bluuebunny/crossref_metadata_2025 huggingface dataset:
Bortezomib and Thalidomide Treatment of Newly Diagnosed Patients with Multiple Myeloma - Efficacy and Neurotoxicity.
Authors: Borrello Ivan , Ferguson Anna , Huff Carol Ann , George Shirley , Biedryzcki Barbara , Cornblath David , Chaudhry Vinay
Published: October 2019

Introduction: As single agents, Bortezomib (V) and thalidomide (T) have activity in less than 50% of newly diagnosed myeloma (MM) patients whereas combinations with dexamethasone (D) with T (TD) and V (VD) demonstrated response rates of 63% and 85%, respectively. We sought to examine the anti-myeloma activity of VT, a steroid-free regimen. Considering the neurotoxicity of both agents, we examined the baseline neuropathy and progression of neurotoxicity post-VT.

Methods: Eligible patients had untreated Salmon-Durie Stage II/III MM and an ECOG performance status ≤2. V was given 1.3mg/m2 i.v. on days 1, 4, 8, 11 every 21 days. T was started at 50mg daily and increased weekly to 150mg. Patients were treated for a minimum of 4 (evaluable), but to a maximum of 8 cycles. The neurologic evaluation utilized the reduced Total Neuropathy Score (rTNS), a validated scoring system combining symptoms, signs and nerve conduction studies. The rTNS was measured at baseline and after every 2 cycles. rTNS scores range between 0 to 32.

Results: 30 patients have been enrolled and 27 are evaluable for response. Median age is 59 (37–83), b2-microglobulin 3.3 (1.2–36.9), and albumin 3.6 (2.3–5.2). The mean dose of V was 1.11mg/m2 and of T 110 mg/d. A dose reduction for ≥1 agent was required in all patients. Treatment was discontinued in 5 patients due to neurotoxicity, and 1 due to disease progression. A ≥ 50% reduction in M-spikes was observed in 82% of patients with 31% achieving undetectable levels. Median time to best response was 5 cycles (range 2–8). Clinical evidence of baseline peripheral neuropathy (PN) was noted in 15%, whereas 67% had abnormal baseline skin biopsies. By cycle 5 all patients developed PN: rTNS 2–8, (grade 1) 50%; rTNS 9–16 (grade 2) 31%; rTNS 17–24 (grade 3) 15%; and rTNS 25–32 (grade 4) 4%. There was no correlation between severity of PN and the cumulative V/T doses. Neuropathic symptoms improved with T/V dose reductions. Additional common adverse events (≥ grade 2) included fatigue 57%, constipation 52%, generalized pain 44%, and leg cramps 23%. Although the incidence of thromboembolic events with steroid-containing regimens ranges between 15–20%, no deep vein thromboses occurred in this study.

Conclusions: The VT combination achieved an 82% response rate in previously untreated multiple myeloma patients. No DVTs occurred with this steroid-free combination. This study established the baseline PN in newly diagnosed, untreated myeloma patients and demonstrated progression of neuropathy post-VT therapy. Future studies utilizing neuropathic preventive agents, lower doses of VT, or V in combination with other agents may yield similar disease responses with reduced neuropathic toxicities.
Genetic Heterogeneity in MALT Lymphoma.
Authors: Streubel Berthold , Raderer Markus , Chott Andreas
Published: October 2019

Translocations t(11;18)/API2-MALT1, t(14;18)/IGH-MALT1, and t(1;14)/IGH-BCL10 are known to occur in MALT lymphoma and affect a common signalling mechanism, resulting in the constitutive activation of the NF-kB pathway. However, genetic rearrangements are unknown in the majority of MALT lymphoma. In the current study, we investigated the karyotypes of 28 MALT lymphoma of various anatomical sites. Conventional cytogenetics and FISH identified aberrations in 26/28 MALT lymphoma. Balanced translocations were found in 21 cases. IGH was rearranged in the majority of cases with balanced translocations (n=20/21); 1/21 case had novel t(6;7)(q25;q11). IGH partner genes involved MALT1, FOXP1, BCL6 and 4 new chromosomal regions on chromosome arms 1p, 1q, 5q, and 9p. Long-distance inverse polymerase chain reaction identified three novel partner genes on 1p (CNN3), 5q (ODZ2), and 9p (JMJD2C). FISH assays were established and confirmed LDI-PCR results. Real-time quantitative RT-PCR showed up-regulation of the novel genes in the translocation-positive cases. Immunohistochemistry for MALT1, BCL10, FOXP1, and NF-kB was performed in all cases and demonstrated that NF-kB and FOXP1 were not activated in the majority of the cases. Our study shows that MALT lymphoma are genetically heterogenous (at least 10 different translocations) and do not share NF-kB as unifying pathway.
Lenalidomide (L) in Combination with Dexamethasone (D) Improves Survival and Time to Progression in Elderly Patients (pts) with Relapsed or Refractory (rel/ref) Multiple Myeloma (MM).
Authors: Chanan-Khan Asher Alban , Weber Donna , Dimopoulos Meletius , Chen Christine , Niesvizky Reuben , Wang Michael , Belch Andrew , Attal Michel , Spencer Andrew , Prince Miles , Olesnyckyj Marta , Zeldis Jerome , Yu Zhinuan , Knight Robert
Published: October 2019

Introduction: Treatment of elderly pts (age >65 years) with rel/ref MM remains a challenge due to concurrent comorbid conditions and poor tolerability to chemotherapy limiting therapeutic options. Recently, 2 phase III randomized clinical trials (MM-090 and MM-010) demonstrated superiority of the LD combination over D alone in previously treated MM pts. We examined the clinical benefit of LD combination in elderly pts enrolled in these 2 clinical trials.

Methods: This is a retrospective data analysis of pts enrolled on the MM-090 and MM-010. All elderly pts (>65 years) were identified and parameters of clinical outcome (overall response, OR; time to progression, TTP; overall survival, OS) recorded. To determine the clinical benefit of L in elderly pt population, we compared the group of elderly pts who received LD combination with those who received D + placebo as well as with the group of younger pts who received the LD combination in these studies.

Results: Collectively, 704 pts were enrolled with 285 identified as elderly. In the elderly group, 146 pts were randomized to LD combination and 139 to D + placebo. There were no significant differences in baseline characteristics of these two groups. In the elderly group who received LD, median time from diagnosis was 3.3 yrs (range 0.5–14.7) and 77.4% had received ≥ 2 prior therapies (prior D and/or thalidomide in 69% and 32% respectively). Using the Blade criteria, the overall response (ORR) was significantly better in pts on combination treatment vs. D alone (58.9% vs. 20.9%; p<0.001). On an intent-to-treat analysis, the median time to progression (TTP; primary endpoint) for pts treated with D alone was 20 vs. 60 wks on combination (p<0.001) and the median overall survival was 79 wks and has not been reached (p=<0.001) in the combination group, respectively. We then compared the elderly pts with the younger pts who received LD combination. The ORR and median TTP was (58.9% vs. 60.9%) and (60 wks vs. 47.3 wks), respectively. The OS was 128 wks in the younger pts and is not reached in the elderly.

Conclusion: L when combined with D improves ORR, prolongs TTP and OS in elderly pts with rel/ref MM and thus offers an important treatment option for this pt population. This clinical benefit of L achieved in the elderly is comparable to that in the younger pts and thus is irrespective of age of the pt treated. The availability of L as an oral formulation provides an added benefit in the management of elderly pts population.
Aberrant Immunoglobulin Class Switch Recombination and Switch Translocations in Activated B Cell-Like Diffuse Large B-Cell Lymphoma.
Authors: Lenz Georg , Nagel Inga , Siebert Reiner , Sanger Warren , Wright George W. , Zhao Hong , Rosenwald Andreas , Muller-Hermelink Hans-Konrad , Gascoyne Randy D. , Campo Elias , Jaffe Elaine S. , Smeland Erlend B. , Fisher Richard I. , Kuehl W. Michael , Chan Wing C. , Staudt Louis M.
Published: October 2019

To elucidate the mechanisms underlying chromosomal translocations in diffuse large B-cell lymphoma (DLBCL), we investigated the nature and extent of immunoglobulin class switch recombination (CSR) in these tumors. We used a Southern blot assay to detect legitimate and illegitimate CSR events in 92 primary tumor samples of either the activated B-cell-like (ABC; n = 50), germinal center B cell-like (GCB; n =31), and primary mediastinal B cell lymphoma (PMBL; n = 11) subgroups of DLBCL. We could clearly distinguish ABC DLBCL from GCB and PMBL by the phenotype shown in these experiments. The frequency of legitimate CSR was significantly lower in ABC DLBCL compared to GCB DLBCL and PMBL (30% vs. 58% vs. 64%; p = 0.012 and p = 0.035 respectively). In contrast, illegitimate switch recombination events were more frequent in ABC than in GCB (56% vs. 32%; p = 0.037). Similarly, ABC DLBCL had a higher frequency of internal deletions within the switch μ region compared to GCB DLBCL and PMBL (48% vs. 13% vs. 0%; p = 0.0012 and p = 0.003, respectively). ABC DLBCLs also had frequent deletions within Sγ (66%) and Sα (26%). Sequence analysis revealed ongoing Sμ deletions within ABC DLBCL tumor clones, with 4–19 separate deletions occurring per case. In addition, Sμ deletions were accompanied by ongoing segmental duplications and apparent AID-dependent somatic mutations in Sμ, consistent with failed attempts at AID-mediated CSR. Unexpectedly, short fragments derived from multiple chromosomes were interspersed within the deleted Sμ regions. Furthermore, aberrant switch recombination was responsible for translocations in ABC DLBCLs involving the known oncogenes BCL6 and MYC. In addition, we were able to identify a novel translocation involving the switch γ3 region and the gene encoding the B-cell transcription factor SPIB in the ABC cell line OCI-Ly3. These findings suggest that ABC DLBCLs have abnormalities in the regulation of CSR that predispose to chromosomal translocations.
A Systematic Review and Meta-Analysis of Thalidomide in Patients with Previously Untreated Multiple Myeloma.
Authors: Hicks Lisa , Haynes Adam , Reece Donna E. , Walker Irwin , Herst Jordan A. , Imrie Kevin
Published: October 2019

Background: Multiple myeloma disproportionately affects the elderly and is currently an incurable malignancy. New therapies for myeloma, particularly oral therapies, are urgently needed.

Objectives:

To determine if thalidomide with or without other agents, improves response rate (≥ 50% reduction in monoclonal protein), survival, and/or progression in patients with previously untreated myeloma. To determine the frequency and significance of major adverse events associated with thalidomide in this setting.

Methods: A literature search of Medline (1966–June 2006), Embase (1980–June 2006), the Cochrane Library, abstracts from the annual meetings of the American Society of Hematology (1999–2005) and the American Society of Oncology (1999–2006) was completed with a pre-specified search strategy. No language restrictions were applied. Randomized controlled trials of induction thalidomide (any dose, any duration) for adults with previously untreated multiple myeloma were included. Trials of exclusively maintenance therapy were excluded. Two reviewers independently extracted data. The methodological quality of selected trials was assessed and summarized. Weighted data was expressed as relative risk, risk difference, number needed to treat (NNT), and number needed to harm (NNH). A random-effects model was used.

Results: Six eligible studies involving almost two thousand patients (N=1875) were identified and meta-analyzed. Two studies were published and four were reported in abstract form only. Five studies reported overall response rate (ORR); the four largest trials reported statistically significant improvements in ORR with the addition of thalidomide to standard therapy. The weighted relative risk of responding to a thalidomide-containing regimen versus control was 1.50 (95% CI 1.21 to 1.86). The NNT to achieve one additional response with thalidomide was 4 (95% CI 2.9 to 8.3). Two trials reported improvements in EFS/PFS. One trial reported an improvement in OS. The risk of VTE, peripheral neuropathy, and constipation was consistently elevated with thalidomide such that for every 50 patients treated with a thalidomide-containing-regimen, one could expect 12 to 13 additional patients to respond, 4 additional patients to develop VTE (NNH 12.5; 95% CI 8.3 to 20), 2 additional patients to develop peripheral neuropathy (NNH 25; 95% CI 16.7 to 50), and 4 additional patients to develop constipation (NNH14; 95% CI 10 to 25). In our analyses, prophylactic anticoagulation appeared to decrease, but not abolish, the risk of VTE with thalidomide.

Conclusions: Thalidomide improves response rate and possibly progression free and overall survival in patients with previously untreated myeloma. It also increases the incidence of VTE, neuropathy, constipation and other adverse events. Further studies are required to confirm the survival advantage seen in one study, and to determine the optimum strategy for VTE prophylaxis.
Treatment Associated Venous Thromboembolism (VTE) and Survival in Multiple Myeloma Patients.
Authors: Zangari Maurizio , Cavallo Federica , Fink Louis M. , Barlogie Bart , Bolejack Vanessa , Burns Michael J. , Anaissie Elias , Hollmig Klaus A. , Mohiuddin Abid , Pineda-Roman Mauricio , Tricot Guido J.
Published: October 2019

Background: Venous thromboembolism (VTE) is a well known cancer complication generally associated with poor survival. We have analyzed the prognostic impact of the development of a thrombotic episode in newly diagnosed multiple myeloma (MM).

Methods: Newly diagnosed patients with symptomatic MM were eligible for enrollment. At registration, patients were randomly assigned to Thalidomide (Thal) or not; both arms received otherwise identical chemotherapy. A total of 668 patients entered the study. The two study arms were well balanced, in terms of age, Ig Subtype, cytogenetic, B2 microglobulin vs CRP. Baseline patients and disease characteristics were well balanced between those individuals who experienced VTE and the others. VTE patients were treated with LMWH followed by coumadin for at least 6 months. A 12 month landmark for VTE was used; group comparisons were performed for EFS and OS using the log rank test.

Results: With a median follow up of 53 months a total of 155 (23%) patients experienced VTE during treatment, 98 (30%) patients in the Thal arm and 57 (16%) in the non Thal arm. The development of thrombotic episode was not a negative factor for EFS (p=.1) or OS (p=0.1) (Figure 1) for the entire group as well as for the Thal/chemotherapy treated group (EF p=0.7, OS p=0.6). Patients who received only chemotherapy and developed a thrombotic episode experienced a significantly longer EFS (p=0.02) (Figure 2), and a similar trend in OS.

Conclusions: OS and EFS survival of myeloma patients treated with chemotherapy ± thalidomide is not affected by VTE development. Patients treated with chemotherapy (but not Thalidomide) who developed thrombosis during treatment experienced significantly longer EFS. Our observation supports a survival benefit associated with anticoagulation therapy in multiple myeloma patients.

Figure 1: OS from 12 months post VAD by VTE Figure 1:. OS from 12 months post VAD by VTE Figure 2: EFS from 12 months post VAD by VTE in the Non Thalidomide arm Figure 2:. EFS from 12 months post VAD by VTE in the Non Thalidomide arm
Pharmacologic Evaluation of Orally Bioavailable Inhibitors of the 20S Proteasome.
Authors: Kirk Christopher J. , Aujay Monette A. , Ho Mark , Jiang Jing , Laidig Guy J. , Lewis Evan R. , Lu Yan , Muchamuel Tony , Woo Tina M. , Sun Congcong M. , Sylvain Catherine , Yang Jinfu , Zhou Han-jie , Bennett Mark K.
Published: October 2019

Clinical application of proteasome inhibitors in the treatment of hematologic malignancies such as myeloma and lymphoma is restricted in part by the necessity of frequent IV administration and would be improved by oral (PO) administration. Selective inhibitors of the protease subunits of the 20S proteasome can be generated from peptidyl aldehydes, boronates, vinyl sulfones, and epoxyketones. Many of these peptide based proteasome inhibitors are cell permeant and capable of systemic proteasome inhibition upon intravenous (IV) administration to experimental animals such as mice and rats. In the cases of the peptidyl boronate bortezomib (Velcade™) and the epoxyketone PR-171, proteasome inhibition can be achieved in patients with IV administration. However, systemic exposure following PO administration of these inhibitors may be limited by several factors including gastric pH, gastric and intestinal peptidases, efflux pumps, biliary excretion and intestinal and hepatic metabolic activities. We have tested over 80 peptide epoxyketones with potent (IC50 <100 nM) in vitro inhibitory activity against the chymotrypsin-like activity of the 20S proteasome for bioavailability following PO administration in mice. Systemic exposure was monitored by measuring chymotrypsin-like inhibition in a number of tissues and an approximation of the relative bioavailability for selected compounds was measured by comparing the pharmacodynamics of IV and PO administration using a range of doses. These compounds were also tested in vitro for solubility, intestinal cell permeability, intestinal and hepatic metabolism, and sensitivity to the multidrug resistance protein 1 (MDR1) efflux pump in order to determine which properties were associated with oral bioavailability. We have found that oral bioavailability in mice is associated with increased intrinsic solubility and metabolic stability and reduced MDR1 sensitivity. Proteasome inhibition following PO administration is rapid, resulting in maximal proteasome inhibition within 15 minutes. Rapid absorption and clearance of selected compounds was also confirmed in mice and rats by pharmacokinetic analysis. Repeated oral administration was well tolerated at doses that resulted in significant (>80%) proteasome inhibition in most tissues. The anti-tumor efficacy of these orally bioavailable proteasome inhibitors are being assessed in both human tumor xenograft and mouse syngeneic models. The results from these studies will enable further pre-clinical development of potent, orally bioavailable proteasome inhibitors for the treatment of malignant diseases.
Zoledronic Acid Early Increases Serum Osteoprotegerin in Early Stage Multiple Myeloma.
Authors: Gozzetti Alessandro , Martini Giuseppe , Salvadori Stefania , Gennari Luigi , Avanzati Annalisa , Franci Beatrice , Campagna Maria Stella , Nuti Ranuccio , Lauria Francesco
Published: October 2019

Myeloma cells are able to induce an imbalance in the OPG/RANKL system in bone environment, and due to osteoclastic activation this is responsible for myeloma bone disease. Recent in vitro and in vivo evidence suggests that bisphosphonates exerts activity on myeloma cells, which could be of therapeutic interest particularly in patients in initial phases of multiple myeloma.

We evaluated 26 patients with smoldering myeloma (stage IA) to assess basal levels of circulating cytokines as OPG, RANKL and macrophage inflammatory protein-1 alpha MIP-1α and serum markers of bone turnover at baseline and at 2, 4, 6 months and compared to 58 healthy subjects as control group. Moreover we treated 16 of those patients with a potent bisphosphonate, zoledronic acid (ZA), to find out if its antiosteoclastic activity can be related to changes in circulating levels of RANKL, OPG and (MIP-1α). Results showed serum levels of OPG significantly lower and an increase of RANKL:OPG ratio in MM patients respect to controls. A significant correlation was found between RANKL/OPG ratio and serum CTX (r=0.45;p<0.05). Patients with MM treated with zoledronic acid did not show significant changes in serum and urinary calcium as well as serum creatinine compared to untreated patients. An early and significant reduction of serum CTX was found in treated patients, while bone ALP decreased later and not significantly. Interestingly ZA increased serum OPG and reduced RANKL:OPG ratio (4,8±2,6 vs 2,9±0,8 ; p<0.05).The positive effect of zoledronic acid on bone tissue was confirmed by the increased in lumbar spine BMD at 6 months (+3.8%± 2.6 vs baseline; p<0.05). Larger studies are urgently needed to better clarify the significance of these findings.
Megakaryocytes Cultured from Patients with IMF Produced Significantly Higher mRNA but Not Protein Levels of Fibrosing Grown Factors.
Authors: Wang Jen-Chin , Chang Tsong H , Goldberg Amit , Novetsky Allan D. , Lichter Steven , Lipton Jeffrey
Published: October 2019

Currently, the prevailing concept concerning the etiology of bone marrow fibrosis in patients with idiopathic myelofibrosis (IMF) is that it results from excessive production of fibrosing growth factors including transforming growth factor beta (TGF-B1), platelet-derived growth factor (PDGF), and fibroblast growth factor (FGF) from megakaryocytes and monocytes. Since megakaryocytes are difficult to isolate from bone marrow in IMF patients, this concept remains speculative. We obtained megakaryocytes (CD41+ cells) from 10-day in vitro culture of blood CD34+ cells in serum-free medium with thrombopoietin and stem cell factors as described and cultured monocytes from isolating blood CD14+ cells. Then quantitative analyses of fibrosing growth factors at the mRNA and protein levels were obtained. mRNA levels were obtained from real-time RT-PCR technique, and protein levels were obtained from ELISA analysis of the supernatant of CD41+ cells cultured 4 h in serum-free medium. The results showed 1) mRNA levels of TGF-B1, PDGF, and FGF produced by the megakaryocytes were significantly elevated in agnogenic myeloid metaplasia (AMM) compared with those in normal controls (p<0.05). While these growth factors were elevated several-fold in AMM compared with other myeloproliferative disorders (MPD) including essential thrombocythemia and polycythemia vera, they were not statistically significant. 2) mRNA levels of TGF-B1 were higher than levels of PDGF or FGF. 3) The mRNA levels of these growth factors produced from CD14+ cells were not significantly elevated in AMM compared with other MPDs or controls; the AMM mRNA levels were significantly elevated only in some patients. 4) The correlation of mRNA levels of these growth factors with the degree of myelofibrosis in AMM was significant with megakaryocytes (r=0.73) but not with monocytes (r=0.23). 5) ELISA analysis of the growth factors from the cultured megakaryocytes showed that, in most of the patients with AMM and other MPDs and in volunteer controls, the growth factors were undetectable, and only a few patients with AMM had significantly elevated protein levels of these growth factors. We conclude thatin IMF, megakaryocytes but not monocytes are the predominant cells producing fibrosing growth factors, andthe failure of finding increased protein levels of these growth factors in the in vitro system suggest that other factors are necessary to initiate translation of these growth factors in the megakaryoctes, and neutrophil emperipolesis with releasing factors may be important in this process.
Early-Polycythemia Vera (e-PV) with Thrombocytosis: Clinical Characteristics, Morphological Features and JAK2V617F Mutational Status Contributing to the Differential Diagnosis with Essential Thrombocythemia.
Authors: Iurlo Alessandra , Gianelli Umberto , Moro Alessia , Bianchi Paola , Fermo Elisa , Vener Claudia , Savi Federica , Franca Radaelli , Bosari Silvano , Zanella Alberto
Published: October 2019

According to the recently published WHO classification, two well-defined stages of PV, known as polycythemic phase and post-polycythemic myelofibrosis, are clearly recognizable. A few years ago, it has been reported that erythrocytosis may develop in the course of ET, and that PV may present in the early stage of the disease with an elevated platelet count, mimicking ET. Recently, it has been suggested that PV could be preceded by an “early” phase of the disease (Thiele et al, 2005), in which the increase in the red cell mass or hemoglobin level are lower than requested for the diagnosis either by the updated diagnostic criteria of the Polycythemia Vera Study Group (PVSG) or by the WHO classification. Very recently, the European Clinical and Pathological (ECP) criteria for the diagnosis of the “early” PV have been published (Michiels et al, 2006).

The aim of this study is to examine the clinical features, the bone marrow biopsies (BMBs) and the JAK2V617F mutational status of 17 e-PV patients. All presented, at diagnosis, the clinical and morphological features of ET, and manifested a well-developed polycythemic phase of PV during the course of the follow-up (median 8.6 yrs; range 2–17 yrs). We compared the study group with 19 cases of PV and 14 cases of ET (according to WHO) as controls. Clinically, e-PV patients revealed at diagnosis increased levels of Hb (e-PV: 15.5g/dl; ET: 13.8g/dl; PV: 16.9g/dl), Hct (e-PV: 45.9%; ET: 41%; PV: 51.8%) and Ptl count (e-PV: 854×109/l; ET: 877×109/l; PV: 691×109/l), splenomegaly (e-PV: 43%; ET: 0%; PV: 61%) and hepatomegaly (e-PV: 53%; ET: 14%; PV: 61%). Morphological examination of the BMBs in e-PV patients demonstrated moderate to marked increase of the BM cellularity (e-PV: 65%; ET: 0%; PV: 73%) and pleiomorphic (i.e. clusters of small to giant) megakaryocytes (e-PV: 83%; ET: 20%; PV: 100%). Moreover, increased (e-PV: 100%; ET: 14%; PV: 100%) and left shifted erythropoiesis (e-PV: 82%; ET: 0%; PV: 79%), and increased (e-PV: 65%; ET: 14%; PV: 100%) and left-shifted granulopoiesis (e-PV: 65%; ET: 0%; PV: 58%) were also found. Mutational status analysis revealed that 15/15 e-PV cases (100%) carried the JAK2V617F mutation (6 homozygous and 9 heterozygous), in comparison to 7/13 (54%) ET (1 homozygous, 6 heterozygous) and to 17/19 (89%) PV (5 homozygous, 12 heterozygous). In conclusion, the results of our study confirm the existence of a “early” phase of PV that may mimic ET. A diagnostic algorithm, useful to differentiate e-PV from ET, may be obtained considering altogether the clinical, morphological and molecular characteristics of each patient.
Distinct Leukemogenic Activity and Imatinib Responsiveness of a BCR-PFGFRα Fusion Tyrosine Kinase.
Authors: Gavrilescu L. Cristina , Cross Nicholas C.P. , Van Etten Richard A.
Published: October 2019

Chromosomal rearrangements of the platelet-derived growth factor receptor alpha (PDGFRA) gene on 4q12 have been described in a subset of patients with idiopathic hypereosinophilic syndrome/chronic eosinophilic leukemia (IHES/CEL). The most common, found in 12–17% of CEL patients, is the FIP1L1-PDGFRA fusion, resulting from a cytogenetically invisible deletion on 4q12 (Cools et al., NEJM2003; 348:1201; Griffin et al., PNAS2003; 100:7830). FIP1L1-PDGFRA+ CEL patients have an excellent response to low-dose imatinib therapy, often accompanied by complete molecular remission (Pardanani et al., Leuk. Res.2006; 30:965). Additional fusion partners of PDGFRA include KIF5B and CDK5RAP2 in CEL, and BCR in atypical chronic myeloid leukemia with eosinophilia and t(4;22)(q12;q11) (Baxter et al., Hum. Mol. Genet.2002; 11:1391). However, the oncogenic activity and imatinib responsiveness of these other activated PDGFRA alleles are unknown. In this study, we assessed the imatinib sensitivity of BCR-PDGFRα and FIP1L1-PDGFRα in hematopoietic cell lines, and compared their leukemogenic activity in a mouse retroviral bone marrow transduction/transplantation model. Like FIP1L1-PDGFRα, BCR-PDGFRα transformed Ba/F3 cells to become independent of IL-3 for survival and growth. In the absence of IL-3, FIP1L1-PDGFRα-expressing Ba/F3 cells were 30-fold more sensitive to imatinib than BCR-PDGFRα (IC50 = 3 nM vs. 90 nM) for inhibition of proliferation and induction of apoptosis. A FIP1L1-PDGFRα N659D mutant (Cools et al., Cancer Cell2003; 3:459) was relatively resistant to imatinib (IC50 = 200 nM), while the corresponding BCR-PDGFRα N659D mutant displayed increased imatinib resistance (IC50 > 2 μM). Inhibition of cellular proliferation correlated with decreased tyrosyl phosphorylation of the respective fusion kinase and of the downstream substrate PLCγ1. In leukemogenesis experiments, recipients of bone marrow transduced with retrovirus expressing either FIP1L1-PDGFRα or BCR-PDGFRα developed fatal myeloproliferative disease, with similar median and overall survival. This was accompanied by significant eosinophilia, relative to the disease induced by BCR-ABL in this model system (Li et al., Blood2001; 97:1442). Treatment of recipients of FIP1L1-PDGFRA- or BCR-PDGFRA-transduced marrow with imatinib (125 mg/kg/day by oral gavage for 20 days) suppressed leukocytosis and prolonged their survival. These results suggest that distinct signaling pathways activated by leukemogenic PDGFRα fusions in hematopoietic progenitors induce eosinophila in vivo. However, different fusion partners of PDGFRα can significantly influence the sensitivity of the fusion kinase to imatinib treatment.
Therapeutic Potential of a Desert Plant Triterpenoid Molecule for Polycythemia Vera.
Authors: Gaikwad Amos S. , Gutterman Jordan U. , Prchal Josef T.
Published: October 2019

Polycythemia vera (PV) is an acquired, clonal stem cell, myeloproliferative, hematological disorder with variable increase in erythrocytes, neutrophils and granulocytes. PV causes significant morbidity and mortality from thrombotic and hemorrhagic complications and has a propensity for leukemic transformation. Phlebotomy, interferon a and myelosuppressive chemotherapy have been the cornerstones of treatment to date. With no specific drugs to treat PV effectively, the development of new therapeutic modalities is important. A somatic mutation in the JAK2 tyrosine kinase (V617F) causing constitutive activation of the JAK/STAT pathway was recently reported in over 80% of PV patients. Based on this observation, we explored the therapeutic efficacy of tyrosine kinase inhibitors (TKIs) such as imatinib (Gaikwad A et al, Blood106: 2601, 2005) and AMN107 for PV. These TKIs showed marginal efficacy in vitro. Research using natural sources has led to important pharmacological targets for cancer therapy. Several plant molecules have either been introduced to the US market or are in late-phase clinical trials. We focused on a natural product called “Avicin D,” which is a plant derived triterpene electrophilic molecule with cyto-protective and anti-inflammatory functions (Haridas V et al, J. Clin. Invest113: 65, 2004; Haridas V et al, PNAS, 98:11557, 2001). Avicin D is reported to initiate selective pro-apoptotic activity in malignant cells (Mujoo K, et al. Cancer Res.61: 5486, 2001) by direct perturbation of mitochondria (Haridas V, et al. PNAS, 98: 5821, 2001). It has also been shown to down-regulate a group of pro-survival, anti-apoptotic proteins that act downstream of cytochrome c release, including HSP70, HSP90 and XIAP (Gaikwad A, et al. Clin. Cancer Res. 11: 1953Gaikwad A, et al. Clin. Cancer Res. 11: 2005). In addition, Avicin D also blocks glycolysis, a key metabolic process that malignant cells exploit for their proliferation and survival (Warburg phenomenon). To examine Avicin D’s therapeutic efficacy for PV, we utilized mouse reporter cells expressing the JAK2 V617F protein. Avicin D inhibited the growth of these cells with an IC50 of 2μM. Interestingly, in cells carrying wild-type JAK2 there was no significant inhibitory effect. In addition, Avicin D showed marked growth inhibition of human erythroleukemic cells (HEL) that harbor the JAK2 V617F mutation at an IC50 of 2μM. We then examined Avicin D’s effect on in vitro expanded native human erythroid progenitor cells. Avicin D showed specific growth inhibition of erythroid progenitor cells from PV patients (IC50 ~3μM) with no significant effect on the normal progenitors. We conclude that Avicin D should be a promising candidate for clinical trials of PV and other disorders that are associated with JAK2 V617F somatic mutations.
A Microelectronic DNA Chip Detects the V617F JAK-2 Allelic Ratio in Myeloproliferative Disorders.
Authors: Di Ianni Mauro , Moretti Lorenzo , Del Papa Beatrice , Ciurnelli Raffaella , De Ioanni Mariangela , Falzetti Franca , Tabilio Antonio
Published: October 2019

A single activating mutation in Janus Kinase (JAK)-2 is detected in 65%–97% patients with polycythemia vera (PV), in 23%–57% with essential thrombocytemia and in 35%–57% with idiopathic myelofibrosis. Recent studies have demonstrated the heterozygote population contains clonal and polyclonal granulopoiesis; clonality correlates with the Jak2V617F allelic ratio. We developed a microchip diagnostic platform (NMW NanoChip Molecular Biology Workstation, manufactured by Nanogen Inc, San Diego, CA) to identify the JAK-2 mutation in granulocyte-derived DNA and determine the allelic ratio. The system enables electronic deposition of biotinylated amplicons to selected pads. Twenty-three DNA samples from patients with PV were analysed by direct sequencing, AS-PCR and microelectronic chip. Sequencing results: a fragment of 345 genomic DNA was amplified by PCR under standard condition. 21/23 samples showed mutated JAK-2, 18/21 presented wild-type and mutated alleles; 3/21 had only the mutated allele. 21/23 samples showed mutated JAK-2. Microelectronic chip results: 21/23 samples showed mutated JAK-2, with a frequency of 91%. All samples were genotyped: at the first analysis 3 were identified as homozygotes; 18 were identified as heterozygotes. The allelic ratio in the 3 homozygotes shows residual wild-type allele due, in our view, to residual polyclonal granulopoiesis which the microchip detects. The allelic ratio of the 18 designated heterozygotes showed different T/G percentages, suggesting different disease stages. Combining microchip results with clonality assays could distinguish true heterozygotes. Overall genotyping was performed in 100% of positive cases. Sensitivity assay (on serial dilution of mutated HEL vs unmutated K562 cell lines) showed the electronic microchip detects the JAK-2 mutation in at least 1% of a total cell mixture. Our system is a fast, reliable and sensitive method alternative to DNA sequencing, AS-PCR or dHPLC. It overcomes the relative low sensitivity of direct sequencing and at the same time provides genotyping data even when sequencing fails or results are hard to interpret. This electronic microchip system is as sensitive as dHPLC but does not require an extra sequencing step for genotyping. Specific advantages over AS-PCR include automated calculation of the allelic ratio and consequently the genotype without any reduction in sensitivity.
Altered Expression and Signalling of Bone Morphogenetic Protein Receptor 1A (BMPR-1A) and Enhanced Hematopoietic Progenitor Cell Growth in Long-Term Stromal Culture Assays from Two Patients with a Myeloproliferative Syndrome and Pulmonary Hypertension.
Authors: Hildebrandt Martin , Bommert Kurt , Rautenberg Kirstin , Ludwig Wolf-Dieter
Published: October 2019

The rare association of pulmonary hypertension and myeloproliferative syndromes (MPS) has been described previously (Garcia-Manero G et al., Am J Hematol1999; 60(2): 130-5, Dingli D et al., Chest2001, 120(3): 801-8), but the mechanisms contributing to this potentially hazardous condition await further elucidation. Bone morphogenetic protein receptors (BMPR) modulate the size of the hematopoetic niche (Zhang J et al., Nature2003, 425(6960): 836-41), with an inhibitory effect on myeloproliferation. In the pulmonary vascular bed, a decreased or deficient expression of BMPR has been shown to result in the proliferation of vascular smooth-muscle cells, asymmetric neointimal hyperplasia in small pulmonary arteries and subsequent pulmonary hypertension (Lane KB et al., Nat Genet2000, 26(1):81-4; Du L et al, N Engl J Med2003, 348(6): 500-9). We hypothesized that, in patients suffering from MPS and pulmonary hypertension, changes in the expression of BMPR and subsequent signalling molecules Angiopoietin-1 (Ang-1) and its receptor, TIE-2, may occur not only in the lung, but also in the bone marrow and correspond with enhanced myeloproliferation in vitro.

Bone marrow stromal cells were cultured frompatients with pulmonary hypertension and MPS (n=2),patients with MPS and no evidence of pulmonary hypertension (n=3) andhealthy controls (n=3).The cultured cells were subjected to Western Blot analysis for the expression of BMP receptors BMPR-1A and BMPR-2, Angiopoietin-1 and TIE-2. Furthermore, a modified long-term culture initiating cell (LTC-IC) assay was established using the cultured bone marrow stromal cells as layers for autologous and allogeneic progenitor cell assays.

The two patients suffering from both, MPS and pulmonary hypertension, showed a diminished expression of BMPR-1A and an enhanced expression of Ang-1 and TIE-2 in cultured stromal cells when compared to patients with MPS alone and to healthy controls. In one of the two patients, a three- to fourfold increase in the number of long-term culture-initiating cells after seeding of CD34-positive cells and of bone marrow mononuclear cells from healthy donors and from the other patients included was observed in modified LTC-IC assays.

Our observations argue in favour of changes in BMP receptor expression and signalling with impact not only on pulmonary hypertension as described before, but also on the emergence of a myeloproliferative state.
Enhancement of Hematopoietic Stem Cell Engraftment and Graft vs. Leukemia Effect Induced by Matched and Haploidentically Mismatched Allograft Using Multi-Donor Stem Cell Transplantation (MDT).
Authors: Shapira Michael Y. , Or Reuven , Resnick Igor B. , Samuel Simcha , Gesundheit Benjamin , Slavin Shimon
Published: October 2019

The aim of allogeneic stem cell transplantation (SCT) is to combine tumor cytoreduction with optimal doses of chemoradiotherapy and to replace host with donor immunohematopoietic cells. Induction of host-vs.-graft tolerance by engraftment of donor stem cells, enables durable engraftment of donor lymphocytes, with subsequent induction of graft vs. leukemia effect (GVL) expected to eliminate all residual chemoradio-resistant malignant cells of host origin by alloreactive donor cells. Relapse shortly post SCT indicates tumor refractoriness and carries poor prognosis.

Multi-donor stem cell transplantation (MDT) is an innovative transplantation strategy aiming at both increasing the number of donor stem cells infused to the recipient to improve the chance of engraftment of at least one of the donors, thus avoiding the need for re-transplantation in case of rejection, while in parallel improving the displacement of residual host cells and GVL effect by stimulation of alloreactive donor T cells as bystander effect to the rejection of 1 donor by the other donor in order.

We describe 4 patients that were treated with MDT following early post allogeneic transplant relapse (n=3, resistant AML) or rejection (n=1). In order to reduce the risk for the patients, and with the assumption that MDT will stimulate anti host immune response leading to engraftment and GVL, we chose to combine transplantation from 2 donors (including the 1st donor in the patients transplanted due to relapse and both parents in the patient transplanted due rejection). The patients received a non myeloablative conditioning treatment. All patients engrafted (2 with the 2nd donor, 1 with 1st donor and 1 from his mismatched mother) with 100% donor chimera. Time to engraftment was short - median 11d (range 10–12). CR was achieved with the appearance of GVHD probably due to stimulation of donor vs. donor (and therefore against host alloantigens). No relapse was documented during follow up. Two of the recipients are currently alive with follow-up ranging 1.5–25m.

Conclusions: The use of MDT induced engraftment and GVL in patients with chemo-immuno-resistant leukemia. In view of the profound anti host activating effect of MDT, we plan to assess its therapeutic potential on engraftment facilitation when reduced intensity conditioning is used in patients in need for SCT lacking an HLA matched related or unrelated donor.
ZAP-70 Expression and Stem Cell Transplantation Results in Patients with CLL.
Authors: Villamor Neus , Salamero Olga , Esteve Jordi , Carreras Joaquim , Moreno Carol , Rovira Montserrat , Aymerich Marta , Bosch Francesc , Carreras Enric , Colomer Dolors , Campo Elías , Montserrat Emili
Published: October 2019

The absence of mutations in the VH gene (UM-VH) and the expression of ZAP-70 by leukemic cells are closely related adverse prognostic factors in CLL. Whether ZAP-70 should substitute VH gene analysis as prognostic predictor in patients with CLL is a matter of debate. We and others have shown that allogeneic but not autologous stem-cell transplantation may overcome the negative impact of the UM-VH on the outcome of patients with CLL. Herein we report on transplantation results based on ZAP-70 expression in leukemic cells. Thirty-seven patients (24 M/13 F; median age: 48 years, range: 29-63) received either an autologous (auto-SCT) (n=22) or an allogeneic (allo-SCT) (n=15) transplant. ZAP-70 was assessed by flow cytometry (19 cases), lymph node immunohistochemistry (10 cases) or both (8 cases). No discrepancies were observed in those cases in which both peripheral blood and lymph nodes were analyzed. Thirty-one patients were ZAP-70 positive and 6 ZAP-70 negative. ZAP-70 was positive in all UM-VH (n=24) and in 4 of 8 mutated VH patients. After a median follow-up of 62 months (range: 4 – 142), 5-year survival is 86% (66% to 100%) for allo-SCT and 50% (28% to 72%) for auto-SCT (p=NS), whereas 5-year disease-free survival (DFS) is 80% (54% to 100%) for allo-SCT and 37% (15% to 59%) for auto-SCT (p=0.03). Seventeen patients have relapsed: 16 of 31 of those being ZAP-70 positive and 1 of 6 of those ZAP-70 negative. In ZAP-70 positive patients the cumulative relapse rate at 5 years is 10% (2% to 65%) in the allo-SCT group and 53% (35% to 81%) in the auto-SCT group (p=0.04) (figure 1a). In addition, ZAP-70 positive patients submitted to allo-SCT have a trend for a longer DFS (76% at 5 years; 46% to 100%) than those receiving an auto-SCT (39% at 5 years; 15% to 63%) (p=0.07) (figure 1b). In summary, among transplanted patients those with ZAP-70 positive CLL have a better relapse rate and DFS if submitted to allo-SCT.

Figure Figure
WT1-Specific CD8+ T Lymphocytes May Participate in the Elimination of Acute Lymphoblastic Leukemia Following Allogeneic Stem Cell Transplantation.
Authors: Rezvani Katayoun , Yong Agnes , Mielke Stephan , Savani Bipin N. , Price David A. , Gostick Emma , Douek Daniel C. , Barrett A. John
Published: October 2019

There is clinical evidence that a graft-versus-leukemia (GVL) effect occurs following allogeneic stem cell transplantation for acute lymphoblastic leukemia (ALL). However, the potency of this GVL effect is often associated with unwanted graft-versus-host-disease (GVHD) and disease relapse remains a major contributor to treatment failure. Wilms’ tumor gene 1 (WT1) is overexpressed in 70–90% of cases of ALL and has been identified as a convenient minimal residual disease (MRD) marker. WT1 is an attractive immunotherapeutic target in ALL because peptides derived from WT1 can induce CD8+ T-cell responses, and being non-allelic, WT1 would be unlikely to provoke GVHD. We investigated whether CD8+ T-cells directed against an HLA-A*0201 restricted epitope of WT1 (WT126) occur in ALL patients during the early phase of immune reconstitution post-SCT (days 30–180). We analyzed CD8+ T-cell responses against WT1 in 10 HLA-A*0201+ ALL SCT recipients and their respective donors using WT1/HLA-A*0201 tetrameric complexes and flow cytometry for intracellular IFN-gamma. We studied the kinetics WT1-specific CD8+ T-cell responses in consecutive samples obtained post-SCT. CD8+ T-cells recognizing WT1 were detected ex vivo in samples from 5 of 10 ALL patients post-SCT but not in patients pre-SCT. WT1-tetramer+ CD8+ T cells had a predominantly effector memory phenotype (CD45RO+CD27−CD57+). WT1 gene expression in pre-SCT and donor samples was assayed by quantitative real-time PCR (RQ-PCR). WT1 expression in PBMC from healthy donors was significantly lower than in patients (median 0, range 0–66 ×10−4 WT1/ABL compared to patients, median 12, range 0–2275 ×10−4 WT1/ABL) (P < 0.01). There was a strong correlation between the emergence of WT1-specific CD8+ T cells and a reduction in WT1 gene expression (P < 0.001) (as depicted below) suggesting direct anti-ALL activity post-SCT. Disappearance of WT1-specific CD8+ T-cells from the blood coincided with reappearance of WT1 gene transcripts, consistent with a molecular relapse, further supporting the direct involvement of WT1-specific CD8+ T-cells in the GVL response. These results provide evidence for the first time of spontaneous T-cell reactivity against a leukemia antigen in ALL patients. Our results support the immunogenicity of WT1 in ALL patients post-SCT and a potential application for WT1 peptides in post-transplant immunotherapy of ALL.

Figure Figure
Cytotoxic T Lymphocyte Responses to PR1 Peptide in Chronic Myeloid Leukemia Patients Inversely Correlate with Proteinase 3 and Elastase Expression but Donor PR1 Responses Determine Survival after Stem Cell Transplantation.
Authors: Yong Agnes S.M. , Rezvani Katayoun , Savani Bipin N. , Eniafe Rhoda , Mielke Stephan , Goldman John M. , Barrett A. John
Published: October 2019

The primary granule proteins (PGP) neutrophil elastase (ELA2) and proteinase 3 (PR3) both contain the nonapeptide PR1 which can induce cytotoxic T lymphocyte (CTL) responses in chronic myeloid leukemia (CML). The relative contribution of PR3 and ELA2 to PR1 expression is not known. We previously found that higher levels of PR3 and ELA2 gene expression in CD34+ progenitor cells were associated with longer survival in CML patients. Eradication of leukemia depends on the elimination of leukemic stem cells which are thought to reside within the CD34+ progenitor cell pool. We therefore studied PGP expression and T cell response to PR1 in 23 CML patients and their HLA-identical family donors prior to T-cell depleted allogeneic stem cell transplantation (SCT) with T-cell add-back on day 45–100 post-SCT. CD8+ T cells and CD34+ progenitor cells were purified from mononuclear cells (MNC) of cryopreserved leukapheresis products from HLA-A*0201+ CML patients. Following reverse transcription of RNA from MNC and CD34+ cells, ELA2 and PR3 gene expression was measured using real-time quantitative polymerase chain reaction (RQ-PCR). To assess PR1-CTL responses, T2 cells were loaded with PR1 peptide (VLQELNVTV) at 0.1, 1, and 10μM for 2h, irradiated and subsequently co-cultured with CD8+ T cells for 3h. PR1-CTL response was measured as interferon-γ mRNA expression (relative to CD8) obtained by RQ-PCR. PR1-specific CTL responses were detected in 5/23 CML patients and 6/16 HLA-identical donors. In CML patients, pre-SCT expression of both PR3 and ELA2 in MNC was strongly correlated with the expression in CD34+ cells (p=0.009 and p=0.0006 respectively). There was an inverse relationship between PR1-CTL response in CML patients pre-SCT and PR3 or ELA2 expression (p=0.02 and p=0.01 respectively). This data suggest that both PR3 and ELA2 expression in CD34+ cells and their progeny are a potential source of PR1. However, high expression of these proteins may result in selective deletion of PR1 T cell clones. The presence of PR1-responses in HLA-identical donors was associated with an improved overall survival post-SCT. However, there was no impact of PR1-response in either CML patients pre-SCT or donors, on time to achieve molecular remission post-SCT but a greater proportion of patients whose donors did not have a PR1-response succumbed to fatal graft-versus-host disease. These findings support post-transplant vaccination strategies with PR1 peptide to eradicate minimal residual disease but in patients who are not eligible for SCT, the lower PR1 response in the presence of high PGP expression suggests that there is a risk that vaccines given to patients with minimal residual disease could induce tolerance.

Figure Figure