saadatzadeh Mohammad R.
Published: October 2019

Fanconi anemia (FA) is a chromosomal instability disorder characterized by a progressive bone marrow (BM) aplasia. Previous studies from our lab and others suggest that enhanced oxidant- and inhibitory cytokine-mediated apoptosis of hematopoietic stem/progenitor cells are important mechanisms in the pathogenesis of BM failure in FA. Recently, we showed that oxidant-induced apoptosis in Fancc −/− murine embryonic fibroblasts (MEFs) was mediated through hyperactivation of the redox-dependent protein, apoptosis signal-regulating kinase 1 (ASK1, Saadatzadeh et al, JBC 2004). Here, we tested whether alterations in ASK1 signaling also participate in the pro-apoptotic phenotype of primary Fancc −/− MEFs and progenitors in response to the inhibitory cytokine, TNF-α. To determine whether Fancc −/− MEFs exhibit altered ASK1 signaling after TNF-α treatment, we examined ASK1 activity using in vitro kinase assays. These studies demonstrated a 2–3 fold increase in TNF-α-induced ASK1 activity in Fancc −/− MEFs compared to WT (n=5). Consistent with ASK1 hyperactivation, we observed an increase in activation of the downstream stress kinases, c-jun N-terminal kinase and p38, in Fancc −/− MEFs compared to WT (n=5). Utilizing a dominant negative ASK1 cDNA to decrease ASK1 activity, we determined that TNF-α-induced apoptosis in Fancc −/− MEFs was ASK1 dependent (n=3, p<0.05). In addition, TNF-α-induced apoptosis in Fancc −/− MEFs was completely blocked by both antioxidants (selenomethionine and N-acetylcysteine, n=3, p<0.001) and the p38 inhibitor SB 203580 (n=6, p<0.001). To examine whether ASK1 has a critical role in the hypersensitivity of Fancc −/− progenitors to TNF-α-induced apoptosis, we crossed ASK1 knockout mice with Fancc mice to generate the following four experimental genotypes; WT, Fancc +/+;ASK1 −/−, Fancc −/−;ASK1 +/+, and Fancc −/−;ASK1 −/−. BM cellularity and progenitors/femur were similar between all genotypes (n=3). Interestingly, TNF-α hypersensitivity of Fancc −/− progenitors was restored to WT levels when ASK1 was deleted (n=3, p<0.05), suggesting that TNF-α-induced hypersensitivity of Fancc −/− progenitors is mediated through ASK1, similar to Fancc −/− MEFs. In addition, TNF-α-induced apoptosis in Fancc −/− progenitors was restored to WT levels after culturing with the p38 inhibitor SB 203580 (n=6, p<0.005). Since ASK1 activity is directly regulated by the cellular redox state, these data support the idea that aberrant redox regulation may be involved in the observed pro-apoptotic phenotype of Fancc −/− cells after exposure to inhibitory cytokines such as TNF-α. Collectively, these data suggest that inhibiting the ASK1 apoptotic pathway in Fancc −/− cells via antioxidants or small molecule inhibitors may protect Fancc −/− hematopoeitic stem/progenitors from an apoptotic fate and delay BM failure.
Hyperacute Graft-Versus-Host Disease: Analysis of Risk Factors, Clinical Manifestations and Outcomes.
Authors: Couriel Daniel R. , Saliba Rima , de Lima Marcos J. , Hosing Chitra , Andersson Borje , De Jesus Jorge , Mickler Kerry , Hsu Yvonne , Shpall Elizabeth J. , Ghosh Shubhra , Champlin Richard
Published: October 2019

Objective: To evaluate risk factors, clinical manifestations and outcome of hyperacute or early acute graft-versus host disease (GVHD), defined as that occurring within 14 days after hematopoietic stem cell (HSC) transplantation.

Methods: A total of 815 consecutive patients transplanted at UT MD Anderson Cancer Center between 1/1998 and 9/2002 were retrospectively analyzed.

Results: Of 381 patients presenting with acute GVHD, 22% (n=83) had biopsy-proven hyperacute GVHD. Grade I GVHD occurred in 12% of these patients, grade II in 53%, grade III in 13% and grade IV in 22%. The proportion of grade II-IV GVHD in this group was significantly higher (88%) than in the 298 patients presenting with acute GVHD between days 15 and 100 (64%, p value <0.001). The majority of patients with hyperacute GVHD had skin involvement (89%), followed by GI (43%), and liver GVHD (19%). Skin involvement was significantly more common (89% vs 76%, p value 0.01), and more severe (stage III or IV 63% vs 32%, p value <0.001) in the hyperacute group. There was no statistical difference in frequency and severity of visceral involvement. Risk factors for hyperacute GVHD were evaluated using Cox proportional hazards model. On univariate analysis, a mismatched (MM) related (HR=4.4, p value<0.001) matched unrelated (MUD) graft (HR=2.4, p value<0.001) and a myeloablative preparative regimen with or without TBI (HR=2.5, p value<0.001) were significantly associated with a higher rate of hyperacute GVHD. These effects remained significant in a multivariate model. Donor’s age greater than 40 years was associated with a lower rate of hyperacute GVHD (HR=0.6,p value=0.05), and there was a trend for increased rates for patients receiving sex mismatched grafts, solid tumor transplants or multiple (>5) chemotherapy regimens before transplant. Age, disease status, GVHD prophylaxis, or stem cell source (BM vs peripheral HSC) were not associated with hyperacute GVHD. Overall mortality was significantly higher within 6 months post transplant for patients with hyperacute GVHD (HR=2.2, p value <0.001) compared to all other patients, or to patients developing acute GVHD after day 14 (HR=1.6, p = 0.009). GVHD-related death was also significantly higher (HR=2.3, p=0.007) in the hyperacute GVHD group.

Conclusions: Hyperacute GVHD occurs in a substantial proportion of patients undersgoing HSC transplant, even prior to neutrophil engraftment. Skin involvement, grades 3–4 GVHD and a higher mortality are common features of this syndrome . Patients at high risk or with a diagnosis of hyperacute GVHD should be included in clinical studies whenever possible.
VPAC1 Receptor-Mediated Regulation of Megakaryopoiesis.
Authors: Freson Kathleen , Thys Chantal , Wittevrongel Christine , De Vos Rita , Vermylen Jos , Hoylaerts Marc F. , Van Geet Chris
Published: October 2019

Identification of the regulatory pathways that direct megakaryopoiesis and platelet production is essential for the development of novel strategies to treat life threatening bleeding complications in bone marrow suppressed patients. We demonstrated that megakaryocytes and platelets express the Gs-coupled VPAC1 receptor, for which both PACAP and VIP are specific agonists. We have further identified a bleeding tendency and found three copies of the PACAP gene in two related patients with severe mental retardation, responsible for elevated PACAP plasma levels and associated increased platelet cAMP concentrations, resulting in strongly reduced platelet aggregation (JCI, 2004, 113, 905). In this study, we have further demonstrated a fundamental role for the VPAC1 signalling pathway during megakaryocyte maturation and platelet formation. Patients with PACAP overexpression have mild thrombocytopenia, a normal platelet survival and relatively small platelets with an MPV of 8.2 fL (normal MPV 9-13 fL). FACS analysis of the patients’ platelets showed reduced expression of GPIX and GPIIIa. Electron microscopy of bone marrow of patients and of mice, specifically overexpressing PACAP in megakaryocytes revealed the presence of early megakaryocyte progenitors but almost not of mature megakaryocytes. Immature megakaryoblasts seemed to have reduced levels of rough endoplasmic reticulum cisternae and free ribosomes. To further study the modulating role of VPAC1 in thrombopoiesis, control mice were therefore subcutaneously injected with neutralizing polyclonal or monoclonal anti-PACAP, anti-VIP or anti-VPAC1 antibodies. Injection of these antibodies in all cases led to increased platelet counts, compared to control antibodies (monoclonal anti-PACAP antibody: 1194 ± 237 x 109 plt/L; control antibody: 722 ± 178 x 109 plt/L; p=0.01, unpaired t-test at day 7 after injection). This strategy was also capable of reducing the drop in platelet count in busulfan treated mice (polyclonal anti-PACAP antibody: 561 ± 121 x 109 plt/L; control antibody: 349 ± 65 x 109 plt/L; p=0.033, unpaired t-test at day 29 or day 18 after respectively antibody and busulfan injection). In addition, bone marrow examination of mice injected with monoclonal anti-PACAP or anti-VPAC1 antibodies revealed an increase in megakaryocyte numbers and showed a marked expansion and mobilization of megakaryocyte progenitor cells. Mice injected with a monoclonal anti-VPAC1 antibody showed an increase of about 50% in bone marrow CFU-MK. In conclusion, we provide evidence that the VPAC1 pathway modulates normal megakaryopoiesis. Further studies are needed to evaluate whether this pathway can be safely manipulated in man in the treatment of thrombocytopenia.
Asn 505 Mutation of the C-MPL Gene, A Cause of Familial Essential Thrombocythemia, Induces the Autonomous Homodimerizaton of the C-Mpl Independent of Ligand Stimulation.
Authors: Ding Jianmin , Komatsu Hirokazu , Iida Shinsuke , Wakita Atsushi , Nitta Masakazu , Miyazaki Hiroshi , Ueda Ryuzo
Published: October 2019

The c-MPL gene and its ligand, thrombopoietin (TPO), regulate the proliferation and differentiation of megakaryocytes and platelets. We previously found a dominant-positive activating mutation of the c-MPL gene as a cause of familial essential thrombocythemia. This mutation (Ser505 to Asn505) was located in the transmembrane domain of the c-MPL. The Ba/F3 cells expressing the mutant allele of Asn505 acquired interleukin-3 (IL-3)-independent survival capacity and the autonomous phosphorylation of Mek1/2 and Stat5b in the absence of IL-3 (Blood 103; 4198, 2004). The mechanism underlying how c-Mpl with Asn505 induces constitutive phosphorylation of Mek and Stat5b remains unknown. However, it is assumed that dimerization of c-Mpl molecules may be accelerated as a result of this specific mutation, as in the cases of mutations of c-kit and Neu at their transmembrane domains. Thus we examined the status of dimer formation of the c-Mpl molecules in the Ba/F3 cells with the Asn505 cells in the presence and absence of IL-3 or Tpo using the cell surface chemical cross-linking assay. As a result, in wild-type (Ser505) c-Mpl-expressing Ba/F3 cells, homodimeric signals of the c-Mpl proteins were detected only in the presence of IL-3 or Tpo, whereas mutant (Asn505) c-Mpl-expressing Ba/F3 cells harbored constitutively homodimeric signals even in the absence of IL-3 or Tpo. These observations suggest that mutant c-Mpl with Asn505 can activate the downstream signals involved in the Tpo-c-Mpl signaling pathway mediated by the formation of autonomous homodimerizaton independent of ligand stimulation. As asparagine (Asn) is well known to provide a strong free energy of association by forming interhelical hydrogen bonds in a hydrophobic environment, the Asn505 mutation in the transmembrane domain of the c-MPL is thought to drive strong interactions (dimerizations) among the c-Mpl molecules, leading to the activation of the Tpo-c-Mpl signaling and consequently autonomously activated megakaryocytopoiesis with the excessive production of platelets. The disulfide-bonded homodimerization may be also induced by the Asn505 mutation. Further investigation of this issue is now in progress.
Combination Therapy with Rituximab and Fludarabine in Waldenstrom’s Macroglobulinemia.
Authors: Treon Steven P. , Branagan Andrew , Wasi Parveen , Emmanouilides Christos A. , Frankel Stanley R. , Lister Andrew , Morel Pierre , Matous Jeffrey , Enschede Sari Heitner , Kimby Eva
Published: October 2019

Rituximab and Fludarabine are active in Waldenstrom’s macroglobulinemia (WM) producing major response rates of 40–70%. Both pre-clinical and clinical studies in related low-grade B-cell malignancies suggest that additive, and even synergistic benefit with combination Fludarabine and Rituximab may result. As such, we conducted this combination study with Rituximab and Fludarabine in WM patients. WM patients who had received < 2 prior therapies, and who had not previously been treated with any nucleoside analogue or Rituximab were eligible. Intended therapy was as follows:

Weeks 1–4 Rituximab (375 mg/m2/week)

Weeks 5, 9, 13 Fludarabine daily for 5 days (25 mg/m2)

Weeks 17, 18 Rituximab (375 mg/m2/week)

Weeks 19, 23, 27 Fludarabine daily for 5 days (25 mg/m2)

Week 30, 31 Rituximab (375 mg/m2/week)

Patients were evaluated at week 12, and if they did not progress were eligible for further therapy and were evaluable for response. 43 WM patients were enrolled with a median age of 61 (range 52–75 yrs), and median prior therapies of 1 (range 0–2). 40/43 patients continued on therapy beyond week 12. Two patients (1 PR, 1 SD) died after completing protocol therapy including one elderly patient who had an influenza pneumonia and another debilitated patient who may have had a secondary malignancy. Delays in therapy due to neutropenia were common, and 58% of patients experienced Grade III/IV neutropenia. Protocol therapy was truncated after the 4th and 5th courses of fludarabine in several patients for persistent neutropenia and/or thrombocytopenia despite delays in therapy and/or use of G-CSF support. Other complications included: Herpes zoster outbreak in 3 of the first 21 patients, before prophylactic acyclovir or equivalent was initiated; parasthesias (n=3); pneumonitis (n=2); development of bladder cancer (n=1) and a high grade lymphoma (n=1); subdural hemorrhage in a patient who had a 3-fold increase in serum viscosity following the first 4 infusions of Rituximab. On an intent to treat basis, 39/43 (90.1%) patients demonstrated a response. Response categories were as follows: CR (n=3); PR (n=32); MR (n=4). One patient remains in stable disease at 20+ months. At a median follow-up of 17 months, 36 of 39 responding patients remain in remission. Rituximab in combination with fludarabine is highly active and produces durable responses in WM.
Performance of Free Light Chain Assays in Clinical Practice.
Authors: Katzmann Jerry A. , Dispenzieri Angela , Abraham Roshini S. , Kyle Robert A.
Published: October 2019

The quantitative assay for free light chains (FLC) is a recently introduced commercial product (FREELITETM, The Binding Site, Ltd.) that has been reported to be sensitive and specific for detecting and monitoring free light chain diseases such as primary systemic amyloidosis (AL), light chain deposition disease (LCDD), non-secretory multiple myeloma (NSMM), and LCMM (light chain multiple myeloma). We have prospectively evaluated the test performance in clinical practice.

Results : In 2003, our Clinical Laboratory received samples for FLC assays from 1020 Mayo Clinic patients. The majority of these patients (88%) had plasma cell disorders (PCD). All 120 patients who did not have PCD had normal K/L FLC ratios. Among these, 52 had non-AL amyloidosis: localized amyloid (23), hereditary (16), senile (6), secondary (3), and amyloid of unknown type (4). The 68 other patients who did not have a PCD were being tested because of peripheral neuropathy, rule out AL, anemia, proteinuria, lymphoproliferative disease, and a number of other indications with small numbers of patients (n=13). Among the monoclonal gammopathy patients were 330 with MM, 269 AL, 115 MGUS, 72 SMM, 22 plasmacytomas, 20 NSMM, 9 macroglobulinemia, 7 LCDD, and a variety of other diagnoses with smaller numbers of patients. The sensitivity of the K/L FLC ratio was 100% in LCDD (7/7) and 70% in NSMM (14/20). The 6 NSMM patients with normal K/L FLC ratios had all been treated with SCT and 5 of the 6 had achieved hematologic remission by bone marrow plasma cells. Among the 110 AL patients who had not been previously treated and who had a FLC assay performed within 120 days of diagnosis, the sensitivity of the K/L FLC ratio was 92% compared to 71% for serum IFE and 84% for urine IFE. Using all 3 assays, there was 99.1% (109/110) sensitivity for detecting monoclonal light chain in AL.

Conclusion : The performance of the FLC assay in this prospective analysis matches the results from published retrospective validation studies.
Diagnostic Imaging of Multiple Myeloma - FDG PET and MRI Complementary for Tracking Short Vs. Long Term Tumor Response.
Authors: Walker Ronald , Jones-Jackson L. , Rasmussen Eric , Miceli M. , Anaissie Elias , Epstein Joshua , Tricot Guido , Shaughnessy John , Barlogie Bart
Published: October 2019

Multiple myeloma results in both diffuse infiltration and/or focal lesions (FL) of the marrow. MRI detects FL long before X-ray. We routinely perform MRI of skull, entire spine, pelvis, sternum and shoulders. MRI-defined number of FL (#FL) is an adverse prognostic variable for both EFS and OS for patients treated according to Total Therapy 2, 2nd in importance only to cytogenetic abnormalities (CA) (univariate and Cox multivariate models using standard prognostic factors, CA, and #FL). Of these patients who also had FDG PET (PET), baseline analysis revealed the same axial skeleton FL as on MRI (r=0.47, p<0.001). Another 268 FL in long bones and 11 sites of extramedullary tumor were seen with PET due to MRI’s restricted field of view. Serial MRI and PET in 59 patients with FL on MRI (Fig. 1) and 123 patients with hyperintense MRI signal on STIR (no FL) revealed that patients without FL normalize MRI and PET scans synchronously with clinical (M-protein and bone marrow) response (nCR/CR) (Fig. 2a). However, in patients with FL, PET normalization follows nCR/CR very closely, while regression of MRI-FL (MRI-CR) lags in proportion to MRI #FL (Fig. 2b). We conclude PET is superior to MRI for detection of extent of disease and monitoring treatment response. However, PET-negative MRI-FL usually contain viable myeloma cells on biopsy which eventually progress. Thus, long term serial MRI is superior to PET in assessing completeness of response.
Targeting Mitochondrial Factor Smac/DIABLO as Therapy for Multiple Myeloma (MM).
Authors: Chauhan Dharminder , Li Guilan , Hideshima Teru , Podar Klaus , Tassone Pierfrancesco , Neri Paola E. , Mitsiades Constantine , Mitsiades Nicholas , Raje Noopur , Kumar Shaji , Schlossman Robert , Sharma Sushil , Munshi Nikhil , Zawel Leigh , Anderson Kenneth C.
Published: October 2019

Mitochondrial signaling is an important component of chemotherapy-induced apoptosis in cancer cells. Stress-inducers trigger the following sequence of events: Smac (second mitochondria-derived activator of caspases)/ DIABLO is released from the mitochondrial inner membrane to cytosol; activated/cytosolic Smac binds to XIAP, a protein belonging to the family of IAPs (Inhibitors of apoptosis proteins), thereby abrogating its inhibitory effects on caspase-9 and resulting in activation of caspase-9-mediated apoptotic cascade. IAPs play a key role in regulating the apoptotic signaling, and overexpression of IAPs confer chemoresistance in various tumor types. Here we show that a low molecular weight synthetic Smac mimetic (Novartis) by virtue of its ability to bind and inhibit IAPs, block the growth of MM cell lines, including those resistant to conventional agents including dexamethasone, melphalan or doxorubicin. Examination of purified patient MM cells demonstrated similar results. In contrast, no significant toxicity of Smac mimetic was noted against normal peripheral blood mononuclear cells. Moreover, Smac mimetic also did not affect viability of MM patient-derived bone marrow stromal cells (BMSCs). Treatment of MM cells overexpressing anti-apoptotic protein Bcl2 with Smac mimetic significantly decreases their viability. Importantly, combined treatment of MM cells with Smac mimetic and Tumor necrosis factor-related apoptosis inducing ligand (TRAIL, Apo-2 ligand) trigger synergistic anti-MM activity, without significant toxicity in normal cells. Finally, Smac mimetic enhances the anti-MM activity of conventional Melphalan. Collectively, these findings provide the rationale for clinical evaluation of Smac mimetic alone and in combination with Melphalan or TRAIL, to enhance MM cell killing, overcome drug-resistance, and improve patient outcome in MM.
Identification of a Novel Toll-Like Receptor-Independent Immunoadjuvant Pathway That Depends upon Programmed Cell Death.
Authors: Hoebe Kasper , Janssen Edith , Beutler Bruce
Published: October 2019

Molecules of microbial origin, and synthetic derivatives of these molecules, have long been used for their immuno-adjuvant effect, and as the key sensors of microbial infection, Toll-like receptors (TLRs) are thought to be essential for adjuvanticity. To the contrary, we now demonstrate the existence of a robust, TLR-independent pathway for adjuvant effect: one that is actually far stronger than the TLR-dependent pathway. Activation of Toll-like receptors (TLRs) and the subsequent production of cytokines such as type I interferon leads to the maturation of dendritic cells (DCs) with upregulation of MHC molecules and costimulatory molecules such as CD40, CD80 and CD86, allowing for optimal interaction between DCs and T-cells. We have determined that TLR signal transduction is minimally dependent upon two adapter proteins, MyD88 and TRIF. In compound homozygous mutant (DKO) mice that lack functional MyD88 and TRIF, there is complete abrogation of all TLR signaling. Such animals therefore comprise a unique model with which to study TLR-independent immune responses. We have now used DKO mice to determine whether an adaptive immune response can be obtained in the absence of TLR signaling. As expected, adjuvanticity obtained via “classical” microbial adjuvants such as complete Freund’s adjuvant or LPS was completely absent in DKO mice. However, subcutaneous administration of syngeneic murine cells expressing ovalbumin and rendered apoptotic by exposure to ultraviolet light resulted in a strong T-cell response in vivo, with impressive production of interferon-g by CD8+ cells and efficient killing of EL-4 cells that expressed CD8-specific OVA peptides, both in wildtype and DKO mice. Adjuvanticity was observed only in the context of apoptosis, in that living cells, not exposed to ultraviolet light before injection, induced little or no response. Moreover, the mixture of the protein antigen with apoptotic cells was insufficient to induce an adaptive immune response; rather, only cells that expressed the protein prior to induction of apoptosis were stimulatory. These results indicate the existence of a specific, cell death-dependent mechanism for adjuvanticity that is TLR-independent and induced by endogenous molecules. We propose that this new adjuvant pathway is of fundamental importance to immune responses at large. We believe that it is required for initiation of the adaptive immune response witnessed in the context of allograft rejection, graft-versus-host disease, and autoimmune diseases as well.
Reduced-Intensity Conditioning (RIC) Followed by Allogeneic Transplantation in Relapsed Lymphomas: Evidence for Graft-Versus-Lymphoma Effect in Low and High Grade Histologies, but Not in Hodgkin Disease.
Authors: Corradini Paolo , Dodero Anna , Bregni Marco , Olivieri Attilio , Farina Lucia , Rizzo Elena , Milani Raffaella , Scime Rosanna , Rambaldi Alessandro , Bonifazi Francesca , Patriarca Francesca , Locasciulli Anna , Bacigalupo Andrea , Gianni Alessandro Massimo , Tarella Corrado
Published: October 2019

Reduced intensity conditioning (RIC) followed by allogeneic stem cell transplantation (SCT) is associated with durable engraftment, low transplant-related mortality (TRM) and clinical remissions in hematologic malignancies. In addition, RIC followed by allograft is feasible also in patients who had failed previous autologous (auto) SCT. Here, we report the outcome of 118 lymphoma pts receiving RIC and allogeneic SCT from HLA-identical sibling donors. Histologic types included in the study were: chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL; n=26), low-grade non-Hodgkin lymphoma (LG-NHL; n=24, 21 follicular NHL), high-grade NHL (HG-NHL, B cell n=28, T cell n=17), Hodgkin disease (HD; n=23). Median age was 50 years (range: 20–69). 47% of pts had refractory disease (46% CLL/SLL; 33% LG-NHL; 44% HG-NHL; 56% HD) and 48% have failed an auto SCT (15% CLL/SLL; 33% LG-NHL; 55% HG-NHL; 82% HD). All pts received debulkying chemotherapy followed by the same RIC regimen with thiotepa (10 mg/kg), fludarabine (60 mg/ms) and cyclophosphamide (60 mg/kg). GVHD prophylaxis consisted of cyclosporine A and short-course methotrexate. All pts engrafted. De-novo acute GVHD developed in 43% of pts and chronic GVHD in 45%. The 2-year TRM rate was 13% and was not influenced by previous auto SCT or hystological type. At a median follow-up of 20 months (range, 6–62 months), 91 pts (77%) are alive (n=69 CR, n=8 PR, n=14 with disease) and 27 died (23%) for disease (n=17) or TRM (n=10). The 2 years PFS rates for CLL/SLL, LG-NHL, HG-NHL, HD were 63%, 78%, 64%, 16%, respectively. Chemorefractory disease at time of SCT was associated with a worse 2-year PFS compared to chemosensitive disease (41% versus 74%, p<0.001). There was a trend toward lower relapse risk for pts developing acute or chronic GVHD (37% versus 50%, p=0.2). The 2-year OS rates for CLL/SLL, LG-NHL, HG-NHL, HD were 77%, 90%, 76%, 56%, respectively. Chemosensitive disease had significant impact on OS (84% 2-year OS compared with 64% for chemorefractory patients; p< 0.007) as well the occurrence of chronic GVHD (87% versus 57%; p<0.0059). Interestingly, previous auto SCT and age > 55 years did not influence OS. Among pts in clinical CR, 24 were suitable for PCR monitoring of residual disease and 17 pts (71%) are in continuous molecular remission. Our study indicates: 1) low TRM in a multicenter phase II study; 2) low risk of relapse and good survival rates in indolent lymphomas; 3) encouraging results in HG-NHL 4) disappointing results in HD