bluuebunny/crossref_metadata_2025 huggingface dataset:
A New Fusion Gene NUP98-IQCG Identified in an Acute T/Myeloid Leukemia with t(3;11)(q29q13;p15) Translocation.
Authors: Pan Qin , Zhu Yong-Jin , Gu Bai-Wei , Cai Xun , Bai Xue-Tao , Yuan Hai-Yang , Zhu Jiang , Chen Zhu , Xue Yong-Quan , Chen Sai-Juan
Published: April 2020

NUP98 has been involved in multiple recurrent chromosome rearrangements in leukemia. We identified a novel fusion between NUP98 and IQ motif containing G (IQCG) from a de novo acute T/myeloid leukemia harboring t(3;11)(q29q13;p15). IQCG has 2 putative coiled-coil domains and an IQ domain. The FG repeat from NUP98 and the coiled-coil domain from IQCG were retained in the fusion protein. We demonstrated that NUP98-IQCG could form homodimer or heterodimerize with either NUP98 or IQCG, bind Co-activators and/or Co-repressors, and show transcriptional activity in vitro. Expression of NUP98-IQCG inhibited 32Dcl3 cell apoptosis induced by Ara-C, and partially blocked granulocyte differentiation induced by G-CSF. Colony forming assay also indicated that NUP98-IQCG was able to stimulate proliferation and partially block differentiation of hematopoietic stem/progenitor cells. We conclude that NUP98-IQCG may play an essential role in leukemogenesis by stimulating progenitor cell proliferation and inhibiting differentiation and apoptosis.
Intensively Timed Induction (ITI) Chemotherapy in Adults with Acute Myelogenous Leukemia (AML).
Authors: Rytting Michael , Verstovsek Srdan , Garcia-Manero Guillermo , Ravandi-Kashani Farhad , Pierce Sherry , Estey Elihu
Published: April 2020

The Children’s Cancer Group (CCG) demonstrated that children with AML have longer event free survival (EFS) and disease free survival (DFS), 42 +/− 7% and 55 +/− 9%, at a minimum of two years off of therapy when randomly assigned to ITI rather than standard chemotherapy (Woods, et al Blood 1996 Jun 15;87(12):4979–89). This led us to administer an almost identical regimen of ITI chemotherapy to 50 adults less than 50 years of age with untreated AML, excluding patients (pts) with inv16 or t(8;21). Each pt who remains alive in 1st complete remission (CR) has been followed for more than 3 years: the risk of relapse decreases sharply after this time (de Lima, et al. Blood 1997 Dec 15;90(12):4719–24). Daunorubicin 20 mg/m2/day intravenously (IV) on days 1–4 and 11–14, cytarabine 200 mg/m2/day IV on days 1–4 and 11–14, etoposide 100 mg/m2/day IV on days 1–4 and 11–14 and dexamethasone 6 mg/m2/day PO days 1–4 and 11–14 were given initially along with intrathecal cytarabine 70 mg on day 1 and 11 (DCTER regimen). G-CSF started on day 7 and continued until the ANC was more than 1000 for 2 days. The day 11 chemotherapy was given unless there was hypotension or clinical colitis. All pts started therapy in a protected environment. Pts in CR after the above chemotherapy repeated the induction course. After recovery, cytarabine 3 g/m2 every 12 hours for four doses was infused on days 0–3 and on days 12–15. L-asparaginase 6000 IU/m2 IV was given at hour 18 on days 3 and 15. The cytarabine and L-asparaginase were repeated upon recovery of the granulocyte count. The study was ongoing from 2/15/02 to 8/10/04. The 50 pts, median age 41 years, represented 62% of all pts eligible during this time period. Table 1 compares results in the 50 DCTER pts with the results in 35 non-core binding factor AML pts, median age 41 years, given idarubicin/cytarabine (IA) for induction and consolidation without intensive timing. The results in the IA pts treated from 1991–1993 were similar to those treated 1994–2001 (proportion alive in CR 16% and 19%). The lower CR rate in pts given DCTER reflected an increased death rate in the first 12 weeks after starting treatment. This was counterbalanced by a higher rate of potential cure, considering all 85 pts (95% confidence interval for the true difference [−.09, .34] suggesting that a difference of 26% is as plausible as no difference since .26 and 0 are each equidistant from the midpoint of the confidence interval). A larger cohort is needed to know if the higher early risk associated with DCTER is potentially justified by a better long-term prognosis.

Table 1 Patients Performance Status >1 Unfavorable Cytogenetics CR Proportion alive in CR at 3 years DCTER 50 14% 24% 69% 30% IA 35 0% 23% 80% 17%
Generic and Branded Low Molecular Weight Heparins Can Be Differentiated Based on Biologic and Pharmacologic Assays.
Authors: Jeske Walter P. , Ackerman Paul , Drenth Amanda , Walenga Jeanine M. , Bakhos Mamdouh
Published: April 2020

Several generic versions of the low molecular weight heparins enoxaparin and dalteparin have become available in different parts of the world and several products are currently under review by the FDA. At this time, there are no regulatory guidelines for defining equivalence of complex biologic drugs. Because of their complexity and hybrid nature (biologic and chemical), the available generic versions may not be acceptable by using currently applicable generic criteria. Due to their complex nature, the pharmacologic effect of these may not be similar to the branded product. Therefore, additional methods are needed to equate these drugs with the branded products. In this study, multiple lots of Lovenox or generic enoxaparin such as Lupenox (Lupin Pharma, India), Dripanina (Ariston, Brazil), Dilutol (Lazar, Argentina), Clenox (Pharmayect, Columbia), and Cutenox (Gland Pharma, India) and generic versions of dalteparin such as Daltehep (Gland Pharma, India) were supplemented to human plasma in the absence or presence of protamine sulfate (PS). Anticoagulant activity was measured using the aPTT, Heptest, PiCT assays. Antithrombin and anti-factor Xa activity were assessed by amidolytic assays. Effects on thrombin generation and the activation of thrombin activatable fibrinolytic inhibitor (TAFI) were determined by functional assay. At prophylactic levels of LMWH, no differences in anticoagulant or antiprotease activities were observed between generic and branded LMWHs. At therapeutic concentrations, significantly higher anticoagulant and antiprotease activities were observed with Lovenox. Assay-dependent variations in PS neutralization of antiprotease and anticoagulant activities were observed at higher concentrations of the generic versions of enoxaparin and dalteparin. Significant differences were noted in the effects of these agents on thrombin generation and the activation of TAFI. Current generic LMWHs possess some differences from branded LMWHs in known biologic properties of heparin. Variations in PS neutralization raise the question as to whether the interaction with other plasmatic proteins also differs from that of the branded drug. These results emphasize a need to consider multiple functional parameters when defining bioequivalence of complex biologic drugs and underscore the importance of further pharmacologic studies involving animal models and human clinical trials. Such approaches can be incorporated in the recommendations to develop guidelines for the approval of the generic versions of low molecular weight heparins.
Outcomes after Autologous Stem Cell Transplantation for Mantle Cell Lymphoma Based on Remission Status and Induction Chemotherapy Regimen.
Authors: Till Brian G. , Gooley Theodore A. , Crawford Nathan , Gopal Ajay K. , Maloney David G. , Petersdorf Stephen H. , Pagel John M. , Holmberg Leona , Bensinger William , Press Oliver W.
Published: April 2020

Mantle cell lymphoma (MCL) has one of the worst prognoses of any subtype of non-Hodgkin’s lymphoma (NHL), with a median survival of 3–4 years in most series. High-dose therapy followed by autologous stem cell transplantation (ASCT) is an increasingly common treatment approach for MCL, and some ASCT studies suggest that less heavily pre-treated patients have a longer duration of progression-free survival (PFS), suggesting that ASCT may lead to more favorable outcomes if used earlier in the course of therapy. We analyzed the outcomes of ASCT with respect to remission status at the time of transplantation and induction regimen used in 56 consecutive transplanted patients with MCL. Twenty-one patients received induction chemotherapy with HyperCVAD with or without rituximab (±R) followed by ASCT in first complete or partial remission (CR1/PR1), 15 received CHOP (±R) followed by ASCT in CR1/PR1, and 20 received ASCT following disease progression. A variety of conditioning regimens were used for ASCT in all 3 groups. Estimates of overall (OS) and progression-free survival (PFS) at 3 years among patients transplanted in CR1/PR1 were 93% and 63%, compared with 46% and 36% for patients transplanted with relapsed or refractory disease, respectively (Figure 1). The hazard of mortality among patients transplanted with relapsed or refractory disease was 6.09 times that of patients transplanted in CR1/PR1 (P=.006). Patients in the CHOP (±R) group appeared to have a higher risk of failure for PFS compared to patients in the HyperCVAD (±R) group, though the difference did not reach statistical significance (hazard ratio [HR] 3.67, P=.11) with the small sample size available. The estimated 3-year PFS was 81% for patients in the HyperCVAD group and 44% for patients in the CHOP group. Patients who received R with induction therapy had a reduced risk of mortality (HR 0.33, P=.05) and failure for PFS (HR 0.28, P=.005) compared to those who did not. In summary, these results suggest that ASCT in first remission leads to improved survival outcomes for patients with MCL compared to ASCT with relapsed or refractory disease, and a HyperCVAD (±R) induction regimen may be associated with an improved PFS among patients transplanted in CR1/PR1.

Figure 1. Kaplan-Meier estimates of overall survival from the time of ASCT, with respect to remission status at ASCT. Figure 1. Kaplan-Meier estimates of overall survival from the time of ASCT, with respect to remission status at ASCT.
BEAM as a Frontline High Dose Chemotherapy Supported by Autologous PBSCT in Elderly Patients (≥60 Years) with Diffuse Large B-Cell Lymphoma (DLBCL): Comparison with Younger Cohort of Patients.
Authors: Ivanov V. , Coso D. , Rey J. , Aurran T. , Stoppa A.-M. , de Collela J.-M. Schiano , Gastaut J.-A. , Blaise Didier , Bouabdallah R.
Published: April 2020

Recent developments in supportive care increase the number of elderly patients available for high dose therapy (HDT) supported by autologous PBSCT. In several clinical trials high dose chemotherapy supported by PBSCT as a part of first-line treatment in young NHL patients with poor prognostic features suggested survival benefit. It is not clear if the same strategy is applicable into the older patients. The Institute Paoli-Calmettes database was reviewed for all DLBCL patients received BEAM followed by PBSCT in two age cohorts: 50–59 years old and >=60 years old between January 1998 and December 2006 (9 years). All patients were HIV-negative and received BEAM intensification as a part of front-line treatment. All of them were grafted in situation of complete or partial response after CHOP or R-CHOP induction. There was no significant difference into the aaIPI score between the groups (aaIPI score ≥ 2: 17 pts (63%) in elderly and 17 pts (61%) in younger group. Factors evaluated included treatment-related mortality (TRM), overall survival (OS) and event-free survival (EFS). Fifty five patients were identified, among them 27 into the older cohort (median age 63 y, range 60–68) and 28 patients into the younger cohort (median age 55 y, range 51–59). The median follow-up for the whole group was 23,5 months (0,5–93 months). TRM into the older group (1/27 pts (3,7%)) was not statistically different from the younger patients (0/28 pts) and was comparable with previously published data. The estimated 5-year OS (figure 1) was 75,5% (95%CI 52–90%) for the older group (curve 1) compared to 85% (95%CI 63–95%) in the younger group (p=0,47) (curve 2). There were 8 events (1 TRM and 7 relapses) in the older group and 3 events (all relapses) in the younger group (3-year EFS (figure 2) 66% (curve 1) vs 85,5% (curve 2), p=0,16 and 5-year EFS 49,4% vs 85,5%, p=0.051). We conclude that front-line autologous PBSCT with BEAM conditioning can be safely performed in patients 60 years old and older with DLBCL after CHOP of R-CHOP induction. The toxicity profile and TRM may not be different from that of younger patients. The high rate of OS in older group is encouraging, but the relapse still remains the important obstacle for durable remission.

Figure Figure
Incorporating Target Immunotherapy into Standard Conditioning Regimen Prior to Autologous Stem Cell Transplantaion (ASCT) Improves the Transplant Outcome for Pateints with Relapsed/Refractroy B Cell Non-Hodgkin Lymphoma (B-NHL).
Authors: Farhat Mohamed I. , Karmali Reem , Gregory Stephanie A. , Venugopal Parameswaran , Kassar Mohamad , Shah Neel , Brooks Terrance , Fung Henry C.
Published: April 2020

Background: Refractory or relapse B-NHL has a poor prognosis with conventional chemotherapy. Autologous stem cell transplant (ASCT) preceded by high dose chemotherapy has been the preferred therapeutic choice for such patients. The majority of the treatment failures occur within one to two years post transplant with disease progression after transplant accounted for most of the failures. The incorporation of targeted immunotherapy (rituximab) into the upfront and relapse setting is becoming of the standard of care for patients with B-NHL. The objective of this study is to evaluate the impact of rituximab (R) on disease free survival (DFS) when added to a standard conditioning regimen -- BEAM (carmustine, cytarabine, etoposide, and melphalan) prior to ASCT.

Methods: A single institution retrospective analysis of 53 patients (pts), whom were heavily pre-treated, underwent ASCT between 08/98 & 07/06. All pts received rituximab in combination with high dose cytoxan for stem cell mobilization. 37 pts received R-BEAM and 16 received BEAM prior to ASCT. Actuarial rate for DFS was estimated from the day of SCT using the Kaplan-Meier method.

Results: The group was predominantly men, 73% and 78%, with a median age of 57 years for both the R-BEAM and BEAM group. With a median follow up of 15.7 months, 13/37 (32%) and 11/16 (64%) pts who received R-BEAM and BEAM respectively developed disease progression after ASCT. The 2-yr actuarial disease-free survivals (figure1) were 60% and 21% for the R-BEAM and BEAM arm respectively (p=0.006).

Conclusion: In this study, the outcome of pts who received R-BEAM compares favorably to those who receive BEAM alone with significant improvement in disease-free survival. Thus, incorporating target immunotherapy into standard conditioning regimen may have altered the natural history of the disease for pts undergoing ASCT for relapsed/refractory B-NHL.

Disease Free survival Disease Free survival
Durable Responses to Lenalidomide (Revlimid®) in Patients with AL Amyloidosis: Follow Up of a Phase II Trial.
Authors: Seldin David C. , Rosenzweig Michael , Finn Kathleen T. , Fennessey Salli , Shelton Anthony , Zeldis Jerome B. , Skinner Martha , Sanchorawala Vaishali
Published: April 2020

AL amyloidosis is a clonal plasma cell dyscrasia in which misfolded immunoglobulin light chains deposit in tissues and produce organ failure and death. Untreated, median survival is short. Melphalan-based regimens can produce hematologic remissions and improvement in organ function; more than 20% of patients treated with high dose melphalan and autologous stem cell transplantation (HDM/SCT) have survived more than 10 years (Blood, in press). The combination of lenalidomide and dexamethasone can also produce partial and complete hematologic responses (Blood2007;109:492–496). Here we report on remission duration and long-term results of treatment in the original 34 patients and an additional 9 patients, with median follow up of 26.5 m. The median age of the 43 patients was 64 (range, 44–84), 70% were male, 67% were lambda isotype, 46% had multi-organ involvement, and 42% had cardiac involvement. 90% had received prior melphalan-based therapy; in 60% this was HDM/SCT. 14% of patients had received thalidomide and 5%, bortezomib. 10% had no prior treatment. Patients were begun at 15 mg lenalidomide per day for 21 days per month; the median tolerated dose was 10 mg. The response rate was 60% (24% CR, 36% PR); an additional 15% of patients had minor responses. Of the 8 patients who achieved a CR, 6 occurred at 3–6 months of treatment, but 2 were late (18m, 19m). 7 of 8 are alive; one died of cardiac allograft rejection. 3 of 8 have relapsed. 5 of 8 maintain remissions for 6–30 m, of which 4 of 8 continue in CR off therapy for 6–21m. Kaplan-Meier survival for all 43 patients is shown. 7 of 8 patients achieving CR had significant proteinuria: in 2 patients (29%), proteinuria resolved (2 g to 120 mg, 8.8 g to 140 mg); in 3 (43%) it improved by 50% or more; and in 2 there has been no change. Thus, lenalidomide can produce beneficial hematologic and organ responses in AL amyloidosis patients, and remissions can be durable off therapy. Further trials should be done to determine how and when to incorporate lenalidomide into treatment protocols for AL amyloidosis.

Figure Figure
Role of Jagged/Notch Signaling in the Cell Fate Determination of Bone Marrow Human Mesenchymal Stem Cells.
Authors: Ugarte Fernando , Ryser Martin F. , Thieme Sebastian , Bornhaeuser Martin , Brenner Sebastian
Published: April 2020

Notch, expressed on hematopoietic progenitors plays a crucial role in hematopoiesis. Mesenchymal stem cells (MSC) express both, Notch and its ligand Jagged and are known to support self renewal of hematopoietic progenitors via cell-cell contact and cytokine secretion. The Jagged/Notch signaling pathway has been implicated in the differentiation process of MSC, however it is not completely understood and current observations are contradictory. In order to analyze the effect of Notch signaling on human MSC differentiation we constructed lentiviral vectors that contained either the GFP-marker gene, hJagged1 IRES GFP, hNotch1 intracellular domain (NICD) IRES GFP or a gene fusion between dominant negative Mastermind1 (MAML1dn - inhibitor of Notch signaling) and the Cherry reporter gene. Primary hMSC that were obtained from bone marrow of 3 different donors were transduced with respective lentivirus vectors to greater than 98%. After exposure to adipogenic and osteogenic differentiation stimuli hMSC differentiation was quantified by Alizarin red or oil red staining, alkaline phosphatase (AP) activity and expression levels of adipogenic or osteogenic markers by Real-time PCR. Jagged1 transduced hMSC demonstrated enhanced calcium phosphate deposits and enhanced AP activity and expression levels in osteogenic differentiation medium, while adipogenic differentiation was strongly inhibited as quantified by oil red staining and low mRNA expression of genes upregulated during adipogenic differentiation (pprY, Fabp4). Similarly, overexpression of NICD induced strong and rapid osteogenic differentiation while inhibiting adipogenic differentiation and reducing cell viability. Moreover, NICD overexpression upregulates the expression of endogenous Jagged1 up to 5-fold. Inhibition of Notch signaling via overexpression of MAML1dn partially blocked the effect of hJagged1 and NICD in co-transduction experiments. In another approach MSC samples obtained from 20 donors with various osteogenic differentiation potential as measured by AP activity were analyzed for Notch1 and Jagged1 expression. While there was no correlation between AP activity and Notch1 levels we observed a significant positive correlation for AP activity and Jagged1 expression. In summary, our data strongly suggest that increased Jagged/Notch signaling enhances the osteogenic differentiation of hMSC while inhibiting their adipogenic fate.
Effect of Pax3 Overexpression on the Engraftment of Mesenchymal Stem Cells in Dystrophic Mice.
Authors: Gang Eun Ji , Darabi Radbod , Bosnakovski Darko , Xu Zhaohui , Perlingeiro Rita
Published: April 2020

Mesenchymal stem cells (MSCs) residing within the bone marrow (BM) not only support hematopoiesis, but also differentiate into multiple lineages, including fat, bone, and cartilage. Because MSCs are multipotent and have a great expansion capacity in vitro, these cells are attractive candidates for clinical applications to repair or regenerate damaged tissues of mesenchymal origin. However, treatment of muscle degenerative diseases with MSCs has been hampered by the poor differentiation of MSCs into the muscle lineage. To date most in vitro methods require the presence of strong non-physiological agents, such as azacytidine. Accordingly, limited engraftment has been observed following the transplantation of BM into dystrophic mice (less than 4%). In the present study we explored the potential of Pax3, the master regulator of the embryonic myogenic program, to promote myogenic differentiation from human BM-derived MSCs. Upon genetic modification with lentiviral vectors encoding Pax3-GFP or GFP only (vector control), MSCs were evaluated for their potential to differentiate towards muscle in vitro. Real time PCR analyses revealed up-regulation of several myogenic regulatory factors in Pax3-transduced MSC cultures in various differentiation conditions, whereas control GFP-MSCs showed undetectable levels. When transplanted into cardiotoxin-injured tibialis anterior (TA) muscles of Rag2−/− γc−/− immunodeficient as well as dystrophic mice, superior engraftment was observed with Pax3-transduced MSCs (31% vs. 12% and 12% vs. 6.5%, respectively). To assess whether such levels of engraftment are therapeutically relevant, functional analyses of transplanted muscles are required. We are currently undertaking these experiments.
Mesenchymal Stromal Cells Derived from Human Non-Small Cell Lung Cancer, Normal Lung Tissue, and Bone Marrow: Differences in Frequency, Cytogenetics, Proliferation Behavior, and Chemosensitivity - Implications for Cancer Therapy.
Authors: Gottschling Sandra , Meister Michael , Jauch Anna , Krause Ulf C. , Seckinger Anja , Hoffmann Hans , Dienemann Hendrik , Anthony Ho D. , Thomas Michael
Published: April 2020

Stromal microenvironment plays a vital role for the induction and maintenance of solid tumors. Knowledge on the nature and impact of the tumor-microenvironment in lung cancer is still inadequate. In this study, we investigated and compared stromal cells (SC) derived from non-small cell lung cancer (NSCLC), normal lung tissue (NLT), and bone marrow (BM) for the presence and properties of mesenchymal stromal cells (MSC). NSCLC-SC and NLT-SC displayed mesenchymal morphology, immunophenotype, and differentiation capabilities implying their MSC character. However, their frequency, proliferation behavior, and chemosensitivity varied significantly from BM-MSC and among each other. Colony forming units-fibroblast (CFU-f) were 300-fold less frequent in bone marrow but exhibited an up to 44-fold higher colony efficiency (CE) than in lung. Compared to NLT, NSCLC specimens were four times enriched in CFU-f possessing an unfailing CE over several passages. According to colony size, the proliferation potential of NSCLC-CFU-f was double that of NLT-CFU-f and did not vary with cell density thus indicating an autonomous growth and lack of feedback regulation. Moreover, NSCLC-SC exhibited a markedly reduced sensitivity to cisplatin with delayed apoptosis and faster recovery from cytotoxic damage. In line with functional alterations multicolor-fluorescence in situ hybridization revealed genetic aberrations and chromosomal instability of NSCLC-SC but not of NLT-SC. These data provide evidence for the presence of tumor-specific MSC in NSCLC and suggest the survival of a particular tumor-stroma after chemotherapeutic treatment.
Role of Imatinib Mesylate in Osteoblastogenesis.
Authors: Tibullo Daniele , Giallongo Cesarina , La Cava Piera , Berretta Salvatore , Stagno Fabio , Chiarenza Annalisa , Palumbo Giuseppe A. , Di Raimondo Francesco
Published: April 2020

Imatinib mesylate (IM), a tyrosine kinase inhibitor, currently used in chronic myeloid leukaemia (CML) may also affect the growth of other cellular systems besides CML cells. It has been reported that IM may affect bone tissue remodeling mainly by an inhibitory activity on osteoclastogenesis. We therefore evaluated possible effects of IM on osteoblatic differentiation of Mesenchymal Stem Cells derived from bone marrow (BMSCs). BMSCs are multi-potent non-haematopoietic progenitor cells that differentiate into osteoblasts, adipocytes, chondrocytes, skeletal myocytes and nervous cells. Culture of normal human BMSCs were treated with osteogenic medium (OM) with or without IM 1 μM. By RT-PCR, we assessed mRNA expression of osteogenic markers such as RUNX2, osteocalcin (OCN) and osteopontin (OPN) at 7, 14, and 21 days of culture. BMSCs treated with OM and IM 1 μM showed increased levels of osteogenic markers mRNA as compared to BMSCs cultured with OM only (RUNX2 ctrl=0,37±0,02 vs OM+IM=0,79±0,06; OCN ctrl=0,36±0,04 vs OM+IM= 0,97±0,03; OPN ctrl=0,94±0,01 vs OM+IM= 1,55±0,13). We also evaluated the OCN levels, and the OPG (osteoprotegerin)/RANKL (receptor activator of nuclear factor-kappa B ligand) ratio (OPG/RANKL ratio) in the surnatant of the culture at day 21 by ELISA assays. OPG/RANKL ratio (OM alone = 55,4±0,4 vs OM+IM = 65,3±0,7; P<0,005) and OCN levels (OM alone = 1,4±0,08 ng/mL vs OM+IM= 4,02±0,21 ng/mL; P<0,005) were increased in BMSCs samples cultured with IM in comparison to control (OM alone). In addition, we examined the OPG/RANKL ratio in serum of 17 CML patients treated for at least two years with IM and we found that it was increased in comparison to normal controls (ctrl=1,0±0,2 vs patients= 3,1±2,63; P= 0,036). In summary, our data show that IM increases osteogenic markers’ mRNA expression in BMSCs and increase the OPG/RANKL ratio both in surnatant of BMSCs cultured with IM and in serum of patients treated with IM, thus indicating that IM potentially favours osteoblastogenesis.
The Mutation Spectrum of CML Patients Resistant to Imatinib: More Heterogeneous Than Just BCR-ABL Kinase Domain Point Mutations?.
Authors: Lundan Tuija , Gruber Franz , Silye Aleksandra , Hjorth-Hansen Henrik , Mikkola Ingvild , Remes Kari , Simonsson Bengt , Gedde-Dahl Tobias , Porkka Kimmo
Published: April 2020

Cells harboring BCR-ABL kinase domain (KD) mutations are the major source for resistance to tyrosine kinase inhibitors (TKI) in chronic myeloid leukemia (CML). A second and third generation of TKIs are in development for inhibition of mutated forms of BCR-ABL. At present, clinical treatment strategies are based on molecular response and mutational profiling. Profiling is usually performed using unbiased methods (sequencing and D-HPLC of PCR products), but mutation specific methods are upcoming (ARMS-PCR, ASO-PCR, ligation-PCR). Here, we describe molecular findings resulting from a collaborative Nordic approach for molecular characterization of imatinib resistant Norwegian (N) and Finnish (F) CML patients. Complementary DNA derived from a total of 87 subjects (60 N and 27 F) with suboptimal or failed response to imatinib (less than 3 log reduction in tumor load after 12 months of treatment or loss of any response) were subjected to BCR-ABL KD sequencing of amino acid residues 240 to 420 (N) and 209 to 498 (F). In addition, single samples were analyzed by multiplex PCR for detection of transcript variants as well as cloning and sequencing of single clones. BCR-ABL KD mutations were found in 35 patients (20 N, 15 F, 40% of the total material). The most frequent findings were ABL KD point mutations with M351T and G250E representing the single most frequent mutations, 25 and 16%, respectively. The most frequently mutated cluster was in the catalytic loop (40% of all mutations), followed by P-loop (38%) and other regions (22%). Mutations in the gatekeeper cluster were extremely rare in our material (one case of a F317L). Additional sequence variations were observed in 6 patients: the L248V associated deletion of exon 5 (n=1), smaller insertion or deletion variants (2–4 basepairs) resulting in truncated proteins (n=2) and ABL exon 7 splicing (n=3). Finally, three cases with imatinib resistance expressed rare BCR-ABL transcript types without any evidence for KD mutations (two cases of e6a2 and one with e19a2 transcript). Imatinib resistance due to T315I BCR-ABL is extremely rare in Nordic CML patients. In addition to known hotspot mutations we identified novel sequence irregularities in association to imatinib resistance. We hypothesize that resistance-associated BCR-ABL isoforms are more heterogeneous than previously thought. Consequently, diagnostic procedures aiming in comprehensive molecular verification of TKI resistance should be designed as unbiased, since methods solely targeting known mutated BCR-ABL variants will miss many patients. However, an early detection of low-level mutated clones will require sensitive, mutation-specific analyses.
P2X7 Receptor Polymorphisms and Clinical Outcomes in Allogeneic Hematopoietic Stem Cell Transplantation.
Authors: Lee Kyung-Hun , Kim Inho , Yoon Sung-Soo , Park Seonyang , Kim Byoung Kook
Published: April 2020

The P2X7 receptor is a key player in processing and release of IL-1 family. We have recently demonstrated that A1513C polymorphism in P2X7 gene is related to the occurrence of infections and survival in HLA-matched sibling allogeneic stem cell transplantation (Lee et al., Haematologica2007;92:651–657). Here we extended our study by examining 3 SNP sites of P2X7 gene (A1513C, H155Y, and H270R) in all transplanted patients with hematological malignancies whose peripheral blood samples clinical data were available. Finally, 220 patients (146 acute leukemia, 30 chronic leukemia, 20 non-Hodgkin’s lymphoma, 19 myelodysplastic syndrome, and 5 multiple myemola) were included in the present study and 3 polymorphism sites of both donors and recipients were analyzed from peripheral blood samples. Donor 1513CC was associated with microbiologically documented bacteremia (56.3% for 1513CC donors vs. 30.9% for 1513AA or AC donors, P=.038). In contrast, recipient H155Y TT genotype was associated with lesser frequency of bacteremia (25.8% for TT recipients vs. 38.6% for CC or CT recipients, P=.055). Overall survival or treatment-related mortality did not differ significantly according to A1513C or H155Y polymorphisms. Recipient H270R GG genotype was significantly associated with shorter progression-free survival after transplantation (median, 567 days for GG recipients vs. 2122 days for AA or AG recipients, P=.042). The differences of overall survival according to H270R polymorphism, however, did not reach statistical significance (median 285 days vs. 314 days, P=.230). We conclude that A1513C, H155Y, and H270R polymorphisms in P2X7 receptor gene are related to the occurrence of infections and recurrences of hematological malignancies after allogeneic stem cell transplantation. Defining the polymorphisms may be helpful in optimal selection of patients and donors.

Progression-Free Survival According to H270R Genotype of Recipients Progression-Free Survival According to H270R Genotype of Recipients
Phenotypic Correction of Adamts13-Deficient Mice by Early Intra-Amniotic Gene Transfer of Lentiviral Vector Encoding ADAMTS13 Genes.
Authors: Niiya Masami , Endo Masayuki , Zoltick Philip W. , Muvarak Nidal E. , Motto David G. , Flake Alan W. , Zheng X. Long
Published: April 2020

ADAMTS13, a member of A Disintegrin and Metalloprotease with ThromboSpondin type 1 repeats (ADAMTS) family, is mainly synthesized in the hepatic stellate cells, endothelial cells and megakaryocytes or platelets. It controls the sizes of von Willebrand factor (VWF) multimers by cleaving VWF at the Tyr1605-Met1606 bond. Genetic deficiency of plasma ADAMTS13 activity results in hereditary thrombotic thrombocytopenic purpura (TTP), also named Upshaw-Schülman syndrome. To develop a potential gene therapy approach and to determine the domains of ADAMTS13 required for recognition and cleavage of VWF in vivo, a self-inactivating lentiviral vector encoding human wild-type ADAMTS13 or variant truncated after the spacer domain (construct MDTCS) was administrated by intra-amniotic injection on embryonic day 8. Direct stereomicroscopy and immunofluorescent microscopic analysis revealed that the green fluorescent protein (GFP) reporter, ADAMTS13 and MDTCS were predominantly expressed in the heart, kidneys and skin. The synthesized ADAMTS13 and truncated variant were detectable in mouse plasma by immunoprecipitation and Western blot, as well as by proteolytic cleavage of FRETS-VWF73 substrate. The levels of proteolytic activity in plasma of mice expressing ADAMTS13 and MDTCS were 5 ± 7% and 60 ± 70%, respectively using normal human plasma as a standard, and this proteolytic activity persisted for at least 24 weeks in Adamts13−/−mice and 42 weeks in wild-type mice tested (the duration of observation). The mice expressing both recombinant ADAMTS13 and MDTCS showed a significantly decreased ratio of plasma VWF collagen-binding activity to antigen and a reduction in VWF multimer sizes as compared to those in the controls. Moreover, the mice expressing ADAMTS13 and MDTCS showed a significant prolongation of ferric chloride-induced carotid arterial occlusion time (9.0 ± 0.6 and 25.2 ± 3.2 min, respectively) as compared to the Adamts13−/− mice expressing GFP alone (5.6 ± 0.5 min) (p<0.01). The ferric chloride-induced carotid occlusion time in Adamts13−/− mice expressing ADAMTS13 was almost identical to that in wild type mice with same genetic background (C56BL/6) (8.0 ± 0.2 min) (p>0.05). The data demonstrate the correction of the prothrombotic phenotype in Adamts13−/−mice by gene transfer to the fetus by viral vectors encoding human wild type ADAMTS13 and the carboxyl terminal truncated variant (MDTCS), supporting the feasibility of developing a gene therapy based treatment for hereditary TTP. The discrepancy in the proteolytic activity of MDTCS between in vitro (Zhang P et al. Blood, 2007 in press) and in vivo in the present study suggests the potential cofactors in murine circulation that may rescue the defective proteolytic activity of the carboxyl-terminal truncated ADAMTS13 protease seen in vitro.
Elevated Ferritin Is Associated with Poorer Survival Following Nonablative Allogeneic Transplantation.
Authors: Mahindra Anuj , Sobecks Ronald , Rybicki Lisa , Brown Stacey , Pohlman Brad , Kalaycio Matt , Dean Robert , Andresen Steve , Sweetenham John , Bolwell Brian , Copelan Edward
Published: April 2020

Iron overload is a well-established adverse prognostic factor for allogeneic hematopoietic stem-cell transplantation in thalassemia and appears to adversely affect prognosis with myeloablative transplantation for other diseases as well. In thalassemia, modification of the preparative regimen improves prognosis in patients who are at high risk due to iron overload. We analyzed a large group of patients who received a nonablative preparative regimen for allogeneic transplantation to determine whether the adverse impact of iron overload was circumvented by nonablative transplantation. Sixty-four consecutive patients undergoing nonablative allogeneic transplantation between 12/00 and 12/06 had pretransplant ferritin levels within 100 days preceding transplantation. Median age was 57 (range, 24–67) years; 24 patients were female. Median interval from diagnosis to transplant was 14.5 months (range, 2.4 to 151.6). Diseases included AML (n=16), NHL (15), MDS (10), myelofibrosis (6), CLL (6), CML (5), MM (3), Hodgkin (2), and ALL (1). Six had undergone prior transplantation. Median ferritin was 961 ng/mL (range, 302–6251). All but two received Flu/TBI preparation. Thirty-nine were sibling and 25 unrelated donors. Recursive partitioning identified significantly better survival for patients with ferritin ≤ 1615 ng/mL (n=45) compared to those with ferritin >1615 ng/mL (n=19; P=.012) as shown:

Figure Figure

Among age, gender, diagnosis, interval from diagnosis to transplant, extent of therapy prior to transplant, ferritin level, albumin, AST, alkaline phosphatase, bilirubin, LDH, risk group, donor source, degree of HLA match, donor to recipient gender, and CD34+ dose, only ferritin >1615ng/ml (P=.010, HR= 2.74), older age (P=.049, HR= 1.63), and prior transplantation (p=.002, HR=5.78) were adverse prognostic factors for survival in multivariable analysis. The poorer survival was related to both a higher incidence of treatment-related mortality and a higher rate of relapse mortality, neither of which reached statistical significance. The addition of albumin, a negative acute phase reactant, did not change the prognostic impact of ferritin.

Conclusion: Similar to patients who undergo myeloablative transplantation, survival of patients who undergo nonablative allogeneic transplantation is adversely affected by elevated ferritin. It is important to assess the influence of specific factors on comparative results of myeloablative and nonablative transplantation. These results should stimulate study of iron chelation prior to allogeneic transplantations.
Validation of a Pretransplantation Assessment of Mortality (PAM) Score for Predicting Risk of Death in the First Two Years after Allogeneic Hematopoietic Cell Transplantation in a Tertiary Transplant Center.
Authors: Chopra Akhil , D’Abreo Nina , Nagaraj Gayathri , Aljumaily Raid , Styler Michael , Topolsky David , Crilley Pamela , Gracely Edward
Published: April 2020

Mortality rates after allogeneic hematopoietic cell transplantation for hematological malignancies remains high. Recently, a 50-point pretransplantation assessment of mortality (PAM) score was developed by clinical investigators at the Fred Hutchison Cancer Research Center utilizing eight pre-transplant clinical variables including age, donor type, disease risk, conditioning regimen, percentage of predicted FEV1, percentage of predicted DLCO, serum creatinine level and serum ALT level. The PAM score performed well to predict risk of death in the first two years after allogeneic hematopoietic cell transplantation. The objective of our study was to validate the PAM score in a retrospective cohort of patients that underwent allogeneic hematopoietic cell transplantation at Hahnemann University Hospital from 1986 until 2006. 400 patients underwent allogeneic hematopoietic cell transplantation from 1986 to 2006. 170 patients were found to be eligible for retrospective analysis since pre- transplant serum alanine aminotransferase (ALT) levels were not available for the remaining 230 patients. The PAM score was calculated for each patient using the scoring system outlined in the original article. The patients were categorized into four cohorts with scores ranging from 9 to 16 for category 1 (probability of death, 25%), from 17 to 23 for category 2 (probability of death, 25% to 50%), from 24 to 30 for category 3 (probability of death, 50% to 75%), and from 31 to 44 for category 4 (probability of death, 75%). Kaplan- Meier survival curves were generated for each of the four categories. Overall mortality was 62.9% at 2 years. PAM scores ranged from 11 to 36. 41 patients were grouped into category 1, 71 into category 2, 48 patients into category 3 and 10 patients into category 4. Mortality at 2 years was 39% in category 1, 56.3% in category 2, 85.4% in category 3 and 100% in the high-risk category. Compared to the low risk group, group 2 had a hazard ratio of 1.64 (p = 0.092, CI: 0.92 to 2.94); group 3 had a hazard ratio of 3.996 compared to the low risk group (p < 0.001, CI: 2.23 to 7.16) and group 4 had a hazard ratio of 6.73 versus the low-risk group (p < 0.001, 95% CI: 3.01 to 15.04). In conclusion, the PAM score successfully classified patients into distinct survival groups (p < 0.001 for the comparison by log-rank test). Higher PAM scores were associated with progressively increased risk of death.

Figure Figure
Transplant Outcome Is Not Affected by Body Mass Index (BMI) in Patients with Haematological Malignancies.
Authors: Nikolousis Emmanouil , Paneesha Shankaranarayana , Holder Kathy , Bratby Lynn , Pillai Srinivas , Lovell Richard , Milligan Don
Published: April 2020

Bone marrow transplantation is frequently used as a consolidation therapy in patients with haematological malignancies to improve outcome. Obese individuals have larger absolute lean body and fat masses than non-obese individuals of the same age, gender and height, which might lead to altered pharmacokinetics of chemotherapeutic agents. Data on the impact of body mass on transplant outcome is conflicting. This study included 254 patients (M: 151; F: 103) with 283 transplant episodes (Autograft: 178, Allograft: 105). 81 patients had myeloma, 61 non Hodgkin’s lymphoma, 38 Hodgkin’s lymphoma, 34 acute myeloid leukaemia, 28 chronic lymphocytic leukaemia, 15 acute lymphoblastic leukaemia, 9 T cell non Hodgkin’s lymphoma, 6 each myelodysplasia and aplastic leukaemia and 1 each acute promyelocytic leukaemia and myelofibrosis. Statistical analysis was carried out using SPSS 13.0 for Windows. Overall survival (OS) and event free survival (EFS) were estimated by the Kaplan-Meier method. At transplantation 47% patients had normal & 52% high BMI. After a median follow-up of 14 month (range:0–75), mean OS in patients undergoing autograft with normal BMI was 52 months as compared to 54 with high BMI. Mean OS in patients undergoing allograft with normal BMI was 27 months as compared to 39 with high BMI (log rank test p value; autograft: 0.74 & allograft: 0.09). Odds Ratio for survival in autograft patients was 1.305 (95% CI: 0.611–2.786). For normal BMI patients OR for survival was 1.175(95% CI: 0.73–1.891), where as for high BMI patients 0.901 (95% CI: 0.678–1.197). Odds Ratio for survival in allograft patients was 0.512 (95% CI: 0.23–1.136). For normal BMI patients OR for survival was 0.751 (95% CI: 0.534–1.057), where as for high BMI patients 1.468 (95% CI: 0.921–2.341). Mean EFS in patients undergoing autograft with normal BMI was 42 months as compared to 50 with high BMI. Mean EFS in patients undergoing allograft with normal BMI was 26 months as compared to 48 with high BMI (log rank test p value; autograft: 0.4 & allograft: 0.3). This study shows that high BMI does not adversely impact on either OS or EFS in patients undergoing both autologous and allogeneic transplantation for haematological malignancies. On the contrary, the trend for OS appears to better in patients undergoing allograft with high BMI. We recommend patients with high BMI should not be excluded from having autologous transplantation. Studies with greater number of patients are needed to establish the potential beneficial impact of high BMI on survival in patients undergoing allogeneic transplantation.

Figure Figure
Reduced Intensity Conditioning Is Safe and Effective for Transplantation of Patients with Shwachman Diamond Syndrome.
Authors: Bhatla D. , Davies Stella M. , Harris R.E. , Crockett M. , Shoultz L. , Shenoy S. , Bleesing J. , Hansen M. , Jodele S. , Jordan M. , Filipovich A.H. , Mehta Parinda A.
Published: April 2020

Allogeneic hematopoietic stem cell transplantation is the only potentially curative treatment for the bone marrow dysfunction seen in patients with Shwachman-Diamond Syndrome (SDS). Historically, these patients have fared poorly with intensive conditioning regimens with increased regimen-related toxicity especially involving the heart and lungs. We report our institutional experience with a reduced intensity conditioning protocol in 7 patients with SDS and bone marrow aplasia or MDS/AML. Patient demographics are summarized in Table 1 below. The preparative regimen consisted of Campath-1H for 4 successive days (day -22 to day -19), Fludarabine 30 mg/m2/day for 5 days (Day - 8 to day -4) followed by Melphalan 140 mg/m2 for 1 day (day -3). Four patients received matched related marrow, 2 received matched unrelated peripheral blood stem cells (PBSC) and 1 received matched unrelated marrow. All but one were 8/8 allele HLA matched. Graft-versus-host disease (GVHD) prophylaxis consisted of Cyclosporine(CSA) and Methotrexate in all except one patient who received tacrolimus and mycophenolate mofetil as she was CSA intolerant. All patients established 100% donor-derived hematopoiesis. No major regimen related toxicity was seen except hyperglycemia requiring insulin in one patient and Grade II renal insufficiency in 1 patient which resolved with conservative management. Transplant outcomes are described in Table 2. These data indicate that HSCT with reduced intensity conditioning is feasible in patients with SDS and associated with excellent hematopoietic recovery and modest morbidity.

Table 1. Patient Demographics Patients #1 #2 #3 #4 #5 #6 #7 Age(yrs) 8 10 12 6 3 29 1 Gender Male Male Male Male Male Female Female SBDS mutation 183_184 TA to CT 183_184 TA to CT K62X 183_184 TA to CT 183_184 TA to CT IVS2+2 T to C 183_184 TA to CT Clinical status Transfusion dependence, recurrent infections Cytopenias Cytopenias Dysplasia Cytopenias Acute myeloid leukemia Dysplasia, Cytopenias Marrow cytogenetics del20q12 and del7q31 i(7)(q10) del20q12 del20q12 del20q12 Complex karyotype including monosomy 7,del5q Normal Table 2. Transplant outcomes Patients Patients #1 #2 #3 #4 #5 #6 #7 Days to myeloid engraftment 15 12 15 11 14 14 13 Platelet Recovery(days) 27 39 33 18 68 14 not engrafted Donor Chimerism 100% 100% 100% 100% 100% 99.8% 100% Hospital stay(days) 29 32 35 28 29 22 46 Length of follow-up(days) 758 723 679 385 218 65 33 Acute GVHD Gd II skin None None None None Gd I skin None Lansky scale 100% 100% 100% 100% 100% 90% 60%