bluuebunny/crossref_metadata_2025 huggingface dataset:
Disruption of Gamma Interferon Signaling through the STAT1 Pathway Enhances Alloreactivity While Abrogating GvHD
Authors: Capitini Christian M. , Larabee Shannon M. , Mackall Crystal L. , Fry Terry J.
Published: October 2019

BACKGROUND: Strategies that allow for sufficient alloreactivity to mediate graft-versus-tumor (GVT) effects after allogeneic bone marrow transplant (BMT) while minimizing graft-versus-host-disease (GVHD) remain elusive. Historically, gamma interferon (IFNγ) has been implicated as a critical mediator of both GVT and GVHD pathophysiology, and high levels of IFNγ have been implicated in immunosuppression. Given that immunosuppression is also a hallmark of GVHD, we hypothesized that modulation of IFNγ signaling could have important effects on both GVHD and GVT following BMT. We therefore compared immunocompetence to tumor challenge and tumor vaccination, in the presence or absence of IFNγ receptor (R1) and STAT1 signaling (a primary downstream mediator of IFNγ signaling) on donor bone marrow.

METHODS: We utilized a clinically relevant, minor histocompatibility antigen-mismatched, T cell-depleted BMT model, with delayed donor lymphocyte infusions (DLIs) into thymectomized mice to ensure that T cell recovery occurred exclusively from the DLI, as occurs in most clinical settings during the first 6 months post-BMT. Lethally irradiated female C57BL/6 (B6) × C3H.SW recipients were engrafted with normal B6, B6 IFNγR1-deficient, normal B6129, or B6129 STAT1-deficient bone marrow on day 0 and reconstituted with donor T cells on days 14 and 28 at a dose that induced sublethal GVHD, evidenced by weight loss, histology, and abrogated quantitative immune responses to an HY-directed tumor vaccine. Recipients were then challenged on day 42 with an HY-expressing tumor and followed for survival. Immune reconstitution was assessed by flow cytometry both prior to and after DLI administration.

RESULTS: Mild GVHD prevents vaccine-mediated immune responses and HY-expressing tumor protection in thymectomized recipients. Both IFNγR1 and STAT1-deficient marrow abrogate GVHD. STAT1-deficient marrow increased absolute numbers of splenic plasmacytoid dendritic cells (CD11cint/Class II+/Gr-1+/B220+) prior to DLI administration. In addition, an increase in the percentage and absolute number of splenic regulatory T cells was observed at the time of tumor challenge. Surprisingly, despite the immunosuppressive milieu, recipients of IFNγR1-deficient marrow had enhanced vaccine-mediated tumor protection as measured by overall survival, similar to syngeneic BMT controls.

CONCLUSION: IFNγ signaling is a critical mediator of GVHD pathophysiology, including the immunosuppression associated with this condition. Disrupting IFNγ signaling through the JAK1/STAT1 pathway is a novel strategy to selectively modulate alloreactivity, and results in enhanced anti-tumor immune responses following allogeneic BMT without exacerbating GVHD.
Active Immune Gene Therapy Using ISF35: Responses Associated with Priming for Death Receptor-Induced Apoptosis and Sensitivity to Fludarabine in Patients with CLL and Del 17p
Authors: Wierda William G , Castro Januario , Aguillon Robier A , Sampath Deepa , Jalayer Annette , Prussak Charles , Keating Michael J , Kipps Thomas J
Published: October 2019

CLL cells can be transduced with adenovirus vector to express CD40-ligand (CD154). Ad-ISF35 is the Ad-vector that encodes a novel molecule capable of ligating CD40 on CLL cells. We conducted a phase I clinical trial to evaluate tolerability, toxicities, and clinical activity of a single infusion of autologous ISF35-transduced CLL cells. Doses of 1×108, 3×108, and 1×109 transduced cells were administered. Three patients (pts) were treated at each dose level. All pts had active disease with an NCI-WG ‘96 indication for treatment, 2 were previously untreated and 7 were previously treated. Four of the previously treated pts had del 17p by FISH prior to ISF35. Pts experienced Grade 1–2 flu-like symptoms that resolved within 2–3 days. Infusions were associated with reduction in leukemia counts and lymph node size at all dose levels; there was no dose-response. These responses were associated with in vivo apoptosis of CLL cells demonstrated by Annexin V staining, including cells collected form pts with del 17p. In vivo phenotypic and biochemical changes occurred in bystander leukemia cells of treated pts, again including cells collected from pts with del 17p. These changes included increased expression of CD95 (FAS) and death receptor 5 (DR5). A notable biochemical change included de novo expression of bid, including in cells from pts with del 17p, which lasted several weeks. Bid links the intrinsic and extrinsic apoptosis pathways and facilitates death-receptor-induced killing. Leukemia cell sensitivity to purine analogue-based therapy can be increased following expression of p73 in CLL cells with del 17p or mutated TP53. We demonstrated that ISF35 induced in vivo expression of p73 following infusion of transduced cells. Treatment of patients with del 17p with purine-analogue based regimens resulted in responses, including complete remissions. Increases in T cell counts were also observed at all dose levels; there was no dose-response relationship. T-cell increases peaked at 1–4 wks post-infusion. These results demonstrate that Ad-ISF35-transduced autologous leukemia cells can be given safely up to 1×109 transduced cells, without dose limiting toxicities, and resulting in phenotypic and biochemical changes in bystander cells in vivo that render them able to present antigen and primes them for death-receptor induced apoptosis.
Generating of Human CD4+ and CD8+ T Lymphocytes toward NY-ESO-1 by T Cell Receptor (TCR) Modification and Transduction for Adoptive TCR Gene-Transfer
Authors: Krönig Holger , Hofer Kathrin , Sommermeyer Daniel , Peschel Christian , Uckert Wolfgang , Bernhard Helga
Published: October 2019

The Cancer Testis (CT) antigen NY-ESO-1 is one of the most immunogenic cancer antigens eliciting strong humoral and cellular immune responses in tumor patients and therefore it is a promising candidate antigen for successful adoptive T cell transfer. The aim of our studies is the transfer of autologous T cells re-directed towards CT antigens by T cell receptor (TCR) gene transfer. The first precondition for genetic transfer of CT-Ag-specific TCRs is the availability of tumor-reactive CD4+ and CD8+ T cell clones that express a CT-Ag-specific TCR. Therefore, we generated the autologous CD8+ T cell clone ThP2 through stimulating HLA-A2.1− PBMCs with autologous HLA-A2+DCs loaded with synthetic NY-ESO-1157–165. After two restimulations, FACS-sorting and cloning, the T cell line specifically recognized the NY-ESO-1157–165 peptide and also specifically lysed NY-ESO-1157–165 expressing tumor cells. In addition, we generated NY-ESO-1 specific T helper1 clones from HLA-DR1+ and HLA-DR4+ healthy donors by stimulation of CD4+ T cells with autologous dendritic cells (DC) pulsed with the NY-ESO-187–111 peptide. The specificity of CD4+ T helper cell clones was determined by proliferation assays and IFN gamma ELISPOT through screening with the NY-ESO-187–111 peptide. By limiting dilution of the NYESO- 1-specific T cell populations we succeeded to isolate CD4+ T cell clones, which recognized NY-ESO-1-pulsed target cells and DCs pulsed with NY-ESO-1 protein. The second precondition for TCR gene transfer is the availability of efficient vector systems. Using vectors based upon mouse myelo-proliferative sarcoma virus (MPSV), it was possible to achieve a high transgene expression in the TCR-transduced T cells. Therefore, we cloned the TCR of the HL-A2-restricted NY-ESO-1-specific CTL clone ThP2 in the retroviral vector and documented the correct expression of the TCR-chains using peptide/HLA-multimers following retroviral transduction of peripheral PBMCs. Moreover, the NY-ESO-1 specific lysis of HLA-A2+ NY-ESO-1+ tumor cell lines after transduction in primary T cells was as well effective as the primary T cell clone. Because the expression of naive transgenic T cell receptors in recipient human T cells is often insufficient to achieve highly reactive T cell bulks we modified the TCR of the ThP2 CTL clone by, murinisation, codon optimalization or by introducing cysteins into the constant regions. Afterwards we compared the expression efficiency of the three different modifications on naive T cells by tetramer-staining. We were able to show that codon optimalization leads to an increase in the expression levels of the transgenic TCRs in human CD8+ T cells. The next step is the development of T cell transfer regiments, which are based on class-II-restricted TCR-transduced T cells.
Efficient Gene Marking with a Clinical Gammaretrovirus Vector Expressing MGMTP140K in Baboons and Macaques
Authors: Beard Brian C , Welsh Megan L , Trobridge Grant D , Kiem Hans-Peter
Published: October 2019

Using gene therapy to protect hematopoietic stem cells (HSC) from alkylating agents used in the treatment of malignant disease is an attractive strategy to alleviate prolonged neutropenia and thrombocytopenia that is dose-limiting. Both adult and pediatric patients with glioblastomas urgently need improved therapeutic strategies since even with aggressive treatment the median survival after diagnosis is approximately 12 months. Chemotherapy with the nitrosourea BCNU, the methylating agents procarbazine or temozolomide, and other alkylating agents is effective and can prolong survival but the therapeutic benefit is attenuated due to hematopoietic toxicity which limits dose-escalation of these drugs. We have previously demonstrated in the dog and non-human primate model efficient gene marking, in vivo selection and chemoprotection from temozolomide and BCNU following transplantation with MGMTP140K gene-modified cells. Thus, we wanted to explore the efficacy of autologous transplantation in macaques and baboons using cells gene-modified with an MLV-based gammaretrovirus vector developed for a pending clinical trial. This retroviral vector contains a myeloproliferative sarcoma virus LTR, negative control region deleted, dl587rev primer binding site (MND) vector backbone, which expresses MGMTP140K from the 5′ LTR promoter, and is pseudotyped with the gibbon ape leukemia virus (GALV) envelope produced from Phoenix-GALV packaging cells (PhGALV-MND.GRS.P140K c38). Following transduction the gene marking in pre-infusion colony forming units (CFUs) was 74.3% and 69.8% in the macaque and baboon respectively as determined by CFU-PCR. Intracellular MGMT-staining of cultured baboon cells 4 (76.4%) and 11 (89.9%) days after transduction confirmed high gene transfer levels. Both animals recovered neutrophil and platelet levels within expected time frames relative to historical controls and the average provirus copy number determined by real-time PCR approximately one month after transplantation was 0.14 and 0.72 in the macaque and baboon respectively. Retrovirus integration site analysis in the macaque 90 days after transplantation, and before chemotherapy, confirmed polyclonal hematopoietic reconstitution. There was no indication of progression to a pre-leukemic state. The macaque has been treated twice with O6-benzylguanine (O6BG) and BCNU and the gene marking has stably increased approximately 2.5-fold. Aside from transient elevated liver enzymes following O6BG/BCNU treatment no additional extra-hematopoietic toxicity has been observed. In summary, we have been able to achieve efficient polyclonal gene marking with MGMTP140K gene-modified cells using a vector designed for clinical application in both macaques and baboons and have preliminary evidence of in vivo selection in the macaque. We believe that these large animal studies closely reflect a clinical setting and will help to further improve clinical HSC gene therapy.
MicroRNA Expression in Macrophages-Derived Microvesicles May Contribute to Cellular Survival and Differentiation
Authors: Ismail Noura , Marsh Clay B. , Hunter Melissa
Published: October 2019

Microvesicles (MV) (also know as exosomes) are small membrane-bound vesicles released by numerous cell types that contain proteins, mRNA and microRNA. We found that MV from activated monocytes drove survival and differentiation in naïve cells. We therefore were interested in understanding the content of MV produced by activated mononuclear phagocytes. Purified peripheral blood monocytes were treated in vitro for 24 h with or without the monocyte survival factors, GM-CSF or M-CSF, respectively. Examination of monocytes and macrophages by electron microscopy or culture supernatants by flow cytometry demonstrated that monocytes produced MV, which quantitatively increased upon differentiation. Treatment with GM-CSF resulted in more MV production than M-CSF-treated monocytes. To examine whether MV from differentiated cells induced myeloid maturation, the MV were collected and added to fresh monocytes; only MV derived from GM-CSF treated cells induced differentiation of naïve monocytes into macrophages. We next hypothesized that expression of microRNA contained in the MV modulated differentiation of monocytes. Profiling of MV from GM-CSF and M-CSF derived macrophages revealed only two significantly expressed microRNAs. We found that mir-155 was significantly elevated by two-fold in MV from GM-CSF-treated cells, while mir-340 was significantly increased seven-fold in M-CSF-derived MV. Notably, mir- 223 was the highest expressed microRNA in MV from both GM-CSF and M-CSF-treated cells. Recent data suggest that expression of mir-223 regulates myeloid, granulocytic and osteoclasts differentiation, and has a role in hematopoietic stem cell proliferation. While mir-223 is present in MV from both GM-CSF and M-CSF treated cells, it is possible that the low abundance of MV produce from M-CSF-treated cells resulted in less effective concentration to induce differentiation. In this model, it is also possible that regulation of proteins targeted by the increase in mir-155 and decrease mir-340 in the GM-CSF-derived MV are responsible for myeloid differentiation. Since changes in microRNA expression including mir-223 has been reported in AML, our data suggest that myeloid-derived MV in the peripheral blood containing mir-223 may be altered contributing to leukemogenesis.
Recreating a Human Hematopoietic Stem Cell Niche in Vitro by Unique Stroma Cells from the First Trimester Human Placenta and Fetal Liver
Authors: Magnusson Mattias , Romero Melissa , Prashad Sacha , Van Handel Ben , Aivio Suvi , Huang Andy , Pellegrini Matteo , Mikkola Hanna
Published: October 2019

Expansion of human hematopoietic stem cells (HSCs) ex vivo has been difficult due to limited understanding of their growth requirements and the molecular complexity of their natural microenvironments. To mimic the niches in which human HSCs normally develop and expand during ontogeny, we have derived two unique types of stromal niche cells from the first trimester human placenta and the fetal liver. These lines either support maintenance of multipotential progenitors in culture, or promote differentiation into macrophages. Impressively, the supportive lines facilitate over 50,000-fold expansion of the most immature human HSCs/progenitors (CD34+CD38-Thy1+) during 8-week culture supplemented with minimal cytokines FLT3L, SCF and TPO, whereas the cells cultured on non-supportive stroma or without stroma under the same conditions differentiated within 2 weeks. As the supportive stroma lines also facilitate differentiation of human hematopoietic progenitors into myeloid, erythroid and B-lymphoid lineages, we were able to show that the expanded progenitors preserved full multipotentiality during long-term culture ex vivo. Furthermore, our findings indicate that the supportive stroma lines also direct differentiation of human embryonic stem cells (hESC) into hematopoietic progenitor cells (CD45+CD34+) that generate multiple types of myeloerythroid colonies. These data imply that the unique supportive niche cells can both support hematopoietic specification and sustain a multilineage hematopoietic hierarchy in culture over several weeks. Strikingly, the supportive effect from the unique stromal cells was dominant over the differentiation effect from the non-supportive lines. Even supernatant from the supportive lines was able to partially protect the progenitors that were cultured on the non-supportive lines, whereas mixing of the two types of stroma resulted in sustained preservation of the multipotential progenitors. These results indicate that the supportive stroma cells possess both secreted and surface bound molecules that protect multipotentiality of HSCs. Global gene expression analysis revealed that the supportive stroma lines from both the placenta and the fetal liver were almost identical (r=0.99) and very different from the non-supportive lines that promote differentiation (r=0.34), implying that they represent two distinct niche cell types. Interestingly, the non-supportive lines express known mesenchymal markers such as (CD73, CD44 and CD166), whereas the identity of the supportive cells is less obvious. In summary, we have identified unique human stromal niche cells that may be critical components of the HSC niches in the placenta and the fetal liver. Molecular characterization of these stroma lines may enable us to define key mechanisms that govern the multipotentiality of HSCs.
Poor Outcome for Children and Adolescents with Progressive Disease or Relapse of Lymphoblastic Lymphoma - a Report of the BFM Group
Authors: Burkhardt Birgit , Reiter Alfred , Landmann Eva , Lang Peter J. , Lassay Lisa , Dickerhoff Roswitha , Lakomek Max , Henze Guenter , von Stackelberg Arend
Published: October 2019

Background/Objectives: Little is known about the outcome of pediatric patients with lymphoblastic lymphoma (LBL) who suffer form progressive disease or relapse.

Patients and Methods: We analyzed the pattern of LBL-relapses after current NHL-BFM-frontline therapy between 4/90 and 3/03. Relapse therapy was according to ALL-Relapse-BFM protocols or ALL-BFM protocols for high-risk patients.

Results: 28 of 251 registered T-LBL-patients (11%) and six of 73 pB-LBL-patients (8%) suffered from relapse. Of the 28 T-LBL-patients, one died from infection during relapse-chemotherapy, 18 failed to achieve stable remission and died from disease-progression, and nine reached allogeneic stemcell transplantation (SCT). Two of these nine SCT patients died from transplantation-associated toxicity, three died from disease-progression and four are still alive. The patients are in 2nd remission of their lymphoma for 48, 68, 125 and 131 months respectively after allogeneic SCT. One of the four patients developed colon adenocarcinoma 47 months after SCT. Of the six relapsed pB-LBL patients one died due to toxicity of relapse-chemotherapy, two died from disease-progression after chemotherapy and three received allogeneic SCT. Of these, two died from subsequent disease-progression while one patient is still alive 57 months after allogeneic SCT.

Conclusions: Outcome of LBL-patients with relapse during or after current intensive 1st line therapy is poor. More than 50% of these patients failed to achieve remission to intensive 2nd line chemotherapy. Consolidation by allogeneic SCT may offer a cure for those who reach second remission.
Treatment of Primary CNS Lymphoma in the Elderly, a Multicenter Retrospective Analysis
Authors: Bromberg Jacoline E.C. , Doorduyn Jeanette , Schuurmans Macha , Poortmans Philip , Taphoorn Martin J.B. , Seute Tatjana , Dekker Adriaan W. , Enting Roelien H. , van Imhoff Gustaaf , van Norden Yvette , van den Bent Martin
Published: October 2019

Patients with primary CNS lymphoma (PCNSL) are preferably treated with high-dose Methotrexate (HD-MTX)-based chemotherapy followed by consolidation radiotherapy in many centers. As elderly patients have an increased risk of complications with this approach, they are frequently treated with chemotherapy or radiotherapy alone. Little is known about the efficacy and toxicity of either of these treatments in elderly patients outside clinical studies. We analysed all patients aged 60 or above referred with PCNSL to 5 Dutch centers between 1998 and 2007. 110 patients were identified. We excluded: patients who were not treated because of a poor condition (n=25), patients with EBVrelated NHL (n=3), patients with PCNSL confined to the eyes (n=3), and patients with missing information on follow-up (n=5). The remaining 74 patients had a median age of 65 years (range 60–82), and a median KPS of 70% (range 30–100). Twenty-nine of them were treated with radiotherapy only, 19 with chemotherapy only and 26 with both; 19 of these 26 received radiotherapy after failure of chemotherapy. Median KPS was 70 in both single treatment modality groups and 80 in the group receiving both modalities. The response rate (CR or PR) was 69% (20/29) in patients treated with radiotherapy only and 63% (12/19) in patients treated with chemotherapy only. Timing of both response evaluation and radiotherapy after chemotherapy were highly variable in the group treated with both modalities, therefore these patients are only included in the overall survival analyses. Median overall survival (OS) was 20 months: 7 months for patients treated with radiotherapy only, 23 months for those treated with chemotherapy only, and 31 months for combined modality treatment (p=0.01). The KPS was a significant prognostic factor for as well PFS as OS (p<0.001): median PFS and OS were 3 and 4 months respectively in patients with KPS < 70 and 18 and 25 months in patients with a KPS ≥ 70. Forty of the 45 patients receiving chemotherapy were planned for treatment with a MTX dose of 3g/m2. There were 2 toxic deaths. Ten of the 40 patients received delayed or reduced doses, or aborted chemotherapy because of toxicity. Delayed encephalopathy was reported in 15 patients: 7/30 patients after radiotherapy, 1/19 after chemotherapy only and 7/26 after combined treatment. Five died as a consequence of the encephalopathy.

Conclusion: Performance status is the most important single variable determining prognosis in elderly patients. Overall survival after HD-MTX-based treatment for PCNSL in the elderly appears to approach survival obtained in younger patients, provided the performance status is adequate. Treatment related mortality of HD-MTX-based chemotherapy seems not to be increased in older patients.
Inhibition of Cdk4/6 by PD 0332991 Results in Abrogation of Osteoclast Formation
Authors: Feng Rentian , Huang Xiangao , Roodman G. David , Chen-Kiang Selina , Lentzsch Suzanne
Published: October 2019

Multiple myeloma (MM) is characterized by increased osteoclast activity resulting in bone destruction and development of lytic lesions. Despite overall survival achieved by new treatment modalities, new drugs are required to specifically and effectively inhibit bone destruction. PD 0332991 is a selective small molecule inhibitor of cyclin-dependent kinase (Cdk)4 and Cdk6 with oral bioavailability. We have demonstrated recently that inhibition of Cdk4/6 by PD 0332991 effectively controls MM tumor expansion in animal models and sensitizes chemoresistant MM tumor cells to bortezomib killing despite protection by bone marrow stromal cells. Currently clinical phase I/II trials are ongoing to test the efficacy of the combination of PD 0332991 and bortezomib.

Selective inhibition of Cdk4/6 by PD 0332991 leads to sustained G1 arrest but not apoptosis, and this appears to be cell type-specific. Consistent with these findings, PD 0332991 is well tolerated in MM patients. On this basis, we address the possibility that PD 0332991, alone and in combination with bortezomib, may inhibit osteoclastogenesis, by culturing non-adherent mononuclear bone marrow cells from MM patients cultured in the presence of M-CSF and RANKL for three weeks. Treatment of human osteoclast cultures with PD 0332991 for 3 weeks decreased osteoclast formation in a dose-dependent manner with an IC50 50 nM. The combination of PD 0332991 and bortezomib led to synergistic inhibition of osteoclast formation and completely abrogated osteoclastogenesis with low dose PD 0332991 (25 nM) and bortezomib (2 nM). Furthermore, we show in a time course study that treatment with PD 0332991 for the first week, but not the second or third week, was sufficient to inhibit osteoclast formation. PD 0332991 is the only known selective inhibitor of Cdk4 and Cdk6, which at concentration below 5 uM does not cross react with at least 38 kinases or induce apoptosis. Taken together, our data suggest that by inducing G1 arrest and inhibiting progenitor expansion, PD 0332991 is a powerful and selective inhibitor for osteoclastogenesis. We propose that targeting Cdk4/6 with PD 0332291 in combination therapy is a promising therapeutic strategy to improve bone integrity in MM.
Thalidomide + Dexamethasone as Maintenance after Single Autologous Stem Cell Transplantation Improves Progression-Free Survival (PFS) in Advanced Multiple Myeloma. A Prospective Brazilian Randomized Trial
Authors: Maiolino Angelo , Hungria Vania T. , Oliveira-Duarte G. , Oliveira LC , Mercante DR , Miranda Ecm , Quero A. , Peres AL Miguel , Barros Jc , Tanaka P. , Magalhães RP , Rego Eduardo M. , Nucci M. , Lorand-Metze Irene , Lima CSP , Zalcberg I. , Braggio E. , De Souza Carmino
Published: October 2019

Introduction: Autologous stem cell transplantation (ASCT) remains the mainstay of treatment of multiple myeloma (MM) in patients <65 years old. However, most patients relapse after ASCT suggesting that additional treatment is needed. The Brazilian Multiple Myeloma Group designed a study to evaluate the impact of thalidomide maintenance after ASCT.

Methods: From October 2003 to July 2008, 212 untreated patients <70 years old were enrolled in a prospective randomized multicenter study. All patients signed an informed consent and the protocol was approved by the Ethical Committees of each center. The treatment consisted of 3 phases:

induction with 3–5 cycles of VAD; high-dose cyclophosphamide (4g/m2) plus G-CSF for stem cell mobilization;

(3) melphalan 200 mg/m2 and ASCT. On day +60 post ASCT patients were randomized to receive dexamethasone (40 mg/d × 4 days every 28 days) with (arm A) or without (arm B) thalidomide (200 mg daily) for 12 months or until disease progression.

Results: The median age was 55 years (27–70), 52% were male, the median serum beta-2 microglobulin was 3.66 mg/dl, 33% were ISS stage 3, 36% were ISS stage 2 and 24% had deletion of chromosome 13. In July of 2008, 93 patients (44%) were randomized: 54 in arm A and 39 in arm B. Reasons for non-randomization were: treatment related deaths during phases 1–3 (n= 39), disease progression (n= 22), ineligible or refused ASCT (n= 7), SMD after ASCT (n= 1), protocol violation (n= 3), abandoned (n= 19), and still in phases 1–3 (n= 28). Clinical characteristics of each group were similar. The median follow-up from diagnosis was 15 months. PFS in arms A and B were 42% (95% confidence interval [CI] 22–62) and 25% (95% CI 5–45), p= 0.07. A multivariate analysis that included baseline serum beta-2-microglobulin and deletion of chromosome 13 showed that maintenance with thalidomide was significantly associated with better PFS (hazard ratio 2.43, 95% CI 1.10–5.35, p=0.03). Overall survival was 65% in arm A (95% CI 35–95) and 74% in arm B (95% CI 44–100), p= NS.

Conclusions: A high proportion of MM in Brazil has advanced disease at diagnosis, and this explains the high number of patients who did not reach the maintenance phase. This study shows that the addition of thalidomide to dexamethasone improves PFS after a single ASCT.
Lenalidomide as Salvage Therapy after Allogeneic Stem′Cell Transplantation in Multiple Myeloma
Authors: Lehmann Friederike , El-Cheikh Jean , Zabelina Tatjana , Ayuk Francis , Wolschke Christine , Atanackovic Djordje , Bacher Ulrike , Zander Axel R. , Blaise Didier , Mohty Mohamad , Kroeger Nicolaus
Published: October 2019

Lenalidomide is an immunmodulatory drug, orally administered once daily, which is effective in relapsed multiple myeloma (MM). Here, we evaluated the efficacy and toxicity of lenalidomide in 24 MM patients with relapsed disease after allogeneic stem cell transplantation (allo-SCT). Median age was 59 (range, 37–70) years. The series included 8 females and 16 males. Prior to allo-SCT, all patients were heavily pretreated with a median of 6 chemotherapy cycles including autologous and allo-SCT in all patients. Also, prior to lenalidomide, salvage treatment included donor lymphocyte infusions (DLI) in 18 pts,, thalidomide in 11 pts and bortezomib in 13 pts. Lenalidomide was given at 15mg (n=4) or 25 mg (n=20) orally once daily on day 1–21 every 28 days and in 20 patients in combination with dexamethason.. No prophylactic anticoagulation was used. The median number of completed cycles was 5 (range 2–17). Myelotoxicity according NCI criteria was the primarily and major encountered side effect (leukopenia: 4% grade 4, 21% grade 3, 17% grade 2, thrombopenia: 17% grade 3, 29% grade 2) and led to dose reduction in 54% of the patients. Infectious complications were observed in 50%. Non-hematological toxicity consisted of cramps (n=9), fatique (n=5) and constipation (n=2). Thrombembolic complications (cerebral infarction) were observed in one patient, who received concomitant corticosteroid treatment for acute graft-vs.-host disease (GvHD), but neurological symptoms resolved completely. GvHD of the skin under lenalidomide treatment was seen in 3 patients (one grade 2, and 2 grade 1), with one case occurring shortly after an additional DLI.. Objective remission was achieved in 66% of the patients (CR: 8%, VGPR: 8%, PR: 50%) and stable disease (SD) in 13% of the patients, while in 21% progressive disease was noted. Prior treatment with thalidomide or with bortezomib did not influenced the rate of CR/PR. Surprisingly, patients with del 13q14 achieved a higher CR/PR rate than those without del 13q14 (p=0.02). The median time to progression was 9.7 months (95% CI: 7.5–11.9) and the median overall survival was 19.9 months (95% CI: 17.3–22.5). Lenalidomide is effective in relapsed patients with MM after allo-SCT. Major toxicity is myelotoxicity, which required dose reduction in the majority of patients. The optimal dose of lenalidomide after allo-SCT has to be investigated.
In Vitro and in Vivo Inhibition of JAK2 Signaling by Potent and Selective JAK2 Inhibitor
Authors: Markovtsov Vadim , Tonkin Elizabeth , Fang Shuling , Liu Chiang , Gelman Marina , Lang Wayne , Romero Jason , McLaughlin John , Bhamidipati Somasekhar , Clough Jeffrey , Singh Rajinder , Low Caroline , Reitsma Andrea , Taylor Vanessa , Siu Stacey , Park Gary , Sweeney David , Pine Polly , Torneros Allan , Clemens George , Duan Matt , Daniel Ruby , Payan Donald , Holland Sacha , Hitoshi Yasumichi
Published: October 2019

Recent advances in understanding the role of JAK2 V617F mutation in Bcr-Abl negative myeloproliferative (MPD) diseases pathogenesis opened up a possibility to develop highly targeted therapies against these debilitating ailments. We used a Ba/F3 cell line expressing the V617F mutant of JAK2 to screen a focused small molecule library for potential inhibitors of JAK2 V617F-dependent proliferation. Further extensive SAR of initial hits resulted in identification of R723, a potent and selective JAK2 inhibitor. This molecule is strongly antiproliferative (IC50 130–200 nM) against mouse BaF3 cells used for initial screening as well as against human UKE1 and SET2 cell lines harboring the same mutation. On the other hand, R723 has only weak activity in IL2-dependent (i.e. JAK1/JAK3-dependent) proliferation assays performed with human primary T (IC50 1300 nM) and mouse T-cell leukaemia CTLL2 cells (IC50 600 nM). A 10 to 20 fold cell-based selectivity of R723 was further confirmed by measuring inhibition of constitutive STAT5 phosphorylation in SET2 and BaF3 cells versus inhibition of IL-2 inducible STAT5 phosphorylation in human primary T and mouse CTLL2 cells using FACS-based approach. Compound R723 has low nonspecific antiproliferative activity against JAK2-independent MOLT4, A549 and H1299 cell lines with an IC50 ranging from 4 to 6 uM. The molecule has been also proven to be potent (IC50 of 2 nM against JAK2 in biochemical assay) and highly selective (window of more than 500 fold over JAK1 and 10 fold over JAK3) inhibitor of JAK2 kinase in vitro. Moreover, when tested in biochemical assay against a panel of more than 200 kinases at a concentration of 20 nM (IC90 for JAK2), R723 inhibited none of them. The selectivity of R723 was further confirmed using a variety of cell-based assays probing T-, B- and mast cell activation. Compound R723 was further evaluated in a stress-induced erythropoiesis mouse model, where kinetics of EPO-dependent hematocrit recovery from phenylhydrazine-induced anemia was assessed. Significant delay in recovery was observed at doses of 75 and 100 mg/kg bid indicating strong compound effect on EPOR signaling in vivo. The result could not be attributed to general toxicity effects as 14 day toxicology study did not identify any abnormalities at doses tested. As a result, R723 could become the basis for next generation of potent and selective compounds targeting JAK2-dependent myeloproliferative diseases.
Differential Biological Properties and Signaling Effects of Different JAK2 Alleles
Authors: Mohi M. Golam , Yan Dongqing , Zou Haiying
Published: October 2019

JAK2, a member of the Janus family of non-receptor protein tyrosine kinases, acts as an important mediator of cytokine receptor signaling and plays a critical role in hematopoiesis. Somatic mutations in JAK2 have been identified in a spectrum of myeloproliferative disorders (MPD). A specific point mutation (V617F) in the pseudokinase domain (JH2) of JAK2 has been identified in most patients with polycythemia vera (PV) and 50–60% patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF). Although JAK2V617F is the predominant allele associated with PV, ET and PMF, other activating alleles of JAK2 also have been identified in distinct MPD. Somatic mutations in the exon 12 (linker region between JH2 and JH3) of JAK2 are associated with a distinct type of PV characterized by isolated erythrocytosis. Another activating mutation (T875N) in the kinase domain (JH1) of JAK2 has been identified in a cell line derived from acute megakaryoblastic leukemia (AMKL) patient, and expression of JAK2T875N in murine bone marrow induces a MPD with features of AMKL. Thus, different activating JAK2 alleles are associated with different disease phenotypes. The basis for the differences in the in vivo effects of different JAK2 alleles remains unclear. To understand the biological and signaling effects of these JAK2 mutants, we have generated Ba/F3-EpoR cell lines expressing JAK2WT, JAK2V617F, JAK2 exon 12 mutant K539L or JAK2T875N. Expression of JAK2V617F, JAK2K539L or JAK2T875N in Ba/F3-EpoR cells resulted in IL-3 independence, whereas JAK2WT failed to grow in absence of IL-3. We observed significantly increased proliferation of JAK2T875N expressing cells compared to cells expressing either JAK2V617F or JAK2K539L in the absence of cytokine. Moreover, expression of JAK2T875N caused increased transformation of Ba/F3-EpoR cells in a colony assay. Biochemical analysis revealed that the kinase activity of JAK2K539L was higher than JAK2V617F but comparable to JAK2T875N. More importantly, significant differences in signaling downstream of these JAK2 mutants were observed. Expression of JAK2T875N resulted in increased phosphorylation of Stat3, Stat5, Gab2, Akt, and Erk/MAPK and induction of Pim1 and Pim2 compared to JAK2V617F and JAK2K539L mutants. Although JAK2K539L exhibited more kinase activity than JAK2V617F but it was less potent in activation of downstream signaling molecules, such as Stat5, Gab2, Erk/MAPK. Variable association of mutant JAK2 with EpoR was also observed, with JAK2K539L being the most and JAK2T875N being the least associated with EpoR. These results strongly suggest that the biological properties and signaling effects of JAK2 alleles are different, and they may interact differentially with cytokine receptor, which might contribute to the differences in the disease phenotype.
Follicular Non-Hodgkin Lymphoma Grade 3a and 3b Subtypes Exhibit Similar Clinical Behaviour and Treatment Outcome
Authors: Shustik Jesse , Quinn Michael , Connors Joseph M , Gascoyne Randy D , Skinnider Brian , Sehn Laurie H
Published: October 2019

The clinical usefulness of separating follicular lymphoma (FL) grade 3 into FL grade 3A (FL3a) and 3B (FL3b) subtypes remains controversial. The relative aggressiveness of these two subtypes and their potential curability with anthracycline-based therapy continues to be debated. Discrepancies in the literature may be related to the relatively small number of patients in most series and to the variable inclusion of patients with a diffuse histologic component on biopsy, likely representing early transformation. We present a retrospective review of clinical characteristics and outcome in a large cohort of patients with FL3a and FL3b, as strictly defined by WHO diagnostic criteria.

Patients Patients diagnosed with FL grade 3 in the province of British Columbia (BC) between the years 1982 and 2008 were identified through the BC Cancer Agency Lymphoid Cancer Database. A total of 161 patients with pathology available for central review were included for analysis, and were categorized as FL3a (n=139) or FL3b (n=22) using current WHO criteria. Cases containing a diffuse component on biopsy were excluded.

Results Median age of the entire cohort was 63 years (range, 18–88). There were no significant differences in age, sex, stage, serum LDH levels, or number of extranodal sites between the subgroups. However, there was a trend toward worse performance status in patients with FL3b compared with FL3a (PS 2–4 27% vs. 12%, respectively; p=0.054). Overall, more patients in the FL3b subgroup had a high-intermediate or high-risk IPI score compared with patients in the FL3a subgroup (36% vs. 17%, p=0.028). A significantly higher number of patients with FL3b received an anthracycline-based regimen as front-line therapy (82% vs. 36%, p <0.001) and approximately one third of the entire cohort received rituximab. With a median follow-up of 38 months (range, 1–224), no difference in overall survival (OS) (p=0.79) or disease-specific survival (DSS) (p=0.38) was observed between FL3a and FL3b patients (see Figure below) (5-year OS, 72% vs. 70% and 5-year DSS, 77% vs. 70%, respectively). Analysis limited to the FL3a subgroup showed no OS advantage for patients who received an anthracycline-containing regimen as initial therapy compared with those who did not (p=0.24).

Conclusions Overall no significant differences in clinical outcome were noted between patients with FL3a and FL3b. No plateau in the survival curves was seen, suggesting that neither subtype is curable even with the use of anthracycline-based therapy. This large retrospective analysis, which expands on an earlier cohort from the province of British Columbia, calls into question the clinical relevance of this histological grading designation.

Figure Figure
High-Throughput Sequencing for Diagnosis, Prognosis and Monitoring of Lymphoid Malignancies
Authors: Boyd Scott D , Merker Jason D. , Zehnder James L. , Fire Andrew Z
Published: October 2019

New high-throughput DNA sequencing methods have many advantages for the analysis of rearranged immunoglobulin (Ig) and T cell receptor (TCR) loci in suspected lymphoid malignancies. Instead of primarily analyzing the size distribution of rearranged immune receptors using gel or capillary electrophoresis, sequencing complex nucleic acid pools provides much more complete characterization of the receptor sequences present. Currently, separate assays are used to evaluate Ig and TCR clonality, Ig hypermutation status, and to test for residual malignant cells after treatment. We used multiplexed PCR amplification of immunoglobulin heavy chain loci for high-throughput pyrosequencing with the Roche 454 platform to evaluate the detection of clonal IgH sequences, determination of hypermutation status, and sensitivity of detection of low levels of a known clonal receptor. Our method also evaluated the feasibility of physical “bar-coding” of DNA molecules derived from different samples to enable cost-effective sequencing of sample pools. 23 pooled specimens were sequenced including: known lymphoma specimens (4); peripheral blood leukocytes from normal individuals (4); specimens with indeterminate or discordant results by conventional clonality assays (7); and a dilution series of a clonal CLL/SLL specimen into normal peripheral blood leukocytes. Our initial experiment characterized 522,099 IgH sequences from these specimens, with a mean read length of 242 base pairs. We have employed IgBlast (NCBI) for initial alignment of rearranged IgH sequences to germline V, D and J gene segments. Our data demonstrate that clonal IgH populations can be readily detected and characterized as to their germline or hypermutated status and unique V-D and D-J junctions with this approach. In our dilution series experiment, the clonal receptor sequence could be reliably observed after 1000-fold dilution. Normal patient specimens show expected extensive sequence diversity, and we are exploring methods for optimally summarizing and representing these data. Some PCR artifacts, as well as non-immunoglobulin sequences commonly amplified by current IgH primer sets were also detected, and suggest avenues for further improvement of the methods used for detection and characterization of clonal immune receptors. More broadly, these methods also offer a novel approach to monitoring normal immune responses such as the response of patients to vaccination, and of characterizing the immune system in patients with autoimmune diseases.
LEF1 Is a Tumor Suppressor in T Cell Acute Lymphoblastic Leukemia
Authors: Gutierrez Alejandro , Sanda Takaomi , Winter Stuart , Larson Richard S. , Silverman Lewis B. , Hunger Stephen P. , Sallan Stephen E. , Look A. Thomas
Published: October 2019

In an effort to further unravel the molecular pathogenesis of T cell acute lymphoblastic leukemia (T-ALL), we performed array CGH on diagnostic specimens from 47 pediatric patients with T-ALL. The LEF1 transcription factor is best known as a positive mediator of oncogenic β-catenin signaling, and it is required for the expression of MYC, Cyclin D1 and Survivin in some contexts, including some normal hematopoietic progenitors. Additionally, Lef1 has been shown to be required for the survival of murine Notch-dependent T cell lymphoma cells. We were thus surprised to find highly focal deletions of LEF1 in 10.6 % (n = 5 of 47) of primary T-ALL patient samples, with 3 cases harboring homozygous deletions and 2 harboring heterozygous deletions of this locus. These deletions involved no other known genes. We then sequenced this gene in 45 of these cases and found heterozygous mutations in 3 additional T-ALL samples, including 2 cases with frameshift mutations predicted to truncate the protein prior to its context-dependent activation and HMG box domains, and one case with an Asp85Asn substitution. Seven of the eight samples harboring LEF1 abnormalities also had mutations in the heterodimerization or PEST domains of NOTCH1. In an effort to characterize the biologic consequences of LEF1 loss in T-ALL, we took advantage of expression microarrays that were previously performed on most of these samples. LEF1 loss defines a novel T-ALL subtype characterized by arrest at the CD1-positive early cortical stage, and whose gene expression profile resembles that of HOX11-positive cases, although expression of HOX11 was generally low in these samples. Loss of LEF1 was mutually exclusive to overexpression of TAL1 and of the HOXA/MEIS1 cluster. Interestingly, LEF1-negative T-ALL cases were associated with increased expression of MYC, and gene set enrichment analysis identified a significant association between LEF1 loss and upregulation of MYC target genes. In addition to its role as a transcriptional activator in the setting of active WNT/β-catenin signaling, LEF1 can also act as a transcriptional repressor in some cellular contexts. Experiments are currently underway to establish the mechanism mediating the tumor suppressor activity of LEF1 in T-ALL.
Incidence of Cancer-Associated Venous Thromboembolism (VTE): A Population-Based Cohort Study
Authors: Ashrani Aneel A. , Heit John A. , Crusan Daniel J. , Petterson Tanya M. , Bailey Kent R. , Melton L. Joseph
Published: October 2019

Background: Active cancer without or with chemotherapy is associated with a 4- to 6.5-fold increased risk for VTE 1. However, cancer-associated VTE incidence may be under- or overestimated by reports that linked cancer registry data to an anticoagulation clinic database or to hospital discharge diagnosis codes 2–5.

Objective: To estimate the incidence of VTE in active cancer in a well-defined geographic population that includes the full spectrum of VTE events (including rapidly fatal events) occurring in the community.

Methods: Using the longitudinal resources of the Rochester Epidemiology Project and Mayo Clinic Tumor Registry, we identified all Olmsted County, MN residents with incident VTE by review of their complete medical records in the community 6, and all residents with active cancer (by cancer site), over the 28-year period, 1973–2000. Using residents with active cancer within the three months prior or subsequent to incident VTE as the numerator, and all residents with active cancer as the denominator, we estimated the incidence of VTE in active cancer, both overall and for the subsets of pancreatic cancer, brain cancer and lymphoma.

Results: Over 78,236 active-cancer person-years, 595 Olmsted County residents developed incident VTE. The overall active cancer-associated VTE incidence was 760 per 100,000 person-years (95% CI: 701, 824), about 6.5-fold higher than the general population 7. Over 473, 983 and 4,258 pancreatic cancer, brain cancer and lymphoma person-years, respectively, 51, 18 and 46 residents developed incident VTE. The pancreatic cancer-, brain cancer- and lymphoma-associated VTE incidence rates were 10,784 (95% CI: 8029, 14,179), 1,831 (95% CI: 1085, 2,893) and 1,080 (95% CI: 791, 1,441) per 100,000, respectively. Overall, cancer-associated VTE incidence increased with age, and for all ages, men had a higher VTE rate than women. We observed a much higher pancreatic cancer-associated VTE incidence than previously reported.

Conclusions: The incidence of VTE in individuals with active cancer (especially pancreatic cancer) is higher than the general population. The incidence of VTE in patients with pancreatic cancer is higher than previously estimated.