bluuebunny/crossref_metadata_2025 huggingface dataset:
Disruption of the Crosstalk CXCL12/CXCR4 Chemotactic Pathway To Enhance Melphalan-Induced Apoptosis in Multiple Myeloma Cell Lines.
Authors: Saad Ayman A. , Fortney James , Wang Lin , O’Leary Heather , Gibson Laura , Ericson Solveig
Published: October 2019

INTRODUCTION:

Multiple myeloma cells display functional CXCR4 chemokine receptor that stimulates the migration of these cells toward their natural ligand, CXCL12 (stromal-derived factor, SDF-1a). CXCL12 is secreted by bone marrow stroma. Consistent with their CXCR4 expression, myeloma cells home to the marrow microenvironment, where adhesive interactions promote growth, survival, and confer cell adhesion-mediated drug resistance.

METHODS

U266-B1 cells (ATCC myeloma cell line) were pre-treated with recombinant CXCL12 for 30 minutes prior to the addition of melphalan for up to 72 hours. Both melphalan and CXCL12 were added at 24 hour intervals. Cell lines alone and cell lines with only melphalan or only CXCL12 were used as controls. We have also tested the influence of adding AMD3100, a reversible inhibitor of CXCR4, on myeloma cell survival. U266-B1 cells in media alone were pre-treated with AMD3100 for 24 hours prior to treatment with melphalan for 16 hours. Cell viability following treatment was quantified by flow cytometry assay using Annexin-V-FITC staining. Western blot analysis was used to quantify the apoptotic activity of the cell lines using 4 apoptotic markers: PARP (poly ADP-ribose polymerase), caspase-3, Bcl-2, and Mcl-1.

RESULTS:

Recombinant CXCL12 conferred a protective effect to myeloma cell lines during melphalan treatment. This effect was more pronounced at 72 hours of treatment. Western blot analysis showed diminished expression of the apoptotic markers, cleaved PARP (poly ADP-ribose polymerase) and active caspase-3 in the melphalan-treated cell lines with prior exposure to CXCL12. Additionally, pretreatment with AMD3100 resulted in enhanced apoptosis following melphalan treatment.

CONCLUSION:

Our data showed that CXCL12, a naturally occurring cytokine secreted by bone marrow stromal cells confers a protective effect on myeloma cells against apoptosis. Disruption of this effect by AMD3100 resulted in enhanced melphalan-induced apoptosis of myeloma cells. We are continuing to study this effect with the potential future utility of AMD3100 as a melphalan chemo-sensitizer in the treatment of multiple myeloma.
Expression of Soluble Syndecan-1 or Heparanase by Myeloma Cells Enhances Levels of Angiogenic Growth Factors Present in Endothelial Cells.
Authors: Yang Yang , Frith Ashley Elizabeth , Theus Allison , Macleod Veronica , Sanderson Ralph D.
Published: October 2019

Multiple myeloma is a devastating cancer with a high rate of morbidity and mortality. Our previous in vivo studies demonstrate that both shed syndecan-1 and heparanase can promote myeloma tumor growth, metastasis and angiogenesis. To examine the mechanism underlying this enhanced angiogenesis, human umbilical vein endothelial cells (HUVEC) were cocultured with cells of the CAG myeloma cell line (vector-only controls, CAGcontrol) or CAG cells engineered to express high levels of either soluble syndecan-1 ectodomain (CAGssyn1 ) or heparanase ( CAGHPSE ). After coculture for 48 hours, levels of angiogenic growth factors present in the endothelial cells were examined. The goal was to determine if expression of either soluble syndecan-1 or heparanase by CAG myeloma cells altered growth factor levels relative to those present when control CAG cells were used. Co-culture with CAGssyn1 or CAGHPSE cells did not enhance endothelial levels of FGF-2, while levels of hepatoma-derived growth factor (HDGF) and hepatocyte growth factor (HGF) were elevated in endothelia growing in the presence of CAGssyn1 cells but not CAGHPSE cells or CAGcontrol cells. However, VEGF levels present in endothelial cells were substantially enhanced by the presence of CAGssyn1 (1.9-fold increase) or CAGHPSE cells (1.6-fold increase). Surprisingly, levels of VEGF in conditioned media of cocultures containing either CAGssyn1 or CAGHPSE cells was low. In contrast, when cultured in the absence of HUVECs, VEGF levels were elevated in conditioned media of both CAGssyn1and CAGHPSE cells. Addition of this conditioned media containing high levels of VEGF to HUVECs growing in the absence of CAG cells did not result in an elevation of VEGF levels in the endothelial cells. Together, these experiments suggest that VEGF expression is upregulated in CAG cells expressing high levels of shed syndecan-1 or heparanase and that VEGF becomes associated with the endothelial cells only when they are cultured in the presence of the myeloma cells. This cross-talk between myeloma and endothelial cells may lead to the enhanced angiogenesis that occurs in vivo in tumors formed by myeloma cells producing high levels of shed syndecan-1 and/or heparanase.
CS1: A Potential New Therapeutic Target for the Treatment of Multiple Myeloma.
Authors: Hsi Eric D. , Steinle Roxanne , Balasa Balaji , Draksharapu Aparna , Shum Benny , Huseni Marukh , Powers David , Nanisetti Amulya , Williams Marna , Vexler Vladimir , Hussein Mohamad , Afar Daniel
Published: October 2019

Background: To identify genes upregulated in human memory B and plasma cells, naïve B cell cDNA was subtracted from plasma cell and memory B cell cDNA. One gene that was highly expressed in plasma cells encodes CS1 (CD2 subset 1, CRACC, SLAMF7), a cell surface glycoprotein of the CD2 family. CS1 was originally identified as a natural killer (NK) cell marker. Monoclonal antibodies (mAbs) specific for CS1 were used to validate CS1 as a potential target for the treatment of multiple myeloma (MM).

Methods: Anti-CS1 mAbs were generated by immunizing mice with a protein comprising of the extracellular domain of CS1. Two clones, MuLuc63 and MuLuc90, were selected to characterize CS1 protein expression in normal and diseased tissues and blood. Fresh frozen tissue analysis was performed by immunohistochemistry (IHC). Blood and bone marrow analysis was performed using flow cytometry with directly conjugated antibodies. HuLuc63, a novel humanized anti-CS1 mAb (derived from MuLuc63) was used for functional characterization in non-isotopic LDH-based antibody-dependent cellular cytotoxicity (ADCC) assays.

Results: IHC analysis showed that anti-CS1 staining occurred only on mononuclear cells within tissues. The majority of the mononuclear cells were identified as tissue plasma cells by co-staining with anti-CD138 antibodies. No anti-CS1 staining was detected on the epithelia, smooth muscle cells or vessels of any normal tissues tested. Strong anti-CS1 staining was also observed on myeloma cells in 9 of 9 plasmacytomas tested. Flow cytometry analysis of whole blood from both normal healthy donors and MM patients showed specific anti-CS1 staining in a subset of leukocytes, consisting primarily of CD3−CD(16+56)+ NK cells, CD3+CD(16+56)+ NKT cells, and CD3+CD8+ T cells. Flow cytometry of MM bone marrow showed a similar leukocyte subset staining pattern, except that strong staining was also observed on the majority of CD138+CD45−/dim to + myeloma cells. No anti-CS1 binding was detected to hematopoietic CD34+CD45+ stem cells. To test if antibodies towards CS1 may have anti-tumor cell activity in vitro, ADCC studies using effector cells (peripheral blood mononuclear cells) from 23 MM patients and L363 MM target cells were performed. The results showed that HuLuc63, a humanized form of MuLuc63, induced significant ADCC in a dose dependent manner.

Conclusions: Our study identifies CS1 as an antigen that is uniformly expressed on normal and neoplastic plasma cells at high levels. The novel humanized anti-CS1 mAb, HuLuc63, exhibits significant ADCC using MM patient effector cells. These results demonstrate that HuLuc63 could be a potential new treatment for multiple myeloma. HuLuc63 will be entering a phase I clinical study for multiple myeloma.
A Novel, Mercaptoketone-Based HDAC Inhibitor, KD5170 Exerts Marked Inhibition of Osteoclast Formation and Anti-Myeloma Activity In Vitro.
Authors: Feng Rentian , Hager Jeffrey H. , Hassig Christian A. , Phar Shawn A. Scranton , Payne Joseph E. , Mapara Markus Y. , Roodman David , Lentzsch Suzanne
Published: October 2019

Multiple Myeloma (MM) is characterized by the malignant proliferation of plasma cells and high osteoclast (OCL) activity resulting in increased bone destruction and the development of lytic lesions. Ideally, novel therapeutic agents directed at MM should not only target the myeloma cell, but also OCL activity. Multiple, chemically distinct, Histone Deacetylase (HDAC) inhibitors have shown promising anti-myeloma activity in pre-clinical studies. The majority of the HDAC inhibitors in clinical development employ either benzamide or hydroxamate chemistry. Through an uHTS biochemical screen of 600,000 compounds and subsequent medicinal chemistry efforts, a novel mercaptoketone series of HDAC inhibitors has been developed. The most potent compounds in this series exhibit nanomolar activity in both biochemical and cell-based assays. To determine the clinical potential of this series in treating MM we have profiled the lead compound, KD5170 in assays that monitor osteoclastogenesis and proliferation and apoptosis of MM cells.

To evaluate the effects of KD5170 on the formation of OCL from mononuclear hematopoietic precursors, we utilized a human bone marrow culture system from MM patients to generate OCLs by in vitro RANKL/M-CSF stimulation. KD5170 treatment resulted in a significant dose dependent (0.5–8 10−6M) inhibition of osteoclast formation. In addition, RPMI-8226 cell proliferation was significantly suppressed by KD5170 at 3 10−6M as measured by [3H]-Thymidine-incorporation. Furthermore, we observed that KD5170 induced dose- (0.05–1 10−6M) and time-dependent (2–24h) apoptosis of U266 MM cells, as measured by Hoechst/PI double staining to identify distinct stages of apoptosis and to differentiate between apoptotic and necrotic cell death. Significantly, less than 5% of the cells were necrotic (PI-positive) indicating active engagement of the apoptotic pathway. KD5170 treatment (1 10−6M, 72 h) also markedly decreased the viability of primary CD138+ bone marrow cells from MM patients, as determined by trypan blue exclusion. In contrast, the viability of CD138− and mononuclear bone marrow cells from MM patients and normal healthy donors were unaffected, suggesting targeted selectivity.

Our studies identify KD5170 as a promising new HDAC inhibitor that inhibits both osteoclast formation as well as the proliferation of MM cells. Furthermore, pronounced anti-proliferative activity of KD5170 on the MM cell line U266, suggests that a subset of MM may be the ideal target of this agent. U266 harbors a translocation involving 11q13 raising the possibility that this chromosomal alteration and Cyclin D1 dysregulation may have a role in mediating KD5170 sensitivity and as such will be a focus of future studies.
Effects of a Novel Histone Deacetylase Inhibitor, PXD101, When Used as Monotherapy or in Combination with Bortezomib on Tumor Growth in Mouse Models of Human Multiple Myeloma.
Authors: Campbell Richard A. , Sanchez Eric , Chen Haiming , Turker Lauren , Trac Olivia , Li Mingjie , Shalitin Dror , Gordon Melinda S. , Pang Shen , Bonavida Benjamin , Said Jonathan , Jeffers Michael , Qian Xiaozhong , Lichenstein Henri S. , Berenson James R.
Published: October 2019

Histone deacetylase (HDAC) inhibitors represent a new mechanistic class of anti-cancer therapeutics that inhibit HDAC enzymes and have been shown to have anti-proliferative effects in cancer cells (including drug resistance subtypes), induce apoptosis, inhibit angiogenesis, and sensitize cancer cells when combined with other available anti-cancer therapies. PXD101 is a novel investigational small molecule drug that selectively inhibits HDAC enzymes. In recent preclinical studies, PXD101 has been shown to have the potential to treat a wide range of solid and hematological malignancies either as a monotherapy or in combination with other active agents. In this study, we evaluated the activity of PXD101 on multiple myeloma samples when used as monotherapy or in combination with the proteasome inhibitor bortezomib. In vitro experiments indicated that PXD101 pretreatment (20 mM; 3h) sensitized RPMI-8226 human multiple myeloma cells to subsequent bortezomib exposure (5 nM; 72h). To examine PXD101 and bortezomib in vivo, two mouse models of human multiple myeloma were utilized (LAGλ-1 and LAGκ-1B). LAGλ-1 was generated from a patient resistant to melphalan therapy and LAGκ-1B from a patient who progressed on bortezomib treatment (Campbell et al, International Journal of Oncology 2006). SCID mice were implanted with LAGλ-1 or LAGκ-1B tumor fragments into the left superficial gluteal muscle. Tumors were allowed to grow for 14 days at which time human IgG levels were detectable in the mouse serum, and mice were randomly assigned into treatment groups. Groups consisted of Vehicle only, PXD101 alone (40 mg/kg), bortezomib alone (0.5 mg/kg), or PXD101 (40 mg/kg) + bortezomib (0.5 mg/kg). In one cohort, PXD101 and bortezomib were administered twice weekly (M, Th) and in another cohort PXD101 was administered 5 days a week (M-F) and bortezomib twice weekly (M, Th). When administered, PXD101 was given i.p twice daily and bortezomib once daily intravenously. The results of these animal experiments will provide preclinical information on the activity of PXD101 monotherapy and PXD101/bortezomib combination therapy on drug-resistant myeloma samples, and may help to define the optimal schedule for potential clinical evaluation of this drug combination.
Total Therapies (TT) for Newly Diagnosed Patients with Multiple Myeloma (MM): Significant Benefit in High-Risk Disease with Cytogenetic Abnormalities (CA) over the Course of 17 Years.
Authors: van Rhee Frits , Bolejack Vanessa , Anaissie Elias , Tricot Guido , Hollmig Klaus , Zangari Maurizio , Pineda-Roman Mauricio , Mohiuddin Abid , Hoering Antje , Crowley John , Barlogie Bart
Published: October 2019

Background: In 1989, TT with tandem autotransplants (AT) was developed - in analogy to St. Jude’s TT program for childhood ALL - to advance the treatment outcome of MM patients. New agents and treatment principles were introduced as we moved from TT1 to T2 to TT3, mainly to address the inferior outcome in high-risk patients, introducing post-AT consolidation chemotherapy and thalidomide in TT2 and treatment in between chemotherapy cycles/transplants plus bortezomib in TT3. The consistently high-risk associated with the presence of metaphase cytogenetic abnormalities (CA) had been recognized in TT1. We now examine the benefit of intensifying therapy over time in patients with and without CA.

Patients and Methods: According to our MM data base, 979 newly diagnosed patients had at least 1 AT; 181 patients were enrolled prior to 1999 and 798 patients thereafter (566 on TT2 and 232 on TT3). Univariate and multivariate analyses were performed to examine the impact on overall survival (OS) of time of protocol enrollment in the context of pre-treatment laboratory parameters

Results: Pre-treatment features of the 2 cohorts were comparable in terms of frequencies of CA and serum levels of B2M, CRP and creatinine. The recent cohort (after 1999) had higher percentages of patients aged >65yr (22% vs 8%) and with LDH>190U/L (32% vs 7%, p<.001) but lower proportions with albumin<3.5g/dL (2% vs 9%, p<.001) and Hb<10g/dL (19% vs 50%, p<.001). Median OS was superior in the 798 patients enrolled after 1999 compared to the 181 prior to 1999 (not reached vs 72mo, p=.007). Other univariately significant adverse features included CA, B2M>=3mg/L, CRP>=6mg/L (all p<.001); age >=65yr (p=.002), creatinine>1.5mg/dL (p=.007), albumin<3.0g/dL (p=.018) and male gender (p=.049). On multivariate analysis, the following adverse variables entered the model: CA (HR=2.19, p<.001), B2M>=3mg/L (HR=1.79, p<.001), CRP>=6mg/L (HR=1.48, p=.002), age >=65yr (HR=1.44, p=.015), enrollment pre-1999 (HR=1.38, p=.026) and male gender (HR=1.27, p=.047). Importantly, a significant benefit was apparent for the one-third of patients with CA who received TT post-1999 (median not reached versus 4yr, p=.02), whereas there was a trend apparent for those without CA (Figure).

Conclusion: Pursuit of the TT approach has indeed improved the outcome of the one-third of high-risk patients with CA, with a 5-yr OS rate of ~55% post-1999 versus 35% pre-1999.

OS By Enrollment Periods  Total Therapies 1/2/3 OS By Enrollment Periods  Total Therapies 1/2/3
Melphalan (M), Prednisone (P) and Lenalidomide (R) Combination (MPR) for Newly Diagnosed Multiple Myeloma Patients Who Are Not Candidates for Stem Cell Transplantation.
Authors: Roy Vivek , Leif Bergsagel P. , Jacob Allred , Philip Greipp R.
Published: October 2019

Lenalidomide is an orally active immunomodulatory drug effective in the treatment of multiple myeloma. We evaluated the toxicity and efficacy of MPR combination in a phase I/II study. Patients with newly diagnosed symptomatic multiple myeloma who were not candidates or refused stem cell transplantation were eligible. Treatment consisted of M and P on days 1 – 4 and R on days 1 – 21 in a 28 day cycle. All patients received aspirin 325 mg a day. Routine antibacterial prophylaxis was not used. Patients were accrued in cohorts of 3 at dose level 0 (M 5mg/m 2/d P 60 mg/m2/d R 10 mg/d) and level 1 (M 8mg/m2/d P 60 mg/m2/d R 10 mg/d). Patients were followed for at least 2 cycles to assess Dose Limiting Toxicity (DLT). G/GM-CSF use was not allowed in the first 2 cycles unless DLT had occurred. DLT was defined as occurrence of at least grade (G) 4 hematologic toxicity, G3 febrile neutropenia, or G 3 non-hematologic toxicity in the first two cycles. Failure to start the next cycle within 7 days of day 1 due to toxicity also constituted DLT.

Results: 7 patients have been accrued (4 at dose level 0 {one patient had to be replaced} and 3 at dose level 1). 4 were males. ECOG performance status was 0 or1 (2 and 4 patients). Median age was 74 yrs, range 72 – 85. Between 3 and 10 cycles (median 4) have been administered at the time of this reporting. The following G3 or higher toxicities were encountered and considered at least possibly related to treatment: 2 patients at level 0 had G3 neutropenia. Of the three patients at dose level 1, there was 1 G4 neutropenia, 1 G4 thrombocytopenia, 1 G3 hyperglycemia and 1 G4 vascular access device related thrombosis. Responses were defined by EBMT/IBMTR criteria. 1 had a complete remission (after 3 cycles), 4 had partial remission (at least 50% reduction in monoclonal protein) and 1 had minimal response (after 3 cycles). Due to 3 DLT’s in dose level 1, dose level 0 was chosen for phase II of the study.

Conclusion: M 5 mg/m2, P 60 mg/m2 and R 10 mg combination is well tolerated and appears effective in patients with multiple myeloma. These results are similar to the experience reported by Palumbo et al (Blood2005, 106(11):abst 785) although a stricter definition of DLT was used in our study. Escalation of R dose in combination with M 5 mg/m2, P 60 mg/m2 may be possible and improve responses. Updated data will be presented.
Deep Vein Thrombosis in Myeloma: Estimate of Prevelance and Recommendations for Therapy Based upon a Survey of Members of the International Myeloma Working Group (IMWG).
Authors: Durie Brian G.M. , Richardson Paul , Palumbo Antonio , Dimopoulos Meletios A. , Cavo Michele , Hajek Roman , Joshua Douglas E. , Shimizu Kazuyuki , Tricot Guido J. , Gertz Morie , Tosi Patrizia , Vesole David H. , Hussein Mohamad A. , Ludwig Heinz , Goldschmidt Hartmut , Miguel J.F. San , Sonneveld Pieter , Rajkumar S. Vincent
Published: October 2019

Purpose: Deep vein thrombosis (DVT) is increasingly recognized as a major complication of thalidomide (T; Thal) and lenalidomide (R; Rev) based therapy for multiple myeloma. Estimated incidence varies widely in the literature. There is no consensus on specific risk factors, indications for thromboprophylaxis or the agent of choice for DVT prophylaxis. The purpose of this study was to develop a consensus among members of the IMWG.

Patients and Methods: A survey was electronically mailed to 67 IMWG members on July 21, 2006 and consisted of 24 multiple-choice questions.

Results: Twenty-three IMWG members (34%) sent in their responses by the cut-off date. All (100%) felt that DVT was an important question, but most (95%) did not avoid Thal/Rev based therapy because of the DVT risk. Overall results are summarized in the Table below. A majority of physicians felt that concomitant therapy with high dose dexamethasone (90% of physicians), alkylators (55%), doxorubicin/liposomal doxorubicin (90%), and erythropoietin (75%) increase the risk of DVT associated with Thal or Rev based therapy. Aspirin (81 mg or 325 mg per day) was the preferred choice of prophylaxis when necessary. However, 65% of respondents preferred warfarin to a therapeutic INR or LMW heparin if either doxorubicin or liposomal doxorubicin was added to the regimens. Most (95–100%) felt that DVT prophylaxis was not indicated for: VAD, MP, bortezomib +/− dexamethasone regimens. Seventy-five percent felt it to be safe to resume therapy with Thal/Rev after therapeutic anticoagulation. Most (95%) believed randomized trials were needed.

Conclusion: DVT is recognized as a major clinical problem associated with Thal/Rev containing regimens. Most respondents felt that the DVT risk did not warrant any prophylaxis when either Thal or Rev were used as single agents. Aspirin was the preferred form of DVT prophylaxis for most Thal/Rev containing regimens, except when doxorubicin/liposomal doxorubicin or melphalan were added. Additional responses will be analyzed both electronically and by conference call prior to the meeting.

Therapy Estimated incidence of DVT (% respondents) Choice of thromboprophylaxis (% respondents) Single agent thal <5% (61%) None (65%) Single agent Rev <5% (80%) None (65%) Thal-Dex 10–20% (52%) Aspirin (60%) Rev-Dex 10–20% (48%) Aspirin (60%) MPT 10–20% (65%) Aspirin (40%) R-MP 5–10% (57%) Aspirin (70%)
A Phase 1 Dose Escalation Study of a Fully Human, Antagonist Anti-CD40 Antibody, HCD122 (Formerly CHIR-12.12) in Patients with Relapsed and Refractory Multiple Myeloma.
Authors: Bensinger William , Jagannath Sundar , Becker Pamela S. , Anderson Kenneth C. , Stadtmauer Edward A. , Aukerman Lea , Fox Judith , Girish Sandhya , Bilic Sanela , Guzy Serge , Solinger Alan , Dort Sherri , Wang Yongyu , Hurst Deborah
Published: October 2019

HCD122 is a novel, fully human, IgG1 antagonistic monoclonal antibody targeting the CD40 receptor. This antibody blocks CD40-mediated signaling and is a potent mediator of antibody-dependent cellular cytotoxicity (ADCC). Previous preclinical investigation confirmed expression of CD40 on myeloma cells in the majority of patients and reported antitumor activity of HCD122 against multiple myeloma cells ex vivo (Tai, Y et al. Cancer Res2005; 65(13): 5898–5906). This ongoing phase 1 study will determine the maximum tolerated dose of CHIR-12.12 in multiple myeloma patients (pts) who are relapsed or refractory after at least one prior therapy. Planned dose levels are 1, 3 and 10 mg/kg administered IV once weekly for 4 weeks. Each dose group will enroll 3–6 pts to evaluate safety, pharmacokinetics (PK) and clinical response. To date, 9 pts have been treated at 2 dose levels: 3 pts at 1 mg/kg and 6 pts at 3 mg/kg. Median patient age is 65 yrs (46–81 yrs); median number of prior therapies is 3 (2–12). No dose limiting toxicity (DLT) occurred at the 1mg/kg dose level. At 3 mg/kg, 1 DLT of grade 4 thrombocytopenia occurred in 1 pt. No other grade 3 and 4 lab abnormalities and adverse events have been reported. In 7 pts with available data, infusions were well tolerated, with easily managed grade 1–2 toxicities, primarily chills (5 pts), nausea (3 pts), pyrexia (2 pts), and arthralgia (2 pts) mainly reported during the first infusion. Preliminary PK analysis showed more than dose proportional - increase in Cmax and AUC at the 3 mg/kg dose level compared to the 1 mg/kg dose level. At the 3 mg/kg dose, antibody accumulation occurred week-to-week; the mean Cmax after the fourth infusion on Day 22 was 126.1 mg/mL(range 52 – 195 ug/mL) and HCD122 levels were measurable up to Day 57 and in one patient up to Day 99. One week after the last 3 mg/kg dose, trough levels ranged from 28 to 109 mg/mL. Of the 3 pts at 1 mg/kg, one showed stable disease (SD) for >23 weeks and two had progressive disease (PD) by week 5. Of the 6 pts at 3 mg/kg, one had partial response (PR) at week 9 and was confirmed at week 15, one had SD for > 5 weeks, and 4 had PD at week 5. One pt with PD terminated the study before final safety evaluation, and must be replaced before assessment of the 3mg/kg dose level is complete. Thus, in preliminary studies, HCD122 appears to be safe and well tolerated to date at doses of 1 mg/kg and 3 mg/kg weekly for 4 doses and shows promising anti-myeloma activity. Enrollment is continuing to determine MTD.
Kit Signaling Regulates Mitf Expression in Mastocytosis.
Authors: Lee Youl-Nam , Noel Pierre , Shahlaee Amir , Carter Melody , Kapur Reuben , Wayne Alan , Metcalfe Dean D. , Takemoto Clifford
Published: October 2019

Mastocytosis is a heterogeneous disease arising from abnormal proliferation of mast cells. Activating mutations in codon D816 of the tyrosine kinase receptor, c-kit, are found in the majority of adult patients with systemic mastocytosis, an aggressive form of the disease. Constitutive activation of the Kit signaling pathway is critical to the transformed phenotype, and thus understanding how this pathway regulates downstream events is of great importance. A number of transcription factors are also essential to mast cell development, including the Microphthalmia-associated transcription factor (Mitf). We examined Mitf expression in bone marrow biopsies from nine patients with systemic mastocytosis by immunohistochemistry; we found that Mitf is highly expressed in all cases with the D816V mutation. In contrast, Mitf is not highly expressed in non-malignant mast cells in the bone marrow from patients with aplastic anemia and leukemia, suggesting thatMitf expression is regulated by Kit-dependent signalsMitf may play a role in the transformed phenotype of mastocytosis.We show that in normal mast cells, Kit signaling markedly upregulates Mitf expression. In both normal and malignant mast cells, pharmacologic inhibitors of Kit, and the downstream kinase, PI3K, block Mitf expression. To examine whether Mitf is required for transformed phenotype from constitutive Kit signaling in mast cells, we have used a shRNA-expressing lentivirus to knockdown Mitf expression in mastocytosis cell lines. We found that silencing of Mitf markedly impaired growth in proliferation and colony forming cell assays. This work demonstrates a link between two critical factors, Kit and Mitf, in the development of malignant mast cell disease.
Concomitant Myelofibrosis and Plasma Cell Disorders: The Mayo Clinic Experience.
Authors: Mesa Ruben A. , Rajkumar S. Vincent , Schwager Susan , McClure Rebecca , Powell Heather , Lasho Terra , Kyle Robert A. , Tefferi Ayalew
Published: October 2019

BACKGROUND: Bone marrow fibrosis might represent either a primary myeloid malignancy such as myelofibrosis with myeloid metaplasia (MMM) or a reactive phenomenon associated with other malignancies or inflammatory/infectious processes. The development of myelofibrosis in the context of a plasma cell dyscrasia is uncommon and incompletely understood from the standpoint of both pathogenesis and clinical implications. We sought to determine the origin and clinical ramifications of this phenomenon based on a consecutive series of patients with plasma cell disorders (PCD).

METHODS: A retrospective search of institutional databases was performed for clinical cases of PCD with the concomitant observation of myelofibrosis. Bone marrow aspirates and trephines from the time of diagnosis of both PCD and myelofibrosis were reviewed. Clinical history was abstracted for clinical manifestations as well as disease course. DNA from archived cytogenetic pellets, when available, from the time of diagnosis, was screened for the presence of JAK2V617F.

RESULTS: Among 7587 patients with a PCD seen over the last 30 years at our institution, 29 (0.3%) displayed concomitant myelofibrosis (median age 59 years, range 27–77; 52% males). The specific PCD was multiple myeloma in 25 patients, smoldering myeloma in 2, plasmacytoma in 1, and heavy chain disease in 1. Four patients (14%) had organomegaly and bone marrow histological features most consistent with MMM. Otherwise, myeloproliferative disorder (MPD)-characteristic features were infrequent and included leukocytosis and/or thrombocytosis in only 2 patients. None of the patients displayed thrombohemorrhagic complications. Archived DNA was available in 7 cases without either clinical or bone marrow histological evidence of a MPD and yet revealed the presence of JAK2V617F in 6 of the 7 cases (86%). In addition, one other patient without MPD phenotype displayed a monosomy 5 abnormality. To date, 2 patients, one with and one without a MPD phenotype underwent leukemic transformation. All other causes of death were related to PCD-associated complications. Median survival (Kaplan-Meier) for the patients with multiple myeloma was 50 months. Survival stratified by the myeloma international staging system (ISS) was similar or better than expected (published controls of 62, 44, and 29 months): median survivals for this series of 73, 51 and 37 months for stage I, II, and III disease, respectively.

CONCLUSIONS: Myelofibrosis that accompanies a PCD often represents an underlying MPD and does not negatively impact the natural history of the associated PCD.
The Jak2 V617F Oncogene Associated with Polycythemia Vera Requires a Functional FERM Domain for Transformation and for Expression of the Myc and Pim Proto-Oncogenes.
Authors: Sattler Martin , Crowley Brian J. , Gonneville Jeffrey R. , Ho Jeremy , Hudon Heidi H. , Podar Klaus , Royer Yohan , Constantinescu Stefan N. , Griffin James D.
Published: October 2019

The V617F activating point mutation in Jak2 has been shown to be associated with a proportion of patients with myeloproliferative disorders, including polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis. In normal hematopoietic cells, Jak2 signals only when associated with a growth factor receptor (such as the Epo receptor), but the role of receptors in transformation by Jak2V617F has been controversial. Constructs expressing wild type or Jak2V617F, with and without a point mutation in the FERM domain (Y114A) required for receptor binding, were introduced into BaF3 cells already expressing a human Epo receptor. These cells were then evaluated for growth factor independence, response to growth factors, and activation of several critical Jak2 signaling pathways. We found that, whereas BaF3-EpoR cells are readily transformed by Jak2V617F to Epo-independence, the addition of the FERM domain mutation blocked transformation to factor independence. Also, the FERM domain was required for induction of reactive oxygen species by Jak2V617F. Further, while cells expressing Jak2V617F had constitutive activation of STAT5, cells expressing Jak2 V617F/Y114A did not, suggesting that signaling is defective at a very proximal level. In addition, Jak2V617F induced expression of the Myc and Pim proto-oncogenes, which are known to cooperate in the transformation of hematopoietic cells. The expression of both proteins was dependent on a functional FERM domain. Finally, we evaluated the role of active STAT5 in transformation of BaF3 cells by introducing a STAT5 mutant that is constitutively activated, and found that activation of STAT5, by itself, was sufficient to induce Jak2V617F-dependent downstream targets believed to be critical for transformation, including Myc and Pim. Overall, our results suggest that constitutive activation of Jak2 requires an intact FERM domain for a transforming phenotype, and is necessary for activation of the major target of Jak2, STAT5. These results also suggest that small molecules that target Jak2, the FERM domain, or STAT5 would have activity in diseases associated with V617F mutations.
Responses to Thalidomide Combined with Prednisone Therapy for Myelofibrosis with Myeloid Metaplasia.
Authors: Qian Shenxian , Tan Junfeng , Shi Pengfei , Gao Gaqian
Published: October 2019

Myelofibrosis with myeloid metaplasia (MMM) is currently classified as a classic (i.e. not yet molecularly defined) myeloproliferative disorder (MPD), along with essential thrombocythemia (ET) and polycythemia vera (PV). All three MPDs represent stem-cell-derived clonal myeloproliferation that, in the case of MMM, is accompanied by an intense bone marrow stromal reaction that includes collagen fibrosis, osteosclerosis, and angiogenesis. To present the results of a long-term analysis of 2 sequential phase 2 trials of thalidomide (alone or in combination) for palliation of myelofibrosis with myeloid metaplasia (MMM). We analyzed (March 2003 to August 2005) initial and long-term outcomes from 33 patients with symptomatic MMM who had enrolled in either our thalidomide single-agent trial (n=12) or our trial of low-dose thalidomide (50 mg/d) combined with prednisone (n=21). Among the 33 study patients, 18 (54%) showed some improvement in their clinical course. Response rates for specific end points included improvements in anemia (12 of 33 [36%]), thrombocytopenia (8 of 12 [66%]), or splenomegaly (5 of 30 [17%]). The combination of low-dose thalidomide and prednisone, as opposed to single-agent thalidomide, was better tolerated and more efficacious. After a median follow-up of 17 months (range, 9–27 months), 10 of 33 patients (33%) showed an ongoing response, including 8 patients in whom protocol treatment has been discontinued for a median of 21 months (range, 16–27 months). Durable treatment responses were documented for only anemia and thrombocytopenia. Treatment response was not affected by the baseline status of bone marrow fibrosis, angiogenesis, osteosclerosis, cytogenetics. Unusual drug effects, all reversible, included leukocytosis (8 patients) and/or thrombocytosis (6 patients). Thalidomide (alone or combined with prednisone) is an effective first-line treatment of symptomatic anemia or thrombocytopenia in MMM. Thalidomide-based therapy has the potential to produce durable responses in MMM-associated cytopenias, even after discontinuation of the drug.
Cytogenetic Abnormalities in Essential Thrombocythemia: Prevalence and Prognostic Significance.
Authors: Wolanskyj Alexandra P. , Gangat Naseema , Schwager Susan M. , Ketterling Rhett P. , Tefferi Ayalew
Published: October 2019

Objectives: We conducted a study to describe the cytogenetic findings as well as clinical correlates and long-term prognostic relevance of abnormal cytogenetics at the time of diagnosis of Essential thrombocythemia (ET), in terms of clinical presentation, disease transformation into more aggressive myeloid disorders, and life expectancy.

Patients and Methods: The study cohort consisted of a consecutive group of patients with ET who fulfilled the World Health Organization (WHO) diagnostic criteria, in whom bone marrow biopsy was performed at diagnosis, with interpretable cytogenetic analysis obtained in all cases.

Results: A total of 403 patients were studied (median age, 56 years; median follow-up 64 months). The prevalence of abnormal cytogenetics at presentation was 6.7 % (27 of 403). The most common cytogenetic anomalies identified included trisomy 9 (4 patients), trisomy 8 (3 patients) and deletion 20q (3 patients). Parameters at diagnosis that were significantly associated with abnormal cytogenetics included palpable splenomegaly (p=0.03), current tobacco use (p=0.04); venous thrombosis (p= 0.019), extreme thrombocytosis (i.e., platelet count >1500 × 109/L, p = 0.03) and anemia with a hemoglobin of less than 10 g/dl (p=0.02); but did not include Jak2 mutation status, nor advanced age (≥ 60 years). During follow up, patients with abnormal cytogenetics were more likely to experience venous thrombosis (p=0.02) but not shorter survival [figure 1], transformation to AML, MDS or MMM, nor a greater requirement for cytotoxic therapy.

Conclusion: Cytogenetic anomalies at presentation are relatively uncommon in ET, and do not predict a greater predilection towards evolution into more aggressive myeloid disorders, nor inferior survival.

Fig. 1 Overall Survival According to Normal vs Abnormal Karyotype Fig. 1. Overall Survival According to Normal vs Abnormal Karyotype
Ex-Vivo Expansion and mRNA Transfection of Cord Blood Derived Natural Killer Cells with Preserved Cytotoxicity.
Authors: Tuncer Hande H. , Betancur Monica M. , Boissel Laurent H. , Friedman Robb S. , Klingemann Hans G.
Published: October 2019

Natural Killer (NK) cell-mediated cytotoxicity can control leukemia relapse while protecting patients from graft-versus-host disease (GVHD) after allogeneic stem cell transplant. Cord blood (CB) is rich in NK cells with similar properties of proliferation and cytotoxicity as adult blood NK cells. Hence these cells are attractive for developing strategies to eliminate residual disease after cord blood transplant. In this study, CB mononuclear cells were cryopreserved after CD3 depletion performed by immunomagnetic microbead selection (Miltenyi Biotec, Auburn, CA). After thawing, cells were plated for NK expansion, with or without a feeder layer of irradiated cord mesenchymal (UCM) cells either from the same (autologous) or from an unrelated (allogeneic) cord donor, with or without IL-2 (1000 IU/ml), IL-15 (10 ng/ml), IL-3 (10ng/ml) and Flt3 (10ng/ml). At a median of 19 days of culture (range 14–21), there was significantly higher expansion (range 3.5–72 fold) of CD56+/CD3− cells with the UCM feeder layer and cytokines compared to controls (mean 21.2 ± 20.8 fold increase vs 1.6 ± 0.9 fold increase with feeder layer only and 1.8 ± 0.89 fold increase with cytokines only, p=0.039 and p=0.041 respectively). There was no significant difference in NK expansion between autologous and allogeneic UCM feeder layers (29.6 ± 26.8 vs. 12.8 ± 8.9 fold, p=0.243). Expanded CB-NK cells were then tested for cytotoxicity against K562 cells using a calorimetric assay with PKH67-GL. CB-NK cells expanded either with autologous or allogeneic UCM feeder layers displayed enhanced cytotoxicity compared to controls plated with cytokines only (91.78±0.7% vs. 82.5±1.8%, p=0.003 and 89±2.3% vs. 83.7±0.18%, p=0.056, respectively). In order to test whether expanded CB-NK cells can be transfected for ultimately targeting malignant cells, we electroporated expanded CB-NK cells with mRNA produced from plasmid GFP-DNA by in vitro transcription. Flow cytometry was used to detect viability, which was 94%, 92% and 93% for non-transfected, GFP-DNA and GFP-mRNA samples respectively. GFP-mRNA expression at 24 hours was significantly higher (range 36.6–50.8%, mean 42.8±5.2%) compared GFP-cDNA controls (mean 4.2±0.35%, p<0.001). Mean GFP-mRNA expression was 35%, 31% and 16.5% at 48, 72 and 144 hours respectively. In summary, CB-NK cells can be effectively expanded with a feeder layer of UCM cells while preserving cytotoxicity and can also be genetically modified by mRNA transfection.
Alloreactivity of Natural Killer Cell Clones Against Childhood Precursor-B Lymphoblastic Leukemia Cells Is Determined by Differential KIR Expression.
Authors: Feuchtinger Tobias , Pfeiffer Matthias , Pfaffle Alexander , Telschik Heiko-Manuel , Handgretinger Rupert , Wernet Dorothee , Schumm Michael , Lotfi Ramin , Lang Peter
Published: October 2019

Relapse after allogeneic stem cell transplantation of patients with acute lymphoblastic leukemia (ALL) remains a major problem. A beneficial impact of alloreactive NK cells has been reported for myeloid malignancies, but has been questionable for B-lineage-ALL. Here we show that the majority of NK cell clones from healthy donors could effectively lyse primary blasts from a representative childhood precurser-B-ALL and demonstrate the relevance of differential surface expression of KIRs (killer cell immunglobulin like receptors CD158a, CD158b and CD158e) for antileukemic alloreactivity. More than 79% of clones exerted specific lysis >40% against primary ALL blasts. Mismatch of effector/target HLA-C type (KIR-ligand incompatibility) did not correlate with antileukemic alloreactivity, although non-malignant lymphoblastoid cell lines were lysed according to KIR-ligand incompatibility. In contrast differential surface expression of the three major KIRs showed significant impact on the antileukemic activity against precursor-B-ALL blasts. NK clones with none of the three KIRs or a single KIR that recognized no ligand, were not inhibited by the targets and exerted higher lysis (p = <0.03) in comparison to NK clones with expression of one or more KIRs with a ligand on the ALL blasts. In conclusion the differential surface expression of KIRs is relevant to predict NK cell activity against childhood lymphoid leukemia.
Functional Characterization of a Continuous Natural Killer Cell Line, KIL, In Vitro and in Adoptive Immunotherapy.
Authors: Tsai Schickwann , Leukel H. Christopher , Morita Chikako , Heikens Marc J. , Cao Thai M.
Published: October 2019

We have recently established a continuous natural killer (NK) cell line, KIL (for Killer Lymphocyte), from a coculture of normal murine whole bone marrow (WBM) and the OP-9 stromal cell line expressing the Notch ligand, Jagged2, in the presence of stem cell factor and IL-7 (Dehart et al. Blood2005;105:3521). A clonal prototype, KIL C.2, was derived from a C57BL/6 mouse and has the CD3−NK1.1+CD122+ phenotype, expresses the NKG2D activating receptor, and proliferates extensively upon in vitro stimulation with IL-2 (20ng/ml). We now show that KIL C.2 stimulated with IL-2 for 48 hours produce large quantities of interferon-gamma (10,300 ± 1850 pg/ml) and modest amounts of tumor necrosis factor-alpha (200 ± 60 pg/ml). IL-4 and IL-10 secretion was below background levels. We then evaluated the cytolytic potential of KIL C.2 using a FACS-based flourolysis assay using GFP-labeled target cells. We observed that KIL C.2 potently lyse allogeneic A20 (H2d) lymphoma tumor targets without prior activation. In addition, KIL C.2 is moderately lytic against two syngeneic (H2b) targets: a myeloma cell line, 5T33MM, and BLL C.2, a tumorigenic leukemia/lymphoma cell line that was established from a WBM culture and has the CD19+CD25+CD43+sIg− pre-B-cell phenotype (S.T. and T.M.C., unpublished data). To study the mechanism of cytolysis we performed killing assays in the presence of anti-NKG2D (MI-6) or anti-NK1.1 (PK136) blocking antibodies. Blockade of either NK1.1 or NKG2D reduced cytotoxicity against BLL C.2 targets by 50%. BLL C.2 has the C57BL/6 background, thus does not express H60 but expresses the other NKG2D ligands Rae1 at high levels and Mult1 at moderate levels as determined by quantitative RT-PCR. In contrast, blocking NK1.1 and NKG2D had no effect on cytolysis of A20 suggesting the presence of alternative activation pathways such as those mediated by MHC class I alloantigen receptors. To examine the in vivo tumoricidal potential of KIL C.2, we used a C57BL/6 → BALB/c T-cell depleted (TCD)-WBM transplantation and A20-luciferase tumor model and monitored tumor progression using the Xenogen bioluminescence imaging system. BALB/c mice lethally irradiated and rescued with 2.5 × 107 TCD-WBM along with A20-luciferase inoculation uniformly died from tumor. Mice given TCD-WBM and 1 × 106 donor splenocytes did not develop tumor but died from lethal graft-versus-host disease (GVHD). In contrast, mice given TCD-WBM and two injections of KIL C.2 at doses of 1 × 107 per mouse were protected from tumor progression without developing signs of GVHD. These results show that KIL C.2 is a highly functional NK cell line with the capacity for proliferation, cytokine secretion, and cytotoxicity against syngeneic as well as allogeneic tumors. As such, KIL C.2 provides a useful cell line model for studies of NK cell biology and suggests a potentially effective strategy for NK cell-based immunotherapy.
Elevated MCV in Patients with Hereditary Hemochromatosis.
Authors: Mikhail Sameh E. , Phatak Pradyumna D.
Published: October 2019

Background: Previous studies have reported that patients with Hereditary Hemochromatosis have higher red cell volumes than normal controls. The relationship of this abnormality to the degree of iron overload and to HFE genotype has not been systematically studied.

Method: We retrospectively compared the mean corpuscular volume (MCV) of patients with Hereditary Hemochromatosis with that of an age-, race- and sex-matched control population from the primary care clinic at our institution. We studied the relationship of MCV at diagnosis with serum transferrin saturation, serum ferritin level, phlebotomy-mobilizable iron stores and HFE genotype in our patients.

Results: The study population included 290 patients who were treated for Hereditary Hemochromatosis at our center. 122/290 were homozygous for C282Y; the mean MCV of this group was significantly elevated at 95.2 fL (control group was 89.2 fL; p<0.001) Among all patients with phlebotomy-mobilizable iron greater than four grams, regardless of HFE genotype, the mean MCV was 94.8 fL (significantly greater than control, p<0.001); there was no significant difference between those who are homozygous for the C282Y HFE mutation and those who are not. Among C282Y homozygous individuals, there was no significant correlation between the MCV versus serum transferrin saturation, serum ferritin and phlebotomy-mobilizable iron (r= 0.07, 0.207 and −0.005 respectively). In patients with mobilizable iron greater than four grams who were not C282Y homozygous there was weak correlation between the MCV versus serum transferrin saturation and serum ferritin (r= 0.39 and 0.37 respectively) and no significant correlation versus phlebotomy-mobilizable iron (r= 0.04).

Conclusion: Our study confirms the presence of elevated MCV levels in patients with Hereditary Hemochromatosis. The mean MCV was high in both C282Y homozygous individuals as well as those with elevated levels of body iron (>4 g) who were not homozygous for the C282Y HFE mutation. For the most part, the elevated MCV did not correlate with measures of body iron stores although there was a weak correlation with serum transferrin saturation and serum ferritin among iron-loaded individuals who were not homozygous for C282Y. Further studies of the factors that influence MCV in these patients may provide insights into the derangements of iron utilization that may occur in iron overload syndromes.