bluuebunny/crossref_metadata_2025 huggingface dataset:
Elevated HIF-1α Levels in CLL B Cells May Explain Their Autocrine VEGF Secretion.
Authors: Lee Yean K. , Shanafelt Tait , Chan Denise , Giaccia Amato , Mukhopadhyay Debabrata , Taccioli Cristian , Calin George , Croce Carlo , Kay Neil E.
Published: October 2019

We and others previously reported that leukemic CLL cells spontaneously secrete VEGF and that VEGF can enhance the leukemic B cell apoptosis resistance via unknown mechanisms. Here, we report elevated VEGF secretion is associated with CLL B cell over-expression of hypoxia inducible factor-1 alpha (HIF-1α), a transcription factor that is a key regulator of VEGF synthesis. Immunostaining analysis confirmed CLL B cells in blood and bone marrow over-expressed both HIF-1α and VEGF. The high HIF-1α levels were not linked to proline residue (402/564) mutations in oxygen dependent degradation domains; absence of proline hydroxylation dependent enzymes (PHD1, 2, 3, and FIH); or to pVHL mutation (exon 1, 2 and 3); or aberrant transcription of HIF-1α. However, pVHL, a regulator of HIF-1α degradation, was found to be lower in CLL B cells and unable to physically interact with HIF-1α in CLL B cells. Nuclear extraction assay revealed that HIF-1α interacted with its co-factor, p300/CRP in B cells, indicating that HIF-1α was transcriptionally active. We hypothesized that reduction of pVHL expression in CLL B cells could be related to repression by micro RNA function which is known to be aberrantly expressed in CLL. We focused on miR-92-1 known to be over-expressed in CLL B cells and it has pVHL as a putative target. In a luciferase assay of transfected human megakaryocytic cell line, MEG-01, we found a direct effect for miR-92-1 on pVHL, with significant repression of luciferase activity of VHL expression by miR-92-1 compared to both control vectors and a mutated target mRNA sequences. This result indicated that wild-type miR-92-1 was able to interact directly with the 3′ UTR sequence of pVHL and subsequently repress pVHL expression. To further confirm this finding, we did transient transfections of either the CLL cell line Mec-1, primary CLL B cells or normal B cells with a sense oligo miR-92-1 (both Mec-1 and CLL B cells) or wild-type miR-92-1 plasmid (normal B cells). We found the level of flow detectable pVHL lower when compared to non-transfected cells. Because we were interested in the relationship of HIF-1α to CLL B cell survival we studied if chemotherapeutic drugs can downregulate HIF-1α in CLL B cells. To do this we studied flavopiridol a drug that has very promising activity in CLL. Our results indicated that flavopiridol does down-regulate HIF-1α levels in CLL B cells. This latter change was associated with induction of apoptosis in CLL B cells. Overall, our data show that elevated VEGF secretion is related to constitutively elevated HIF-1α levels and is an explanation for the VEGF-based autocrine pathway found in CLL B cells. We also show that elevated HIF-1α levels could be partially related to depressed pVHL with a unique mechanism for its repression: the over-expressed microRNA, miR-92-1 in CLL-B cells.
Bi-Directional Notch Signaling Via Signal-Sending and Signal-Receiving Microdomains of the Partnered Immunological Synapses during Th:DC Interaction.
Authors: Vyas Yatin M. , Parness Jerome , Rodeberg David , Huang Winifred
Published: October 2019

The Notch pathway regulates adaptive immune responses, yet the temporal development of a specific molecular anatomy underlying the directionality of Notch signaling, central to cell-fate decisions, remains unknown. Using the development of the functional immune synapse (IS) of the human physiological T-helper lymphocyte (Th): Dendritic cell (DC) interaction as our model, we followed the temporal accumulation of Notch signaling components, unprocessed and processed, in the developing ThIS and the apposed DCIS of these cells by 2D and 3D immunofluorescense microscopy. Downstream Notch targets in both cell types were followed, as well. We demonstrate that Th-Notch1 receptor and DC-Notch ligands (Delta-like1, Jagged-1) cluster in their apposed central-supramolecular-activation-clusters (cSMAC), whereas DC-Notch1 receptor and Th-Notch ligands cluster in their apposed peripheral-SMAC in an anti-parallel arrangement to that seen in the cSMAC. The resultant accumulation in both cell types of processed nuclear Notch receptor, its ligands, as well as HES-1 and phosphorylated-STAT3, supports antiparallel, reciprocal Notch signal propagation in the DC-to-Th direction via the cSMAC and Th-to-DC direction via the pSMAC. The imposed asymmetric recruitment of the components of Notch pathway, therefore, provides a novel bi-directional route by which the partnered ThIS and DCIS regulate Notch-mediated immune responses. Our data indicate that terminally differentiated immune cells communicate bidirectionally using unidirectional Notch signaling platforms that are spatially segregated into reciprocally signaling microdomains. Significantly, our observations of bidirectional Notch signaling indicate that the heterologous Th:DC interaction is cooperative, requiring reciprocal information transfer across both cell types to mount an appropriate immune response.
The Bcl-2 Family Inhibitor ABT-263 Shows Significant Anti-Tumor Efficacy in Models of B Cell Non-Hodgkin’s Lymphoma.
Authors: Shoemaker Alex R. , Mitten Michael J. , Oleksijew Anatol , O’Connor Jacqeuline M. , Wang Baole , Ackler Scott , Joseph Mary , Zhang Haichao , Nimmer Paul , Xiao Yu , Tse Christin , Frost David J. , Fesik Stephen W. , Rosenberg Saul H. , Elmore Steven W.
Published: October 2019

ABT-263 is an orally bioavailable small molecule inhibitor of Bcl-2 family proteins with a Ki of ≤ 1 nM against Bcl-2, Bcl-XL, and Bcl-w. Non-Hodgkin’s B-cell lymphomas represent clinically relevant disease targets for this molecule due, in part, to strong expression of Bcl-2 often associated with various types of NHL (frequently involving a t(14;18) translocation including the Bcl-2 locus). ABT-263 exhibits sub-micromolar in vitro activity against a variety of NHL cell lines. DoHH-2 and WSU-DLCL2 are two B-cell NHL lines harboring the t(14;18) translocation that exhibit differential in vitro sensitivity to ABT-263. Granta-519 is a mantle cell lymphoma line with the characteristic t(11;14)(q13:q32) translocation resulting in overexpression of cyclin D1. ABT-263 has an EC50 of approximately 150 nM in the Granta-519 cell line. Here we present efficacy data evaluating the activity of ABT-263 in several NHL xenograft models. ABT-263 has significant in vivo anti-tumor efficacy in established flank tumor models both as monotherapy and in combination with cytotoxic agents. The efficacy of ABT-263 at 100 mg/kg/day, p.o., q.d. ×21 was evaluated as monotherapy and in combination with etoposide, vincristine, modified CHOP, R-CHOP, bortezomib, rapamycin, and rituximab. Results show that ABT-263 significantly inhibits tumor growth as a monotherapy (~50–60% tumor growth inhibition) and enhances the efficacy of these cytotoxic agents in combination therapy. Statistically significant enhancement of tumor growth inhibition was observed for each combination relative to monotherapy treatment. Efficacy was maintained even when therapy was initiated on larger (~500 mm3) tumors. Combinations of ABT-263 + rapamycin and ABT-263 + rituximab result in complete regression of a significant percentage of established B cell lymphoma tumors for a sustained period of time in vivo. The combination of ABT-263 + R-CHOP resulted in complete regression of 100% of the tumors in the mantle cell lymphoma model. The strong in vitro potency and tumor regressions seen in vivo suggest that ABT-263 has great potential for the oral treatment of NHL B-cell lymphomas.
Low Dose Prothrombin Complex Concentrates Reverse Oral Anticoagulation with Minimal Complications.
Authors: Bagot Catherine N. , Kothari Jaimal , Patel Raj K. , Arya Roopen
Published: October 2019

Prothrombin complex concentrate (PCC) is widely used for the reversal of warfarin in the context of life threatening haemorrhage or emergency surgery. For these indications, it is considered superior to fresh frozen plasma (FFP), in terms of viral safety, efficacy and speed of administration. Dosage of PCCs for coumarin reversal (30–50IU/kg) is based on that required for patients with haemophilia B. This dose is frequently associated with thrombosis, particularly in the presence of liver disease. Factors underlying the prothrombotic potential of PCCs include the activation of clotting factors during the manufacturing process, coexisting co morbidities in the patient and repeated administration leading to clotting factor accumulation in vivo. The risk of thrombosis has led to a reluctance to administer PCCs, despite proven efficacy, in patients with arterial or venous thromboembolism, sepsis and liver disease. Recent work suggests that PCCs can effectively reverse anticoagulation when administered in much lower doses. We collected data on 38 patients (mean age 70 years) who received Beriplex (Aventis Behring) to reverse anticoagulation either for life threatening haemorrhage or emergency surgery. Patients were receiving warfarin for atrial fibrillation (50%), recurrent thromboembolic disease (18.5%) and mechanical heart valves (13%). 52.5% received Beriplex following haemorrhage, 26.5% to correct the INR prior to emergency surgery and 2.5% for an excessively elevated INR. 87.6% received 500IU Beriplex with the remainder receiving 1000IU. Of 38 patients, 18 out of 29 received Vitamin K. Mean INR fell significantly (p<0.01) following Beriplex administration (mean INR pre-Beriplex 4.8, post-Beriplex 1.8). 81% had an INR <2 following Beriplex. Of patients with an INR <8 receiving one vial, 64% corrected their INR to <1.4 (96% patients had an INR <2). 59% of patients had additional risk factors including ischaemic heart disease, recurrent thromboembolism, sepsis or abnormal liver function. No patients had thrombotic complications. Three had evidence of DIC following administration of Beriplex, all in association with sepsis and no patient experienced bleeding complications at the time of surgery. Bleeding was well controlled in those experiencing life threatening haemorrhage except in two patients, who presented with massive intracranial bleeds. This data indicates that PCCs should be considered in all patients on warfarin who require urgent anticoagulation reversal, at a dose of 500IU only. PCCs at a fixed low dose appear to correct anticoagulation effectively based on both clinical and laboratory parameters, without the need for repeated administration. Importantly, PCCs at this lower dose were not associated with thromboembolic events. Low dose PCCs therefore provide patients with a safe and effective antidote to warfarin.
A Multicenter Study of a Gemcitabine-Based Regimen in Relapsed or Refractory Hodgkin’s Lymphoma Patients.
Authors: Validire Patricia , Fermé Christophe , Brice Pauline , Diviné Marine , Gabarre Jean , Bouabdallah Krimo , Fitoussi Olivier , Chaoui Driss , Pacquement Hélène , Soussain Carole , Carde Patrice , Sahli Rafika , Decaudin Didier
Published: October 2019

The aim of this study was to assess the efficacy of a gemcitabine-based regimen in pretreated Hodgkin’s lymphoma (HL) patients. Relapsed or refractory HL patients treated with gemcitabine, used alone or in combination with other cytotoxic agents, were retrospectively reviewed. Fifty-five patients were included in the study. Initial characteristics before gemcitabine administration were: Ann Arbor stage III–IV: 84%; International Prognostic Score less than 3 in 20/43 cases (47%); thirty-one primary refractory patients at the end of first-line therapy (56%); median number of previous chemotherapy regimens of 3. Twenty-nine patients received gemcitabine alone with a median starting dose of 750 mg/m2 per injection (range: 180–1250 mg/m2); Gemcitabine was administered at a starting dose of 1000 mg/m2 per injection (range: 500–1250) in combination with vinorelbine in 10 patients, oxaliplatin in 13 patients, and other drugs in 3 patients, with a median of 6 injections (range: 1–18). Overall response rate was 20% with 11% of complete remission. On univariate analysis, two adverse factors at progression were significant for response to gemcitabine-based regimen: stage III–IV disease and hemoglobin level less than 10.5 g/dl. In conclusion, the two identified prognostic factors for response to gemcitabine are part of the International Prognostic Score of HL, suggesting that response to gemcitabine is mainly influenced by the specific prognostic factors of HL. Moreover, with an ORR of 29%, our results of the gemcitabine administered alone regimen are not different from those reported in the literature. In contrast, the results of the various series of HL patients treated by gemcitabine-combined regimens, mainly with cisplatin or derivatives, vinorelbine, ifosfamide, doxorubicin, and prednisone, are very different due to different patient characteristics. In heavily pretreated cases, as in our study, the ORR was 26%; inversely, in patients who had received only one or two lines of chemotherapy, the ORR varied between 64% and 82% with 9% to 54% of complete remissions. This discordance can probably be explained by the prognostic impact of previous treatment lines in the response to gemcitabine. This observation emphasizes the possible interest of using gemcitabine earlier in the treatment of Hodgkin’s lymphoma, namely at the time of first relapse or after first-line treatment in primary refractory HL patients.
Treatment of Extranodal Marginal Zone Lymphoma and Primary Cutaneous B Cell Lymphoma with Rituximab: A Single Institution Experience.
Authors: Chen Robert , Peterson Glen , Vucurevich Diana , Lee Choon-kee , Robinson William , Myint Han
Published: October 2019

Extranodal marginal zone lymphoma is a low grade B cell lymphoma. It comprises about 5% of all non-hodgkin’s lymphoma. They typically present in the lung, thyroid, breast, lacrimal and salivary gland, orbit, and skin. Primary cutaneous B cell lymphoma is an extremely rare form of lymphoma with 3 histological variants: marginal zone, follicular, and diffuse large B cell. In general, they have a high remission rate with overall survival up to 80% at 10 years. At the University of Colorado Health Sciences Center, we have seen ten such cases from July 2005 to July 2007. There is no standard treatement for either extranodal marginal zone lymphoma or primary cutaneous B cell lymphoma, but prior reports have shown long term survival with surgery, radiation, and chemotherapy. Most institutions use radiation therapy as the main modality after excisional biopsy. There is one report by Gitelson et al (leuk lymphoma 2006) using single agent rituximab as the only modality. Out of the ten cases at our institution, six cases were primary cutaneous B cell lymphoma (4 marginal zone, 1 follicular, one diffuse large B cell), and four cases were extranodal marginal zone lymphoma. Four out of six cases of primary cutaneous B cell lymphoma and three out of four of extranodal marginal zone lymphoma was treated with single agent rituximab. Two out of the six cases of primary cutaneous B cell lymphoma and one out of four of extranodal marginal zone lymphoma was treated with radiation. Patients were given induction therapy rituximab at the dose of 375mg/m2 IV weekly x 4 weeks and maintenance therapy at 375 mg/m2 IV every two to three month interval for 8 cycles. 7 patients were given rituximab single agent. All achieved CR (100%) immediately after induction therapy at 1 month. They were also given maintenance treatment. No relapses has occured at the medium follow up of 7 month (3–24 month). Our preliminary result supports the recent report of single agent rituximab. Even though extranodal marginal zone lymphoma is a rare disorder and primary cutaneous B cell lymphoma even more rarer, we feel that a well designed prospective randomized trial would determine the role of rituximab for this disease.
Phase II Study of 2-Weekly CHOP+Rituximab Followed by Yttrium 90 Ibritumomab Tiuxetan (Zevalin) in Patients with Previously Untreated Diffuse Large B Cell Lymphoma (DLBCL).
Authors: Venugopal Parameswaran , Gregory Stephanie A. , Obrien Teresa , Drasga Ray , Bayer Robert
Published: October 2019

Interval reduction from 3 to 2 weeks (CHOP-14; Pfreundschuh et al, Blood, 2004), addition of Rituximab to CHOP-21 (R-CHOP-21, Coiffier et al NEJM 2002) and consolidation with radioimmunotherapy following CHOP chemotherapy have all been shown to improve the outcome of DLBCL. We report the results of a Phase II clinical trial designed to evaluate the safety and efficacy of combination of the above, using 2-weekly CHOP+Rituximab followed by consolidation with Zevalin in patients with untreated DLBCL.

Patients and Methods: Patients with previously untreated DLBCL with measurable disease, age>18 years and performance status 0-2 were eligible. Patients were required to have adequate marrow, liver and kidney functions. Patients with transformed lymphoma were excluded. Patients received standard CHOP along with Rituximab 375mg/m2 IV on day 1. Treatment was repeated every two weeks for a total of 6 cycles. Zevalin was administered within 6–8 weeks after the last dose of CHOPR therapy.

Results: 14 patients were enrolled. The median age was 59.5 years (range 48–82), and 6 patients were men. All patients had an ECOG performance status of 0 or 1. 3 (21%) had stage II, 5 (36%) stage III and 6 (43%) had stage IV disease. IPI was 2 in 13 (93%) and 3 in 1 (7%). Bone marrow showed lymphoma in two patients only. LDH was elevated in 9 patients. All patients except 2 completed all 6 cycles of CHOP+R. One patient withdrew consent after 5 cycles and one patient with multiple lung masses could not be continued on therapy after two cycles because of prolonged rehab. Following CHOP+R alone in patients who received a minimum of 4 cycles of CHOP+R (13), 8 patients achieved CR and 5 achieved PR. Following consolidation with Zevalin, three patients in PR were converted to CR. After a median follow-up of 17.5 months, OSS is 14/14 (100%). 11 patients (79%) remain in CR. One patient progressed and proceeded to stem cell transplant; one came off study after 2 cycles of CHOP+R and remains with evidence of disease; and the third patient, who achieved PR with CHOP+R is waiting restaging after Zevalin. Most common adverse events were hematologic. Grade 3 Neutropenia was observed in 10 patients and grade 4 in 7. Grade 3 thrombocytopenia was observed in 3 and grade 4 in 1. Non-hematologic toxicities included: grade 3 pneumonia in 3, DVT in 1, grade 4 pneumonia in 1, grade 3 fever in 2 and grade 4 fever in 1.

Conclusion: Consolidation with Zevalin radioimmunotherapy following dose dense CHOP+R therapy is well tolerated, effective and able to convert PRs to CRs.
Novel Link between PI 3′ Kinase/p38 Map Kinase Pathway in Dasatinib (BMS354825) Mediated Effect in BCR-ABL Expressing Cells.
Authors: Lumby Crystal , Singh VeerPal , Reddy Shrisha , Sivess-Franks Sue , Dowell Jonathan , Verma Amit , Parmar Simrit
Published: October 2019

Background: Dasatinib (BMS354825), a dual Src/Abl tyrosine kinase inhibitor, exhibits potent antileukemic effects in vitro and in vivo. Despite the well-established role of BMS354825 in the treatment of imatinib-resistant chronic myelogenous leukemia (CML), the molecular mechanisms that result in generation of antileukemic responses remain unknown.

Methods: BCR/ABL (wild type and those carrying mutations: E255K, H396P, Y253F, M351T and T315I) expressing murine BAF3 cell lines were exposed to varying concentrations of BMS354825 for variable times to evaluate for effect on phosphorylation/activation of p38 MAP Kinase and PI3′ Kinase pathway in presence or absence of pharmacologic inhibitors of p38 MAP Kinase and mTOR respectively.

Results: In the present study we provide evidence that BMS354825 induces phosphorylation of the p38 MAP Kinase, and activation of its kinase domain, in BCR-ABL expressing cell lines except for those carrying T315I mutation. We also identify the kinases MapKapK-2 which is upregulated in response to BMS354825 as shown by increased phosphorylation of hsp27. Importantly, pharmacological inhibition of p38 MAP Kinase by SB203580 reverses the growth inhibitory effects of BMS354825 on primary leukemic CFU-GM progenitors from patients with CML (see figure). Moreover, SB203580 leads to impairment of the BMS354825 mediated antiproliferative effects in both wild type and mutated CML lines except for those carrying T315I. On the other hand, BMS354825 leads to dephosphorylation of p70S6 Kinase and its downstream effector pathway including down regulation of ribosomal S6. We also report that the pharmacological inhibition of mTOR by Rapamycin augments the growth inhibitory effects of BMS354825 on primary leukemic CFU-GM progenitors from CML patients (see figure). Furthermore, pharmacologic inhibition of p38 MAP Kinase by SB203580 led to reversal of the BMS354825 mediated dephosphorylation of p70 S6 Kinase demonstrating that it maybe downstream of p38 MAP Kinase activation. Altogether, our data establish that activation of the p38 MAP Kinase signaling cascade plays an important role in the generation of the effects of BMS354825 on BCR-ABL expressing cells.

Conclusion:

We have identified a novel crosstalk mechanism between the p38 MAPK and the PI3′ Kinase pathway which is unique to the effect of BMS354825 in CML. p38 MAP Kinase pathway may play an important role in developing of resistance to BMS354825 in CML. mTOR inhibition may augment effect of BMS354825 in CML and its role in combination with BMS354825 should be explored in resistant disease.

Figure Figure
Vitamin D Receptor (VDR) Polymorphisms in Chronic Lymphocytic Leukaemia (CLL) Patients.
Authors: Kwa Faith , Kapuscinski Miroslav , Cole-Sinclair Merrole
Published: October 2019

Chronic Lymphocytic Leukaemia (CLL) is a lymphoproliferative disorder in which leukaemic B cells are arrested in the G0/G1 phase of the cell cycle, resulting in their prolonged lifespan. The clinical course of CLL is highly variable with survival ranging from a few months to decades. Thus, a good prognostic tool is needed to help predict the course of the disease. The pathogenesis of CLL is unknown but it has been associated with the “escape” of B cells from apoptosis, possibly due to overexpression of anti-apoptotic genes and downregulation of pro-apoptotic genes. The expression of these genes is influenced by alterations in chromatin structure through a change in DNA methylation (chromatin remodelling) mediated by multi-protein complexes. The Vitamin D receptor (VDR), which forms part of one such chromatin remodelling complex, has been shown to be linked to the susceptibility to and outcome of other malignancies (e.g. prostate cancer). We hypothesised that VDR polymorphisms (FokI, TaqI, BsmI and ApaI) may be linked to CLL pathogenesis and/or might be a marker of CLL progression, given the immunomodulatory actions of Vitamin D. The genotypes of 34 patients with CLL were determined by either allele-specific PCR (FokI) or PCR/restriction digestion analysis (TaqI, BsmI and ApaI). Clinical data including disease stage (Rai), other prognostic markers (e.g. lymphocyte doubling time) and therapy status were recorded. Statistical analysis including Wilcoxon, Chi-square and Spearman correlation tests were performed. Initial analysis-to-date has not shown significant difference in allelic distribution between CLL patients and a healthy control population. There was no statistically significant association between the 4 VDR polymorphisms and the clinical parameters of CLL (all p>0.05). A larger cohort of CLL patients is currently under study to determine any such potential correlation.
Fifth Succesfully Retreatment with Alemtuzumab in Patient with Chronic Lymphocytic Leukemia.
Authors: Egyed Miklos , Kollar Balazs , Karadi Eva , Rajnics Peter , Magyarlaki Tamas , Kereskai Laszlo
Published: October 2019

Before the introduction of Alemtuzumab, the prognosis of fludarabine-refractory chronic lymphocytic leukemia (CLL) was very poor. This humanised anti-CD52 monoclonal antibody was found to be effective in CLL patients with additional high-risk prognostic markers. In our case report, we present the courses of treatment with Alemtuzumab in a CLL patient with del q11, elevated ZAP-70 and CD38 levels. He was first admitted to our department because of CLL in 1996. He received high dose Chlorambucil, later fludarabine courses between 1996–2002. Because of early disease progression five courses of Alemtuzumab were administered between 2003–2007. The treatment resulting nodal partial remission lasting about 10–11 months. No CTC 3–4 grade side effects were registered, nor hospitalization was necessary. The patient responded favorably to each course of repeated therapy with single agent Alemtuzumab, suggesting, that Alemtuzumab retraitment is the choice of therapy for fludarabine refractory CLL patients.
Newly Diagnosed Multiple Myeloma Patients with 1q21 Amplification Treated by Autologous Stem Cell Transplantation Have Shorter Progression-Free Survival Interval.
Authors: Nemec Pavel , Greslikova Henrieta , Kuglik Petr , Filkova Hana , Zaoralova Romana , Vranova Vladimira , Smejkalova Jana , Vidlakova Petra , Oltova Alexandra , Hajek Roman
Published: October 2019

Amplification of chromosome band 1q21 as well as increased expression of CKS1B gene in this area is a frequently mentioned prognostic factor for patients with multiple myeloma (MM). Total 39 newly diagnosed multiple myeloma patients (median age: 56 years) enrolled in Faculty Hospital Brno, Brno, Czech Republic, were examined for 1q21 amplification status. All patients received 4 cycles of vincristine, adriamycin and dexamethasone (VAD) as induction and one course melphalan 200mg/m2 followed by autologous stem cell transplantation (ASCT). The median follow-up from treatment start was 16.1 (range: 2.2–44.0) months. All the “end-point” intervals and treatment responses were assigned by IMWG criteria. Plasma cells were identified by cytoplasmic light-chain immunofluorescence followed by fluorescence in situ hybridisation (cIg-FISH). Amplification of 1q21 (Amp(1q21)) was assigned utilizing labelled BAC clone (RP11-205M9) DNA probe. Cut-off level for Amp(1q21) was established to 10% of total amount of cells with additional signals detected. Amp(1q21) was found in 41% (16/39) patients. Clinical parameters valid for patients with Amp(1q21) versus patients lacking Amp(1q21) were as follows: overall response rate (ORR) achieved 87.5% (14/16) vs. 91.3% (21/23) patients (p=0.404); overall survival (OS) median was 22.4 months vs. not yet reached (p=0.022); time to progression (TTP) median was 16.1 months vs. not yet reached (p=0.010); progression-free survival (PFS) median was 15.6 months vs. 25.2 months (p=0.023) and duration of response (DOR) median was 15.9 months vs. not yet reached (p=0.048). There were found statistical significant difference in all named “end-point” intervals (OS, TTP, PFS and DOR) between patients with/without Amp(1q21) but not in ORR. In conclusion, patients with Amp(1q21) treated by ASCT have significant shorter PFS median (15.6 months) when compared with patients lacking Amp(1q21) with PFS median 25.2 months (p=0.023). This findings is in accordance with previously published work (Chang et al., 2006).
Hepatocyte Growth Factor Expression in Bone Marrow Microenvironment Is Critical for Progression of MGUS to Myeloma.
Authors: Ramasamy Karthik , Schey Stephen , Mufti Ghulam J. , Farzaneh Farzin , Calle Yolanda
Published: October 2019

Hepatocyte growth factor (HGF) augments proliferation, survival and increases migration of plasma cells in vitro and in addition it induces haemangiogeneis and lymphangiogenesis. C-met (HGF receptor) is expressed on plasma cells and HGF is constitutively elevated in serum of patients with myeloma (MM) and MGUS patients. We investigated the distribution of HGF in bone marrow biopsies to better understand its role in progression of monoclonal gammopathy.

Materials and methods: Bone marrow trephines were analysed by immunohistochemistry of paraffin embedded bone marrow (BM) trephine specimens. Trephines were dewaxed without antigen retrieval. Plasma cell enumeration was done using CD38 staining (Novocastra, UK). HGF (Goat antihuman IgG, R&D systems) staining was performed using Vectastain® ABC kit with DAB reactivity. Slides were analysed using an Optical microscope (Zeiss Standard 20, Germany).

Results: Archived BM specimens of MM (n= 7), MGUS (n= 5) and normal controls (n=6) were stained in duplicate. Trephines were graded from Grade 1–3 (weak, moderate, strong) based on the intensity of staining. The localisation of staining to paratrabecular, perivascular and intertrabecular areas was analysed. Interstitial and cytoplasmic staining patterns were analysed. HGF staining localised to paratrabecular regions was observed in all normal individuals with staining intensity of Grade 1–2 in 3/6(50%) and Grade 3 in 3/6(50%). In 4/5(80%) patients with MGUS a distinct intertrabecular pattern of staining was observed in discrete regions, where plasma cells were localised. Intensity of staining was Grade 2 in 3/5(60%) and Grade 3 in 2/5(40%) MGUS patients. Plasma cell percentages were between 5–20% in MGUS patients. In MM patients we found a correlation between the plasma cell counts and the degree of HGF staining. In 2/7 (28.5%) patients plasma cell percentages ranged between 5% and 15% and Grade 1–2 discrete paratrabecular staining was observed with none or low perivascular staining. In 5/7 (71.5%) MM patients plasma cell percentages were >20% and Grade 3 confluent staining with interstitial and cytoplasmic location of HGF was observed. In addition, patients with Grade 3 staining presented a strong perivascular localisation of HGF.

Conclusion: HGF distribution in bone marrow microenvironment of MGUS and MM patients is non random. We observed that MGUS patients have a discrete intertrabecular pattern and MM patients have a confluent interstitial pattern of staining with strong perivascular localisation. Other groups have shown that high HGF levels increase angiogenesis. We hypothesise that HGF localisation and pattern of distribution in BM microenvironment leads to neo angiogenesis and progression from MGUS to MM. Ongoing studies are evaluating this hypothesis.
The mTOR Inhibitor RAD001 (Everolimus) Inhibits Myeloma Tumor Growth In Vivo and Enhances the Anti-MM Effects of Bortezomib and Arsenic Trioxide.
Authors: Campbell Richard A. , Sanchez Eric , Steinberg Jeffrey , Share Michael , Wang Joseph , Li Mingjie , Leuchter Richard , Chen Haiming , Shalitin Dror , Pang Shen , Said Jonathan , Berenson James R.
Published: October 2019

The mammalian target of rapamycin (mTOR) is an intracellular protein that acts as a central regulator of multiple signaling pathways (IGF, EGF, PDGF, VEGF, amino acids) that mediate abnormal growth, proliferation, survival and angiogenesis in cancer. mTOR is a critical component of the PI3K/Akt pathway, a key cell survival pathway that is dysregulated in many cancers including multiple myeloma (MM). mTOR is an important therapeutic target because it is a “rate-limiting” bottleneck in the key signaling pathway that regulates cell survival, proliferation, and angiogenesis. RAD001 (everolimus) is a novel oral mTOR pathway inhibitor. Recent data suggests that RAD001 has direct effects on tumor cell proliferation and may have antiangiogenic activity due to inhibition of tumoral VEGF production and effects on vascular endothelial and smooth muscle cell biology. We first evaluated the in vivo effects of single agent RAD001 in mice bearing the human MM tumor LAGλ-1. Tumor-bearing mice receiving RAD001 at 3, 10, or 30 mg/kg once daily five times per week (M-F) via oral gavage showed marked inhibition of tumor growth at all doses (P<0.0001) and reduction of paraprotein levels (P<0.0001) compared to mice receiving placebo. Next, we evaluated the in vivo anti-MM effects of RAD001 in combination with arsenic trioxide (ATO) and RAD001 in combination with bortezomib. Each severe combined immunodeficient (SCID) mouse was implanted with a fragment (2 – 4 mm3) of the LAGλ-1 tumor into the left hind limb muscle. The tumors were allowed to grow for 10 days at which time human IgG levels were detectable in the mouse serum, and mice were blindly assigned into treatment groups. Tumor-bearing mice received RAD001 at 1, 3, or 10 mg/kg once daily five times per week via oral gavage, ATO (3 mg/kg) once daily five times per week via intraperitoneal injection, bortezomib (0.5 mg/kg) once daily twice weekly via intravenous injection, or a placebo. Mice receiving RAD001/ATO combination therapy exhibited decreased hIgG levels and tumor volume compared to those mice receiving single agent and placebo therapy. Additionally, those mice receiving the RAD001/bortezomib combination treatment displayed similar MM tumor growth inhibition including decreased hIgG levels and tumor volume as the RAD001/ATO-treated mice. These preliminary in vivo results are encouraging with combination RAD001+ATO and RAD001+bortezomib therapy for the treatment of MM and additional studies are being performed to further optimize the clinical development of RAD001 in combination with other anti-MM treatments for patients with MM.
Complete Remission Achievement during Long-Term Maintenance with Bortezomib Based Therapy in Multiple Myeloma: A Case Report.
Authors: Crescenzi Sabrina Leonetti , Piccioni Anna Lina , Bagnato Antonino , Venditti Adriano
Published: October 2019

The treatment of multiple myeloma (MM), a B-cell neoplasm characterized by a clonal expansion of plasma cell in the bone marrow, remains unsuccessful in a significant proportion of patients. The majority of MM patients fail to achieve a complete response (CR) with standard therapy. Bortezomib is a new drug that has been shown to induce a complete response (CR) in 10% of pre-treated patients and continues to show benefit in relapsing/refractory myeloma treatment. We report a case of 72 year-old women with IgAl and IgGK MM who resulted refractory to two different line therapies. Patient was diagnosed with MM in March 2005 following the admission to our clinic for bone pain and weight loss. Laboratory blood tests were: total protein 9,4 gr/dl; IgAl and IgGK monoclonal spike (IgG: 917; IgA: 2990, IgM<17, Ks: 253, ls: 997 gr/dl); Beta2 microglobulin 2,7 mg/L; Ca 9,7 mg/dl. Mild pancytopenia was found. No renal failure and significant proteinuria were observed, Bence Jones were negative. Bone scans revealed diffuse lytic lesions to spine with a vertebral fracture (D12). Bone marrow biopsy showed 60% of plasma cells, with atypical characteristics. Initial treatment consisted of melphalan, prednisone and pamidronate that were given for a total of four cycles. Patient achieved a stable disease according to International Myeloma Working Group criteria. Then the patient underwent idarubicine based regimen without any response indicating refractory MM. In November 2005, a 3-week cycle schema with Bortezomib (1,3 mg/m2 i.v. on day 1,8,11), Melphalan (10 mg PO on day 1,3,5) and Dexamethasone (4 mg i.v. on day 1,8,11,15) was started. The patient achieved a partial response (PR) after 8 cycles of this combination therapy .No therapy related Grade 3–4 neuropathy and trombocytopenia were observed. The patient experienced a mild HZV reinfection. In June 2006, a maintenance regimen with Bortezomib (1,3 mg/m2 i.v. on day 1,8) Melphalan (6 mg PO on day 4,11) and Dexamethasone (4 mg i.v. on day 1,8,15) was initiated considering the positive response and the absence of toxicity from the primary previous therapy. Each cycle was repeated every three weeks for a total of 16 courses, until June 2007. Efficacy assessment (including bone marrow biopsy) was performed every three months and showed a sustained decrease in plasma cell infiltration and a persistent reduction of M serum protein.Very Good PR and CR were achieved after 10 and 16 maintenance courses, respectively. This case demonstrates that a Bortezomib-based regimen is effective and feasible in refractory MM and, interestingly, supports the hypothesis that a long-term maintenance therapy may improve the quality of response. This treatment approach should be evaluated in a larger patient cohort and in prospective randomised trials.
TEL-AML1 Dysregulates the TGFβ Pathway: A Basis for Pre-Leukaemic Stem Cell Selection.
Authors: Ford Anthony , Palmi Chiara , Bueno Clara , Knight Deborah , Cardus Penny , Hong Dengli , Cazzaniga Giovanni , Enver Tariq , Greaves Mel
Published: October 2019

TEL-AML1 fusion is the most frequent, defined genetic abnormality in paediatric cancer and is usually an early or initiating and pre-natal event in childhood acute lymphoblastic leukaemia (ALL). Transformation results in the generation of a persistent pre-leukaemic clone, which converts to frank ALL post-natally (at 1-15years) following the acquisition of secondary genetic alterations. The mechanism by which the transcriptional dysregulation imposed by TEL-AML1 impacts on the pre-leukaemic phenotype and disease natural history is unknown. Using regulated expression of TEL-AML1, we show that, in murine progenitor cells, this protein blocks the TGFβ response pathway including the critical regulator of cell cycle inhibition, p27. This inhibition facilitates the selective expansion of otherwise more slowly expanding TEL-AML1 progenitor cells. The mechanism of action of TEL-AML1 appears to operate downstream of SMAD2/3 phosphorylation (by TGFβ signalling) and involves binding of a transcriptional repressor complex to TGFβ target genes. In transgenic Eμ TEL-AML1 mice, we show that early pro-B cells (but not pre-B cells) are selectively expanded and appear less sensitive to the inhibitory effects of TGFβ than equivalent wild-type cells. Finally, we show that expression of TEL-AML1 in human cord blood progenitor cells leads to the expansion of a candidate pre-leukaemic stem cell population with an early B lineage phenotype (CD34+/CD38-/CD19+) that has a selective growth advantage in the presence of TGFβ.
Association of Neurodevelopmental Abnormalities Is a Common Clinical Character in Japanese Patients with Severe Congenital Neutropenia Due to HAX1 Deficiency.
Authors: Okada Satoshi , Ishikawa Nobutsune , Shirao Kenichiro , Tsumura Miyuki , Satoh Takashi , Ohno Yoshinori , Yasunaga Shin’ichiro , Ohtsubo Motoaki , Takihara Yoshihiro , Kobayashi Masao
Published: October 2019

Severe congenital neutropenia (SCN) is characterized by early childhood onset of profound neutropenia and recurrent life-threatening infections. The bone marrow usually shows a reduced number of mature myeloid cells with an arrest at the promyelocyte-myelocyte stage. SCN includes a variety of hematological disorders caused by the different genetic abnormality. Genetic analysis in individuals with SCN indicates that 60% of cases are attributable to heterozygous mutation in ELA2, encoding neutrophil elastase. Recently, homozygous mutation in HAX1 encoding HAX1, mitochondrial protein associates with HS1 which is a substrate for Src family tyrosine kinase, was identified in individuals with autosomal recessive forms of SCN and the original Kostmann family in Sweden. In this study, we examined involvement of HAX1 and ELA2 in 17 Japanese cases with SCN. Ten patients had heterozygous mutations, including two novel mutations in ELA2. Five patients had homozygous or compound heterozygous mutations in HAX1. A homozygous single-base-pair substitution at nucleotide 256 (256C>T) HAX1 mutation causing a nonsense change R86X was detected in three patients. Two sibling cases showed compound heterozygous mutation of 256C>T and 59-bp deletion at nucleotide 376–434. This 59-bp deletion was a novel mutation leading to a frameshift and a premature stop codon at nucleotide 441–443 (R126fsX128). All heterozygous carriers had no detectable phenotype. HAX1 protein was undetectable in both homozygous and compound heterozygous patients by immunoblot analysis of leukocytes. Notably, all patients with HAX1 deficiency showed failure to thrive and mild to severe developmental delay. Further, three patients carrying homozygous R86X mutation had epilepsy. Two of three patients had complex partial seizures, and another had atypical absence. An Iranian patient carrying homozygous R86X mutation of HAX1 was described to show failure to thrive in the original report of HAX1 deficiency, and three of six cases of the original Kostmann family showed neurological deficits. In contrast, no SCN patients with a heterozygous mutation in ELA2 displayed the association of neurodevelopmental abnormalities. Thus, R86X mutation in HAX1 is a common abnormality in Japanese SCN patients with HAX1 deficiency and may lead to neurodevelopmental abnormalities as well as severe myelopoietic defects. Because HAX1 is a ubiquitously expressed gene, further accumulation of the patients should be needed to characterize the clinical features of HAX1 deficiency.
Significant Disease Burden in Paroxysmal Nocturnal Hemoglobinuria Patients with Lower Levels of Hemolysis, Mild Anemia and Minimal Transfusion: Clinical Improvement with Eculizumab Therapy.
Authors: Bessler Monica , Schrezenmeier Hubert , Maciejewski Jaroslaw P. , Hill Anita , Rollins Scott A. , Young Neal S. , Luzzatto Lucio
Published: October 2019

Paroxysmal nocturnal hemoglobinuria (PNH) is a debilitating and life-threatening disease. However, disease burden in patients with lower lactate dehydrogenase (LDH), higher hemoglobin and minimal transfusion support has not been determined. The terminal complement inhibitor, eculizumab (Soliris™), was shown to significantly reduce hemolysis, improve anemia and fatigue, and reduce thrombosis in a broad PNH population. We examined the long-term efficacy of eculizumab in multiple patient subgroups. Efficacy data included hemolysis (as assessed by LDH), transfusion requirements, fatigue (measured by Functional Assessment of Chronic Illness Therapy-Fatigue instrument, FACIT-Fatigue), and thrombosis. Data were analyzed by lower baseline hemolysis (LDH <1490 U/L, n=46), mild anemia (Hgb ≥10.5 g/L, n=54), and minimal transfusion (0 or 1 episodes in prior yr, n=21) among 187 PNH patients who had been enrolled in eculizumab trials (Phase 2 Pilot, Phase 3 TRIUMPH and SHEPHERD studies) and who continued to receive eculizumab in a long-term extension trial (median duration, 22 months). Eculizumab provided significant clinical benefit to patients regardless of baseline degree of hemolysis, anemia, or transfusion requirement (Table). Fatigue improved with eculizumab across all baseline LDH quartiles. Of interest, reduced hemolysis with eculizumab improved fatigue even in patients with only mild anemia and little or no transfusion requirement, suggesting that lowering hemolysis improves fatigue, independent of anemia. Fatigue scores increased during the first 6 mos of treatment and continued to improve throughout the last six months of treatment. Transfusion requirements also maintained during long-term treatment; patients requiring >1 transfusion in the year prior received a median of 8.5 units in the 6 months prior to eculizumab, 2.0 units in the first 6 months of treatment, and 0 units in the last 6 months of treatment. In patients with evidence of marrow failure (n=25; platelets <100,000/mm3 and no prior thrombosis) compared with pre-treatment, hemolysis (LDH) was reduced by 1217±211 U/L, median units transfused were reduced from 8 (mean 9.2±1.2) to 0 (4.4±1.4) and fatigue improved by 8.1±2.1 points (p≤0.001 for each) during the last six mos of eculizumab therapy. Thromboembolism (TE) was examined in all eculizumab trial patients (N=195). The TE rate was elevated in the pre-treatment period for patients with lower baseline hemolysis, mild anemia and minimal transfusion; eculizumab reduced TE in these patient subgroups by 58% (p=0.009, n=48), 84% (p<0.001, n=55) and 100% (p=0.063, n=22), respectively. In conclusion, patients who may be expected to have less severe disease suffer from significant disease burden, and long-term treatment with eculizumab provides substantial clinical improvement.
ENMD-1198 in Combination with Vincristine Shows Synergistic Activity in Leukemia Cells and Prolonged Mouse Survival in Human Leukemia Xenografts.
Authors: Liaw Tracy T.Y.E. , Burke Patricia P.A. , Chen X. , LaVallee Theresa T.M. , Lock Richard , Kavallaris Maria
Published: October 2019

ENMD-1198 (2-methoxyestra-1, 3, 5, (10), 16-tetraene-3-carboxamide), an analogue of 2 methoxyestradiol (2ME2 or Panzem®), is a microtubule destabilizing agent that binds to the colchicine-binding site of β-tubulin. ENMD-1198 has shown anti-angiogenic and anti-proliferative activities in several tumour models and is currently being evaluated in a Phase 1 clinical trial. To date however, the efficacy and mechanisms of action of ENMD-1198 in leukemia are not well-studied or fully understood. Hence, in order to assess the efficacy of ENMD-1198 in leukemia, a clinically relevant model of primary human ALL cells xenografted into immuno-deficient (NOD/SCID) mice was used (1). Three human ALL xenografts (ALL3, ALL7 and ALL19) that exhibit intrinsic differences in response to vincristine (VCR) (1) were treated with 100 mg/kg ENMD-1198 by gavage (daily for 28 days), commencing treatment when engraftment rates reached 1% human CD45+ in mouse peripheral blood. Treatment with ENMD-1198 significantly increased the mouse survival rates compared to vehicle control in all three xenografts (Leukemia Growth Delay (LGD) for ALL3 = 17.3 days, p < 0.005; ALL7 = 21.5 days, p < 0.005; ALL19 = 16.7 days, p < 0.005). Interestingly, ALL7, the least sensitive xenograft to vincristine, showed the best response to ENMD-1198 with a growth delay factor of 21 days. To determine whether the combination of ENMD-1198 and VCR has therapeutic advantages in leukemia, anti-proliferative studies of the drug combination in CCRF-CEM leukemia cells were carried out. A synergistic effect was observed when ENMD-1198 and VCR were combined. The effect of this drug combination was further examined in the ALL human xenograft mouse model. Mice inoculated with ALL7 xenograft were treated with 50 mg/kg ENMD-1198 (daily for 28 days) and 0.5 mg/kg VCR (weekly for 4 weeks) by intraperitoneal injection. The drug combination significantly prolonged mouse survival rates compared to single ENMD-1198 and VCR treatments (LGD 35.19days; p < 0.005). Functional analysis of the drug combination treatment in CCRF-CEM cells in vitro showed that the cells arrested at G2/M followed by sub-G1 (apoptosis) phase, with decreased HIF-1α and JAK-2 proteins. Apoptosis in vitro was associated with increased DR5, active caspase 3 and cleaved PARP proteins in CCRF-CEM cells treated with the combination. In summary, ENMD-1198 alone, and in combination with VCR, has shown promising results in the treatment of preclinical models of leukemia.