bluuebunny/crossref_metadata_2025 huggingface dataset:
The Tyrophostin Adaphostin (NSC680410) Inhibits Multiple Myeloma Bone Marrow Angiogenesis In Vitro and In Vivo.
Authors: Podar Klaus , Zhang Jing , Raab Marc S. , Vallet Sonia , Fulciniti Mariateresa , Sattler Martin , Ikeda Yutaka , Chauhan Dharminder , Anderson Kenneth C.
Published: April 2020

Our own and other previous studies demonstrate marked anti-proliferative activity of the tyrophostin adaphostin (NSC680410) in a variety of hematologic malignancies including chronic myelocytic leukemia (CML), chronic lymphcytic leukemia (CLL), acute myelocytic leukemia (AML), and Multiple Myeloma. Here we show that adaphostin (NSC680410), similar to bortezomib, additionally inhibits tumor angiogenesis within the MM bone marrow (BM) microenvironment. This effect is elicited both indirectly by inhibition of VEGF production and secretion in MM cells, as well as directly by abrogation of endothelial cell growth. Specifically, adaphostin triggers marked downregulation of nuclear c-Myc expression in MM cells. Both adaphostin, as well as specific downregulation of c-Myc using siRNA, lead to a decrease in cobalt chloride- induced Hif-1alpha- expression and Hif-1alpha activity, as evidenced by western blot analysis and expression of Hif-1alpha- driven luciferase, respectively. Indeed secretion of the Hif-1alpha target gene VEGF is markedly inhibited in a dose- and time- dependent manner. Importantly, neither knockdown of c-Abl expression nor exogenous overexpression of caspase- cleavage- induced c-Abl fragment abrogates drug- induced Hif-1alpha downregulation or inhibition of its activity. Taken together, these results indicate the existence of a c-Myc/ Hif-1alpha- dependent, but c-Abl- independent, pathway modulating MM cell production and secretion of VEGF. In contrast, we demonstrate a direct antiangiogenic effect of adaphostin on endothelial cells, similar to H2O2, is mediated via c-Jun upregulation, inhibition of cell proliferation, and the induction of cell apoptosis. Moreover, our data further demonstrate activity of adaphostin within the BM microenvironment. Adaphostin, similar to bortezomib, significantly inhibits VEGF secretion triggered by adhesion of MM cells to BMSCs and endothelial cells. Consequently, conditioned medium derived from adaphostin- treated co-cultures markedly inhibits endothelial cell growth and tubule formation in a dose- dependent manner. Finally, we confirmed these in vitro results using an in vivo xenograft mouse model of human MM. Specifically, western blot analysis, as well as immunohistochemistry, demonstrate marked downregulation of both Hif-1alpha and CD31 in tumors isolated from adaphostin- treated animals versus control animals, confirming the in vivo antiangiogenic effect of adaphostin. Similar effects were obtained using a SCIDhu mouse model as well as a significant decrease of MM- related bone disease, due to anti- VEGF activity of adaphostin. Taken together, these data provide the rationale for the clinical evaluation of adaphostin to target both MM cells and the BM milieu to improve patient outcome in Multiple Myeloma.
Activated CMRF-56 Selected Blood Dendritic Cells: A Potential Anti-Myeloma Vaccine.
Authors: Freeman Jennifer L. , Vari Frank , Hart Derek N.J.
Published: April 2020

Despite recent advances in therapy, such as stem cell transplantation and new drugs including proteosome inhibitors, multiple myeloma (MM) remains incurable for most patients. Immune therapy using dendritic cell (DC) vaccination to generate anti-myeloma cytotoxic T cell (CTL) responses targeting residual disease is a promising, potentially curative strategy that would be compatible with other therapies. Blood DC isolated using the monoclonal antibody (mAb) CMRF-56, can induce CTL responses (J. Immunotherapy. 2005; 28:322). We have established the ability of CMRF-56 blood DC to migrate in vitro and induce CTL responses to MM antigens and developed clinical scale production of the CMRF-56 blood DC preparations. Migration of CMRF-56 blood DC toward a CCL21 gradient was significantly improved by addition of granulocyte macrophage colony stimulating factor (GM-CSF) and prostaglandin E2 (PGE2) (n=5, p<0.05) and exceeded that of activated MoDC. Activation of CMRF-56 blood DC with GM-CSF also optimal for induction mature phenotype (n=4, p<0.05), survival of blood DC after 48 hours culture (n=3, p<0.01), and generation of CTL (n=5, p<0.05). Addition of PGE2 made no significant difference to CTL induction. GM-CSF activated CMRF-56 blood DC loaded with U266 lysate induced polyclonal anti-myeloma CTL responses capable of 38–61% specific lysis of U266 cells in a 51Cr release assay. To determine specificity, we examined the CTL specific for known MM tumour antigens and could identify CTL recognising Muc1 and HM1.24 HLA-A*201 epitopes in 3/3 donors tested. CMRF-56 blood DC derived from both healthy and MM donors were pulsed with HLA-A*201 restricted peptides derived from MM tumour antigens including Muc1 and HM1.24/BST2, along with novel antigens such as cancer testis antigen (CT)-7 (MAGE-C1) and B cell maturation antigen (BCMA). Results from healthy donors have shown that CTL specific for all above mentioned antigens can be induced with 4/8 responding to HM1.24, 2/8 to Muc1, 4/8 to CT7 and 1/6 to BCMA and these CTL lysed U266 cells specifically. In parallel, we have developed and validated clinical scale production of CMRF-56 blood DC preparations using clinical grade biotinylated CMRF-56 mAb and the CliniMACS system. On average over 100 million CMRF-56 blood DC were recovered from immunoselected apheresis products from both healthy (n=10) and MM donors (n=4) with average blood DC purities of 31% and 42% respectively. These results indicate that CMRF-56 blood DC can be produced on a clinical scale, can migrate toward secondary lymphoid chemokine CCL21 in vitro and can induce anti-myeloma CTL responses to both known and novel MM antigens. These data supports the use of CMRF-56 blood DC preparations loaded with peptide antigen as a potential anti-myeloma vaccine for patients with minimal residual disease.
The Novel, Orally Available Multi-Kinase Inhibitor BAY 73-4506 Targets Myeloma Cells and Their Bone Marrow Microenvironment.
Authors: Raab Marc S. , Breitkreutz Iris , Klaus Podar , Zhang Jing , Blotta Simona , Christensen Olaf , Hideshima Teru , Chauhan Dharminder , Wilhelm Scott , Anderson Kenneth C.
Published: April 2020

Multitargeted treatment approaches have been shown to be more effective than single agent therapy in multiple myeloma (MM). In addition, agents targeting not only the MM cells directly but also their microenvironment, like bone marrow stromal cells (BMSCs), endothelial cells, and osteoclasts (OCLs) causing enhancement of tumor cell growth, angiogenesis, and MM bone disease, respectively, are promising new treatment modalities for this still non-curable disease.Here we investigated the novel, orally available multi-kinase inhibitor BAY 73-4506, currently in phase I clinical trials, for its therapeutic effect in MM. BAY is a potent inhibitor of angiogenic (VEGFR 1-3, PDGFR-b), as well as oncogenic, kinases (cKIT, RET, FGFR, Raf). We first tested the ability of BAY to suppress MM cell proliferation and survival in a wide array of MM cell lines (MM.1S, RPMI 8226, NCI H929, OPM2, KMS11, KMS 18, INA6, U266, KMS12BM, S6B45), including those resistant to conventional chemotherapeutics (MM.1R, Dox40, LR5). Our data show that BAY is active in all cell lines tested in a low micromolar range equivalent to concentrations achieved in patient plasma during the first clinical trial in solid tumors. Importantly, BAY also overcomes the growth advantage conferred in a BMSC-MM, as well as an endothelial cell-MM, coculture system. BAY treatment abrogates MEK, ERK and AKT phosphorylation in a time and dose dependent manner, followed by induction of apoptosis, evidenced by Annexin staining and DNA fragmentation. Since VEGF signaling pathway is a potent inducer of angiogenesis and BAY targets VEGFR 1-3, we examined anti-angiogenic properties of BAY. This compound inhibits endothelial cell growth and endothelial cell tubuli formation in vitro at concentrations less than 1mM; moreover, BAY markedly inhibits the VEGF-induced cell migration on fibronectin. Activation of MAP kinase is a critical event during OCL differentiation, activation, and survival; BAY inhibits osteoclastogenesis, evidenced by blockade of M-CSF/RANKL-triggered differentiation of mononuclear cells to TRAP-positive osteoclasts, an important marker of osteoclastogenesis. Finally, combination treatment of BAY with dexamethasone shows synergistic effects on MM cell growth and survival. These in vitro experiments on the effects of BAY on MM tumor cells directly, in co-culture with endothelial or BMSCs, as well as on osteoclast differentiation, provides the basis for its evaluation in a murine model of human MM to confirm these promising in vitro effects of this novel multi-kinase inhibitor, finally leading to clinical evaluation to improve patient outcome.
Transgenic Expression of JAK2V617F Causes Myeloproliferative Disorders in Mice.
Authors: Xing Shu , Zhao Zhizhuang Joe
Published: April 2020

Recently, an acquired mutation of tyrosine kinase JAK2 was found in most patients with myeloproliferative disorders (MPDs) including polycythemia vera (PV), essential thrombocythemia (ET), and idiopathic myelofibrosis. We have generated transgenic mice expressing the mutation enzyme, JAK2V617F, in the hematopoietic system driven by the promoter of the vav gene. The mice are viable and fertile. One line of the transgenic mice expressed a lower level of JAK2V617F and displayed elevated blood cell counts, while the other line expressed a higher level of JAK2V617F and exhibited a marked increase in blood counts and developed phenotypes that closely resembled human ET and PV. The latter line of mice also developed marrow and spleen fibrosis as the animal aged. In general, the transgenic mice had megakaryocytic hyperplasia in the bone morrow and extramedullary hematopoiesis resulting in splenomegaly, and their serum erythropoietin level was also significantly reduced. In vitro colony assays demonstrated that transgenic mice possessed an increased number of hematopoietic progenitor cells in peripheral blood, spleen, and bone marrow and that these cells displayed hyper-sensitivity to growth factors and cytokines. The data prove that JAK2V617F is a cause of MPDs. Our study thus provides a permanent mouse system for further study to define the pathological role of JAK2V617F and to develop treatment for MPDs.
JAK2
V617F Mutational Load in Patients with Polycythemia Vera (PV) Measured by Peripheral Blood DNA Is Associated with Disease Severity.
Authors: Silver Richard T. , Vandris Katherine , Wang Y. Lynn , Christos Paul J. , Adriano Fernando , Jones Amy V. , Cross Nicholas C.P.
Published: April 2020

Different methods using peripheral blood RNA (Vannucchi AM, et al. Leukemia. 2007,1–8), or archival bone marrow DNA (Tefferi A, et al. Leukemia. 2007,1–2) have yielded varied results correlating allele burden with severity and duration of disease. We therefore aimed to determine whether JAK2V617F allele burden correlated with certain parameters of disease. At our institution, 105 patients were diagnosed according to the criteria of the Polycythemia Vera Study Group. We grouped their JAK2V617F allele burdens into quintiles. DNA from peripheral blood was analyzed using pyrosequencing. For those patients whose allele burden was <5%, a sensitive ARMS (amplification refractory mutation system) assay was performed to demonstrate the presence of the JAK2V617F allele. Duration of disease was assessed from onset of symptoms. Spleen size was measured in cm below the midpoint of the left costal margin in the midclavicular line and categorized as not enlarged, slightly (1–3 cm), moderately (4–9 cm), or grossly enlarged (>9 cm). Thrombotic events were recorded within 5 years of JAK2V617F determination. There were 52 men and 53 women. The patients ranged in age from 35 to 88 years, median 60 years. The median duration of disease was 7.4 years (range: 0.2 - 36.6 years), and the median duration of follow-up after JAK2V617F determination was 12 months year (range: 1 - 43 months). The mean mutant allele burden was 46.0% (s.d. ± 29.7%). The fifth, and highest quintile had a mean mutant allele burden of 90.2% (s.d. ± 5.8%); the lowest quintile had a mean mutant allele burden of 9.9% (s.d. ± 6.3%). JAK2V617F did not correlate with age, gender, hematocrit and platelet count at diagnosis, or rate of phlebotomy prior to cytoreductive therapy. Increasing JAK2V617F burden did correlate with higher WBC at diagnosis (P=0.02), degree of splenomegaly (P<0.0001), presence of marrow fibrosis (P=0.03), and longer duration of disease (P=0.001). There was a trend for a higher JAK2V617F allele burden among patients with venous compared to arterial thrombosis. When the subset of patients who had JAK2V617F testing performed within 5 years of diagnosis (N=35) was examined, trends similar to those we had reported for all 105 patients were found, but no definitive statement can be made because of the small sample size. We noted a JAK2V617F allele burden of more than 80% during the course of the illness was associated with a significant disease phenotype. This is the first report of increased marrow fibrosis associated with high JAK2V617F burden. The importance of using quantitative JAK2V617F for assessing allele burden is stressed because patients with an increased allele burden may be candidates for anti-JAK therapy.
Frequency of JAK2 Exon 12 Mutations in JAK2 Exon 14 V617F-Negative Patients: High Frequency in ET Patients.
Authors: Duke Veronique M. , Gurunlian Nicole , Yogashangari Bi , Colley Tom , McNamara Christopher , Hoffbrand A. Victor , Foroni Letizia
Published: April 2020

The identification of the G to T point (V617F) mutation in exon 14 of the JH2 domain of the Janus kinase 2 gene (JAK2) has recently simplified the diagnosis of many patients affected with a variety of myeloproliferative disorders (MPDs). More recently JAK2 exon 12 mutations have been described in these disorders; although the role of these mutations is as yet unclear. There have been only two investigations which have identified the presence of exon 12 mutations in clinical diagnosis among polycythaemia vera (PV) (Scott et al NEJM 2007, Pardanani et al Leukaemia 2007). A total of 725 patients diagnosed clinically with a variety of MPDs were investigated for both the frequently occurring exon 14 V617F and for the rarer exon 12 mutations.

Methods: Peripheral blood samples from all patients were screened for the V617F mutation by gene-scaning on an ABI 3130 fragment analyser. 478 of these patients were further screened for the exon 12 mutant allele by denaturing high performance liquid chromatography (Transgenomic WAVE®) and direct sequencing.

Results: Of 725 patients screened for the V617F mutation 204 (28%) were found to be heterozygous (HET) and 30 (4%) homozygously (HM) mutated while 491 (68%) were wildtype (WT). 478 were further tested for the exon 12 mutation and eight were found to carry mutations. Two of the patients had previously described mutations while three novel mutations were described in the remaining six patients. Only one of these was in the PV group while the majority of exon 12 mutations clustered in the essential thrombocythemia (ET) (4/8) and Budd Chiari patients (2/8). All exon 12 mutations were identified at very low level.

Conclusions: Combined rapid gene-scanning and WAVE analysis allows for the characterisation of a large cohort of patients diagnosed clinically to have a variety of MPDs. This helps with the correct diagnosis of a frequently difficult group of patients. Detection of exon 12 mutations should be used in patients who are WT for V617F, including patients in the ET and the Budd Chiari syndrome groups. Both V617F and exon 12 mutation analysis tests should be included in primary screening in the routine management of MPD patients.

Diagnosis Total WT for V617F (mutant exon 12) HET for V617F (mutant exon 12) HM for V617F (mutant exon 12) Results of Exon 14 V617F and exon 12 mutation screening Budd Chiari/PVT 60 42 (2) 16 (0) 2 (0) Polycythemia Vera (PV) 187 112 (1) 63 (0) 12 (0) Reactive PV 28 28 (0) 0 (none tested) 0 (none tested) ET 198 123 (4) 71 (none tested) 4 (none tested) MPD 123 87 (1) 29 (none tested) 7 (none tested) Inspecified diagnosis 129 99 (none tested) 25 (none tested) 5 (none tested) TOTAL 725 491 (8) 204 (0) 30 (0)
Targeting CDK4/6 and the Cell Cycle in Combination with Bortezomib in the 5T33MM Myeloma Model.
Authors: Menu Eline , Garcia Josefina , Huang Xiangao , Di Liberto Maurizio , Toogood Peter , Chen Isan , Vanderkerken Karin , Chen-Kiang Selina
Published: April 2020

Multiple myeloma (MM) is ultimately fatal in part because no effective cell cycle-based therapy has been available to control tumor cell proliferation. Loss of cell cycle control in MM cells stems from coordinated deregulation of Cdk4-cyclin D1 or Cdk6 (Cdk4)-cyclin D2. PD 0332991 is the first orally bioactive small molecule that potently and specifically inhibits Cdk4 and Cdk6. It represents a promising cell cycle-based therapy for myeloma owing to its ability to potently inhibit Cdk4/6 and induce G1 cell cycle arrest in primary human myeloma cells in BM stromal cell co-cultures and to control tumor progression in a xenograft model. However, the efficacy of PD 0332991 in the presence of an intact immune system is unknown. To optimize therapeutic targeting of Cdk4/6 with PD 0332991, we investigated the effectiveness of PD 0332991 in inhibiting Cdk4/6 and controlling myeloma tumor progression in the immunocompetent, bone migrating 5T33MM model. By quantitative real-time PCR analysis, we found that these myeloma cells express a normal plasma cell transcription program such as upregulated Blimp-1 and loss of Bcl-6 expression. However, they proliferate aggressively due to Cdk4 overexpression and impaired p27Kip1 expression, thereby mimicking relapse disease in human myeloma. PD 0332991 potently inhibits Cdk4/6 phosphorylation of Rb in primary 5T33MM cells and induces G1 cell cycle arrest, both in vivo and ex vivo. Accordingly, treatment with PD 0332991 significantly prolongs the survival of tumor-induced 5T33MM mice; a mean of 35 days in the PD 0332991-treated group (N=9) versus 25 days in the vehicle-treated group (N=9, p< 0.003). These findings demonstrate for the first time that PD 0332991 targets Cdk4/6 and controls tumor expansion in the presence of an intact immune system. To further optimize Cdk4/6 targeting in MM, we combined PD 0332991 with bortezomib, a cytotoxic drug widely used in MM treatment. Pretreatment of 5T33MMvt cells with PD 0332991 for 48 hours markedly augmented bortezomib killing, to the same level of twice the dose of bortezomib when it was used alone. These studies of PD 0332991 in the immunocompetent 5T33MM model demonstrate that, as a consequence of Cdk4/6 inhibition and induction of G1 arrest, PD 0332991 sensitizes MM cells to killing by a cytotoxic agent. Targeting Cdk4/6 in combination therapy, therefore, represents a novel and promising strategy for myeloma treatment.
Analysis of Mutant cMPL in Philadelphia Chromosome-Negative Myeloproliferative Disorders (Ph−MPDs) Using a Novel High-Sensitivity Assay.
Authors: Samuelson Scott J. , Swierczek Sabina , Parker Charles J. , Boucher Kenneth , Jelinek Jaroslav , Prchal Josef
Published: April 2020

cMPL is a gene encoding for the thrombopoietin receptor that is essential for thrombopoiesis and contributes to pluripotent hematopoietic stem cell expansion. A gain of function cMPL mutation, MPLW515L, was identified in myeloid cells from patients with primary myelofibrosis (PMF). Subsequent studies identified a second gain of function mutation, MPLW515K, in PMF and essential thrombocytosis (ET). The prevalence of MPLW515L and MPLW515K mutations was 5% in PMF and 1% in ET. No mutant cMPL was detected in Polycythemia Vera (PV). The methods utilized in these assays were sensitive to mutant frequencies of >3–5%. We developed a rapid, sensitive, quantitative real time PCR assay based on a unique primer design wherein allelic discrimination was enhanced by the synergistic effect of a mismatch in the −1 position, and a locked nucleic acid nucleoside at the −2 position of the allele-specific primers. An assay of similar design can detect G1849T mutation of JAK2 in <0.1% mutant allele in peripheral blood granulocyte (Nussenzveig Exp Hematol20073:32). We hypothesized that a similar high sensitivity assay would increase detection of mutant cMPL in Ph−MPDs. We analyzed genomic DNA from peripheral blood granulocytes of 197 MPD patients and found that 10/197 (5.1%) carried one of the two cMPL mutations. Further, 5 of these patients were also JAK2V617F positive. cMPL mutations were detected in 1/78 (1.3%) PV patients, 3/56 (5.4%) ET patients, 4/49 (8.2%) PMF patients, and 2/11 (18%) MPD-Unspecified patients. W515L accounted for 9/10 cases, with W515K accounting for only 1. Of the ten positive samples, five (including the patient with PV) had ≤1% mutant alleles. To confirm the validity of our assay, we tested DNA from 96 normal controls. Neither W515L nor W515K was detected (p=0.03 compared to samples from the Ph−MPD patients). Additionally, when DNA from megakaryocytic colonies from a patient with 0.70% mutant alleles was analyzed, 12.5% of colonies were found to be heterozygous for cMPLW515L. These studies demonstrate the sensitivity and accuracy of our assay and show that cMPL activating mutations are more common in ET than previously reported. Mutant allele frequency appears greater in megakaryocytic cultures perhaps indicating a proliferative advantage for the cMPL-mutant clone. That mutant cMPL and JAK2V617F can be found in the same patient demonstrates the molecular heterogeneity of Ph−MPDs and emphasizes the need for prospective studies designed to determine the relationship between genotype and clinical phenotype. Scott J. Samuelson and Sabina Swierczek contributed equally to this project.
Pruritus in Polycythemia Vera Is More Prevalent in Non-Smokers and Is Independently Associated with a Lower Risk of Arterial Thrombosis.
Authors: Gangat Naseema , Strand Jacob J. , Lasho Terra L. , Li Chin-Yang , Pardanani Animesh , Tefferi Ayalew
Published: April 2020

Background: Pruritus in polycythemia vera (PV) has been associated with a high JAK2V617F allele burden (Tefferi et al. Cancer. 2006;106:631, Vannucchi et al, Leukemia2007, in press). However, there is limited information regarding clinical correlates of pruritus per se in PV. Accordingly, we conducted a large (n=418) retrospective study in PV to accurately assess the prevalence and severity of pruritus as well as its relationship with presenting clinical features, bone marrow JAK2V617F allele burden, and prognosis.

Methods: The study cohort consisted of a consecutive group of patients with PV who fulfilled the World Health Organization (WHO) diagnostic criteria and in whom information regarding the presence or absence of pruritus at diagnosis was fully documented. Pruritus was graded as being mild, moderate, or severe based on careful review of patient history.

Results:

Prevalence and severity of pruritus: A total of 418 patients fulfilled the above stipulated criteria for study inclusion (median age of 60 years; 56% males; median follow-up of 73 months). Of these, 131 (31%) experienced pruritus at time of initial diagnosis: mild in 97 patients (74%), moderate in 22 (17%), and severe in 12 (9%). Most patients with mild pruritus (n=92) received no specific therapy for pruritus whereas all 12 patients with severe pruritus required treatment; all 7 patients who received treatment with paroxetine responded. Pruritus and JAK2V617F allele burden: Quantitative measurement of JAK2V617F was performed in 64 patients using genomic DNA from archived bone marrow obtained at the time of PV diagnosis. Mutational frequency was 97%; median mutant allele burden was 25.4% (range 0.2 - 93.3%). Among the 62 V617F-positive patients, 24 (39%) had pruritus; the proportion of patients in the upper and lower quartile allele burden ranges were 50% and 0% in the presence of pruritus and 18% and 34% in its absence (p=0.002). Clinical correlates of pruritus: Pruritus was more prevalent in non-smokers (35% vs. 19%; p=0.004) and non-diabetics (33% vs. 16%; p=0.04) and was associated with a lower rate of arterial thrombosis at diagnosis (8% vs. 17%; p=0.01) as well as during follow-up (16% vs. 30%; p=0.003).

These significant associations remained intact during multivariable analysis. Pruritus did not have an impact on the rate of leukemic or fibrotic transformation.

Conclusions: Pruritus in PV is independently associated with a lower risk of arterial thrombosis despite high JAK2V617F allele burden and is also more prevalent in non-smokers. These observations suggest that pruritus might be a surrogate for an underlying platelet pathology with functional relevance.
A Phase 2 Study of Vorinostat (Suberoylanilide Hydroxamic Acid, SAHA) in Relapsed or Refractory Indolent Non Hodgkin Lymphoma. A California Cancer Consortium Study.
Authors: Kirschbaum Mark H. , Zain Jasmine M. , Popplewell Leslie , Pullarkat Vinod , Nademanee Auayporn P. , Delioukina Maria L. , Pulone Bernadette , Matsuoka Deron , Frankel Paul , James Zwiebel A. , Forman Stephen J. , Newman Edward , Gandara David
Published: April 2020

Background: The indolent (follicular, marginal zone and mantle cell) lymphomas tend to recur with decreasing intervals of remission after standard chemotherapy. New modalities of treatment are necessary. Vorinostat (SAHA), an orally administered hydroxamic acid histone deacetylase inhibitor with activity against class I and II deactylases with preclinical and clinical activity against various forms of lymphoma, is being studied in patients with relapsed or refractory indolent lymphoma. We report the results at the first interim analysis, after accrual of the first 17 evaluable patients according to the two-stage trial design.

Methods: Patients with relapsed or refractory follicular, marginal zone, or mantle cell lymphoma are eligible. Vorinostat is dosed at 200 mg po twice daily for 14 consecutive days on a 21 day cycle. CT scanning is performed after every three cycles, as is marrow biopsy for those with marrow involvement at time of entry into study. Patients may have received up to four prior chemotherapy regimens including Zevalin or Bexxar; previous transplant is allowed.

Results: 17 eligible patients (8 female, 9 male) were accrued prior to interim analysis (1 ineligible patient due to wrong histology is excluded from the analysis). The median age at diagnosis was 61 (34–78) years. All patients had progressed or failed to respond to chemotherapy and/or rituximab. Five patients were taken off study due to progression, two came off study due to toxicities (one for fatigue and diarrhea after 10 cycles and the other for diarrhea and dizziness after 2 cycles). One patient stopped therapy due to intercurrent illness, one came off for alternate therapy, 1 completed the treatment per protocol with a CR, and 7 patients remain on treatment. One patient had grade 4 ANC and thrombocytopenia, and one patient had grade 4 thrombocytopenia. Both patients were able to get subsequent cycles. Grade 3 toxicities attributable to study drug were thrombocytopenia, neutropenia, anemia, and fatigue. By the current Cheson criteria, there were 4 patients who achieved complete remission (CR), and 2 patients who achieved partial remission (PR). The two patients with PR as best response subsequently progressed (one at 189 days, one after 462 days). Four other patients are on therapy with stable disease, one patient with stable disease now for over 420 days.

Conclusions: The histone deacetylase inhibitor vorinostat shows promising activity against follicular and marginal zone lymphoma, refractory to chemotherapy and rituximab. The target response for stage 1 (4/17) was met and stage 2 is open for accrual.
Identification of Vacuolar H+- ATPase as a Target for Myeloma Therapeutics.
Authors: Zhu Yuan Xiao , Xin Chung , Ben Liang Lian Sheng , Chng Wee-Joo , Trudel Suzanne , Stewart Keith
Published: April 2020

From a high throughput RNAi screen of the human druggable genome targeting the KMS11 cell line, we identified the suppression of the vacuolar H+- ATPase (V-ATPase) family as cytotoxic to myeloma cells. In the screen, two oligos against each gene for the V-ATPase subunits ATP6V1A and ATP6V1B1 resulted in suppression of cell growth (50% and 60% inhibition of cell viability respectively). We further confirmed this result using both lentiviral shRNA knockdown and two small molecule inhibitors specific for V-ATPase. Silencing of ATP6V1A by lentiviral shRNA knock-down in KMS11 and in OPM1 myeloma cell lines caused 75–80% reduction of cell viability at 5 days post infection (measured by MTT assay). Consistent with this result, the V-ATPase specific inhibitors, bafilomycin A1 and REATA 203, both inhibited the growth of a genetically heterogeneous and standardized panel of 14 human myeloma cell lines in vitro with an IC50 ranging from 2.2 – 8.9 nM (mean 5.25 nM) for bafilomycin A1 and 46–1594 nM (mean 542.5 nM)) for REATA 203. We further demonstrated that patient samples (n=10) were sensitive to 20nM bafilomycin A1 which induced a mean of 58% of MM cells to undergo apoptosis (range 10% to 93%) after 24 hours of treatment. Similar to bafilomycin A1, treatment of primary patient-derived MM cells with 500 nM REATA 203 for 72 hours resulted in a mean 69% apoptosis (range 24% to 97%). In contrast, non-myeloma cells (the CD138- fractions of the bone marrow samples) were less sensitive - mean 9% apoptosis (range from 0% to 34%) under the same treatment conditions. Of high interest, however, unlike most drugs we have studied in pre-clinical myeloma models, the cytotoxicity induced by bafilomycin A1 in MM cell lines is abrogated by co-culture with patient bone marrow stromal cells but is not affected by IL-6 or IGF-1 treatment. Dexamethasone- or melphalan-resistant MM cell lines were also highly sensitive to both bafilomycin A1 and REATA 203. In a xenographic JJN3 mouse model, bafilomycin A1 suppresses and delays growth of tumor in a dose-dependent fashion. Gene expression analysis of normal-donor bone marrow plasma cells (n=19), primary tumor samples from MM patients (n=107) and normal somatic tissues demonstrates ubiquitous expression of most subunits of V-ATPase, however, some subunits are preferentially expressed in myeloma cells compared with normal plasma cells, including ATP6V1F (84% vs. 11%), ATP6V1E1 (29% vs. 5%), ATP6V1G2 (17% vs. 0%) and ATP6V0E 2 (36% vs. 16%). In conclusion, our data indicate that vacuolar H+-ATPase inhibitors are of interest as potential therapeutics for MM.
Imatinib Mesylate (Gleevec®) Produces Responses in Patients with Relapsed/Refractory Waldenstrom’s Macroglobulinemia.
Authors: Soumerai Jacob D. , O’Connor Kelly E. , Ioakimidis Leukothea M. , Patterson Christopher J. , Ciccarelli Bryan T. , Leduc Renee , Hunter Zachary R. , Nelson Marybeth , Roccaro Aldo M. , Leleu Xavier , Hatjiharissi Evdoxia , Adamia Sophia , Xu Lian , Ghobrial Irene M. , Treon Steven P.
Published: April 2020

Waldenstrom’s macroglobulinemia is a B-cell disorder characterized by the infiltration of lymphoplasmacytic cells (LPC) in the bone marrow (BM), along with an IgM monoclonal gammopathy. Characteristic of WM is an increased number of mast cells (MC) which are found in association with LPC, and stimulate LPC growth through several TNF-family members including CD40L, APRIL and BLYS (Ann Oncol17:1275; Blood104:917A). As such, we have targeted MC in WM. One important growth and survival factor for MC is stem cell factor (SCF), which signals through CD117. Imatinib mesylate blocks SCF signaling through CD117, and induces apoptosis of WM BM MC and LPC, both of which highly express CD117 (Blood2004; 104:314b). As such, we performed this Phase II study of imatinib mesylate in patients with relapsed and refractory WM. Intended therapy consisted of imatinib mesylate which was initiated at 400 mg po qD over the first month, and subsequently dose escalated to 600 mg po qD for up to 2 years. Dose de-escalation to 300 mg po qD was permitted for toxicity. Twenty-eight patients were enrolled, 27 of whom are eligible for evaluation at final analysis. Median age was 61 (range 33–80 years), and median prior therapies was 2 (range 1–5). Twenty-four and four patients had relapsed and refractory disease, respectively. With a median follow-up of 6.3 months for all evaluable patients, median serum IgM levels declined from 3,110 to 2,530 mg/dL at best response (p=0.002). On an evaluable basis, 7/27 (26%) and 2/27 (7%) of patients attained a ≥25% and ≥50% decrease in serum IgM, respectively. Responses were prompt, and occurred at a median of 2.1 months. The median time to progression for responding patients was 8.4 (range 2–15 months). Hematological toxicities were the most common feature and ≥grade 2 toxicities included anemia (n=12), leucopenia (n=5), edema (n=3), thrombocytopenia (n=2) and neutropenia (n=1). Tryptase levels, which measure mast cell burden, declined in all patients for whom pre- and post-serum was available from 6.7 (range 2.2–10 ng/ml) to 3 (range 1–5.5 ng/ml) (p<0.001) though did not predict response to therapy.The results of this study demonstrate that imatinib mesylate is an active salvage therapy for WM and is associated with decreases in mast cell burden in patients with WM.
IELSG Phase II Studies of Bortezomib in Malt Lymphomas.
Authors: Conconi Annarita , Lopez-Guillermo Armando , Martinelli Giovanni , Rigacci Luigi , Vitolo Umberto , Zinzani Pier Luigi , Luminari Stefano , Cavalli Franco , Zucca Emanuele
Published: April 2020

The International Extranodal Lymphoma Study Group (IELSG) is coordinating two phase II multicenter studies aimed to assess the antitumor activity and safety of bortezomib treatment in patients (pts) with relapsed or refractory extranodal marginal zone B-cell lymphoma of MALT-type. The IELSG25A study is evaluating bortezomib in pts previously treated with one prior systemic treatment, and the IELSG25B pts previously treated with more than one prior treatment. Bortezomib 1.3 mg/m2 is administered on days 1, 4, 8, and 11 of a 21-day cycle, for up to 6 cycles. Response and progression are determined by International Workshop Criteria. As of July 2007, 18 pts have been enrolled in the studies: 11 (61%) patients were male, median age was 62 years (range, 38–77). At time of enrolment, the Ann Arbor stages distribution was the following: stage I=5 pts (28%), stage II=5 pts (28%), stage IV=8 (44%). In 10 pts primary gastric localization was present, in 5 cases primary skin, in 2 cases primary subcutaneous, in 1 primary lung lymphoma. All patients had ECOG PS=0; in 2 cases (11%) elevated serum LDH was reported. More than 1 site of extranodal localization was present in 4 pts (22%). Median number of prior therapies was 2 (range, 1–3). Median follow-up was 16 months. Nine pts were assessed for response at the end of treatment plan: 3 pts had a CR (33%); 4 a PR (45%) and 2 SD (22%). Five additional pts, with ongoing therapies, have been evaluated after the first two courses of therapy: one pt achieved a CR and 2 pts a PR, in 2 cases a SD was observed. The safety profile of bortezomib is similar to that observed in multiple myeloma and other subtypes of non-Hodgkin lymphoma. The most relevant adverse events grade 3 or higher were peripheral neuropathy and fatigue. One death, non-related to treatment, was observed during the early follow up. These preliminary results suggest that bortezomib is active and safe in relapsed or refractory MALT lymphomas and encourage us to complete the studies accrual.
Vorinostat (Suberoylanilide Hydroxamic Acid, SAHA) Provides Prolonged Safety and Clinical Benefit to Patients with Advanced Cutaneous T-Cell Lymphoma (CTCL).
Authors: Duvic Madeleine , Olsen Elise A. , Breneman Debra , Pacheco Theresa R. , Parker Sareeta , Vonderheid Eric C. , Ricker Justin L. , Rizvi Syed , Chen Cong , Boileau Kathleen , Cooley Paulette , Geskin Larisa J.
Published: April 2020

Background: Vorinostat is a histone deacetylase inhibitor that has been approved by the US FDA for the treatment of cutaneous manifestations in patients with cutaneous T-cell lymphoma (CTCL) who have progressive, persistent or recurrent disease on or following 2 prior therapies.

Patients and Methods: A Phase IIb, open-label trial of oral vorinostat 400 mg daily until progressive disease (PD) or intolerable toxicity was conducted in patients with advanced CTCL. Patients must have received at least 2 prior therapies and recovered from all prior treatment-related toxicities. The primary end point was the objective response rate in patients with stage IIB or higher CTCL as measured by the severity weighted assessment tool. Time to response (TTR), duration of response (DOR), pruritus relief, safety, and tolerability were also evaluated. Patients who had received vorinostat therapy for at least 2 years as of August 1, 2007 were further evaluated in this post-hoc subset analysis.

Results: Six of 74 patients originally treated in the study have received vorinostat therapy for at least 2 years as of 8/1/07, including 5 responders (1 complete responder, 4 partial responders) and 1 with prolonged stable disease (Figure 1). The median age was 65 years (range, 57–74), the median number of prior systemic therapies was 2.5 (range, 1–5), and the median time from CTCL diagnosis to enrollment was 1.8 years (range, 0–5.9). The most common drug-related adverse experiences were diarrhea (100%), fatigue (67%), nausea (50%), and alopecia (50%). Grade ≥ 3 drug-related adverse experiences were anorexia (n = 1), pulmonary embolism (PE, n = 1), and thrombocytopenia (n = 1). The PE (Day 144) was a serious adverse experience (SAE) that resolved within 7 days, and this patient is continuing on therapy as of Day 848. The only other SAE was a rash (Day 925). Two patients discontinued due to PD and 4 are continuing on therapy as of August 1, 2007.

Conclusions: Vorinostat has demonstrated prolonged safety and clinical benefit in patients with advanced CTCL.

Figure 1. Time on study of patients on vorinostat therapy for ≥ 2 years (available data as of August 1, 2007). Figure 1. Time on study of patients on vorinostat therapy for ≥ 2 years (available data as of August 1, 2007).
A Panel of Artificial Antigen-Presenting Cells (AAPC) Expressing Prevalent HLA Alleles Permits Generation of Cytotoxic T Cells Specific for Both Subdominant and Dominant Viral Epitopes for Adoptive Therapy.
Authors: Hasan Aisha , Kollen Wouter J. , Selvakumar A. , Trivedi D. , Sadelain M. , O’Reilly Richard J.
Published: April 2020

Adoptive transfer of antigen specific T cells can be effective in treating viral infections complicating allogeneic hematopoietic stem cell transplant (HSCT) recipients. However, in practice, generation of T cells is often limited by insufficient supply of autologous antigen presenting cells; therapeutic activity in HLA disparate patients may also be impaired if the immuno- dominant T cells generated are restricted by HLA alleles not shared by the host. AAPCs have theoretical advantages for T cell therapies in terms of sustained supply and capacity to selectively stimulate T cells restricted by HLA alleles shared by donor and host. However, to date, only AAPC systems expressing HLA A*0201 have been characterized. Accordingly, we established a panel of AAPC consisting of NIH 3T3 mouse fibroblast cells, each transduced to express β2- microglobulin and a prevalent HLA class-I allele, specifically HLA A*0201, A*0301, A*2402, B*0702, B*0801 or C*0401, as well as the human co-stimulatory molecules B7.1, LFA-3 and ICAM-1. Novel promotor sequences were introduced to secure stable high expression of the allele on the AAPCs. Sensitization of T cells from seropositive donors with AAPCs expressing each of these alleles (4-8 donors/allele), either loaded with overlapping 15-mer peptides spanning the CMVpp65 sequence or transduced to express the CMV pp65 protein, resulted in 12-35 fold expansions of CD8 + T cells exhibiting CMV pp65 epitope-specific, HLA restricted activity, as quantitated by peptide -HLA tetramer binding, epitope specific production of interferon gamma, and cytotoxic activity against peptide loaded or CMV infected targets. Although both peptide pool loaded and transduced AAPCs induce CMV pp65 epitope specific T cells, yields were higher when transduced AAPCs were employed. In studies of T-cells from 5 donors when sensitized with either peptide pool loaded autologous dendritic cells (DC) or HLA sharing AAPCs, sensitization with DC selectively induced T-cells specific for 1-2 immunodominant CMV pp65 epitopes. In contrast, while sensitization with a panel of peptide loaded or transduced AAPCs expressing shared HLA alleles elicited responses to the same dominant epitopes, we could also regularly generate comparable cytotoxic T cell responses to subdominant epitopes which were either not produced or only present at low frequencies in T cells sensitized with autologous DC. Thus, this panel of AAPCs stably expressing a series of HLA alleles which, in aggregate, are detected in 70% of the patients referred for HSCT, can be employed for rapid generation of CMV-pp65 specific T cells of desired HLA restriction for adoptive therapy.
Serum Evaluation of a Panel of 10 Cytokines in Patients with Follicular Lymphoma: High Serum Level of TGF-β Correlates with Good Prognosis, Independently of the FLIPI Score.
Authors: Ghesquieres Herve , Labidi Sana Intidhar , Caux Christine , Chabaud Sylvie , Gargi Therese , Chassagne Catherine , Sebban Catherine , Biron Pierre , Blay Jean-Yves
Published: April 2020

The course of follicular lymphoma (FL) can be predicted using the FLIPI score but additional prognostic biomarkers are required. The immune microenvironment plays an important role in the physiopathology of FL. As cytokines are involved in the interaction between tumor cells and immune bystander cells, it is relevant to evaluate their prognostic value in FL. Nine cytokines (IL-1ra, IL-6, IL-7, IL-10, IL-13, TNFα, VEGF, PDGF, β-FGF) were measured by multiplex ELISA whereas TGF-β was measured by ELISA sandwich technique in the sera of 60 patients treated for FL (31 at diagnosis, 12 in complete remission (CR) and 21 at first relapse) and 20 healthy persons used as controls. All cytokines but β-FGF were detectable. High IL-6 levels were correlated with increased LDH levels. Increased levels of IL-10 at diagnosis were correlated with advanced stage (p=0.05), bone marrow involvement (p=0.05) and high FLIPI (trend, p=0.07). Levels of IL-10 were higher in FL patients at any time of the disease (diagnosis, relapse or CR) than in controls (respectively p<0.001, p=0.0001 and p=0.04). Levels of TGF-β were significantly lower in FL patients in relapse than in patients in CR (p=0.01) or controls (p=0.001). With a median follow-up of 9 years, overall survival (OS) rates in all patients were 69.9% and 57.5% at 6 and 10 years, respectively. Univariate analysis using clinical prognostic factors (β-2 microglobulin rate, stage, FLIPI score) showed that patients at diagnosis with elevated levels of IL-10 (HR=1.005, p=0.026) and decreased levels of TGF-β (HR=0.929, p=0.01) had shorter survival. In multivariate analysis, Ann Arbor stage I-II and high levels of TGF-β were independent prognostic factors associated with better survival. An elevation by 1ng/ml of TGF-β levels reduced the risk of death by 7%. In follicular lymphoma, a high level of TGF-β is an independent prognostic factor correlated with better survival. As immune microenvironment cells are correlated with survival, we propose to analyse the relationship between immunohistochemical staining of these cells and serum cytokine levels in patients. This is currently being investigated.
Promoter Methylation of Multiple Genes in Diffuse Large Cell Lymphoma (DLCL), Follicular Lymphoma (FL) and Bening Follicular Hyperplasia (BFH).
Authors: Paz-Carreira Jose M. , Losada Raquel , Garcia-Rivero Arantxa , Alvarez Augusto , Bal Fernando , Deben Guillermo , Alba-Losada Jesus
Published: April 2020

Transcriptional silencing of tumor suppressor genes, associated with DNA methylation, is a common epigenetic event in leukemias and myelodisplastic syndromes. Much less is known about the specific methylation changes that occur in DCLC, FL and their normal counterpart BFH. Methylation of cyclin dependent kinase inhibitors is more common in high grade lymphomas and may be an important step in the progression and transformation of follicular lymphoma. We determined the methylation status of 7 tumor suppressor genes in 31 patients with B-cell malignancies. Seven patients with benign follicular hyperplasia were used as normal controls. The target genes chosen are potentially involved in B-cells malignancies and encoding proteins implicated in apoptosis regulation (death associated protein kinase, DAP-K), Jak/STAT3 signalling pathway (SHP1), hormonal response (RARb), DNA repair (O6-methilguanine-DNA methyltranferase, MGMT), cell cycle control (p14ARF and p15INK4b) and detoxification of environmental xenobiotics such as doxorubicin (glutathione S-transferase P1, GSTP1). Genomic DNA extracted from paraffin-embedded samples of thirty one B-cell malignancies (twenty six DLCL and five FL) were analyzed by methylation-specific polymerase chain reaction to determine promoter hypermethylation of DAP-k, SHP1, Rarβ, MGMT, p14, p15 and GSTP1. Samples were obtained mostly from lymph nodes; spleen, stomach, skin, and parathyroid gland biopsies were also included. Specimens were collected at diagnosis before specific therapy. Diagnosis was based on morphology and immunohistochemistry analysis. All cases were matched for age, sex and ethnic origin. DAP-k promoter methylation occurred with similar frequency in DLCL (100%), FL (100%) and BFH (86%). SHP1 was methylated in 75% of DLCL, all FL and the 7 patients with BFH. RARb was methylated in all DLCL and FL patients and 85% of the BFH. Sixteen (50%) DLCL, three (60%) FL and none BFH patients showed MGMT methylation. Promoter hypermethylation of p14 and p15 was detected in 11 (42%) and 13 (50%) DLCL, 2 (40%) and 3(60%) FL, 3(42%) and 5(71%) BFH patients respectively. Methylation of GSTP1 was absent in all samples. Inactivacion of DAP-K, SHP1 and Rarβ is present in B-cell malignancies, DLCL and FL, and BFH. Therefore, it may represent a physiologic event conferring a temporal survival advantage necessary for a BFH response. With our data methylation of Cyclin dependent kinase inhibitors such as p14 and p15 is not a differential pathogenic event in DLCL with respect to FL or BFH. Finally, the absence of GSTP1 methylation in DLCL and FL questions its relevance in B-cell neoplasia development.