bluuebunny/crossref_metadata_2025 huggingface dataset:
Treatment of Graft Failure with Fludarabine (Flu), Antithymocyte Globulin (ATG) and a Second Allogeneic Stem Cell Transplantation (SCT).
Authors: Jabbour Elias J. , Shahjahan Munir , Rondon Gabriela , Anderlini Paolo , Giralt Sergio , Couriel Daniel R. , Champlin Richard E. , Khouri Issa F.
Published: April 2020

Failure to engraft after SCT is a life-threatening complication. In a pilot trial, we assessed the feasibility of a second SCT after conditioning with Flu and ATG. Primary graft failure was defined by the inability to achieve any donor cell engraftment and to sustain an absolute neutrophil count (ANC) >0.5 x 109/l, for 3 consecutive days by day 28 after SCT. Secondary graft failure was defined as recurrent neutropenia with an ANC < 0.5 x 109/l for at least 7 consecutive days. Between July 1998 and April 2003, nine patients (pts) with various hematological malignancies (chronic myeloid leukemia [CML] n=5, acute myeloid leukemia [AML] n=2, myelodysplastic syndromes [MDS] n=2) were prospectively studied. Their median age was 38 years (range, 33-75). Eight had a primary and one had a secondary graft failure after conditioning with Bu/Cy (N=3), Flu/Bu (N=1), Melphalan/Flu (N=3), and Cy/Thiotepa/TBI (N=2). Cultures for CMV, Parvo-virus and HSV-6 were all negative. Median time between the first and the second SCT was 54 days (range, 31–140 days). All pts received Flu 25 mg/m2 intravenously (IV)/day on days −5 to −1, and ATG 20 mg/kg/day IV on days −3, −2, −1. Transplantation was provided on day 0. Tacrolimus was used for graft-versus-host disease (GVHD) prophylaxis. Eight pts received their second SCT from the peripheral blood of same donor [matched siblings (sib) =3, one-antigen mismatched sib =2, two-antigen mismatched sibling =1, haplo-identical sibling=1] whereas one had a cord blood (CB) transplant. The median dose of CD34+cells infused was 8 x 106/kg. Three pts had early death and were not evaluable. Six other pts, including the one who received a CB, had engrafted with 100% donor chimerism. Five recovered an ANC> 500 at a median of 18 days (range, 9–42 days). Complete disease response was documented in 4 pts including 3 with CML who achieved molecular response, and one with MDS. Three pts had grade 2, and three grade 3–4 acute GVHD. Median survival time of all pts was 7 months from their first SCT (range, 2–24 months). We conclude that Flu/ATG is a feasible regimen for second SCT as treatment for graft failure, allowing engraftment of donor hematopoiesis. GVHD was a major problem, and future studies should include more intense immunosuppressive prophylaxis.
Successful Engraftment of Autologous CD34+ Stem Cells after High-Dose Therapy and Fixed Dose (6 mg) Pegfilgrastim.
Authors: Marcacci Gianpaolo , Petti Maria C. , Ferrara Felicetto , Storti Sergio , D’Arena Giovanni , Palombi Francesca , Palmieri Salvatore , Giordano Giulio , Pinto Antonio
Published: April 2020

High-dose (HD) therapy plus Autologous Stem Cell Transplantation (ASCT) is a milestone treatment program for most hematologic malignancies. Daily subcutaneous injections of G-CSF (filgrastim/lenograstim) until ANC > 500/μl are routinely administered following ASCT, in order to accelerate hematopoietic recovery and reduce neutropenic complications. Pegfilgrastim has been shown to have similar efficacy when compared to G-CSF for chemotherapy-induced neutropenia, but little is known about its use in the ASCT setting. We used a 6 mg fixed dose of Pegfilgrastim on day +4 following ASCT in 46 patients (22 M/24 F; median age 56 yrs; r 22-70 yrs) with multiple myeloma (25 pts) and relapsed or refractory Hodgkin’s and non-Hodgkin’s lymphoma (21 pts). Patients received peripheral CD34+ stem cells (median number 4.4 x 106/Kg; r 1.8 – 11.8) harvested after mobilizing chemotherapy (cytoxan, vinorelbine/cytoxan, R-IEV, IGEV, R-ICE) and G-CSF. Standard conditioning regimens (HD-Melphalan or BEAM) were used. Engraftment results were compared to those from a historical control group of 182 patients (median age 56 yrs; r 16-74 yrs) who had received HD-Melphalan or BEAM and ASCT (median CD34+ cells 7.6 x 106/Kg, r 1.8–4.6) supported by G-CSF (5 μg/kg/day from day +5 until ANC>500/μl). Median number of days to ANC>500/μl were comparable between the Pegfilgrastim (10, r 8-15) and G-CSF (11, r 7–22) groups, as well as the median number of days to PLT>20,000/μl (Pegfilgrastim = 12, r 9–20 vs G-CSF= 12, r 7–29). Overall infectious rates, including FUO and documented infections, were of 48% and 39% for Pegfilgrastim and G-CSF groups, respectively (p=NS). Median number of days on i.v. antibiotics were 0 (r 0–18) and 6 (r 0–13) for the Pegfilgrastim and G-CSF groups, respectively. No significant differences in the incidence of bone pain, intensity of transfusion support and length of hospital stay were documented between the two groups. These data indicate that a fixed (6 mg) single-dose of Pegfilgrastim is safe and effective to accelerate engraftment after ASCT. No significant differences with G-CSF were apparent as to engraftment times and overall infectious complications. Growth factor costs were however in favor of Pegfilgrastim (Euro 690 vs 892 for a median of 10.5 G-CSF doses).
Hematopoietic Cell Transplantation as Therapy for Pediatric Patients with Isolated CNS Relapse.
Authors: Harker-Murray Paul , Tolar Jakub , Tan Ye , MacMillan Margaret L. , Wagner John , Orchard Paul J.
Published: April 2020

Treatment options for pediatric patients with isolated central nervous system (CNS) relapse of acute lymphoblastic leukemia (ALL) include intensive chemotherapy followed by craniospinal irradiation and hematopoietic stem cell transplantation (HSCT). However, there is little information regarding outcomes of HSCT in this situation. At the University of Minnesota 15 patients <18 years of age underwent allogeneic HSCT for ALL with isolated CNS relapses between 1990 and 2004. Leukemic subtypes included B cell (7), pre-B cell (6), T cell (1), and ALL NOS (1). All patients were in remission at the time of transplantation; 9 patients were in CR2, 5 patients were in CR3, and 1 patient was in CR5. Median duration of CR1 in all patients was 21.9 months (range 8.5–57.7) with median time from diagnosis of ALL to CNS relapse 23.4 months (range 8.9–127.7) in all patients. For patients transplanted in CR2, the median duration of 1st CR was 16.2 months (range 8.5–30.1). For all patients, transplantation was performed following a relapse limited to the CNS. Stem cell sources were related in 8 cases (5 HLA-matched marrow, 2 mismatched marrow, 1 matched cord blood) and unrelated in 7 (3 mismatched URD marrow, 2 5/6 matched cord blood, 2 4/6 matched cord blood). Preparative regimens included total body irradiation (TBI) and cyclophosphamide (Cy) for 12 patients, and Cy, TBI, VP16 for 3 patients. Grade II-IV GvHD occurred in 7 patients (47%; 95% CI 21–72) and grade III-IV in 3 patients (20%; 95% CI 0–40) by day 100. All patients were alive 1 year following transplantation. With a median follow-up of 7 years (range 1 to 19.4), the estimated 5 year survival is 78% (95% CI 51–100%). Of the 2 patients that died, 1 transplanted in CR2 died of relapse, while another transplanted in CR2 died of ARDS. Both were recipients of related donor grafts. These data demonstrate hematopoietic stem cell transplantation is a viable therapeutic option for pediatric patients with isolated CNS relapse, and suggests that a larger multi-institutional analysis should be undertaken.
Histone Deacetylase 3 (HDAC3), a Component of N-CoR-TBL1/R1 Chromatin Remodeling Co-Repressor Complex, Is Recruited to the Target Gene Promoters by PML-RARα To Repress the Transcription In Vivo.
Authors: Tomita Akihiro , Atsumi Akihide , Kiyoi Hitoshi , Naoe Tomoki
Published: April 2020

PML-RARα is a chimeric transcription factor deeply associated with acute promyelocytic leukemia (APL). PML-RARα plays an important role in the aberrant transcription repression on the target genes of wild type retinoic acid receptors (RARα). Pharmacological concentration of all-trans retinoic acid (ATRA) induces transcription de-repression on several target genes, and results in terminal differentiation of APL cells. However, the detailed mechanisms of transcription repression by PML-RARα in vivo are still unclear. Here we demonstrated that histone deacetylase 3 (HDAC3), one component of the N-CoR (nuclear receptor co-repressor)-TBL1/R1 (transducin beta-like protein 1/relating protein) transcription repressor protein complex, is a key regulator of the transcription repression by PML-RARα in vivo. Using immunoprecipitation (IP) assay, we first demonstrated that PML-RARα physically interacted with N-CoR/HDAC3 in vivo in the absence of ligand. The interaction was dissociated by adding ATRA in the dose dependent manner. Next we showed, using chromatin immunoprecipitation (ChIP) assay, that N-CoR/HDAC3 co-repressor complex was recruited to the endogenous target gene promoters (RARβ and CYP26) through PML-RARα. The neighboring histone H4 was de-acetylated and the gene expression was significantly repressed. When HDAC3 protein is knocked down by RNA interference in PML-RARα-presenting cells, the endogenous target gene expression was significantly activated. Almost the same results were also obtained when performing the luciferase reporter assay using RARβ and CYP26 promoter reporter vectors. Previously, we have shown that N-CoR-TBLR1 is recruited to the target gene promoter through PML-RARα in the absence of ligand, resulting in the transcription repression. Consistent with these data, it is strongly suggested that N-CoR/HDAC3/TBLR1 co-repressor complex is closely related to the aberrant transcription regulation by PML-RARα in APL cells. Furthermore, we also confirmed that PLZF-RARα, which is expressed in ATRA resistant APL cells, interacted with N-CoR/HDAC3/TBLR1 in ligand independent manner. These insights provide not only the basic mechanism of transcription repression by leukemia-related chimeric transcription factors, but also the new molecular targets for the transcription therapy for leukemia.
Vascular Endothelial Growth Factor (VEGF) Is a Critical Autocrine Survival Factor in Acute Promyelocytic Leukemia (APL).
Authors: Kini Ameet R. , Roychowdhury Shantanu , Dziuma Alla , Tallman Martin S. , Nagabhushan Moolky
Published: April 2020

We have previously demonstrated (Kini AR et. al. Blood.2001;97:3919–24) that VEGF is expressed in APL and is regulated by all-trans retinoic acid (ATRA). In this study, we examined the role of VEGF as a autocrine survival factor in APL, by using siRNA to VEGF in cultured NB4 APL cells.

The siRNA molecules were transfected into the APL cells by electroporation and resulted in almost complete (>95%) knock-down of VEGF expression. The VEGF knock-down resulted in significant apoptosis (>50%) within 96 hours. To verify the specificity of the VEGF siRNA, we used a siRNA molecule with a single nucleotide mismatch mutation, as well as a scrambled siRNA molecule. These control siRNA molecules did not result in VEGF knock-down, nor did they cause apoptosis, confirming the specificity of the VEGF siRNA and its effect.

In contrast to the striking induction of apoptosis by VEGF knock-down, external blocking of VEGF and VEGF receptors by addition of neutralizing antibodies did not cause apoptosis. In addition, the concentration of VEGF within the cells was over 100-fold higher than the external VEGF concentration. These results suggest that VEGF mediates its actions through an internal autocrine mechanism. Intriguingly, immunfluorescence studies showed that VEGF is strongly expressed in the nucleus, suggesting a novel nuclear role for VEGF.

To study the possible role of the VEGF receptors in internal loops we knocked-down the VEGF receptors using siRNA molecules. VEGF receptor knock-down did not affect survival of the APL cells. To summarize, we demonstrate that VEGF is a survival factor for APL cells, and acts independent of the VEGF receptors through an internal autocrine mechanism. These results indicate that targeting internal VEGF may be an effective therapeutic strategy for APL.
G-CSF and GM-CSF Have Opposite Effects on the Growth and Survival of Cells Containing Abnormal RUNX1.
Authors: Mortazavi Fariborz , Sharma Arun , Baraoidan Marie , Ibanez Vinzon , Yang SiJun , ShriHari Kadkol , Saunthararajah Yogen
Published: April 2020

Primary leukemia cells can respond very differently to cytokines such as G-CSF and GM-CSF, demonstrating marked increases in colony-forming ability to one but not the other (Park et al, Blood1989; 74: 56–65). To examine if such differences might correlate with underlying molecular abnormalities, we examined the growth and survival response of hematopoietic cells containing abnormal RUNX1 variants (a frequent occurrence in leukemia and myelodysplastic syndrome) to treatment with G-CSF and GM-CSF. In the RUNX1-ETO containing cell-line Kasumi-1, GM-CSF decreased colony forming ability whereas G-CSF preserved or increased it. The 32Dcl3 murine myelomonocytic cell-line terminally differentiates in response to G-CSF or mGM-CSF. We transduced 32Dcl3 with RUNX1-ETO or Empty Vector: G-CSF promoted the growth and survival of 32D RUNX1-ETO but caused 32D Empty Vector to terminally differentiate and die. In contrast, 32D RUNX1-ETO cultured in mGM-CSF demonstrated accelerated terminal differentiation and death compared to 32D Empty Vector. Many of the RUNX1 variants seen in leukemia and myelodysplastic syndrome, including RUNX1-ETO, lack the RUNX1 C-terminal transcription regulating domain (TRD). In human CD34+ hematopoietic cells transduced with RUNX1 with TRD deleted (CD34 RUNX1noTRD), colony forming ability was retained even after >40 days of culture in IMDM 10% FBS, SCF 10ng/ml, IL-3 30ng/ml, G-CSF 100ng/ml (CD34 Empty Vector lost colony forming ability after 14 days). However, culture in media supplemented with 100ng/ml GM-CSF instead of G-CSF significantly impaired colony forming ability of CD34 RUNX1noTRD compared to CD34 Empty Vector. In conclusion, RUNX1 variants seen in acute myeloid leukemia and myelodysplastic syndrome promote growth and survival in G-CSF but accelerate differentiation and death in response to GM-CSF. The molecular basis for this effect appears to relate to the presence or absence of the RUNX1 C-terminal transcription regulating domain. These findings may have clinical relevance.
Voriconazole and Liposomal Amphotericin B (Ambisome) Effectively Prevent Mold Infections in Patients (pts) with Acute Myelogenous Leukemia (AML) Following Remission Induction Chemotherapy (RIC).
Authors: Mattiuzzi Gloria N. , Estey Elihu H. , Hernandez Mike , Cabanillas Maria E. , Giles Francis , Cortes Jorge E. , O’Brien Susan , Verstovsek Srdan , Kantarjian Hagop M.
Published: April 2020

Invasive fungal infections (IFI) are a frequent cause of morbidity and death in pts with AML and high-risk myelodysplastic syndrome (HR-MDS). Because early diagnosis of IFI is difficult, antifungal prophylaxis (AFP) including mold-active agents has become an important strategy to reduce morbidity and mortality in this patient population and is routinely used at MDACC for AML and HR-MDS pts undergoing RIC. We retrospectively compared the efficacy and safety of 6 AFP regimens (Sept 97- July 04) among 659 evaluable pts with newly diagnosed AML and HR-MDS who received RIC and had been enrolled in our prospective AFP trials. See regimens listed in Table below. There were no significant differences among the 6 regimens with regard to key baseline characteristics (age, gender, diagnosis, cytogenetics, type of RIC, Zubrod PS, WBC count, non-fungal infection and protected environment) and median days of AFP. 37 pts (5.6%) developed IFI (yeast 3 %; mold 2.6%). No mold infections were observed among pts randomized to AMBI or VORI. With the exception of VORI, which was significantly more effective than IV ITRA (p =0.03), all comparisons of efficacy among the AFP regimens were not significant. Drug discontinuation was the highest with IV VORI (21%) and ABLC (18%). VORI was more toxic than IV ITRA, Caspo, and F+I (p=0.023, 0.001 and 0.031 respectively). VORI toxicity was reversible and consisted of visual and/or auditory hallucinations and elevation in serum bilirubin. There was a trend toward developing VORI toxicity if baseline bilirubin levels were elevated (OR=4.9; p=0.10).

We conclude that the rate of IFI in AML and HR-MDS pts undergoing RIC given mold-active AFP is 5.6 %. VORI and AMBI effectively prevented mold infections. VORI was more effective that IV ITRA but was associated with a high rate of reversible drug-related adverse events.

ABLC (n=131) AMBI (n=69) F+I (n=67) IV ITRA (n=225) CASPO (n=106) VORI (n=61) ABLC: Amphotericin B Lipid Complex: 2.5 mg/kg IV three times/week; AMBI: Liposomal Amphotericin B: 3 mg/kg IV three times/week; F+I: Fluconazole: 400 mg (tab)/d + Itraconazole: 200 mg (caps)/d; IV ITRA: IV itraconazole: 200 mg BID X 2 d, then 200 mg IV/d; CASPO: Caspofungin: 50 mg IV/d; VORI: Voriconazole: 400 mg IV BID x 2 d, then 300 mg IV BID. Median age, years (range) 65(21–87) 63(36–83) 57(19–84) 62(17–89) 65(22–82) 59(23–79) Zubrod ≤ 2 (%) 127(97) 69(100) 65(97) 214(95) 101(95) 61(100) Median days AFP (range) 17(3–32) 14(3–28) 16(3–44) 20(3–41) 21(3–38) 21(3–34) Breakthrough IFI (%) 7(5) 3(4) 3(5) 17(8) 7(7) 0     Yeast (%) 2(2) 3(4) 1(1) 11(5) 3(3) 0     Mold (%) 5(4) 0 2(3) 6(3) 4(4) 0 Drug-related AFP DC (%) 24(18) 10(14) 5(7) 23(10) 4(4) 13(21)
Fondaparinux Combined with Intermittent Pneumatic Compression (IPC) Versus IPC Alone in the Prevention of Venous Thromboembolism after Major Abdominal Surgery: The Randomized APOLLO Study.
Authors: Turpie Alexander G.G. , Bauer Kenneth A. , Caprini Joseph A. , Comp Philip P. , Gent Michael , Muntz James E.
Published: April 2020

Background: The selective factor Xa inhibitor fondaparinux has been shown to be at least as effective and as safe as the low-molecular-weight heparin dalteparin for venous thromboembolism (VTE) prevention after major abdominal surgery (Agnelli GA, et al. Br J Surg, 2005, In press). The benefit of fondaparinux in addition to intermittent pneumatic compression (IPC) in VTE prevention after abdominal surgery has not been evaluated.

Objective: We performed a randomized, double-blind, placebo-controlled, superiority trial to compare the efficacy and safety of fondaparinux in conjunction with IPC versus IPC alone in patients undergoing major abdominal surgery.

Methods: Patients aged at least 40 years undergoing abdominal surgery of at least 45 minutes were included. Patients at highest risk of VTE, requiring pharmacologic prophylaxis in addition to IPC were excluded at the investigator’s discretion. Patients were randomized to receive either fondaparinux 2.5 mg or placebo once daily subcutaneously for 5 to 9 days, starting 6 to 8 hours postoperatively. All patients received IPC. The primary efficacy outcome was the composite of deep-vein thrombosis detected by mandatory bilateral venography, or documented symptomatic deep-vein thrombosis or pulmonary embolism up to day 10. The main safety outcome was major bleeding during the treatment period. A blinded independent committee adjudicated all these outcomes. Follow-up lasted 32 days.

Results: Of the 1309 patients randomized between November 2001 and October 2004 (fondaparinux+IPC, n=650; placebo+IPC, n=659), 842 (64.3%) were evaluable for efficacy. The treatment groups were comparable with regard to VTE risk factors, demographic and surgical characteristics; 82% had at least one VTE risk factor (over and above being at least 40 years old and undergoing abdominal surgery). Fondaparinux significantly reduced the VTE rate from 5.3% (22/418) with placebo to 1.7% (7/424), an odds ratio reduction of 69.8% (95% CI: 27.9 to 87.3; p=0.004). Similarly, fondaparinux significantly reduced the proximal deep-vein thrombosis rate from 1.7% (7/417) to 0.2% (1/423; p=0.037). Major bleeding occurred in 1.6% (10/635) and 0.2% (1/650) of fondaparinux- and placebo-treated patients, respectively (p=0.006). None of the bleeding events were fatal or involved a critical organ.

Conclusions: Fondaparinux combined with IPC was significantly more effective than IPC alone for VTE prevention after major abdominal surgery. Although the bleeding risk was increased with fondaparinux compared with placebo, this risk was low and consistent with that observed in previous fondaparinux studies in surgical patients.
Farnesyl Transferase Inhibitor (Tipifarnib, Zarnestra; Z) in Combination with Standard Chemotherapy with Idarubicin (Ida) and Cytarabine (ara-C) for Patients (pts) with Newly Diagnosed Acute Myeloid Leukemia (AML) or High-Risk Myelodysplastic Syndrome (MDS).
Authors: Cortes Jorge , Garcia-Manero Guillermo , O’Brien Susan , Verstovsek Srdan , Estrov Zeev , Giles Francis , Ravandi Farhad , Wright John , Kantarjian Hagop
Published: April 2020

Tipifarnib is a non-peptidomimetic farnesyl transferase inhibitor (FTI) with clinical activity in hematologic malignancies, including AML, MDS and CML. Pre-clinical data suggests that tipifarnib may be synergistic with some chemotherapeutic agents. We designed a phase I/II study where pts age 15 to 60 years with previously untreated AML or high-risk MDS (blasts >10%) received induction with Ida 12 mg/m2/d on days 1–3, ara-C 1.5 g/m2 IV over 24 hours daily on days 1–4 (days 1–3 only if age ≥ 60 years) and Z with first cohort (7 pts) receiving 200mg orally twice daily (BID), and all others 300 mg BID x 21 days every 28 days. Pts achieving CR received consolidation (5 courses) was with Ida 8 mg/m2/d, days 1–2, ara-C 0.75 g/m2/d, days 1–3, and Z 300 mg BID x14 days every 4–6 weeks. Maintenance was with Z 300 mg BID x 21 d every 4–6 wk for 6 months. We have treated 23 pts, median age 51 yrs (19–59 yrs). Eighteen pts are evaluable for response and 13 (72%) have achieved CR and 1 CRp. Response in evaluable pts by cytogenetics was: 5/8 (83%) for diploid, 4/5 with −5/−7, 1/2 with t(8;21), and 3/5 with other abnormalities. Response by Flt3 was 11/14 (79%) for unmutated Flt3, 1/3 for mutated Flt3. Response by age (excluding t(8;21)) was 6/7 (86%) if ≤50yrs, 7/9 (78%) if >50yrs. With median follow-up of 13 weeks, 1 pt has relapsed after 14 weeks and 1 died (in CR) after 18 wks. The most common grade 3 adverse events include diarrhea in 7 (30%) and hyperbilirrubinemia in 6 (26%), both transient. Thirteen pts (59%) have required treatment interruptions/dose reductions during induction. We conclude that Z combined with Ida and ara-C induces a high rate of CR in AML, with increased incidence of diarrhea and hyperbilirrrubinemia. Comparison with chemotherapy alone is warranted..
Clofarabine and Clofarabine Plus Low-Dose Cytarabine (ara-C) as Induction Therapy for Patients (pts) ≥ 60 Years with Newly Diagnosed Acute Myeloid Leukemia (AML).
Authors: Faderl Stefan , Ravandi Farhad , Ferrajoli Alessandra , Garcia-Manero Guillermo , Estrov Zeev , Thomas Deborah A. , O’Brien Susan , Verstovsek Srdan , Wierda William , Cortes Jorge , Giles Francis J. , Kwari Monica , Jarmon Rita , Kantarjian Hagop M.
Published: April 2020

Optimal induction therapy for pts ≥ 60 years (yrs) with AML remains undefined. Clofarabine is a purine deoxyadenosine analog with single agent activity in AML. Due to its metabolic and pharmacokinetic properties it is also an ideal agent for biochemical modulation strategies with other nucleoside analogs such as ara-C. In previous trials we reported our experience with clofarabine + intermediate doses of ara-C for salvage and frontline AML pts. To further explore clofarabine in AML, we investigated a lower dose schedule of clofarabine +/− low-dose ara-C (LDAC) in pts ≥ 60 yrs and previously untreated AML. Pts were adaptively randomized (based on history of response in each arm) to receive either clofarabine (C) at 30mg/m2/d i.v. daily d1-5 or clofarabine at 30mg/m2/d i.v. daily d1-5 + LDAC at 20mg/m2/d s.c. daily d1-14. Pts in remission received consolidation therapy q4-7 wks with clofarabine daily on d1-3 +/− LDAC daily on d1-7. Pts with ECOG PS ≥ 3 and/or a history of NYHA ≥ grade 3 heart disease were not eligible. Pts were admitted to a laminar airflow room (“protective environment”) for the duration of the induction. All pts received antibiotic prophylaxis consisting of levaquin, valacyclovir, and itraconazole (or equivalents). No antifungal prophylaxis was given during the days of the clofarabine infusions. Fifty-four pts have been enrolled (C 14; C+LDAC 40). Median age: 71 yrs (range 60–83). An antecedent hematologic disorder (MDS, CMML, NHL) or other malignancy was present in 28 pts (52%). Twenty-four pts (44%) had abnormal cytogenetics of whom 11 (20%) had −5, −7, or 11q23 abnormalities. Flt3 ITD/mutations were found in 7 pts (14%). Forty-four pts are evaluable for response (C 12; C+LDAC 32). Twenty-four pts (55%) achieved CR and 1 (2%) CRp for an overall response rate (ORR) of 57%. Median time to CR: 34 days (25–78). Response by treatment arm: 1) C (n=12): 5 (42%) CR; 2) C+LDAC (n=32): 19 (59%) CR, 1 (3%) CRp, ORR 62% (p=NS for CR rate). Most toxicities were ≤ grade 2 and included nausea/vomiting, diarrhea, fatigue, skin rashes and temporary elevations of transaminases and serum bilirubin. Myelosuppression was ubiquitous and myelosuppression-associated infectious complications were frequent. Nine pts (17%) died during induction (6/40 on C; 3/14 on C+LDAC), of whom 2 died early (≤ 14 days of treatment start): 71 yo female with E. coli sepsis on d12 (C); 83 yo male with pulmonary hemorrhage and pneumonia d14 (C+LDAC). In summary, clofarabine and clofarabine + LDAC show activity in older pts with AML. Its role in elderly AML induction needs to be evaluated further. Myelosuppression-associated complications remain frequent. An update of responses and time-to-event parameters will be presented.
Loss of Heterozygosity on Chromosome 6q14-q24 Confers Poor Outcome on Children and Adolescents with T-Cell Lymphoblastic Lymphoma.
Authors: Burkhardt Birgit , Bruch Jochen , Mueller Franziska , Salzburg Janina , Harbott Jochen , Reiter Alfred
Published: April 2020

Deletions of chromosome 6q have been reported in a variety of hematological malignancies. Regarding their biological and prognostic impact, however, the data available are not conclusive. Therefore the current study focused on 60 pediatric and adolescent patients all diagnosed with T-cell lymphoblastic lymphoma (T-LBL), a clearly defined histological sub-entity of NHL, and all treated uniformly according to the protocol NHL-BFM 95. Deletions of chromosome 6 were analyzed by loss of heterozygosity (LOH) analysis of 25 microsatellite markers located on chromosome 6q14-q24 and the prognostic impact of LOH on 6q was evaluated after combing the results with clinical data of the patients.

Selection criteria for this study were 1) treatment according to multicentre protocol NHL-BFM 95, 2) T-cell immunophenotype of LBL, 3) germ line control and tumor DNA available and 4) three or more informative markers for the analysis of LOH. High-molecular weight germ line DNA was extracted from blood and bone marrow smears or frozen cells. Tumor DNA was isolated from tumor touch imprints or cytospin preparations of malignant effusions or frozen tumor cells. Paired normal and tumor DNA samples from each patient were amplified by PCR, after which fragment length analysis was performed on an ABI PRISM 3100 Genetic Analyzer.

From 04/95 until 03/03 a total of 186 patients were diagnosed with T-LBL and treated according to the protocol NHL-BFM 95. The probability of event free survival (pEFS) for these 186 patients was 79±3% (median follow-up of 4.5 (0.5–9.1) years). 60 of the 186 T-LBL patients were evaluable for LOH analysis, including 19 out of the total 25 of T-LBL patients who suffered from tumor failure. Fragment length analysis comparing germ line DNA and tumor DNA was performed for all 25 microsatellite markers per patient resulting in a total of 832 successfully analyzed markers for the whole group of 60 patients. The status of these 832 results was LOH in 10%, retention of heterozygosity in 55%, homozygosity in germ line control and tumor DNA in 25% and microsatellite instability in 9% of results respectively. In 19 out of the 60 evaluable patients (32%) LOH of at least one microsatellite marker was detected. LOH was observed in 13 out of the 19 evaluable patients with tumor failure compared to 6 out of the 41 patients without tumor failure. The probability of disease free survival (pDFS) (5y) for the 19 patients with LOH was 24±11% compared to 85±6% for the 41 patients without LOH (p <0.0001). A critical region of deletion could be assigned to microsatellite markers D6S1284 and D6S1716. pDFS (5y) was 0% for patients with LOH involving these markers (n=7) compared to 82±7% for patients without LOH at these markers (n=31) (p <0.0001). We conclude that LOH on chromosome 6q14-q24, especially of markers D6S1284 and D6S1716, confers poor outcome on children and adolescents with T-LBL.
VENUS - A Study To Validate Centrally Adjudicated Venous Ultrasound Against Venography after Major Orthopaedic Surgery.
Authors: Schellong Sebastian M. , Beyer Jan , Halbritter Kai , Schmidt Benjamin , Turpie Alexander G.G. , Eriksson Bengt I. , Misselwitz Frank , Kälebo Peter
Published: April 2020

Mandatory, bilateral, centrally adjudicated venography is currently required to assess the incidence of deep vein thrombosis (DVT) in confirmatory trials of new antithrombotic agents. Non-invasive and less cumbersome methods would be welcomed by both patients and investigators. Centrally adjudicated complete compression ultrasound (CCUS) of all leg veins could become an alternative to venography. Despite the fact that no formal validation of venography has ever been performed, CCUS will have to be externally validated against this ’gold standard’ to gain general acceptance and regulatory approval. A substudy of two recent phase IIb trials of a novel, oral anticoagulant for the prevention of venous thromboembolism in patients undergoing elective hip or knee arthroplasty was undertaken to validate CCUS against venography. The studies had similar designs and methodology, and were designed to allow the pooling of data. Mandatory bilateral venography was performed 7±2 days after surgery; CCUS was performed later the same day, with the sonographers blinded to the venography result. All sonographers were required to have completed a training course, and to have obtained personal certification for the standardized examination and documentation procedure. Venograms and CCUS videos were adjudicated centrally by two independent readers at separate locations; discrepancies were resolved by consensus reading. From the 1347 patients participating in the two trials, 870 matching pairs of evaluable venograms and CCUS videos were obtained. Observed prevalences of any DVT, including muscle vein thrombosis, were 19.1% with venography, and 13.0% with CCUS. Sensitivity and specificity values [with 95% confidence intervals (95% CI)] for detecting DVT with CCUS are shown in the table. Although false positives did not severely impair specificity, the small number of true positives detected by CCUS resulted in remarkably low sensitivity, in particular for proximal DVT. Based on these external validation figures, centrally adjudicated CCUS is not a viable technique to replace venography for the screening of DVT early after major orthopaedic surgery in confirmatory trials of novel antithrombotic agents.

DVT Sensitivity [% (95% CI)] Specificity [% (95% CI)] Any 27.4 (21.2, 33.6) 95.8 (94.8, 96.8) Proximal 13.0 (−0.7, 26.8) 99.2 (98.9, 99.7) Distal 25.9 (19.7, 32.1) 96.2 (95.3, 97.2)
Analysis of KIR Framework Loci in Patients with NK-Type Lymphoproliferative Disease of Granular Lymphocytes (LDGL).
Authors: Zambello Renato , Scquizzato Elisa , Teramo Antonella , Facco Monica , Pavan Laura , Miorin Marta , Trentin Livio , Agostini Carlo , Semenzato Gianpietro
Published: April 2020

To investigate whether a bias toward the presence of genes coding for activating KIRs were present in patients with NK-type lymphoproliferative disease of granular lymphocytes (NK-LDGL), by multiplex PCR analysis in this study we evaluated the KIR genotype in 35 patients with NK-LDGL and in 50 normal subjects. According to the published data in the Caucasian population, all individuals were positively scored for the framework loci (KIR2DL4+, KIR3DL2+ and KIR3DL3+). All genotypes were characterized by a gene encoding a group 1 HLA-C-specific inhibitory KIR, which was either KIR2DL1, KIR2DL2 or KIR2DL3 and at least contained one activating KIR gene. The KIR genotype analysis revealed that patients genotypes were largely represented by type B haplotype (34/35) that is characterized by the presence of multiple activating KIRs. The same KIR genotype was often shared between NK-LDGL patients. These genotypic profiles were not present in the group of controls and are infrequent (1,2%) or not still found in the Caucasian population. In contrast with literature data reporting that the frequency of haplotype A in homozigous state represents the most common condition in the Caucasian population (accounting for 1/3 of normal individuals), we surprisingly found that also almost all the controls studied (49/50) were equipped with the type B haplotype.

When the genotypic frequency was considered, the most frequently gene content found was 9 in controls and 11 or 14 in patients, indicating that patients genotypes contained a higher number of genes than controls. This picture was almost entirely due to the high variability of the activating genes number. As far as the activating genes are concerned, although the number of control individuals analyzed was still low, the frequency of KIR2DS1, KIR2DS2, KIR2DS4, KIR1D and KIR3DS1 was found to be higher in patients than in controls. This indicates that genotypic profiles of NK-LDGL patients are characterized by an increased presence of activating KIRs.

Consistent with these findings, the analysis of the KIR genes repertoire in NK-LDGL patients clearly indicates that the susceptibility to this disease is influenced by the KIR genotype and in particular by the presence of a considerable number of activating KIR genes. Moreover, we observed that healthy individuals scored as controls (all coming from the north-east of Italy) showed genotypes with a higher number of activating genes as compared to other Caucasian populations. This observation suggests that a genetic predisposition to development of NK-type LDGL might occur among the population of north-east of Italy and in turn this could explain the high frequency of NK-type LDGL in this Italian region.
Rapid, Sustained B Cell Receptor Signaling in Lymphoma B Cells Differs from Normal Signaling in Tumor Infiltrating Nonmalignant B Cells.
Authors: Irish Jonathan M. , Czerwinski Debra K. , Nolan Garry P. , Levy Ronald
Published: April 2020

The B cell receptor (BCR) drives life and death signaling throughout B cell development, and dysregulation of BCR signaling might be expected to play a role in aberrant proliferation of lymphoma B cells. We have previously used flow cytometry based cell signaling profiles to identify patterns of altered signaling in acute myeloid leukemia that were informative of clinical outcome (Irish et al., Cell, 2004). Here we used a similar signaling profiles approach to compare BCR signaling in normal and lymphoma B cells. However, in addition to comparing follicular lymphoma (FL) B cells with peripheral blood B cells from normal donors, we also interrogated signaling within individual non-tumor B cells infiltrating FL tumor biopsies. By staining for CD20 and BCR light chain isotype (κ vs. λ), we could distinguish tumor and normal B cells within each patient biopsy. Following crosslinking of BCR heavy chains (shared by tumor and non-tumor B cells), we measured phosphorylation of Syk and Btk proteins, as markers of early BCR signaling activity, and Erk1/2 and p38, as markers of downstream BCR signaling effector activity. The BCR signaling network in FL tumor B cells was activated more rapidly than infiltrating non-tumor B cells, achieved greater levels of per-cell signaling, and sustained high levels of signaling over a period of hours. In lymphoma B cells, BCR-mediated Btk and Erk1/2 phosphorylation could reach the normal maximum in as little as 4 minutes, which was much more rapid than the 30–60 minutes required for peak signaling in non-tumor B cells. Strikingly, the timing and magnitude of BCR pathway protein phosphorylation we measured in non-tumor B cells within tumor biopsies was the same as that of normal, mature B cells from peripheral blood. These results suggest that the altered BCR signaling we identified in lymphoma is cell-intrinsic and associated with lymphomagenesis, as opposed to being a general change in tumor microenviornment affecting all B cells within a biopsy. FL tumor B cells from different patients were distinguished by the degree and number of changes to BCR signaling, such that variable profiles of lymphoma signaling kinetics distinguished each patient from the consistent signaling of normal B cells. These results identify cell-intrinsic changes to BCR signaling that may contribute to immortalization of lymphoma B cells and suggest that single cell profiles could identify lymphoma specific BCR-mediated signaling responsible for clinical outcomes.
A Human SIL-SCL Fusion Gene Displays a Development Block at the CD4− CD8− to CD4+CD8+ Transition in Transgenic Mice.
Authors: Cheng Yue , Zhang Zhenhua , Slape Chris , Aplan Peter D.
Published: April 2020

Chromosomal rearrangements leading to dysregulation of the SCL gene, located at chromosome 1p32, is a common event in the development of human T-cell acute lymphocytic leukemia (T-ALL). In the most common form of SCL gene rearrangement, an interstitial deletion of approximately 90 kb brings SCL under the control of regulatory elements that normally govern expression of the ubiquitously expressed SIL (SCL interrupting locus) gene, which is located directly upstream of SCL. To investigate the consequences of this event, we reproduced this gene alteration by using cre-mediated recombination. A BAC clone containing both human SIL and SCL genes was isolated and loxP sites were cloned into the intron 1 region of both the SIL and SCL genes, corresponding to the sites at which recombination leading to an interstitial deletion occurs in human T-ALL patients. This BAC clone was used to generate transgenic SILloxloxSCL mice. The SILloxloxSCL mice were bred to mice that express the cre recombinase in the thymus (Lck-cre mice). The BAC transgene was recombined between the two loxP sites in over 50% of the thymocytes from double transgenic Lck-cre/SILloxloxSCL mice, faithfully recapitulating the event seen in human T-ALL patients, and bringing the SCL gene under the direct control of SIL regulatory elements. Aberrant SCL gene expression was verified by RT-PCR. Using FACS analysis, we found that mice carrying both the SILloxloxSCL transgene and the Lck-cre transgene have decreased CD4+/CD8+, CD4+/CD8−, CD4−/CD8+ and increased CD4−/CD8− thymocytes compared to transgene-negative mice or mice that carried the SILloxloxSCL transgene but not the Lck-cre transgene. These findings were detected in mice from 6 to 15 months of age. Interestingly, increased numbers of CD44+ thymocytes can also been identified in SILloxloxSCL/Lck-cre mice. In the spleen, the SILloxloxSCL/Lck-cre mice show a marked reduction in the number of mature CD4+ or CD8+ cells. These results indicate an accumulation of immature T-cells in mice transgenic for both the SILloxloxSCL transgene and the Lck-cre transgene, and demonstrate that conditional activation of SCL under control of SIL regulatory elements can impair normal T-cell development.
Transallelic Effects at the Cyclin D1 Loci in Mantle Cell Lymphoma.
Authors: Epner Elliot M. , Hui Liu , Jin Wang , Jing Huang , Bruce Clurman E.
Published: April 2020

Cyclin D1 is a gene commonly deregulated in B cell malgnancies. Translocation between chromosomes 11 and 14 (t;14) are found in most patients with mantle cell lymphoma (MCL) and 15–20% of patients with multiple myeloma (MM). Gene targeting experiments in cyclin D1 overexpressing MCL and MM were carried out and genetic variants isolated which had lost the IgH translocated or inserted chromosomes. These clones no longer expressed cyclin D1 but expressed high levels of cyclin D3. Analysis of DNA methylation patterns in these clones has demonstrated that the translocated chromosome exerts a long distance trans DNA hypomethylating effect on the normal chromosome (transvection) at the cyclin D1 locus and at least 100 kilobases upstream. Thus, in the absence of the translocated chromosome, the normal cyclin D1 locus is densely DNA methylated. Reintroduction of the translocated chromosome by somatic cell hybrid fusion induced transhypomethylation in trans. This long distance trans hypomethylating effect was also demonstrated in a MM cell line (U266) which contains an insertion rather than a translocation of IgH sequences, suggesting that 3′ Cα IgH sequences are sufficient for this effect. Combined FISH/immunofluorescent antibody labelling studies have shown the presence of both the normal and translocated chromosome 11’s at the outer nucleolar membrane, where they are tethered by the proteins CTCF and nucleophosmin, as demonstrated using chromatin immunoprecipitation assays. Allelic ChIP studies showed the presence of CTCF and nucleophosmin on both the translocated and normal chromosome 11(s). Tethering of the translocated and nontranslocated chromosomes by CTCF/nucleophosmin at the nucleolus provides a mechanism for pairing and long distance DNA transhypomethylation.
Defining Second-Hit Genetic Abnormalities Involved in the t(12; 21) TEL-AML1 Acute Lymphoblastic Leukemia by Array-Based Comparative Genomic Hybridization.
Authors: Tsuzuki Shinobu , Karnan Sivasundaram , Horibe Keizo , Matsumoto Kimikazu , Kato Koji , Sugita Kanji , Nakazawa Shinpei , Seto Masao
Published: April 2020

TEL (ETV6)-AML1 (RUNX1) chimeric gene fusion is the most common genetic abnormality in childhood acute lymphoblastic leukemias (ALL). Evidence suggests that this chimeric gene fusion usually occurs in utero during fetal hematopoiesis and most probably constitutes an initiating or first-hit mutation that is necessary but insufficient for the development of overt, clinical leukemia. In a search for additional secondary and postnatal genetic events that could be linked to leukemia development, we applied a genome- wide array- CGH technique to 24 TEL-AML1 leukemia samples and two cell lines (REH, KOPN41) and found that at least three chromosomal imbalances were involved in all patient samples and cell lines. Recurrent regions of chromosomal imbalances (found in > 10% of clinical samples) were gain of chromosomes 10 (17 %) and 21q (25 %) and loss of chromosomes 2p11 (100 %), 12p13.2 (87 %), 9p21.3 (29 %), 9p13.2 (25 %), 12q21.3, (25 %), 3p21 (21 %), 6q21 (17 %), 4q31.23 (17 %), 11q22-q23 (13 %) and 19q13.11-q13.12 (13 %). The two cell lines showed gain of 21q22.12-qter and loss of 2p11.2, 9p21.3, 12p13.2, and 12q21.3. Among these, six regions of loss (2p11, 3p21, 4q31.23, 9p13.2, 12q21.3 and 19q13.12) have not been identified previously by conventional CGH in TEL-AML1 leukemia. Representative genes involved in the regions of loss were Igkappa (2p11), TEL (12p13.2), p16INK4a/ARF (9p21.3), Pax5 (9p13.2), BTG1 (12q21.3), LIMD1 (3p21), AIM1 and BLIMP1 (6q21), NR3C2 (4q31.23), ATM (11q22-q23), and PDCD5 (19q13.11-q13.12), while the region of gain at 21q contained RUNX1. These findings suggest that, in addition to TEL previously reported as deleted, genes involved in cell cycle regulation, p53 pathways and apoptosis are also often deleted. Our array-CGH obtained data may provide further insights into the molecular basis for the development of TEL-AML1 leukemia.
Characterization of the CATS Protein Which Interacts with the Leukemogenic Fusion Protein CALM/AF10 Using Mono-Clonal Antibodies and Fluroescent Protein Tags.
Authors: Archangelo Leticia Fröhlich , Krause Alexandre , Hölzel Michael , Kremmer Elisabeth , Deshpande Aniruddha , Buske Christian , Bohlander Stefan K.
Published: April 2020

The CATS gene was first identified in a yeast two hybrid screen as CALM interacting protein expressed in thymus and spleen. The CATS interaction region of CALM is contained in the leukemogenic fusion protein CALM/AF10, which is found in acute myeloid leukemia, malignant lymphoma and acute lymphoblastic leukemia. We performed co-immunoprecipitation experiments to confirm CALM-CATS interaction found in the yeast system. RT PCR analysis showed CATS expression in patient cells and cell line carrying the CALM/AF10 rearrangement. Further expression analysis showed CATS expression in different cell subpopulation from the murine thymus (CD4+, CD8+ and CD4+/CD8+) and bone marrow (cKit+, Sca 1+/CKit+, Ter119, Gr1, CD8+, Mac1) but nearly absent in B220 positive cells. Interestingly, there was a high CATS expression in CALM/AF10 expressing B220+ leukemic cells in a CALM/AF10 murine bone marrow transplant model. Several monoclonal antibodies against the C-terminus of human CATS were generated. These antibodies recognize both the human and the murine CATS protein. Using these antibodies we could show a high expression of CATS in different human leukemia, lymphoma and solid tumor cell lines, as well as in normal proliferating cell lines (HEK293 and WI38), but not in T-cell lines (TYRF8 and JB4). Using protein lysates from cell cycle synchronized cells (Hela and U2OS) a clear cell cycle dependent regulation of CATS protein levels was demonstrated. Moreover, Western Blot analysis shows that serum stimulation after serum starvation leads to increased expression of CATS in the glioblastoma cell line T98G. Immunofluorescence shows that CATS is localized mainly to the nucleus. Coexpression of CFP-CATS with YFP tagged CALM or CALM/AF10 was able to markedly increase the nuclear localization of both CALM and the CALM/AF10 fusion protein. This effect of CATS is stronger on the YFP-CALM/AF10 fusion protein than on the CALM protein. When fused to a GAL4 DNA binding domain, CATS acts as a strong repressor of transcription in reporter gene assays. Our results indicate that the subcellular localization of CALM and CALM/AF10 could depend in part on the presence of CATS with a greater fraction of CALM or CALM/AF10 being present in the nucleus in cells with high CATS expression (e.g. lymphoid cells). The CALM-CATS interaction might thus play an important role in CALM/AF10 mediated leukemogenesis.