bluuebunny/crossref_metadata_2025 huggingface dataset:
Defined Bone Marrow Niche Components Mediate the In Vitro Resistance of Acute Myeloid Leukaemic Stem and Progenitor Cells (LSPC) to Cytosine Arabinoside.
Authors: Mony Ullas , Jawad Mays , Russell Nigel H. , Pallis Monica
Published: October 2019

The majority of Acute Myeloid Leukaemia (AML) patients respond to remission-induction chemotherapy, but many patients will suffer disease relapse following induction therapy due to the presence of minimum residual disease (MRD) cells in the bone marrow. Relapse is underpinned by outgrowth of leukaemic stem and progenitor cells (LSPC). We aimed to establish a methodology to distinguish normal from leukaemic SPC, to optimise maintenance of the LSPC phenotype in 48 hour culture and to quantify viable LSPC following culture with and without drugs. The CD34+ CD38− CD123+ high phenotype was used to distinguish LSPC. CD123 fluorescence intensity was measured using fluorescence standards. Miltenyi CD 133 and CD34 coated beads were compared as enrichment strategies. The concentration of LSPC at the start of 48h culture ranged from 0.2 – 16 × 104/ml (median 3.4 × 104/ml). We compared various culture conditions aimed at maintaining these cells in culture without differentiation (i.e. without loss of phenotype), including serum, immobilized fibronectin, SCF, IL-3, IL-6, GM-CSF and angiopoietin-1. We used two flow cytometric assays in parallel for analysis of LSPC survival: in the first assay, viable bulk cells were enumerated using the dye 7-AAD along with an internal standard for cell counting. In the second assay, cells were labelled with CD34FITC, CD123PE, 7-AAD and CD38APC in order to quantify LSPC as a percentage of viable cells. We found that serum-free culture medium, fibronectin-coated wells and a cocktail of IL-3, IL-6, SCF, and angiopoietin-1 was the most successful at maintaining the concentration of CD34+ CD38− CD123+ cells in culture for 48h (median 0% change), although there was considerable variation between samples. We then examined the effect of these niche conditions on the sensitivity of LSPC to cytosine arabinoside (Ara-C). Our data indicates that the survival of LSPC treated with 500 ng/ml Ara-C was 50 per cent of untreated cell survival in the presence of fibronectin and cytokine support (n=9), but 33% per cent of untreated cells survived without this support. Moreover, under these conditions LSPC were less sensitive to Ara-C (median 50% survival, n=10) than unselected cells (median 26% survival). We have defined a system for assessing the in vitro chemosensitivity of LSPC from AML samples to anti-leukaemic agents and have validated the assay by showing that phenotypically-identified LSPC as well as niche components fibronectin and cytokines mediate in vitro resistance to cytosine arabinoside.
Novel Markers for the Isolation of Primary Bone Marrow Derived MSC with Multi-Lineage Differentiation Capacity.
Authors: Buhring Hans-Jorg , Battula Venkata L. , Treml Sabrina , Kanz Lothar , Vogel Wichard
Published: October 2019

We have previously described a novel culture protocol to grow MSC from bone marrow (BM) and non-amniotic placenta (PL) with an immature phenotype and multi-lineage differentiation capacity (1). Using the low affinity nerve growth factor receptor (CD271) (2) as a key marker for isolation of MSC derived from femur shaft bone marrow cells (BM-MSC) of patients undergoing a total hip replacement, we could identify two CD271+ distinct populations: CD271dull and CD271bright cells. Two-color flow cytometer analysis revealed that only the CD271bright population coexpressed the mesenchymal markers CD10, CD13, CD73, and CD105, but was negative for CD45. CD271dull cells were positive for CD45 and HLA-DR but negative for the other markers. To analyze the mesenchymal stem cell potential, colony-forming-unit fibroblast (CFU-F) assays were performed. Not surprisingly, the CFU-F were exclusively detected in the CD271bright but not in the CD271dull fraction. By screening a battery of antibodies against known and unknown antigens, we identified several reagents that selectively detected the CD271brightCD45- population but no other bone marrow cells. These markers included the PDGF-RB (CD140b), the embryonic stem cell marker TRA-1-49, the clustered markers HER-2/erbB2 (CD340), the recently described W8B2 antigen (3), as well as the cell surface antigens defined by the antibodies W1C3, W3D5, W4A5, W5C4, W5C5, W7C6, 9A3, 58B1, F9-3C2F1, and HEK-3D6. In conclusion we identified several novel markers for the prospective isolation and characterization of BM-MSC.
Phase I/II, Randomized, MultiCenter, Dose-Ascension Study of the p38MAPK Inhibitor Scio-469 in Patients with Myelodysplastic Syndrome (MDS).
Authors: Sokol Lubomir , Cripe Larry , Kantarjian Hagop , Sekeres Mikkael A. , Parmar Simrit , Greenberg Peter , Goldberg Stuart , Bhushan Vikas , Shammo Jamile , Hohl Raymond , Li Yu-Ping , Lowe Ann , Zhu Joy , List Alan
Published: October 2019

Pro-apoptotic cytokines generated in the medullary microenvironment potentiate ineffective hematopoiesis in MDS. Preclinical investigations indicate that inhibition of p38α mitogen-activated protein kinase (MAPK) suppresses inflammatory, angiogenic and pro-apoptotic cytokine induction from marrow stromal cells. SCIO-469, is a novel highly selective p38αMAPK inhibitor. SCIO-469 was studied in a phase I/II, multicenter trial in patients (pts) with IPSS low and intermediate-1 risk MDS. The primary objective was to determine the safety and tolerability (NCI CTCAE) of the agent; secondary objectives were to evaluate erythroid response rate, hematologic improvement, bone marrow and cytogenetic response according to IWG MDS response criteria and the relation to biological correlates. Between 2005–2006, 62 pts were enrolled. At the time of abstract submission, 29 pts had preliminary response assessment. Demographic features include IPSS Low (45%) or Int-1 (55%) risk MDS, median age 71.5 years (range 48–88); WHO phenotype: RA 9%, 5q- syndrome 8%, RCMD 32%, RARS 13%, RCDM-RS 13%, RAEB-I 11%, MDS-u 9%, MDS/MPD 4%. Among these, 91% failed erythropoietin treatment (tx) and 11% had prior chemotherapy; 79% received RBC transfusions within 8 wks prior to study tx. Pts were randomly assigned to one of two lower dose levels: 30 mg TID (15 pts) and 60 mg TID (15 pts) and later to a 3rd dose level of 90 mg TID (15 pts) after initial confirmation of drug tolerance. Since maximal tolerated dose (MTD) was not achieved, a 4th dose level (120mg TID) was added with 17 pts accrued, none of whom are included in the response data. Responses were evaluated after 16 weeks of tx, with responders continuing SCIO-469 for up to 78 weeks. Fifteen grade 3 or 4 adverse events were observed in 10 pts: 1 jaundice, 1 hyperamylasemia, 1 hyponatremia, 1 atrial FIB, 1 hypotension, 2 cancer (melanoma, colon), 2 pneumonia, 3 neutropenia, 1 lower extremity edema, 1 pyrexia, and 1 abdominal pain. Other than three grade 3 or 4 neutropenia, no significant hematologic toxicities were observed. The MTD for SCIO-469 was not reached. Preliminary response was analyzed in pts completing >16 weeks of treatment [N=29]: Erythroid: major 1, minor 2; Platelet: major 1, minor 0; Neutrophils: major 3, minor 0, with no relation to dose. BM responses evaluated in 25 pts yielded only 1 partial response. Progression occurred in 4 pts. Tx is ongoing with pending analysis of correlative biological studies. Our preliminary analysis indicates that SCIO-469 is generally well tolerated and modestly active as monotherapy in low/int-1 risk MDS at dose levels 30–90 mg po TID.
Clinical Relevance of Cardiac Iron Overload Estimated by MRI T2* in Regularly Transfused Low Risk MDS.
Authors: Ferte C. , Ernst O. , Beyne-Rauzy O. , Chaury M.P. , Brechignac S. , Brauner M. , Mirault T. , Kanaan K. , Bourgeois E. , Cambier N. , Quesnel Bruno , Fenaux Pierre , Rose Christian
Published: October 2019

Background: Cardiac iron overload is the first cause of mortality in thalassemia. In MDS, a causal relationship between cardiac iron overload and death is not as well established and heart complication may be of intricate origins in these elderly pts. Cardiac MRI T2* allows accurate measurement of heart iron and is influenced by iron content only (and not by other cardiac diseases). T2* value < 20ms is clearly associated with cardiac iron overload. A few reports (Winder et al ASH 2005, Chacko J BJH2006;133: supp1) showed no cardiac iron overload by T2* in small numbers of multitransfused MDS. We performed a similar analysis in our transfused low risk MDS pts.

Methods: We prospectively evaluated by MRI both cardiac T2* according to Anderson (Eur Heart J 2001) and liver iron content (LIC ) according to Gandon (Lancet 2004) and cardiac function by echocardiography in multitransfused low risk MDS pts. Cardiac MRI T2* was also assessed in 33 controls.

Results: 21 MDS were analyzed, 9M/12F. Median age: 75 years ( 50–83); FAB : RA= 3, RAS=13, RAEB = 3, CMML n=1, unclassified n=1. Karyotype: fav n= 1, Int n= 18, failure n=1. IPSS: low n= 10, Int I n= 10, unavailable n=1. Median interval from MDS diagnosis and first transfusion was 40 and 24 months respectively. At inclusion, median number of RBC units transfused was 81 (range 6–282, and greater than 100 in 8 pts). Median serum ferritin level was 2152 ng/ml and 11 patients were on chelation therapy (CT). 9/21 pts had cardiac symptoms and were on cardiac therapy. LVEF was below normal (55%) in 3/21 cases. Left ventricular telediastolic diameter LVTD was above normal (normal 53 mm) in 6/14 pts evaluated. Median LIC was 350 micromoles/g/dw (95–898 ). Median Cardiac T2* was 27 ms (8–74) and did not differ significantly from controls (T2*=27ms+/−6.4). No correlation was found between cardiac T2* and ferritin, LIC, LVEF, time from MDS diagnosis. However 3/21 pts had cardiac iron overload with T2* < 20 (18ms,15ms,8ms respectively). LVEF and number of RBC units transfused of these pts was respectively 69%,51%,33% and 119,150,282 RBC units. Two of them were on iron CT, one of them since 8 years. The last 2 pts had clinical signs of cardiac failure unexplained by other causes and both had increased LVTD.

Conclusions: Although cardiac T2* did not differ significantly in transfused MDS and in controls, 3 heavily transfused (all in the 8 patients who had received >100 RBC units) had clear cardiac iron overload, clinically relevant in 2 of them and not correlated with higher liver iron overload. Differences between our study and previous studies could be due to the higher number of transfused RBC units in our pts with abnormal T2*, as compared to a median of 50 in the study of Winder, but further studies are required to confirm this finding.
Immunosuppressive Therapy in Children with Refractory Anemia: Results of Japanese Childhood MDS Study Group Trial (MDS99).
Authors: Hasegawa Daisuke , Yagasaki Hiroshi , Ohtsuka Yoshitoshi , Inoue Masami , Kikuchi Akira , Ohara Akira , Tsuchida Masahiro , Kojima Seiji , Manabe Atsushi , Nakahata Tatsutoshi
Published: October 2019

Myelodysplastic syndrome (MDS) is a hematopoietic stem cell disorder and uncommon in childhood. Especially, refractory anemia (RA), which is a subgroup of MDS without an increased number of blasts, is quite rare in this age group. Although hematopoietic stem cell transplantation (HSCT) is thought to be a curative therapy for pediatric MDS, it may cause severe complications and mortality. Several reports have shown encouraging results with immunosuppressive therapy (IST) in adult patients with RA. We report the outcome of 12 children with RA enrolled on a prospective multicenter trial conducted by Japanese childhood MDS study group. In this study, a child who was suspected of having RA required repetitive bone marrow aspiration at 6–8 weeks intervals. If the disease was stable and blood transfusion was not urgent, the patient could be monitored closely without any therapy. If physicians decided to start therapy due to progression of cytopenia, the patient received IST consisting antithymocyte globulin (ATG), cyclosporine (CyA) and methylprednisolone (mPSL). Of the 12 children, 9 received IST (IST group), 2 were followed without treatment (observation group) and one underwent HSCT without IST. Seven children showed hematological response in the IST group, and a response rate was 77.8%. Of note, 1 patient with monosomy 7 showed complete cytogenetic response after IST and remained in remission. One patient became refractory to IST after relapse and underwent bone marrow transplantation (BMT) from a human leukocyte antigen (HLA) 1-antigen mismatch relative, and she is alive without disease on 351 days after HSCT. One patient received BMT from an HLA-matched unrelated donor without IST because he had monosomy 7, but he relapsed and died from disease progression. Neither of 2 patients in the observation group experienced disease progression. There were 8 children who showed chromosomal abnormalities, including monosomy 7 and trisomy 8, 7 of whom received IST and 6 children showed hematological response. No severe adverse events related to IST were reported in this study. Eleven of the 12 patients are alive after a median follow-up of 1,319 days. The probability of survival at 5 years was 88.9%, which was superior to our previous retrospective analysis of children with RA. We conclude that IST for children with RA seems an effective modality and warrants an international clinical trial.
A Phase II Study of Nilotinib, a Novel Inhibitor of c-Kit, PDGFR, and Bcr-Abl, Administered to Patients with Systemic Mastocytosis.
Authors: Hochhaus Andreas , Ottmann Oliver G. , Lauber STephanie , Hughes Timothy , Verhoef Gregor , Schwarer Anthony P. , Gratwohl Alois , Rafferty Teresa , Resta Debra , Gattermann Norbert
Published: October 2019

Systemic mastocytosis is a clonal disorder associated with a constitutive activation of the c-kit tyrosine kinase based on point mutations and is characterized by mast cell infiltration of extracutaneous organs. Nilotinib is a novel aminopyrimidine which potently inhibits Bcr-Abl, as well as the PDGF-R, and c-kit tyrosine kinases. Preclinical data demonstrated the activity of nilotinib against D816V mutated c-kit in biochemical and cellular assays. This Phase II study was designed to evaluate the safety and efficacy of nilotinib administered at an oral dose of 400 mg twice daily to patients with systemic mastocytosis defined by specific disease criteria and with a clinical indication for treatment. Data are available for 60 patients (34 male, 26 female). The median age is 51 years (range, 29 to 79). Of the 60 patients 31 (52%) had extramedullary involvement at baseline. In 30/36 patients investigated (83%) D816V c-kit mutation was found by D-HPLC and/or conventional sequencing in bone marrow or extracutaneous organs. Two patients showed the c-kit I798I polymorphism. Treatment is ongoing for 38 (63%) patients; 22 (37%) have discontinued; ten (17%) for adverse events, seven (12%) withdrew consent, and one (2%) each for disease progression and lost to follow-up. There were two (3%) deaths related to disease progression. Based on investigators’ assessment of serum tryptase, bone marrow mast cell counts and improvement of clinical symptoms 12 patients (20%) had a documented clinical response including two (3%) complete, five (8%) incomplete, four (7%) minor, and one partial response. Adverse events occurring in >15% of patients included nausea in 28 (47%), headache in 26 (43%), fatigue in 25 (42%), vomiting in 22 (37%), diarrhea in 21 (35%), pruritis in 16 (27%), and rash in 15 (25%) patients, dizziness and muscle spasms in 14 (23%) patients each, bone pain in 12 (20%), pyrexia and myalgias in 11 (18%) patients each, and dyspnea, constipation, increased ALAT, and arthralgias in ten (17%) patients each. Most side effects occurred early after initiation of nilotinib therapy and were successfully treated with H1- and H2-blockers and/or corticosteroids, indicating a mast cell degranulation syndrome. Overall the most frequent Grade 3/4 adverse events included diarrhea in four (7%) patients, and thrombocytopenia and headache in three (5%) patients each. The data suggest that nilotinib has clinical activity and an acceptable safety and tolerability profile in patients with systemic mastocytosis with constitutive c-kit activation. Individual molecular characterization will help to guide targeted therapy in this disease.
Phase II Preliminary Results of SGN-30 (Anti-CD30 mAb) in Patients with CD30+ Lymphoproliferative Disorders.
Authors: Duvic Madeleine , Kim Youn , Reddy Sunil , Forero-Torres Andres , Pinter-Brown Lauren C. , Rarick Mark U. , Zic John , Worobec Sophie , Korman Neil , Kuzel Timothy , Bohjanen Kimberly , Flessland Karen , Barton Jeremy , Sievers Eric
Published: October 2019

SGN-30 is a monoclonal antibody directed against the CD30 antigen, a member of the TNF receptor super-family. It has been evaluated in phase II clinical trials in patients with systemic Anaplastic Large Cell Lymphoma (ALCL) and Hodgkin’s lymphoma (HL). SGN-30 has shown good tolerability and objective tumor responses in patients with systemic ALCL. This multicenter, single arm phase II study, originally designed to evaluate the tolerability and activity of SGN-30 in patients with relapsed/refractory primary cutaneous ALCL (pcALCL), was expanded to include other symptomatic CD30-positive lymphoproliferative disorders: CD30+ Lymphomatoid Papulosis (LyP) or CD30+ Large Cell Transformation of Mycosis Fungoides (t-MF) without systemic involvement. Seventeen patients (14 male, 3 female) with median age of 63 years (range 33–79) have been enrolled. The median number of prior therapies was 3 (range 1–4). All patients had received at least 1 prior systemic therapy. SGN-30 was administered at a dose of 12 mg/kg by 2-hour infusion every 2–3 weeks. Up to 3 cycles (each comprised of 6 doses of SGN-30) were given. Evaluation of response was determined by Physician’s Global Assessment (PGA) which was performed 2–3 weeks after each infusion. The best response assessment per indication is shown below:

One Grade 3 pruritis and one Grade 3 increase in the number of neoplastic lesions were reported; both were considered to be unlikely related to SGN-30. There was one SAE in which an 80 year-old male died from complications of pneumonia, MI and aortic stenosis. This SAE was considered unrelated to the study drug. Preliminary data from this phase II study indicates that SGN-30 is well tolerated and demonstrates clinical antitumor activity.

Lymphoproliferative Disorders CR PR SD PD Pending Evaluation *Duration of CR: 256+ days **Duration of PRs: 56, 72, 84, and 271 days ***One patient with concurrent LyP received a single dose of SGN-30, 9 days after discontinuing methotrexate, experienced a worsening of his disease, and came off the study with PD. pcALCL (n=10) 5** 3 2 pcALCL/t-MF(n=1) 1* t-MF(n=2) 2 t-MF/LyP(n=3) 1 1 1 LyP(n=1) 1***
Natural Course of the Subsequent Pregnancy after a Single Loss in Women with and without the Factor V Leiden or Prothrombin 20210A Mutations.
Authors: Coppens Michiel , Folkeringa Nienke , Teune Margreet , Hamulyak Karly , van der Meer Jan , Prins Martin H. , Büller Harry R. , Middeldorp Saskia
Published: October 2019

In previous case control studies factor V Leiden and the prothrombin 20210A mutation have been associated with pregnancy loss, in particular recurrent and late (>12 weeks of gestation) losses in women carriers1. The prognosis of a second pregnancy after an initial late pregnancy loss in women with inherited thrombophilia is uncertain; the live birth rate reported in different cohort studies varied from 11 to 98%2–4. This has led to a debate about whether low-molecular-weight heparin should be prescribed to women with thrombophilia and pregnancy loss, despite the absence of evidence about its effectiveness from placebo-controlled trials.

We determined the live birth rate of the 2nd pregnancy following a first loss in a large family study that included patients with documented venous thromboembolism or premature atherosclerosis and carriership of the factor V Leiden or prothrombin 20210A mutations and their 1st degree relatives. Ectopic or terminated pregnancies and pregnancies lost due to known chromosomal abnormalities were excluded, as well as pregnancies during which anticoagulants were used. A total of 993 women (498 carriers) who had had 2809 pregnancies (1420 in carriers) were analyzed. After an early pregnancy loss in the 1st pregnancy, the live birth rate of the 2nd pregnancy was 77% (95%CI 62–87) in carriers and 76% (95%CI 57–89) in non-carriers. After a late pregnancy loss in the 1st pregnancy, the live birth rate of the 2nd pregnancy was 68% (95%CI 46–85) in carriers and 80% (95%CI 49–94) in non-carriers (OR 0.9, 95%CI 0.5–1.3).

We conclude that the pregnancy outcome in women with either factor V Leiden or the prothrombin 20210A mutation is fairly similar to the outcome in women without these mutations. After a late pregnancy loss in the first pregnancy, the live birth rate in the second pregnancy tended to be somewhat lower for carriers than for non-carriers. The live birth rates for both carriers and non-carriers in this study was substantially higher than in some other cohorts, which may be due to the fact that the cohort was not selected based on obstetric indications. Given the observed favourable prognosis after a first pregnancy loss even in women with known thrombophilia and a late loss, low-molecular-weight heparin to improve pregnancy outcome should not be used before results from ongoing randomized placebo controlled trials have shown its effectiveness and safety.
Marked Activity of Bortezomib, Rituximab, and Dexamethason in Relapsed and Refractory Mantle Cell Lymphoma.
Authors: Drach Johannes , Kaufmann Hannes , Pichelmayer Oskar , Sagaster Verena , Seidl Sonja , Chott Andreas , Zielinski Christoph , Raderer Markus
Published: October 2019

Bortezomib (B) belongs to a new class of anti-cancer agents, the proteasome inhibitors, and has documented activity in multiple myeloma and mantle cell lymphoma (MCL). Preclinical studies suggest that B has synergistic activity with rituximab (R), which provides a rationale for the exploration of treatment combinations. We have initiated a phase II study in relapsed/chemotherapy refractory MCL to evaluate the activity and safety of B in combination with R and dexamethasone (BORID). A treatment cycle consists of B at 1.3 mg/m2 administered on days 1, 4, 8, and 11, R at 375 mg/m2 administered on day 1, and dexamethasone 40 mg orally on days 1 to 4. Cycles are repeated every 3 weeks for a total of 6 treatment cycles. Patients (pts) with progressive MCL after at least one prior line of therapy (including CHOP or a CHOP-like regimen) are eligible. We have now enrolled 14 pts at a median age of 69 years (range, 48 to 75 years) after a median of 3 lines of prior therapies (range, 1 to 6) including R in 12 pts, high-dose therapy in 4 pts, and thalidomide in 7 pts. Median time between start of frontline therapy and study inclusion was 43 months (range, 11 to 98 months). Severe adverse events (> grade II) included infections (herpes zoster in 2 pts, bacterial pneumonia, mucosal candidiasis), peripheral neuropathy (3 pts), fatigue (2 pts), vasculitic skin infiltrates in 3 pts, and hyponatremia in 1 pt. Thrombopenia (< 50 G/L) occured in 2 pts. All adverse events were managable by standard means of supportive care and prolongation of the treatment interval between cycles. Of 12 pts evaluable for efficacy, 9 have achieved a response (3 CR, 6 PR), and 2 pts experienced stable disease. Pts in CR were also negative for disease activity by PET scanning. Skin infiltrates (histologically proven T-cell infiltrates) preceded achievement of CR in 2 pts. All 3 pts who achieved a CR are still progression-free at 12 months after treatment initiation. Among pts in PR, 3 pts have relapsed (progression-free survival 14, 11, and 6 months, respectively), and 3 pts are still progression-free beyond 6 months from initiation of treatment. In summary, the BORID treatment regimen has promising activitiy and managable toxicity in patients with heavily pretreated MCL, and development of a vasculitic rash may be an early indicator of a favorable and durable response.
Mature Results of a Prospective Trial of Rituximab in Patients (Pt) with Post Transplant Lymphoproliferative Disorder (PTLD) Unresponsive or Ineligible for Reduced Immunosuppression (RIS).
Authors: Sharman Jeff P. , Tsai Donald E. , Twist Clare J. , Horwitz Steven M. , Jones Carol D. , Horning Sandra J.
Published: October 2019

Background: PTLDs are typically B-cell neoplasms occurring as uncommon but serious complications of reduced T-cell immune surveillance associated with organ transplantation. RIS benefits only a subset of PTLD patients and cytotoxic therapy may be poorly tolerated. Therefore, in October 1998 we initiated a prospective study of rituximab in patients who failed or were unable to receive RIS and now report mature results.

Methods: Patients with CD20+ PTLD were eligible if they had failed to completely respond to RIS or RIS was contraindicated and had Karnofsky performance status >60, age 3–70 years (y), measurable disease, and no change in immunosuppression for at least 2 weeks and no cytotoxic therapy within 4 weeks. Rituximab was given as 375 mg/m2 weekly x 4 with disease evaluation at 1, 3, 6, 9, 12 and 18 months. Response data, survival curves, and the impact of clinical and pathological factors were evaluated.

Results: 24 of 26 enrolled pt were eligible and evaluable. Median age was 42y with 5 <17 y, 18 were male, and 14 progressed on RIS. Median time to PTLD from transplant was 47 months (m) (8 <24 m). 17/22 were EBV+, 17 were large cell or Burkitt histology, and 10 PTLD occurred in the allograft site. Response rate was 63% (46 %CR, 17% PR) and CRs were durable (1/11 progressed). With median follow-up of 65 m (range 44–82), outcomes at 5 y are: overall survival 48%, freedom from progression 41% and failure-free survival 21%. 7 pt died without progression, yielding 5 y cause-specific survival of 69%. Nine of 13 pt with disease progression were successfully salvaged with second-line therapy. In univariate analysis PTLD characteristics did not significantly correlate with outcome but 2/2 Burkitt pt quickly progressed.

Conclusions: Rituximab provided effective, durable treatment for ~40% of pt failing RIS in this series of mainly late PTLD and a majority of pt progressing after rituximab could be treated successfully. However, overall and failure-free survival reflect significant co-morbidity in this population.
Inhibition of GSK-3 Induces Apoptosis of CLL Cells by Abrogating NFkB Nuclear Activity.
Authors: Ougolkov Andrei V. , Bone Nancy , Billadeau Daniel , Lee Yean K. , Kay Neil E.
Published: October 2019

Chronic lymphocytic leukemia (CLL) represents one of the most common hematological malignancies and is characterized by the expansion of a clonal B cell population. Commonly, CLL is defined as a disease of failed apoptosis of the leukemic B cells, and it is this feature that makes them refractory to common chemotherapeutic agents designed to kill CLL cells. The mechanism controlling apoptosis in CLL is complex and influenced by numerous factors, including NFkB-mediated expression of antiapoptotic proteins, such as Bcl-2 and XIAP. We recently demonstrated that glycogen synthase kinase-3 (GSK-3) b positively regulates NFkB-mediated gene transcription and cell survival in pancreatic cancer cells (Cancer Research 2005. 65:2076).

Similar to pancreatic cancer, we have found that pharmacological inhibition of GSK-3 results in decreased expression of NFkB target genes Bcl-2, Mcl-1 and XIAP in CLL B cells. In addition we noted a dose dependent increase in apoptosis levels for both the MEC1 cell line and primary CLL B cell patient samples. Using CLL blood B cells, we have found that both GSK-3b and NFkB accumulate in the nucleus of CLL cells, but not normal blood B cells. In addition, we observed that inhibition of GSK-3 leads to a decrease in NFkB-mediated gene transcription, without affecting the nuclear accumulation of NFkB. However, using chromatin immunoprecipitation, we found that NFkB binding to its target gene promoters (XIAP, Bcl-2) is significantly affected in CLL B cells treated with a GSK-3 inhibitor. These results suggest that GSK-3 regulates the nuclear activity of NFkB in CLL cells by affecting the binding of p65/p50 to the promoters of a subset of its target genes. Our results establish that inhibition of GSK-3 induces apoptosis of CLL cells and suggest GSK-3b as a potential therapeutic target in the treatment of CLL.
A Phase II, Open Label Study of AT-101 in Combination with Rituximab in Patients with Relapsed or Refractory Chronic Lymphocytic Leukemia.
Authors: Castro Januario E. , Olivier Loria J. , Robier Aguillon A. , Danelle James , Carlos Suarez J. , Bi-Ying Yeh , Thomas Kipps J.
Published: October 2019

Chronic lymphocytic leukemia (CLL) cells express high-levels of Bcl-2 and related anti-apoptotic proteins that collectively can enhance leukemia-cell survival and drug-resistance. AT-101 is an orally active BH3-mimetic that can inhibit the anti-apoptotic activity of Bcl-2-family-member proteins (e.g. Bcl-2, Bcl-XL, Mcl-1) and induce CLL cells to undergo apoptosis. Furthermore, we found that AT-101 also can enhance the cytotoxicity of rituximab for CLL cells in vitro. As such we conducted a phase 2 trial to evaluate the safety and activity of AT-101 when used together with rituximab to treat 12 patients who had relapsed/refractory CLL. Patients received AT-101, at 30 mg/d, for 21 or 28 days during each of three 28-day cycles. Rituximab was administered at 375 mg/m2 for 12 doses (total dose = 4,500 mg/m2) on days 1, 3, 5, 8, 15, 22, 29, 31, 33, 40, 57, 59, 61. The first dose of rituximab was given over two days to minimize infusion-related adverse events. The patients’ median age was 61.5 years (range 43–81). Nine patients were high risk and 3 were intermediate risk based on the modified Rai classification and had received a median of 2 prior regimens (range 1–8). Six patients had leukemia cells that expressed ZAP-70 and/or unmutated immunoglobulin variable region genes and 4 patients had either 11q deletions or complex cytogenetics. Six patients interrupted treatment due to adverse events, most of which were transient and without residual complications. Grade 1–2 gastrointestinal effects (e.g., nausea, vomiting) occurred in 11 patients, 2 of whom had grade 3/4 ileus. Six patients experienced treatment-associated fatigue (grade 1–2 in 5 and grade 3 in one). Other than ileus and fatigue the only grade 3/4 event noted was neutropenia. One patient without neutropenia died while undergoing treatment from community-acquired bacterial pneumonia[j1]. Pharmacokinetic studies demonstrated that the average Cmax of AT-101 was 565 ng/ml (280 – 805 ng/ml) at a Tmax of 3.1 hours (1.7 – 5.6 hrs.). Correlative science studies performed on leukemia cells isolated at various times after treatment demonstrated leukemia-cell apoptosis in vivo, with maximum levels seen at times when we observed peak drug levels of AT-101. Eight patients had completed the study and had full response evaluation at the time of this abstract’s submission. The overall response rate was 38% [CRu (2); PR (1); SD (3); PD (2)]. Four of eight patients (50%) had significant reductions in leukemia cell counts and splenomegaly and 5 of 8 (63%) had reductions in lymphadenopathy. AT-101 in combination with Rituximab has apparent activity in patients with relapsed-refractory high-risk CLL. Additional enrollment is planned using alternate AT-101 schedules in an attempt to increase peak plasma concentrations (and potentially activity) and reduce GI toxicity.
Risk Factors for Late Infections after Allogeneic Hematopoietic Stem Cell Transplantation from a Matched Related Donor.
Authors: Robin Marie , Porcher Raphaël , De Castro Araujo Renato , de Latour Régis Peffault , Devergie Agnès , Rocha Vanderson , Larghero Jérome , Espérou Hélène , Adès Lionel , Gluckman Eliane , Ribaud Patricia , Mary Jean-Yves , Socié Gérard
Published: October 2019

After allogeneic hematopoietic stem cell transplantation (HSCT), late infections represent a major cause of morbidity and mortality but little has been previously reported. In a retrospective cohort study, late infections incidence was determined in 196 long-term survivors after matched related HSCT. Only patients transplanted for aplastic anemia, chronic myeloid leukemia (CML) and acute myeloblastic leukemia (AML) were included in this study. Median follow-up was 8 years. Among 30 patients who died beyond the first year, 9 patients died from graft-versus-host disease (GVHD) and 10 from infections. Bacterial late severe infections occurred in 30 patients, yielding an 8-year cumulative incidence of 15%. Late invasive fungal infection occurred in 8 patients corresponding to a cumulative incidence of 3.6%. Most viral infections were hepatitis C and VZV and overall late viral infection incidence was 35%. We identified 3 risk factors for bacterial infections in multiple analysis: CMV status (positive recipient and negative donor), irradiation based conditioning regimen and extensive chronic GVHD within the first year. Extensive chronic GVHD was the only risk factor of non-HCV viral infection in patients transplanted for AML or CML. Thus, late life threatening infections may occur in nearly a fourth of late survivors even after matched related transplantation and are associated not only with chronic GVHD but also with irradiation and to CMV status prior to transplantation.
Lung Resection for Invasive Pulmonary Aspergillosis in Neutropenic Patients with Hematologic Malignancies: Long Term Results in Thirty Two Cases.
Authors: Gastinne Thomas , Touzeau Cyril , Vigouroux Stéphane , Le Gouill Steven , Mahé Béatrice , Dubruille Viviane , Delaunay Jacques , Chevalier Patrice , Guillaume Thierry , Sagan Christine , Gay-Andrieu Françoise , Germaud Patrick , Despins Philippe , Moreau Philippe , Harousseau Jean-Luc
Published: October 2019

Invasive pulmonary aspergillosis (IPA) is a major cause of morbidity and mortality in neutropenic patients. Despite adequate medical treatments, such as amphotericin B or voriconazole, the prognosis of IPA remains poor. Nevertheless, recent studies suggest that the outcome of IPA is improving due to early diagnosis (CT scan, antigenemia), use of new antifungal agents (Azols and Echinocandins), and possibly in some cases to early surgical resection. We here report a retrospective one center study of 32 cases of IPA treated by surgical treatment from 1988 to 2005.

Between 1988 and December 2005, thirty-seven consecutive patients underwent lung resection for suspected IPA. They were 22 men and 15 women, with a median age of 49.2 years (15 – 74). The underlying diseases were AML, ALL, aggressive lymphoma and myeloma in 26, 7, 2 and 2 cases, respectively. Surgery was planned after hematologic recovery from the last course of chemotherapy during which IPA was diagnosed, either possible in 18 cases, probable in 18 cases or proven in 1 case (Ascioglu, CID 2002). Surgery consisted in 1 pneumectomy, 6 bilobectomies, 18 lobectomies, 8 wedge resections and 4 lobectomies with wedge resections. No perioperative deaths occurred and the median duration of post surgical hospitalisation was 13 days. Six patients presented surgery related complications: three patients required prolonged pleural drainage, one had a section of the phrenic nerve, one presented a major bleeding and last patient had a pneumopathy. The diagnosis of definite IPA was confirmed in 32 cases. Immediately after surgery, 26 of the 32 patients were able to receive subsequent intensive chemotherapy courses, including 15 stem cell transplant (SCT), either auto (5) or allogenic (10). In all cases, patients subsequently received parenteral antifungal therapy. During these new intensive chemotherapy courses, recurrent aspergillosis was observed in only 2 cases, inducing 1 death from brain localization. Overall, with a median follow-up of 8.3 years (1–18), 40.5% of the patients are alive and the main cause of mortality was relapse, but not IPA.

In conclusion, early surgical resection together with antifungal therapy allows definite diagnosis of IPA, prevents from IPA recurrence and early death due to hemotysis, with very few peri-operative complications. All the more, it allows subsequent high-dose chemotherapy to treat the underlying hematologic disease. Surgery might be a treatment of choice to a define group of patient.
Tacrolimus Versus Cyclosporine for Graft-Versus-Host Disease Prophylaxis in Pediatric Patients Undergoing Matched Unrelated Donor Hematopoietic Stem Cell Transplants. A Pediatric Blood and Marrow Transplant Consortium Study..
Authors: Atlas Mark P. , Yanik Gregory , Goyal Rakesh
Published: October 2019

Calcineurin inhibitors form the backbone of graft versus host disease (GVHD) prophylaxis in hematopoietic stem cell transplantation. In the adult literature tacrolimus was demonstrated superior to cyclosporine in preventing grade II–IV acute GVHD in both related and unrelated donor transplants; however, there is no data comparing their efficacy in the pediatric population. In a multi-institutional trial, we prospectively evaluated the clinical data on 102 patients receiving either cyclosporine/methotrexate or tacrolimus/methotrexate based prophylactic regimens for 6/6 matched unrelated donor, transplant. Conditioning regimens were fully ablative; no T-cell depletion was permitted; cord blood donors were excluded. Patients were required to receive either cyclosporine or tacrolimus, but the choice was per investigator preference. The two arms were reasonably balanced: 59.8% of patients received cyclosporine and 40.2% of patients received tacrolimus. Rates for maximum grade II–IV acute GVHD were 37.7% for cyclosporine and 39% for tacrolimus (p = 0.89). Rates for maximum grade III–IV acute GVHD were 19.6% for cyclosporine and 24.4% for tacrolimus (p = 0.57). Incidence of chronic GVHD in 97 evaluable patients was 37.9% in 58 patients who received cyclosporine and 35.8% in 39 patients who received tacrolimus (p = 0.84). Survival at 1 year post-transplant was similar in both groups: 59.2% for cyclosporine and 51.2% for tacrolimus (p= 0.31). Toxicity analysis is ongoing. In pediatric matched unrelated donor transplantation, the efficacy of tacrolimus/methotrexate and cyclosporine/methotrexate based regimens for prophylaxis of GVHD are not significantly different.
Graft Versus Host Disease Prophylaxis with Everolimus and Tacrolimus in Patients with Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukaemia (AML) Receiving Allogeneic Peripheral Blood Stem Cell Transplantation (PBSCT).
Authors: Platzbecker Uwe , Pabst Caroline , Kiani Alexander , Schetelig Johannes , Wermke Martin , Helwig Anett , Ehninger Gerhard , Bornhäuser Martin
Published: October 2019

Introduction: The use of a calcineurin-inhibitor in combination with methotrexate is the current standard in the prophylaxis of graft versus host disease (GVHD). Everolimus is a newly developed m-TOR inhibitor, which, besides a potent immunosuppressive action including the stimulation of regulatory CD4+foxp3+ T-cells (Tregs), seems to mediate anti-neoplastic effects in MDS and AML.

Methods: We report results of a prospective study investigating for the first time a combination of everolimus (days 0–56) with tacrolimus (starting day 0) in 16 patients with MDS (RCMD n=3, RAEB-1 n=3, RAEB-2 n=3, CMMOL-1 n=1, CMMOL-2 n=1, MDS/AML n=1) or de novo AML (n=4) undergoing allogeneic myeloablative conditioning (busulfan 16 mg/kg over 4 days, fludarabine 120 mg/m² over 4 days) followed by a median of 7.0 x 106/kg CD34+ peripheral blood stem cells (PBSC) from related (n=2) or unrelated donors (n=14). It is of note that 5 unrelated donor/recipient pairs displayed one allel-mismatch whereas all others were matched in 10 out 10 HLA characters. The median age of the patients was 61 years (range 47–69) and the majority (n=7) of MDS patients were classified INT-2 or HIGH according to IPSS.

Results: All patients engrafted a median of 14 days (platelets) and 17 days (neutrophils) after transplant. On day 21 and 56 after PBSCT the median number of CD4+foxp3+ cells in the blood was not significantly different from normal donors (patients, n=5: 3.2 and 2.3 x 104/ul, controls n=4: 3.7 x 104/ul) Nevertheless, the rate of acute GVHD was moderate with five patients (31 %) developing acute GVHD grade II and only one patient experiencing grade IV GVHD after cessation of immunosuppression due to thrombotic-thrombocytopenic purpura (TTP). Decrease of thrombocytes together clinical signs of TTP were seen in two additional patients while four patients developed VOD of the liver, which was fatal in one case. Extensive chronic GVHD was seen in 50 % of evaluable patients. Mucositis CTC grade III was observed in 5 patients only. The total day 100 mortality rate was 19 % and currently eleven out of sixteen patients (69%) are alive and in remission.

Conclusion: Everolimus and tacrolimus are highly efficient in preventing GVHD after unrelated PBSCT in older patients with MDS and AML, which seems not to be mediated by an increase in Tregs. Nevertheless, side effects associated with thrombotic microangiopathy might be more prevalent compared to other regimens.
Low Incidence of Cytomegalovirus Infection Associated with Rapid Cell-Mediated Immune Reconstitution after Cord Blood Transplantation Using Full Conditioning Regimen Containing 12Gy Total Body Irradiation.
Authors: Takahashi Satoshi , Ooi Jun , Tomonari Akira , Tsukada Nobuhiro , Konuma Takaaki , Kobayashi Takeshi , Sato Aki , Tojo Arinobu , Watanabe Nobukazu , Nakauchi Hiromitsu , Asano Shigetaka
Published: October 2019

The one of crucial questions in cord blood transplantation (CBT) is whether naïvity of cord blood lymphocytes could gain antigen-specific cellular immunity during early phase of transplant. Cytomegalovirus (CMV) infection is serious clinical problem in allogeneic transplant recipients and T cell immunity has known to have an important role in control of virus replication and prevention.

During 1998 and 2006, 111 adults has received myeloablative regimens including 12 Gy of total body irradiation followed by CBT and a standard cyclosporine and methotrexate combination as GVHD prophylaxis in our institute. Patients also received intravenous immunoglobulin from day −3 to day 120 if the immunoglobulin level in the serum was less than 500 mg/dl. CMV antigenemia assay was performed twice a week after neutrophil recovery until day 120. Once CMV antigenemia is positive, patients received 5 mg/kg ganciclovir (GCV) once daily for at least 2 weeks as preemptive therapy. Ninety-two patients achieved engraftment with full donor chimerism and survived without disease relapse at the time of 120 days after CBT (82.8%). None of these 92 recipients had CMV disease during first 4 months after CBT.

We have investigated the association of CMV reactivation status and their immune reconstitution process for 4 months after CBT in 39 patients who received CBT from 2002 to 2006 in our institute. CMV-specific CD4+ and CD8+ T cell recoveries were assessed by detection of interferon-g (IFN-g) producing cells with CMV antigen stimulation using intracellular cytokine staining. The positive was defined as more than 0.1% IFN-g positive cells among CD4+ or CD8+ T cell population.

Six of 39 patients were CMV sero-negative and 33 patients were sero-positive. None of 6 CMV sero-negative receipients and 31 of 33 CMV sero-positive recipients observed CMV reactivation and received GCV therapy within the first 4 months.

CMV-specific CD4+ T cells were detected in 30 of 31 recipients with positive CMV antigenemia (% positive: 55% at 1 month and 85% at 2 month), on the other hand, CMV-specific CD8+ T cells were detected in 14 out of 31 cases (% positive: 14% at 1 month and 22% at 2 month), both of which were comparable to post-bone marrow or peripheral blood transplants (CMV-specific CD4+ T cells were detected 18 of 21 recipients with positive CMV antigenemia and CMV-specific CD8+ T cells were detected in 12 out of 21).

These data suggest that post-thymic naive T lymphocytes in cord blood might obtain memory and effector function in vivo with antigen-specific manner during early phase of post-transplant.
Generation of Multi-Specific CTL Lines Using Overlapping Pentadecapeptide Pools of Aspergillus f16 and Adenovirus Hexon Antigens Presented on Epstein Barr Virus-Transformed B Cells as Antigen Presenting Cells.
Authors: Zhu Fenlu , Davies Barbara , Margolis David A. , Keever-Taylor Carolyn A.
Published: October 2019

Allogeneic hematopoietic progenitor cell transplantation (HPCT) is the optimal treatment for several hematological malignancies, aplasia or other disorders of the hematopoietic or immune system. Post HPCT, patients undergo a prolonged period of immune incompetence during which they are highly susceptible to viral and fungal infections. Epstein Barr Virus (EBV), Adenovirus (Ad) and Aspergillus fumigates are among the most common and lethal infections during this period. We have described the use of overlapping pools of pentadecapeptides pulsed onto monocyte-derived dendritic cells (DC) to generate cytolytic T-cell (CTL) lines specific for an A. fumigatus-derived antigen, Asp f16 (Ramadan et. al., 2005, J Clin Immunol139:257; Ramadan et. al., 200 J Clin Immunol 140:81). We have further demonstrated in 5 donors, the generation of CTL lines recognizing peptides contained within the conserved region of the Adenovirus hexon protein using a similar approach (Keever-Taylor, et. al, Cytotherapy 8, Suppl 1, 122). To reduce the need for large numbers of DC to prime and expand the CTL cultures, we modified our procedures to use peptide pool (pp)-pulsed EBV-transformed B lymphoblastoid cell lines (BLCL) as antigen presenting cells (APC) following 2–3 rounds of initial priming with pp-DC. This approach results in lines that are predominately reactive to Asp f16 or Ad hexon, and which are moderately reactive to EBV. We expanded our studies to determine the feasibility of generating CTL lines that recognize all three infectious agents. Peripheral blood lymphocytes (PBL) from donor RD0309 were primed weekly ×3 with autologous DC (10:1 PBL to DC ratio) pulsed with a pool of both Asp f16 (104 individual peptides) and Ad hexon (105 individual peptides) pentadecapeptides, then switched to Asp/Ad-pulsed BLCL as APC. The cultures were screened one week following restimulation for lysis of non-pulsed (np-BLCL) and Asp f16-pp-pulsed (Asp-BLCL) and Ad hex-pp-pulsed (Ad-BLCL) BLCL targets. Weak reactivity to Ad-BLCL was detected after 3 primings with DC alone that increased following one round of Asp/Ad-BLCL priming. Asp f16-specific CTL activity was not clearly detected until one week after 3DC plus 3BLCL primings, at which time there was also clear reactivity to np-BLCL targets (26.4% np-BLCL, 37% Asp-BLCL, and 53.9% Ad-BLCL at 50:1), indicating CTL activity to all three infectious agents. With an additional priming, Aspergillus specific reactivity exceeded that towards Adenovirus and EBV-specific reactivity declined. These data show that a pool of overlapping peptides from two different antigens presented on DC followed by BLCL as APC can result in multi-specific cell lines that can be potentially used in adoptive immunotherapy protocols for the treatment or prevention of common opportunistic infections following HPCT.