bluuebunny/crossref_metadata_2025 huggingface dataset:
Lenalidomide (L) in Combination with Dexamethasone (D) Significantly Improves Time to Progression (TTP) in Non-Stem Cell Transplant Patients (pts) with Relapsed or Refractory (rel/ref) Multiple Myeloma (MM): Analysis from MM-009 and MM-010 Randomized Phase III Clinical Trials.
Authors: Chanan-Khan Asher Alban , Yu Zhinuan , Weber Donna , Chen Christine , Niesvizky Ruben , Spencer Andrew , Attal Michel , Belch Andrew , Prince Miles , Olesnyckyj Marta , Knight Robert , Zeldis Jerome , Dimopoulos Meletius
Published: October 2019

INTRODUCTION: Although autologous stem cell transplant (ASCT) has demonstrated high overall survival (OS) and progression free survival (PFS) in MM pts, for many pts this may not be an option due to comorbid conditions, advance age, aggressive refractory disease or personal preferences. Two phase III studies demonstrated superiority of the LD combination over D alone in rel/ref MM pts resulting in approval of L by the FDA. To evaluate the utility of LD combination in non-transplant pts we examined all pts who were enrolled on the MM-090 and MM-010 clinical trials and compared the efficacy and safety profile of LD combination among pts with no prior ASCT (NT) vs. those who had a prior ASCT (PT).

METHODS: Data set from the 2 phase III trial was queried and pts with or without a previous ASCT identified. Clinical outcome (overall response rate, ORR; time to progression, TTP; overall survival, OS) was compared between the two groups.

RESULTS: Of the 704 pts enrolled 353 (210 in the PT and 143 in the NT group) were randomized to receive LD. Patients demographic are summarized in Table 1. The median dose for L in both group was 25mg. Using the Blade criteria the ORR and CR rates in the PT vs. NT group were 63% (CR =13%) and 55% (CR=16%) p=0.12, while the median TTP was 44.1 and 61.4 (p=0.13), respectively.

Toxicity: Although a higher incidence grade 3/4 neutropenia in the PT vs. NT group (38.1% vs. 27.3, p<.05) was noted the incidence of grade 3/4 thrombocytopenia were comparable (5.7% vs. 6.3%). The incidence for toxicity associated discontinuation of therapy in each arm was 12.9% and 19.6%.

CONCLUSION: Our analysis showed no difference in ORR and CR rates in the 2 groups though there was a trend towards prolong TTP in NT pts. Although there was a slightly higher incidence of grade 3/4 neutropenia, overall there was no impact of prior ASCT on treatment outcome with LD. An interesting observation was that the median time from first pathologic diagnosis was the same for both groups; this observation is encouraging as they offer advantage to pts who may not have a chance to benefit from ASCT and supports the rationale to investigate L based therapies early on in the course of treatment.

Characteristics of patients treated with LD combination. Prior Transplant Group (PT) No Prior Transplant (NPT) P value Median age (range) 59.5 (33–77) 69 (38–86) <0.001 Sex (%) M/F 62.4/37.6 55.2/44.8 0.187 Stage III (%) 60 70.6 0.184 B2M 3.1 4.1 <0.05 Time from first pathologic diagnosis in years (range) 3.4 (0.6–15.7) 2.9 (0.4–14.7) 0.098
Update on Phase I Clinical Trial of IPI-504, a Novel, Water-Soluble Hsp90 Inhibitor, in Patients with Relapsed/Refractory Multiple Myeloma (MM).
Authors: Siegel David , Jagannath Sundar , Mazumder Amitabha , Vesole David , Borrello Ivan , Sydor Jens , Wright Jim , Patterson Jon , Normant Emmanuel , Adams Julian , Anderson Kenneth , Grayzel David , Richardson Paul
Published: October 2019

Introduction: IPI-504 is a novel, water-soluble Heat Shock Protein 90 (Hsp90) inhibitor. It is the chemical reduction product of 17-AAG, a well-characterized Hsp90 inhibitor. 17-AAG is currently in clinical trials, but suffers from very poor aqueous solubility and must be administered to patients in either suboptimal DMSO formulations, or newer cremophor or emulsion formulations. In contrast, IPI-504 exists as a hydrochloride salt which is soluble in water in excess of 200 mg/mL. Therefore, IPI-504 is 4000-fold more soluble than 17-AAG. It has previously been shown that IPI-504 inter-converts with 17-AAG and exists in a pH and enzyme-mediated dynamic redox equilibrium which has been observed in human clinical trials as well. In preclinical animal studies, IPI-504 has demonstrated in vitro and in vivo anti-tumor effects in a variety of cancers including xenograft and orthotopic models of multiple myeloma (MM).

Methods: An open-label phase I dose-escalation trial of IPI-504 infused on days 1, 4, 8 and 11 of a 21 day cycle is being conducted in patients (pts) with relapsed/refractory MM. IPI-504 is administered to pts in 250cc of normal saline over thirty minutes. The primary objectives of the study are to determine the maximum tolerated dose (MTD) and characterize the pharmacokinetic (PK) and pharmacodynamic (PD) marker effects of IPI-504. Levels of Hsp70 protein were measured as a marker of IPI-504’s biologic activity in peripheral blood leukocytes. Toxicity is evaluated by NCI CTCAE (v3.0) and response by EBMT. An accelerated dose titration design was employed with dose escalation of 100% for the first three dose levels with 1 pt / cohort. The first three pts received 22.5, 45 and 90 mg/m2 respectively. Per protocol, once the 150 mg/m2 level was achieved, dose escalation converted to a modified Fibonacci schema with 3 pts per cohort. Subsequently dose escalation occurred in increments of 66%, 50%, 33% and 25%.

Results: Currently, 15 pts (11 female / 4 male), average age 61.3 years, have been treated. IPI-504 has been well-tolerated at doses ranging from 90 to 300 mg/m2. Patients continue to be enrolled at the 400 mg/m2 dose level. Dose limiting toxicities (DLTs), defined as grade 4 or greater hematologic toxicity and/or grade 3 or greater non-hematologic toxicity within the first 21 days of treatment, have not been observed to date. Dose escalation will continue until a recommended phase 2 dose is identified. Analysis of PK, safety, tolerability, and biologic response to IPI-504 is ongoing. The above support the planning of further clinical exploration of IPI-504 including combination with agents such as bortezomib that have proven synergistic in animal models of MM.
Phase II Trial of SCIO-469 as Monotherapy (M) or in Combination with Bortezomib (MB) in Relapsed Refractory Multiple Myeloma (MM).
Authors: Siegel David S. , Krishnan A. , Lonial S. , Chatta G. , Alsina M. , Jagannath S. , Richardson P. , Hohl R.J. , Lust J.A. , Bensinger W. , Carrum G. , Moreb J. , Simic A. , Barlogie B. , Maziarz R.T. , Anderson K.C. , Lin J. , Lowe A. , Vetticaden S. , Zhu J.
Published: October 2019

Background: p38a mitogen-activated protein kinase (MAPK) mediates production of proinflammatory cytokines and other factors including PGE-2 induced RANKL and IL-1 induced IL-6. SCIO-469 inhibits p38a MAPK blocking synthesis of Il-1ß, PGE2, VEGF, MIP-1α and TNFα thus reducing in vitro MM tumor growth and survival, and drug resistance.

Objectives: Assess efficacy, safety, tolerability of SCIO-469 as M or MB in patients (pts) with relapsed and refractory multiple myeloma.

Methods: Pts were treated with SCIO-469 alone (60 mg po tid)and assessed on Days 15, 30, 52 and 73. Bortezomib (1.0–1.3mg/m2 d 1,4,8,11 q21d) was added to M for PD or SD. Pts who had clinical benefit on either M or MB at Day 73 were allowed to continue on extension study for up to a total of 168 d, to further evaluate efficacy and saftey of M or MB.

Results: 62 pts were enrolled: 39 M, 23 F, median age was 63 years (range 48–84), D-S stage at Dx: I (6%), II (32), III (60%%); median time from diagnosis to treatment 3.1 years; median # prior Rx = 5; including 29% with > 6; 71% prior transplant. 27 had received prior bortezomib and 17 were considered refractory. All 62 pts received study drug; 34 pts received MB at some time during the treatment period. 5 of the 28 (18%) pts treated with M and 25 of 34 (74%) pts treated with MB continued on the extension study. Median time on study was 77.5 d (range 7–304) for M and 180.5 d (range 34–275) for MB. Best response (EBMT; Blade 1998) to M was stable disease in 24% of pts; the best response to MB was 9/34 PR (26%), 2/34 MR (6%),3/34 SD (9%). Median time to progression (TTP) was 50 days for M compared to 140 days for MB.

Safety/Tolerability: Adverse events on M were tabulated separately from those on MB. Neutropenia was minimal, < 3% with either regimen; anemia (47 vs 29%) and thrombocytopenia (26 vs 16%) were more prevalent with MB vs M, respectively. Epistaxis, fatigue and diarrhea were seen in > 15% of pts receiving M. Nausea, vomiting, diarrhea, constipation, abdominal pain, fatigue, pyrexia, anorexia, URI, arthralgia, muscle cramp, dizziness, peripheral neuropathy and headache occurred in > 15% pts receiving MB. 10 pts died on study, 9 during primary phase and 1 during extension phase. 3 deaths were the result of sepsis, the remaining 7 were due to progressive disease.

Conclusions: SCIO-469, 60 mg tid, as monotherapy or in combination with Bortezomib was well tolerated. Although there were no objective responses in M (compared to 32% (11/34) in MB-including 4 patients who had failed prior bortezomib), 24% had stable disease at the end of monotherapy. Further studies to refine the dose of SCIO-469 as monotherapy and of SCIO-469 in combination with other agents are warranted.
In Utero Is Superior to Ex Utero Cord Blood Collection.
Authors: Shpall Elizabeth J. , Karandish Safa , Sadeghi Tara , Hosing Chitra , Garcia Christopher J. , Cherry Michael T. , Walker Julia A. , Denman Corliss R. , Saliba Rima M. , Champlin Richard E. , Fernandez-Viña Marcelo , Cano Pedro , McMannis John D.
Published: October 2019

Cord blood (CB) cells are being used increasingly as a source of hematopoietic support in patients lacking human leukocyte antigent (HLA)-matched family or unrelated donors. 1124 CB units were collected, based on obstetrician preference, with the placenta either in utero or ex utero. If logistically feasible, both an in utero collection followed immediately by an additional ex utero collection once the placenta was delivered was performed. We compared the distribution of the collection parameters shown in the following table, using non-parametric tests [data expressed as median (range)]:

Athough the volume of CB collected was highest with the sequential in utero plus ex utero method, the number of total nucleated cells (TNCs) and CD34+ cells was similar for both the in utero procedures and higher than with the ex utero alone procedure. The number of CB units that had microbial contamination was similar for all three groups: ex utero-0 CB units, in utero-1 CB unit and in utero plus ex utero-1 CB unit. There were no clinical adverse events associated with any of the collection procedures.

Conclusion: In utero collections are safe and result in CB units with significantly higher volumes, TNCs and CD34+ cells than ex utero collections, with no difference in the microbial contamination rate.

CB Parameters Ex Utero (N=390) In Utero (N=334) In+Ex Utero (N=400) In+Ex vs Ex (p) In+Ex vs In (p) In vs Ex (p) Volume (ml) 76.5 88.5 94 <0.0001 0.03 <0.001 (35–199) (45–185) (42–207) Tot Nucleated cells (xE7) 94 115 119 <0.001 0.4 <0.001 (20–267) (36–399) (41–393) CD34+cells (xE6) 2.9 3.4 3.1 0.2 0.16 0.01 (0–30) (0–34) (0–55)
Clinical-Scale Combined CD4/CD8 Depletion for Donor Innate Lymphocyte Infusion (DILI).
Authors: Kunzmann Volker , Smetak Manfred , Weissinger Florian , Birkmann Josef , Einsele Hermann , Wilhelm Martin
Published: October 2019

The ability to selectively deplete or enrich cells of specific phenotype by immunomagnetic selection procedures holds significant promise for application in adoptive immunotherapy protocols. In vitro and in vivo studies demonstrated that MHC-independent effector cells of the innate immune system such as natural killer (NK) cells and γδ T cells have potent anti-tumor activity without inducing Graft-versus-Host disease (GvHD). However, current clinical-scale approaches to reduce the risk of GvHD usually use T-cell depletion (such as CD34+ selection or CD3+ depletion). However, both procedures also deplete γδ T cells which may be advantageous in mediating Graft-versus tumor effects and augmenting innate immune response against infections. Here we present a new method for depletion of T cells with potential GvHD reactivity and simultaneously enrichment of innate lymphocytes (NK cells and γδ T cells) from standard leukapheresis products by using a single-step immunomagnetic protocol which efficiently depletes CD4+ and CD8+ αβ T cells under good manufacturing conditions (GMP). Efficiency of CD4+ and CD8+ T cell depletion from (unstimulated) leukapheresis products (n=6) containing up to 2.0 × 1010 white blood cells, was demonstrated by 4-color flow cytometric analysis (mean log depletion of CD4+ cells: 4.12 (3.17–4.9); mean log depletion of CD8+ cells: 3.77 (2.97–4.54)). In addition to efficient depletion of CD4+ and CD8+ cells, immunomagnetic CD4/CD8 depletion resulted in enrichment of NK cells and γδ T cells (mean NK cell recovery: 38%(19–72), mean γδ T cell cell recovery: 50%(34–79)). In vitro assays of the final product demonstrated that NK cells and γδ T cells preserved their proliferative and cytotoxic capacity. We conclude that simultaneous depletion of CD4+ and CD8+ cells is feasible and can be performed in large scale under GMP conditions with sufficient depletion efficacy of αβ T cells and recovery of functionally intact innate effector lymphocytes (NK cells and γδ T cells) for potential use in adoptive immunotherapy studies.
Improved Survival with Adjuvant Donor Lymphocyte Transfusions Following Allogeneic Stem Cell Transplantation after Reduced Intensity Conditioning for High-Risk AML.
Authors: Schleuning Michael , Schmid Christoph , Koenecke Christian , Hertenstein Bernd , Baurmann Herrad , Schwerdtfeger Rainer , Kolb Hans-Jochem
Published: October 2019

Albeit clinical experience is limited adjuvant transfusion of donor lymphocytes (aDLT) may reduce the risk of relapse after allogeneic stem cell transplantation. We report our data on aDLT in high-risk AML. Cells were given within a prospective protocol that contained a sequence of chemotherapy, reduced intensity conditioning for allogeneic transplantation, and aDLT. For aDLT, patients had to be in CR at least 120 days from transplantation, off immunosuppression and free of GvHD. 30% of patients (n=46) alive at day +120 fulfilled the criteria for aDLT. They had been transplanted (24 from matched unrelated donors) for refractory (n=11) or relapsed leukemia (n=24) or in CR1 because of unfavourable cytogenetics (n=7) or other unfavourable criteria (n=2) or in CR2 with unfavourable cytogenetics (n=2). Twenty-four patients had an unfavourable karyotype, 10 with complex aberrations. Thirty-one patients with similar disease characteristics and fulfilling similar selection criteria (being alive in CR at d +120, no cGVHD and no history of aGVHD> II°) transplanted during the same time period served as control. Of these 10 pts. were transplanted at a center not using aDLT and 21 at the center in Wiesbaden during 2000 and 2002 prior to the introduction of the protocol at this center. The median time from transplant to first aDLT was 160 days (range 71–303). Median follow up of the surviving transfused patients is 594 days (range 113–1920). Nine patients received 1, 18 patients received 2, and 19 patients received 3 transfusions in escalating doses, containing a median of 1×106, 5×106 and 1×107 CD3+ cells/kg at aDLT 1, 2 and 3, respectively. Reasons for giving less than 3 transfusions were GvHD, relapse or refusal of the patient. Induction of GvHD was the main complication; grade II/III acute GvHD developed in 4, and chronic GvHD in 8 patients. So far, 8 (17%) patients have relapsed despite aDLT, as compared to 42% in the control group (Chi-square: p=0.018). Two died of refractory leukemia, whereas 5 achieved a secondary CR following adoptive immunotherapy. At the time of analysis (April 2006), 39/46 patients were alive and all but one patient who is actually receiving adoptive immunotherapy are in CR at a median of 441 days post DLT. The actuarial overall survival two years after transplant is 85% as compared to 53% in the control group and at four years 78% vs. 40%, respectively (p=0.005). In conclusion, aDLT is safe, when given in escalating doses and to a selected group of patients. Results are encouraging, and improved long term survival can be achieved.
A Mouse Model of PR1-CTL Adoptive Cell Therapy Demonstrates Rapid Reduction of Leukemia Burden in Both Bone Marrow and Extramedullary Organs.
Authors: Ma Qing , Wieder Eric D. , Jones Dan J. , Wang Changqing , Li Dan J. , Molldrem Jeffrey J.
Published: October 2019

Cytotoxic T lymphocytes (CTL) specific for a nine amino acid HLA-A2-restricted peptide, PR1 (derived from proteinase 3, P3), are capable of killing leukemia cells and contribute to the elimination of CML. PR1-specific CTL (PR1-CTL) elicited in vitro from healthy donors also can lyse P3-expressing AML blasts. Here, we examined whether PR1-CTL can be adoptively transferred to NOD-SCID/HLA-A2 transgenic mice to eliminate blasts as a preclinical model for PR1-CTL cell therapy for AML. Human AML blasts isolated from bone marrow were transferred into irradiated (200 rad) NOD-SCID/HLA-A2 mice and engrafted in bone marrow at various doses (107, 106, 105) between 2–8 weeks. Human CD45 Ab and mouse CD45.1 Ab were used to identify cells of human origin by FACS and IHC, and human CD33 and CD34 Abs were used to identify AML cells. PR1-CTL were elicited from healthy donors that specifically lysed P3-expressing leukemia. These PR1-CTL were predominantly CD45RA−/CCR7−/CD28+ and maintained an activated effector phenotype following adoptive transfer. Transferred AML blasts were significantly decreased in the bone marrow of mice receiving co-transferred PR1-CTL compared to those that received only AML: 0.5×106 AML cells plus 1×104 PR1-CTL or 0.5×106 AML cells (control) were infused into irradiated mice respectively. Mice were sacrificed at 2–4 weeks post-transfer and tissues were analyzed by FACS and IHC. Bone marrow aspirate from mice that received AML alone had 72–88% blasts in hypercellular marrow, whereas mice that received AML plus PR1-CTL had normal hematopoietic elements and only 10–11% blasts in hypocellular marrow. PR1-CTL also migrated to the secondary lymphoid organs including spleen, peripheral and mesenteric lymph nodes, and in the liver. Leukemia cells also infiltrated extramedullary organs. In the liver, intrasinuosidal and periportal collections of leukemic blasts were associated with vascular damage, and PR-CTL infiltrated these same regions by 2 weeks post-transfer. The PR1-CTL in the bone marrow and liver maintained an effector phenotype similar to the pre-infusion CTL, whereas PR1-CTL in secondary lymph organs such as blood and spleen also contained some cells expressing CCR7 that either reverted to a naïve phenotype or had expanded from the precursor population. By 4 weeks post-transfer, leukemia was no longer evident in the liver, although PR1-CTL remained. In conclusion, we found that PR1-CTL generated in vitro can eliminate the AML cells in NOD-SCID/HLA-A2 mice. PR1-CTL can migrate to sites of disease and maintain their capacity to eliminate leukemic blasts. Surface phenotype of PR1-CTL were consistent with their trafficking pattern in both vascular and end-organ tissues. Our results justify a clinical trial with adoptively transferred PR1-CTL in patients with AML.
Treatment of Established B16 Murine Melanoma Tumors with Tyrp-1 Specific CD4+ Lymphocytes.
Authors: Muranski Pawel , Boni Andrea , Paulos Crystal M. , Irvine Kari R. , Antony Paul A. , Restifo Nicholas P.
Published: October 2019

T-cell mediated response against solid tumors has been mostly associated with CD8+ cytotoxic lymphocytes, which act directly on the MHC class I expressing tumors. In the previously published model, gp-100 melanoma antigen-specific pmel-1 CD8+ T cells required co-administration of IL-2 and vaccine to induce significant regression of poorly immunogenic B16 melanoma in mice. MHC class II restricted CD4+ T-cells (T helpers) may have multiple direct and indirect effects on the immune response, but their role in adoptive cell transfer (ACT) therapy of solid tumors remains mainly undefined and based on highly manipulated models involving foreign antigens. In order to investigate the function of tumor specific CD4+ T-cells we have generated a transgenic mouse expressing a TRP-1 T cell receptor (TCR) directed against class II restricted murine melanocyte differentiation antigen tyrp-1. In vitro expanded TRP1 CD4+ cells secreted Th1-like cytokines upon antigen stimulation and caused direct cytotoxic effect against B16 melanoma. In vivo they mediated a highly effective response against large (>1cm2) B16 melanoma tumors after ACT of as few as 2.5×105 cells/mouse into C57B6 animals, which was associated with a massive tumor infiltration with CD11b+, MAC3+, GR1+ cells. TRP-1 T cells caused partial tumor rejection and prolonged survival in MHC class II−/− hosts implying the ability to directly recognize low level MHC class II on the tumor. This suboptimal effect was significantly enhanced after co-transfer of MHC class II+ APCs into MHC class II−/− hosts allowing for antigen cross- presentation.

Interestingly, Rag1−/− hosts, deficient in all T and B lymphocytes, demonstrated excellent initial response to treatment, but were not cured and succumbed to late relapse of the melanoma. Long-term responses were even more impaired in Rag1−/− γc−/− hosts, while complete and durable cure was observed in TCRα−/−, CD4−/− and C57B6 mice, suggesting involvement of other arms of the adaptive immune system. Similarly, co-transfer of 0.1×106 CD4+ TRP-1 cells and 1×106 CD8+ pmel-1 cells resulted in effective tumor regression, while the same numbers of each cells transferred individually were not sufficient to initiate a rejection. Introduction of tumor-specific CD4+ cells therefore eliminates the previously sine qua non need for co-administration of vaccine and IL-2 for effective treatment with CD8+ pmel-1 cells. Overall, we show that antigen-specific CD4+ T cells are highly effective in mediating the anti-tumor response by causing both the direct anti-tumor effect and by activating innate and adaptive arms of the immune system. These findings suggest that CD4+ T helper cells may play a key role in improving efficacy of ACT immunotherapy as central activators of the anti-tumor response.
A Novel Orally Active Small Molecule Potently Induces G1 Arrest in Primary Myeloma Cells and Prevents Tumor Growth by Specific Inhibition of Cdk4/6.
Authors: Baughn Linda B. , Di Liberto Maurizio , Wu Kaida , Toogood Peter , Louie Tracey , Gottschalk Rachel , Niesvizky Ruben , Cho Hearn J. , Ely Scott , Moore Malcolm A.S. , Chen-Kiang Selina
Published: October 2019

Cell cycle deregulation is central to the initiation and fatality of multiple myeloma, the second most common hematopoietic cancer, although impaired apoptosis plays a critical role in the accumulation of myeloma cells in the bone marrow (BM). Inhibition of Cdk4 and Cdk6 by the Cdk inhibitor, p18(INK4c), is required for the generation of normal, functional plasma cells1. The mechanism for intermittent, unrestrained proliferation of myeloma cells is unknown, but mutually exclusive activation of Cdk4-cyclin D1 or Cdk6-cyclin D2 precedes proliferation of BM myeloma cells in vivo2. These data identify Cdk4 and Cdk6 as key determinants in the loss of cell cycle control in myeloma and suggest that Cdk4/6 may be effective targets for therapeutic intervention. Here we show that by specific inhibition of Cdk4/6, the orally active small molecule PD 0332991 potently induces G1 arrest in primary BM myeloma cells ex vivo, and prevents tumor growth in disseminated human myeloma xenografts. PD 0332991 inhibits Cdk4/6 proportional to the cycling status of the cells independent of cellular transformation, and acts in concert with the physiologic Cdk4/6 inhibitor p18(INK4c). Inhibition of Cdk4/6 by PD 0332991 is not accompanied by induction of apoptosis. However, when used in combination with a second agent such as dexamethasone, PD 0332991 markedly enhances the killing of myeloma cells by dexamethasone. PD 0332991, therefore, represents the first promising and specific inhibitor for therapeutic targeting of Cdk4/6 in multiple myeloma and possibly other B cell cancers.
Inhibition of CKS1B Expression Affects Myeloma Cell Growth In Vivo.
Authors: Chen Bangzheng , Liu Ling , Mitchell Kristin L. , Wu Xiasong , Colla Simona , Zhan Fenghuang , Shaughnessy John D.
Published: October 2019

CKS1B is the regulatory component of the SCF-Skp2 ubiquitin ligase that ubiquinates the cyclin-dependent kinase inhibitor p27kip1 targeting it for proteasomal degradation. Overexpression of CKS1B is associated with poor prognosis in malignancies such as oral squamous cell carcinomas, colorectal carcinoma, breast cancer, prostate cancer, and others. Recently, high expression levels of CKS1B were associated with an aggressive course of Multiple Myeloma (Shaughnessy J. Hematology. 2005;10 Suppl 1:117). We investigated whether inhibition of CKS1B in myeloma cell lines with multiple copies of 1q21 and high expression of CKS1B affects in vivo tumor growth. Myeloma cell lines were transduced with lentivirus conditionally expressing (tet on) CKS1B shRNA and lentivirus constitutively expressing luciferase (for in vivo experiments), or with lentivirus constitutively expressing CKS1B shRNA (for in vitro work). Cells with inducible expression were injected into the human bones of SCID-hu mice, and, once tumors established, expression was induced with doxycycline in the drinking water. Changes in tumor sizes were followed by weekly luminescence imaging. Cells with constitutive shRNA expression were used to determine effects on cell cycle by propidium iodide flow cytometry. Effects of shRNA expression on cytoplasmic and nuclear CKS1B and p27kip1 levels were determined by western blot analysis. Induction of CKS1B shRNA expression in JJN3 and OCI-MY5 cells in SCID-hu mice inhibited tumor growth by 57% and 81% within one week, compared to non-targeting construct. As expected, viable cells recovered from these tumors had low levels of CKS1B and higher levels of p27kip1 proteins. Similar to observations in the MM cell lines JJN3 and OCI-MY5 (see Abstract by Zhan and colleagues), constitutive expression of shRNA in CAG and XG1 cells effectively reduced cytoplasmic and nuclear CKS1B protein levels. This was associated with increased levels of p27kip1 protein, marked (51%) increases of cells in the G1 phase of the cell cycle, and reduced S and G2 (33% and 52%, respectively), compatible with a G1 arrest, and a 76% increase in the number of sub G1 (apoptotic) cells. In contrast to these observations, cells constitutively over-expressing p27kip1 had no change in G1, 50% fewer cells in S, and a 150% increase in G2, indicating G1 and G2 arrests, with no change in the sub G1 fraction. Interestingly, cells over-expressing p27kip1 had lower levels of CKS1B proteins, compatible with the reported proteasomal degradation of the molecule during the later phases of the cell cycle (Hattori T et al, Genes Cells.2003;8:889). These observations suggest that the poor prognosis associated with 1q21 amplifications and high CKS1B expression in myeloma reflects an elevated proliferative state of the tumor cells that results in rapid regrowth of minimal residual disease.
CD4+ Peptide Epitopes from the WT1 Oncoprotein Stimulate CD4+ and CD8+ T Cells That Recognize and Kill Leukemia and Solid Tumor Cells.
Authors: May Rena J. , Pinilla Javier , Korontsvit Tatyana , Zakhaleva Victoriya , Zhang Ronghua H. , Dao Tao , Maslak Peter , Scheinberg David A.
Published: October 2019

Wilms tumor protein 1 (WT1) is a transcription factor over-expressed in several types of leukemias and solid tumors, making it an ideal target for immunotherapy. A number of class I binding WT1 peptides have been identified and shown to stimulate CD8+ T cells. These peptides are being tested as potential cancer vaccine candidates in a variety of clinical trials. However, the induction and maintenance of a robust memory CD8+ cytotoxic T cell response requires CD4+ T cell help. Herein we report the identification of three HLA Class II peptide epitopes of WT1 using the SYFPEITHI and RANKPEP predictive algorithms. Peptides 328–349 and 423–441 are able to stimulate a peptide specific CD4+ response that can recognize WT1 positive tumor cells in multiple HLA-DRB*1 settings, as determined by IFN-gamma ELISPOT assays. Due to the highly polymorphic nature of the HLA class II alleles, such broad reactivity is critical in the development of a universal therapeutic. In addition, we identified a WT1 CD4+ peptide epitope (122–140) that lies within close proximity to a previously identified CD8+ peptide epitope (126–134). Residue 126 was mutated from an Arginine (R) to a Tyrosine (Y) thereby embedding a synthetic immunogenic analog CD8+ peptide that was previously designed to improve immunogenicity and induce a potent CD8+ response. Mutated peptide 122–140 is able to induce a CD4+ and cytotoxic CD8+ WT1 specific T cell response that can recognize the native WT1 epitopes on the surface of human CML and solid tumor cells. Cross-priming experiments demonstrated that APCs pulsed with either CML or mesothelioma tumor lysates can process and present each of the CD4+ peptides identified. These studies provide the rationale for using the three WT1 CD4+ peptides in conjunction with CD8+ peptide epitopes to vaccinate patients with WT1 expressing cancers.
A Novel Strategy for Generation of Human Tumor-Specific T Cell Clones for Adoptive Transfer.
Authors: Lee Seung-Tae , Liu Shujuan , Sukhumalchandra Pariya , Molldrem Jeffrey , Hwu Patrick , Liu Yong-Jun , Kwak Larry W. , Lizee Gregory , Neelapu Sattva S.
Published: October 2019

Adoptive T-cell therapy using donor lymphocyte infusions is a promising approach for treating hematological malignancies. But, efficacy is limited by the induction of graft-versus-host disease. Transfer of tumor-specific T-cell clones could enhance the graft-versus-tumor effect and eliminate graft-versus-host disease. However, isolating antigen-specific T-cell clones by the traditional limiting dilution approach is a time-consuming and laborious process. Here, we describe a novel strategy for rapidly cloning tumor-specific T cells. Lymphoma-specific T-cell lines were generated from two follicular lymphoma patients by repeated in vitro stimulation of lymphocytes isolated from tumor or blood with autologous soluble CD40 ligand-activated tumor cells. After four in vitro stimulations at 10-day intervals in the presence of IL-2 and IL-15, T-cell lines were found to be predominantly CD4+ T cells and produced significant amounts of TNF-a, GM-CSF, and IFN-γ in response to autologous tumor cells. The tumor reactivity was MHC class II restricted suggesting that it was mediated by CD4+ T cells. Staining with a TCR Vb antibody panel, a set of monoclonal antibodies against 24 human TCR Vb families, revealed that certain Vb families were overrepresented in each CD4+ T-cell line. In patient 1, 51% of CD4+ T cells were Vb1 positive, and in patient 2, 27% of CD4+ T cells were Vb8 positive. To clone lymphoma-specific T cells, CD4+ T-cell lines were labeled with CFSE and stimulated with autologous tumor cells. After 9 days of in vitro expansion in the presence of IL-2 and IL-15, CD4+ T-cell lines were stained with an anti-human CD4-APC monoclonal antibody and an anti-human TCR Vb-PE monoclonal antibody for each CD4+ T-cell line. Proliferating Vb1 cells from patient 1 and Vb8 cells from patient 2 were identified by their reduction in CFSE staining, and CD4+TCRV b +CFSEdim cells were sorted by flow cytometer. Monoclonality of the sorted cells was confirmed by PCR using a panel of optimized primers specific for 24 TCR Vb families, by TCR Vb spectratype analysis, and finally, by sequencing the TCR Vb gene used by each T-cell clone. Sorted tumor-specific T-cell clones could be expanded to large numbers using a 14-day rapid expansion protocol with allofeeder PBMCs, and confirmed to retain specificity against autologous tumor cells in a cytokine induction assay. This approach was also successfully used to isolate melanoma-specific CD8+ T-cell clones from two patients. We conclude that this approach is highly reproducible, rapid, and efficient for generating antigen-specific T-cell clones for adoptive T-cell therapy against human malignancies in the autologous or allogeneic setting.
Lenalidomide Strongly Enhances Natural Killer (NK) Cell Mediated Antibody-Dependent Cellular Cytotoxicity (ADCC) of Rituximab Treated Non-Hodkin’s Lymphoma Cell Lines In Vitro.
Authors: Wu Lei , Schafer Peter , Muller George , Stirling David , Bartlett J. Blake
Published: October 2019

Lenalidomide (Revlimid® is approved for the treatment of transfusion-dependent patients with anemia due to low- or intermediate-1-risk MDS associated with a del 5q cytogenetic abnormality with or without additional cytogenetic abnormalities, and in combination with dexamethasone is for the treatment of multiple myeloma patients who have received at least one prior therapy. Encouraging early results suggest a potential for clinical efficacy in B cell non-Hodgkin’s lymphoma (NHL). Potential mechanisms of action include anti-angiogenic, anti-proliferative and immunomodulatory activities. Lenalidomide has been shown to enhance Th1-type cytokines and T cell and NK cell activation markers in patients with advanced cancers. Furthermore, lenalidomide has been shown to enhance rituximab-mediated protection in a SCID mouse lymphoma model in vivo. We have utilized an in vitro ADCC system to assess the ability of lenalidomide to directly enhance human NK cell function in response to therapeutic antibodies, such as rituximab (chimeric anti-CD20 mAb). Isolated NK cells produced little or no IFN-γ in response to IgG and/or IL-2 or IL-12. However, pre-treatment of NK cells with lenalidomide greatly enhanced IFN-γ production by NK cells in a dose-dependent manner. In a functional ADCC assay, NHL cell lines (Namalwa, Farage & Raji) were pre-coated with rituximab and exposed to NK cells pre-treated with lenalidomide in the presence of either exogenous IL-2 or IL-12. After 4 hours in culture the viability of the tumor cells was assessed. Lenalidomide consistently and synergistically increased the killing of tumor cells in a dose-dependent manner and up to >4-fold compared to rituximab alone. Rituximab alone had only a small effect in this model and there was no killing of cells in the absence of rituximab. The presence of either exogenous IL-2 or IL-12 was required to see enhanced killing by lenalidomide. In cancer patients lenalidomide has been shown to increase serum IL-12 levels and is also known to induce IL-2 production by T cells in vitro. Potential mechanisms for enhanced ADCC include increased signaling through NK FCγ receptors and/or IL-2 or IL-12 receptors. However, we found that these receptors are unaffected by lenalidomide, although downstream effects on NK signaling pathways are likely and are being actively investigated. In conclusion, we have shown that lenalidomide strongly enhances the ability of rituximab to induce ADCC mediated killing of NHL cells in vitro. This provides a strong rationale for combination of these drugs in patients with NHL and CLL.
The Effects of Iron Deficiency on Neutrophil/Monocyte Oxidative Burst Response in Children.
Authors: Berrak S.G. , Angaji Meryem , Turkkan Emine , Canpolat Cengiz , Timur Cetin , Demiralp-Eksioglu Emel Z.
Published: October 2019

Background: Microbial killing killing of by phagocytic cells by iron containing myeloperoxidase pathway is part of the immune system. We aimed to investigate the effects of iron deficiency anemia (IDA) on oxidative burst response of phagocytic cells and understand if the effect is reversible after iron supplementation.

Material and methods: There were 57 IDA patients and 31 healthy children as the control group that were aged between 6 months-12 years with similar demographic status. IDA patients were classified according to the severity of their anemia as follows; group 1 (Hb<8 gr/dl), group 2 (Hb≥8 gr/dl and Hb≤10 gr/dl), and group 3 (Hb>10 gr/dl). Neutrophil and monocyte oxidative burst response of each three groups of IDA and control group were compared by flow cytometry. IDA group were given oral iron supplementation (3 mg/kg-Ferrosanol) therapy. On day 15 of iron supplementation therapy neutrophil and monocyte oxidative burst response of each three groups of IDA were rechecked and compared to the control group. In order to minimize the discrepancies observed in functional studies, the oxidative burst index was calculated as the proportion of oxidative burst response in IDA group to the simultaneous oxidative burst response in the control group.

Results: The number of infection episodes observed in the last three months was significantly higher in the IDA group (0–13 episodes) than the control group (1–2 episodes) (p=0.05). Neutrophil (p=0.1) and monocyte (p=0.02) oxidative burst responses in IDA group were found to be less than the control group. On day 15 of iron supplementation therapy, these values were observed to increase to the control group’s values. Evaluation of neutophil and monocyte burst indexes at the diagnosis of IDA revealed that the neutrophil burst indexes in the Hb ≤10 gr/dl group (p=0.006), and monocyte burst indexes in the Hb <8 gr/dl group (p=0.01) were significantly low. The initially observed significant differences in oxidative burst responses disappeared at day 15 of iron supplementation therapy in all hemoglobin groups.

Conclusion: Correction of the differences in oxidative burst response after iron supplementation therapy implies the fact that IDA might be the reason for changes in neutophil and monocyte burst indexes.
Analysis on the Correlation of RPS19 Expression Level to Diamond-Blackfan Anemia; Insights from the Expression Level of RPS19 Point Mutants in Erythroid Cell Lines.
Authors: Kuramitsu Madoka , Hamaguchi Isao , Takuo Mizukami , Masumi Atsuko , Momose Haruka , Takizawa Kazuya , Motizuki Masayo , Naito Seishirou , Yamaguchi Kazunari
Published: October 2019

Mutations in ribosomal protein S19 (RPS19) gene is closely related to Diamond-Blackfan anemia (DBA). We have found point mutation, deletion and insertion in RPS19 gene in DBA patients (Blood, 100: 2724–31, 2002). In our analysis of DBA patients, gene expression of RPS19 from these mutated genomes were down regulated. Retroviral transduction of RPS19 gene to patient bone marrow cells restored the ability to differentiate into erythroid in vitro (Mol Ther., 7: 613–22, 2003). However, the mechanisms of how these mutations in RPS19 associate with anemia remains unknown.

To analyze the mutated genome, we have made constructs of mutated RPS19 gene-expression vectors. From reported 56 different RPS19 mutations, we focused on the missense mutants and tried to characterize them. Flag-tagged twelve missense mutants (V15F, L18R, P47L, W52R, R56Q, S59F, A61Q, R62W, R101H, G120R, G127Q, and G131R) were exogenously expressed in several cell lines by retroviral vectors and were analysed by western blotting or FACS. When these 12 mutants were expressed in erythro-leukemic cell lines K562 and HEL, almost all the mutant proteins (except for G120R) were expressed at significantly low level compared to that of wild type. However, some of these mutants were expressed in human kidney epithelial cell line, 293T cells. These findings suggest that the expression level of RPS19 protein derived from mutated RPS19 genome is dependent on cell types, whether erythroid-lineage cells or others.

In order to analyze the function of RPS19, doxycycline (Dox)-inducible expression of siRNA against RPS19 was induced in K562 cells and observed growth arrest at day 4 after induction of siRNA by Dox. Expression level of RPS19 decreased to one tenth of the normal level. Cell cycle analysis and quantitative-PCR analysis revealed that the growth arrest of K562 cells expressing siRNA against RPS19 was due to the induction of cell cycle arrest at G1 phase (G0/G1 54 % vs 36% ) coincide with the up-regulation of p21 and p57 mRNA level. From these results, we could possibly suggested that, in DBA patients, RPS19 protein expression is not stable and the decreased level of RPS19 protein prolongs G1 phase of cell cycle especially in the course of differentiation on erythroid cells, which may contribute to the retarded production of red blood cells.

Our findings allow to understand the entire feature of DBA-associated RPS19 mutations especially on the erythorid differentiation and may help defining the therapeutic target to improve DBA.
Clinical Characteristics of Patients with Fanconi Anemia in Complementation Group J.
Authors: Dasi-Carpio Angeles , Casado Jose A. , Sanz Guillermo , Rio Paula , Santos Maria-Teresa , Bueren Juan A. , Surralles Jordi , Monteagudo Emilio , Carreras Maria C.
Published: October 2019

Fanconi anemia (FA) is an inherited disorder characterized by chromosomal instability, developmental abnormalities, progressive bone-marrow failure, and cancer susceptibility. To date, twelve complementation groups have been described for FA (A, B, C, D1, D2, E, F, G, I, J, L and M) and eleven associated genes have already been identified (FANCA, FANCB, FANCC, FANCD1/BRCA2, FANCD2, FANCE, FANCF, FANCG/XRCC9, BRIP1/FANCJ, FANCL and FANCM/Hef). The FA complementation group J (FA-J) was first reported in 2004. FA-A is the most common group, accounting for approximately 65% of all affected individuals while FA-J is a rare group, comprising only 1.6% of all FA patients in the International Fanconi Anemia Registry (IFAR). To the best of our knowledge 11 FA-J patients have been reported in the IFAR. Patients with Fanconi anemia show extreme clinical heterogeneity.

Thus, it is of interest to associate the phenotype with the complemantation group, since this could provide predictive information on clinical outcome.

So far, in the Hospital La Fe, 4 patients (two siblings and two unrelated patients) belong to scarce FA-J complementation group. This represents the 4.5 % of the FA patients included in the Spanish Fanconi Anemia Registry. Subtyping was done by viral complementation. Cells from these patients were not complemented with a battery of vectors with FA genes (FANCA, FANCC, FANCG, FANCE, FANCF, and FANCD2). However, the characteristic cellular hypersensitivity to mitomycin C was reverted when a lentiviral vector encoding for BRIP1/FANCJ was used. Moreover, mutational studies confirmed the subtyping of FA-J in these Fanconi patients. The clinical course of the 4 FA-J patienst are shown in the Table.

In conclusion, its noteworthy the high frequency of the FA-J genotype in Spain. The 75% of our patients have major congenital malformations and severe endocrinopathy. All our patients had short stature and 50% were mycrosomic. None of our patients had skin abnormalities, although this is present in 80% of the patients in other registries. Although two of these patients developed aplasia very early (5 years old), FA-J siblings developed severe aplasia at the age of 20 years but none of them developed leukemia or solid tumors.

Clinical course of patients Patient 1* 2* 3 4 * Sibling sex male female male female Aplasia onset (years) 24 20.5 4.3 3.5 Survival (years) 28 31.5 6 alive Short stature Yes Yes Yes Yes Microsomy No Yes Yes No Skin pigmentation No No No No Eyes abnormalities No No microphthalmia Epiphora Skeletal malformations Syndactyly No Abnormal thumbs Abnormal thumbs Kidneys Agenesis, left No No Horseshoe Endocrinopathy failure puberty not available hypotiroidism hypopituitarysm Gastrointestinal malformations No No Esophageal atresia Onphalocele Malignacies No No No No Family cancers Not available Not available Liver Lung, brains and breast
The Changing Map of the Sickle Cell Gene in Kenya.
Authors: Aluoch Joash R. , Aluoch Louise H.M.
Published: October 2019

In a ten year prospective records survey we received reports of sickle cell anemia (SCA) from ethnic groups which initially had no sickle cell trait (SCT) in Kenya. A follow up study was done in 2000 using alkaline Hb-electrophoresis to reassess the current status of SCT in comparison with a study reported in the 1950’s. We found that of the initially unaffected groups, SCT was 8% in the sabaot, 6% in the Taita, 3% in the inland (Mt. Kenya) Kikuyu, 1% in the Turkana, but still absent in the Somali. There was also a shift in %-ages from the 1950 survey in tribes with SCT: 33% in the Suba in stead of 28%, and 22% in the Kambe instead of 35%. We also noted a remarkable difference between cosmopolitan and rural populations in two SCT endemic areas: 15% in Kisumu city against 28% rural neighborhood and 15% in Bungoma town against 21% in its rural population. It appears that sickle cell (HbS) gene distribution and spread is changing in Kenya and that this trend most likely applies to the rest of Africa as well. The findings might be useful in genetic counseling as well as for diagnosis of SCA on clinical grounds in ethnic groups among whom SCA was hitherto considered to be absent.
Cell Free Fetal and Total DNA Levels in Pregnancies at Risk of Sickle Cell Disease and Significant Ethnic Variation.
Authors: Gerovassili Ageliki , Nicolaides Kypros H. , Thein Swee Lay , Rees David
Published: October 2019

Cell free (cf) DNA in maternal circulation is increasingly investigated in pregnancy. We aimed to determine if sickle cell trait women had quantitative differences of cfDNA with controls and if there was an ethnic difference between the cfDNA levels of Northern European and African/African-Caribbean populations. Non-invasive prenatal diagnosis through quantification of fetal and total cfDNA was tested in 33 pregnant women at risk of carrying a fetus affected with sickle cell disease and 124 control pregnancies. Fetal cfDNA assays were based on two Y chromosome specific markers (SRY and DYS14) and total cfDNA was based on the β-globin gene on chromosome 11. Maternal age (MA), gestation age (GA), fetal sex, storage time prior to extraction, maternal weight and body mass index (BMI) before pregnancy were compared to the fetal and total cfDNA levels. No significant difference in the fetal or total cfDNA levels was found between any of the control pregnancies and the sickle cell trait mothers carrying HbAA, HbAS and HbSS fetuses. However, higher levels of total cfDNA, but lower fetal cfDNA levels were observed in the African/African-Caribbean population compared with the Nothern Europeans. A significant variation in cfDNA was found between ethnic groups, which should be taken under consideration in future studies measuring cfDNA.
Bone Density Measurements in Thalassemia Patients with Iron Overload.
Authors: Grosse Regine , Frieling Isolde , Kuehl Bettina , Janka Gritta E. , Fung Ellen B.
Published: October 2019

Since the introduction of an improved blood transfusion and iron chelation therapy in patients with beta-thalassemia major (TM) and intermedia (TI), changes in bone structure and bone deformities are significantly diminished. However, older patients with thalassemia have a higher prevalence for fractures caused by osteoporosis. Additionally, most of these patients suffer from chronic bone pain.

In 13 patients (10 TM und 3 TI, age 8 – 32 years), we have measured the bone mineral density (BMD) by dual-energy x-ray absorption (DXA). All patients, except one, regularly received blood transfusions and iron chelation treatment (deferoxamine, deferiprone, or deferasirox). In the young patient group (n = 5, age: 8 -20 y), a mean Z-score of −1.53 was found (Z-score was calculated from age related BMD). In patients > 20 years (n = 8, age: 21 – 32 years), a mean T-score of −1.66 was observed (T-score was calculated from BMD related to 30 years). Except two patients, all patients had a decreased BMD in comparison to the normal population. One female patient with normal BMD, already received osteoporosis treatment since 5 years due to initially low BMD. Moreover, all patients had a reduced vertebra height in comparison to the normal population.

First results from monitoring the bone density by DXA and the trabecular bone structure by micro-CT, will be reported in all patients after 12 to 18 months under oral supplementation treatment with calcium and vitamin D.