bluuebunny/crossref_metadata_2025 huggingface dataset:
Hematologic Improvement (HI) by TLK199 (Telintra™), a Novel Glutathione Analog, in Myelodysplastic Syndrome: Phase 2 Study Results.
Authors: Raza Azra , Callander Natalie , Ochoa Leonel , Piro Lawrence , Emanuel Peter , Guba Susan , Shapiro Gabriel , Williams Stephanie , Burris Howard , Faderl Stefan , Estrov Zeev , Curtain Peter , Larson Richard , Young Shelby , Brown Gail L.
Published: April 2020

Introduction: Glutathione S-transferase (GST) P1-1 has shown to be an important negative regulator of cellular growth and differentiation. The effect is mediated through binding to Jun kinase (JNK) which causes a decrease in kinase activity. TLK199, a novel analog of glutathione, binds selectively to GSTP1-1 resulting in its dissociation from JNK and subsequent kinase activation. Exposure of hematopoietic progenitor cells to TLK199 led to activation of JNK followed by cellular growth and maturation. TLK199 has shown significant myelostimulant activity in vitro in human bone marrow cell cultures as well as in several in vivo preclinical models of myelopoeisis. In Phase 1, TLK199 treatment resulted in hematologic improvement (HI) in MDS patients at all dose levels.

Methods: The objectives of this multicenter Phase 2 study in MDS were to determine the safety (by NCI-CTC) and efficacy (by modified IWG MDS response criteria) of two dose schedules of TLK199 HCl Liposomes for Injection administered at 600 mg/m2 over 60 minutes by constant rate IV infusion daily x 3 or daily x 5 every 3 weeks. Patients (pts) were treated until lack of response or unacceptable toxicity.

Results: 52 MDS pts (33 M/19 F),(29 RA, 9 RARS, 8 RAEB, 3 RAEB-t, 1 CMML, 2 UK), median age 69 years (range 22–90), received 244+ cycles (1099+ treatments), median 4 (range 1–13+). Thirty-seven pts (71%) were red cell transfusion dependent and 10 pts (19%) were platelet transfusion dependent prior to entry. Pts had failed a median of 1 prior therapy (range 0–6) including: erythropoietin (27/52%), G-CSF (9/17%), thalidomide (10/19%), azacitidine (7/14%), steroids (6/12%), hormones (2/4%), and other therapies (14/27%). Thirty-nine pts were evaluable for efficacy, 32 pts (82%) experienced HI in one or more blood cell lineages, 14 of 16 pts (88%) with trilineage dysfunction, 8 of 13 pts (62%) with bilineage dysfunction, and all 10 pts (100%) with unilineage dysfunction experienced HI. Lineage response was HI-P (14 of 22/64%), HI-N (9 of 27/33%), and HI-E (22 of 35/63%). Responses were accompanied by clinical symptom improvement, decreases in RBC and platelet transfusion requirements including transfusion independence and improvements in bone marrow maturation, differentiation, M/E ratios, and dysplastic morphology. Most common adverse events were mild to moderate acute infusion related reactions commonly seen with liposomal formulations: back pain (9/17%), nausea (8/15%), chills (8/15%), and bone pain (6/12%).

Conclusions: TLK199 is well tolerated and an active agent in all FAB types of MDS. These data support the further clinical development of TLK199 in MDS as well as in other hematologic malignancies characterized by cytopenias.
Long-Term Follow-Up Results of the Combination of Topotecan and Cytarabine and Other Intensive Chemotherapy Regimens in Myelodysplastic Syndrome.
Authors: Jabbour Elias , Kantarjian Hagop , Beran Miloslav , Cortes Jorge , O’Brien Susan , Giles Francis , Pierce Sherry , Shan Jianqin , Plunkett William , Keating Michael , Estey Elihu
Published: April 2020

Background. Higher-risk myelodysplastic syndrome (MDS) is often treated with intensive chemotherapy regimens used for acute myelogenous leukemia (AML). Patients with MDS are often older and may have contraindications to anthracycline-based regimens. Topotecan-cytarabine regimens had shown encouraging results in higher-risk MDS.

Study aims. To analyze the long-term results with topotecan-cytarabine versus other intensive chemotherapy regimens in higher-risk MDS.

Study Groups and Methods. 510 patients with higher-risk MDS (marrow blasts ≥ 10%) treated with intensive chemotherapy were reviewed. Median age was 63 years; 82% had intermediate 2 or high risk MDS; 52% had secondary MDS; 40% had chromosome 5 or 7 abnormalities. Therapy was: topotecan-cytarabine in 77; idarubicin-cytarabine regimens in 270; topotecan-cytarabine and cyclophosphamide in 67; fludarabine-cytarabine in 96. Univariate and multivariate analyses were conducted to evaluate the independent associations of different variables, and most importantly the treatment regimen, with complete response, induction, mortality, and survival.

Results. The overall CR rate was 55%, induction mortality 20%, and 5-year survival rate 8%. The 5-year survival rate was 11% for patients younger than 65 years old, and 17% for patients with a normal karyotype. Among 82 patients younger than 65 years with a normal karyotype, the CR rate was 67%, 5-year survival rate 27%, and 5-year CR duration rate 33%. Topotecan-cytarabine regimens were equivalent to idarubicin-cytarabine regimens in relation to CR rates and survival rates, but were associated with a lower induction mortality. Multivariate analysis confirmed that treatment regimens were not associated with independent significant differences in CR rates or survival. However, topotecan-cytarabine regimens were still selected to be associated with lower induction mortality rates.

Conclusions. Topotecan-cytarabine regimens are equally effective to other AML regimens in higher-risk MDS and may be less toxic. Topotecan-cytarabine should be considered as a reasonable alternative to idarubicin-cytarabine in higher-risk MDS, particularly in older patients with contraindications to anthracyclines.
Trousseau’s Syndrome Revisited: Tissue Factor-Bearing Microparticles in Pancreatic Cancer.
Authors: Zwicker Jeffrey I. , Furie Barbara C. , Kos Cynthia A. , LaRocca Thomas , Bauer Kenneth A. , Furie Bruce
Published: April 2020

Thromboembolic disease is a major complication of malignant disease, and pancreatic cancer ranks among the malignancies associated with the highest rates of thrombosis. Using laser injury in a mouse model of thrombosis, we previously demonstrated that tissue factor-bearing microparticles circulating in normal plasma accumulate in the developing thrombus through an interaction mediated by P-selectin and PSGL-1. Microparticle tissue factor contributes significantly to fibrin propagation within the thrombus. Based upon this observation, we evaluated the hypothesis that elevated numbers of tissue factor-bearing microparticles might be one of the causes of cancer-associated thrombosis. Microparticles are cell-derived vesicular structures under 1000 nm in diameter. Since the light scattering methodology used in commercial flow cytometers cannot determine particle size when the diameter of the particle (200–1000 nm) is the same order of magnitude as the wavelength of the incident light employed (488 nm), we applied a novel instrumentation to determine the size, size distribution, and concentration of tissue factor-bearing microparticles. An NPE Systems Quanta flow cytometer, using impedance to measure particle size, was extensively modified for this microparticle application. Application of 780 nm fluorescent calibration beads yielded a major population from 740 to 820 nm. Application of 520 nm fluorescent beads yielded a major population from 500 to 540 nm. Using a high affinity antibody to tissue factor, tissue factor-bearing microparticles were measured in platelet-poor plasma derived from normal subjects and subjects with surgically unresectable or metastatic pancreatic cancer. Tissue factor bearing microparticles were detected in significantly more subjects with pancreatic cancer (12 of 17, 70%) than healthy individuals in whom tissue factor-bearing microparticles remained below the level of detection in all but a single individual (1 of 11, P=0.005). By comparison, tissue factor-bearing microparticles were not detected in subjects with advanced stage (III or IV) non-small cell lung cancer (N=9). In subjects with advanced pancreatic cancer, the mean concentration of tissue factor bearing microparticles was 444,000 particles/microliter with a mean diameter ranging between 371 nm to 727 nm. The two patients with pancreatic cancer in this study who suffered a recent lower extremity venous thrombosis had high measurable tissue factor-bearing microparticles levels with a mean concentration of 975,000 particles/microliter. These results indicate that tissue factor-bearing microparticles are present in the plasma of many patients with advanced pancreatic cancer and suggest that tissue factor-bearing microparticles may be central to the pathogenesis of cancer-associated thrombosis.
Low Incidence of FIP1L1-PDGFRA in Eosinophilic Patients and Long-Term Experience with Imatinib Therapy.
Authors: Pardanani Animesh D. , Ketterling Rhett P. , Li Chin-Yang , Wolanskyj Alexandra P. , Elliott Michelle A. , Camoriano John K. , Butterfield Joseph H. , Dewald Gordon W. , Tefferi Ayalew
Published: April 2020

Background: The FIP1L1-PDGFRA fusion, an imatinib-sensitive molecular target, is detected in a subset of cases with moderate-to-severe eosinophilia (eosinophil count ≥1500/μL). Using 2 FISH strategies to detect loss of the CHIC2 locus as a surrogate marker of the FIP1L1-PDGFRA fusion, we previously found 14% of cases to be CHIC2-deleted when studying bone marrow smears from 81 adult patients with moderate-to-severe, non-reactive eosinophilia at our institution (Blood 2004, 104:3038–3045). Since then, the FISH test has been made available for routine clinical use both within our institution, and to external physicians as a mail-in test, for the workup of patients with eosinophilic disorders, including systemic mast cell disease (SMCD).

Study design: FISH data, subsequent to the test’s introduction into routine clinical practice (from Nov. 2003), were retrospectively analyzed for CHIC2-deletion. Data from the initial pilot study of 81 patients was not used in this analysis (Table). The imatinib treatment analysis, however, included all CHIC2-deleted patients treated at our institution, including those from the pilot study (Table).

Results:

A total of 741 patients were studied in the post-pilot phase. Of these, 20 cases were found to be CHIC2-deleted, with an overall prevalence of 2.7% (Table). Of the 219 patients seen at our institution, 13 (all male) were found to be CHIC2-deleted (Table) (prevalence=5.9%), and of these, 10 have been treated with imatinib (100 mg/day starting dose) to date. Nine patients are evaluable for response - all achieved a complete clinical and histological remission (7 patients were tested by FISH post-treatment - all achieved cytogenetic remission). One patient received concomitant corticosteroids for co-morbidities and could not be evaluated for response. After a median follow-up of 24 months (range 17 to 39 months), all 9 patients continue to be in remission, with a normal eosinophil count. At last follow-up, one patient had discontinued imatinib and the others were on maintenance therapy at the following dose: 100mg/wk (n=1), 100mg BIW (n=1), 100mg TIW (n=2), 50mg QOD (n=1), and 100 mg/day (n=3).

Conclusions:

FIP1L1-PDGFRA prevalence in non-selected patients with eosinophilia is lower than anticipated from prior studies. Patients with FIP1L1-PDGFRA exhibit durable responses to imatinib therapy. It may be feasible to maintain some patients in remission on an empirically derived, intermittent schedule of low-dose imatinib.

Pilot Data Post-pilot Data (n=741) Total Mayo samples Mayo samples Mail-in samples Number of Patients 81 138 603 822 Number CHIC2-deleted 11 2 18 31 Prevalence 14% 1.4% 3.0% 3.8% Number Imatinib treated 8 2 n/a 10
The V617F JAK2 Mutation Is Uncommon in Cancers and Mutations in STAT5A, STAT5B and the JAK Family Genes Do Not Account for V617F Negative Myeloproliferative Disorders.
Authors: Baxter Joanna E. , Scott Linda M. , Campbell Peter J. , Todd Tony , Stephens Philip , Edkins Sarah , Wooster Richard , Stratton Michael R. , Futreal Andrew P. , Green Anthony R.
Published: April 2020

The JAK2 V617F mutation is present in 97% patients with polycythemia vera (PV) and approximately 50% of patients with essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF). JAK2 is widely expressed and is a critical component of signaling pathways in multiple cell types. It remains unclear to what extent the V617F mutation contributes to the pathogenesis of non-hematological cancers and hematological malignancies other than the classical myeloproliferative disorders.

To determine the frequency with which the V617F mutation occurs in a broad range of human malignancies, we sequenced 486 cell lines derived from over 30 different histological types of solid tumor, together with 132 cell lines derived from hematological malignancies. Only the HEL cell line, which was derived from an erythroleukemia, was positive for the mutation. We then screened a further 211 primary hematological malignancies and found the mutation in 5/90 AML patients, 1/20 myelodysplastic syndrome (MDS) and 1/91 chronic myeloid leukemia (CML) patients. The five V617F positive AML patients were male, significantly older than wild-type AML patients and did not have FLT3-ITD mutation, or the t(8;21), inv(16) or t(15;17) rearrangements. In addition, none of the five patients had a history of a prior overt myeloproliferative disorder (MPD). The V617F positive MDS patient was unusual in that he had trephine features of myelodysplasia with fibrosis, consistent with a myelodysplasia/myeloproliferative overlap syndrome. The V617F-positive CML patient had a twelve-year history of BCR/ABL-negative thrombocytosis prior to presenting with CML.

The molecular basis of the V617F-negative MPDs remains obscure. We therefore screened samples from 24 MPD patients without the V617F allele for alternative JAK2 mutations, and for mutations in other components of the JAK/STAT pathway. All 128 coding exons of JAK1, JAK2, JAK3, TYK2, STAT5A and STAT5B were sequenced in peripheral blood granulocyte DNA from 19 ET and 5 IMF patients that lack the V617F allele. No mutations were detected in these individuals.

Collectively, these data suggest that the JAK2 V617F mutation does not occur in non-haematological cancers, that this mutation is uncommon in myeloid malignancies other than the classic BCR/ABL-negative MPDs, and that the V617F-negative MPDs are likely to reflect mutations in other molecules that modulate the JAK/STAT pathway, or mutations in different signaling pathways.
Glivec/STI571 Treatment Stimulates Megakaryopoiesis and Normalizes PDGF Receptor beta Kinase Expression in Thrombocytopenic Patients with Myeloid Metaplasia with Myelofibrosis.
Authors: le Bousse-Kerdiles Marie Caroline , Desterke Christophe , Guerton Bernadette , Clay Denis , Hasselbalch Hans , Gisslinger Heinz , Thiele Jurgen , Kvasnicka Hans , Barosi Giovanni
Published: April 2020

Myeloid Metaplasia with Myelofibrosis (MMM) is a Philadelphia negative chronic myeloproliferative disorder associated with myeloid metaplasia in the spleen and liver, bone marrow fibrosis and neoangiogenesis. The myeloproliferation is characterized by an increased number of circulating CD34+ hematopoietic progenitors (up to 200 folds) with a prominent amplification of dystrophic megakaryocytes (Mk). Alteration of tyrosine kinase signals is suspected to play a key role in the myeloproliferation and in the increased sensitivity of hematopoietic progenitors to growth factors. Glivec is a small-molecule tyrosine kinase inhibitor of the ABL fusion gene, platelet derived growth factor receptor beta (PDGFRB) and SCF receptor (c-kit). In this study, we investigated the effect of Glivec/STI571 on the megakaryopoiesis and the kinase expression of 11 MMM patients enrolled in a European EUMNET clinical trial. We showed that 50 percents of the treated patients exhibited a tendency to a decrease in the CD34+ cell number from 261/μL to 56/μL with a normalization of the CD34+ cell count (1–10/μL) in 20 percents of these patients (2/10). We also observed a reduction of a CD34+CD41+ Mk subpopulation co-expressing the CD9 tetraspanin that is suggested to be a marker of MMM. Normalization of the CD9 gene expression level was further confirmed by quantitative RT-PCR in MMM mononuclear cells. In contrast, 55 percents of MMM patients (5/9 patients) showed an increase in their platelet count from 191 x 109/L to 282 x 109/L during treatment with Glivec. Interestingly, an increase in the platelet count was also observed in three of the five thrombopenic patients who exhibited an increase greater than 50 x 109/L (p= 0.02) being associated with an augmentation in the number of clonogenic megakaryocytic progenitors (CFU-Mk). The effect of Glivec treatment on megakaryopoiesis was further illustrated by a morphometric analysis on bone marrow histological sections showing a normalization of the size and shape of the dystrophic MMM Mk. We also analyzed by quantitative RT-PCR the effect of Glivec treatment on receptor kinase gene expression in circulating mononuclear cells from MMM patients and demonstrated a normalization of the receptor beta of the PDGF, a cytokine involved in Mk differentiation. Actually, the PDGFB expression that was down-regulated in MMM PBMC before Glivec as compared to normal PBMC (−1.14 +/− 0.76 and − 0.14 +/− 0.42, respectively; p= 0.02) was significantly increased during therapy, reaching a normal level in 60% of tested patients. In conclusion, our results show that Glivec treatment reduced the CD34+ cell number and stimulated the megakaryopoiesis of MMM thrombopenic patients, probably by restoring Mk progenitor differentiation.
Acquired Notch1 Mutations in Murine Precursor-T Leukemia/Lymphoma Models.
Authors: Lin Yingwei , Nichols Rebecca A. , Letterio John J. , Aplan Peter D.
Published: April 2020

NOTCH1 has been implicated in hematopoiesis, T-cell differentiation, and the pathogenesis of precursor T-cell lymphoblastic leukemia/lymphoma (pre-T LBL). Although rare patients with pre-T LBL have chromosomal translocations that activate NOTCH1, it has recently been shown that over 50% of human pre-T LBL samples which did not have chromosomal aberrations involving NOTCH1 had activating mutations in the NOTCH1 heterodimerization (HD) and/or the PEST domain. We examined murine pre-T LBL cell lines as well as primary thymic tumors that arose in SCL/LMO1, OLIG2, OLIG2/LMO1, LMO1, NUP98/HOXD13 transgenic mice, and [p27(− /−) /SMAD3(−/+)] mice for evidence of Notch1 mutations. We also investigated the timing of Notch1 mutation in SCL/LMO1 transgenic mice.

We found that 13/19 cell lines and 29/49 primary thymic tumors had Notch1 mutations in either the HD or PEST domain, but not both. Of the thirteen cell lines with Notch1 mutations, twelve had mutations in the PEST domain. The cell lines with Notch1 mutations were sensitive to gamma-secretase inhibitor treatment, indicating that the mutations were important for the survival of these cells. Of twenty-nine tumors with Notch1 mutations, 23 were in the PEST domain and 6 in the HD. All HD mutations were single base substitutions, whereas all PEST domain mutations were insertion/deletion mutations. Intriguingly, half of the PEST domain mutations mapped to one of two mutational hot spots, suggesting that these regions may be prone to insertion/deletion mutations.

Thymocytes from clinically healthy SCL/LMO1 mice aged 5 weeks showed evidence of oligoclonal T-lymphocyte expansion, but did not have Notch1 mutations and did not form tumors when injected into nude mice (pre-malignant thymocytes), whereas thymocytes from SCL/LMO1 mice aged 8–12 weeks had Notch1 mutations and formed tumors upon transplantation into nude mice. Thus, Notch1 mutations are very frequent secondary events that can cooperate with a wide range of primary events as cells progress from a pre-malignant to a fully transformed state.
Dynamic Alterations in the Expression of P2Y12 Receptor but Not COX-1 in Platelets from Streptozocin-Induced Diabetic Rats.
Authors: Chen Chi , He Zili , Ruby Candice , Arslan Waqas , Kalavar Madhumati , Chou Shyan-Yih , Steier William
Published: April 2020

In patients or animals with established diabetes mellitus, platelets have been shown to be hyperreactive to ADP. This alteration of the platelet function occurs independent of activation of the arachidonate pathway or release of dense granule contents. In the present study, we examined the expression of the platelet ADP-binding receptor, P2Y12, in early stage of streptozocin(STZ)-induced diabetes in rats. Platelet messenger RNA (mRNA) levels for P2Y12 and cyclooxygenase-1 (COX-1) were determined by comparative RT-PCR in 7 control rats on day 0, 6 diabetic rats each on days 2, 3, 4, and 4 diabetic rats on day 7 after intraperitoneal injection of streptozocin (50 mg/kg). The plasma glucose level was 406 ± 10 mg/dl in diabetic rats, significantly greater than 151 ± 12 mg/dl in control rats. After induction of diabetes, the expression of P2Y12 mRNA significantly decreased to 76 ± 9% of the level of control on day 2, 54 ± 6% of control on day 3, reached a nadir of 28 ± 6% of control on day 4, and rose to 43 ± 10% of control on day 7. These dynamic changes of P2Y12 mRNA in platelets reflected the expression of P2Y12 mRNA in megakaryocytes isolated to a high state of purity (>96 %) from rat bone marrow. Specifically, the megakaryocytic P2Y12 mRNA expression in the diabetic rats on days 1, 2 and 3 were 74 ± 8 %, 93 ± 1%, and 117 ± 6% of those in control rats, respectively. Treatment of diabetic rats with insulin reduced the trend of decline in the platelet P2Y12 expression. By contrast, the platelet COX-1 mRNA expression measured on days 2, 3, 4 and 7 after the induction of diabetes was similar to that in the control rats. These results in diabetic rats demonstrate dynamic alterations of the mRNA expression of megakaryocyte-platelet P2Y12, but not of COX-1. The decrease in the P2Y12 receptor expression in early stage of STZ-induced diabetes may serve to attenuate the hyperaggregability of platelets and reduce the risk of vascular thrombosis.
Fibrinogen and von Willebrand Factor-Independent Platelet Aggregation: The Essential Roles of β3 Integrin, Thrombin, and Divalent Ca2+ Cations.
Authors: Yang Hong , Reheman Adili , Chen Pingguo , Hynes Richard O. , Freedman John , Wagner Denisa D. , Ni Heyu
Published: April 2020

It has been documented that fibrinogen (Fg) is required for platelet aggregation. However, we recently found that platelet rich thrombi still formed in mice lacking either Fg (Fg−/−) or both Fg and vWF (Fg/vWF−/−) (Ni et al, JCI106:385–392, 2000). To explore the potential mechanisms of Fg/vWF-independent platelet aggregation, we studied platelet aggregation in vitro in platelet rich plasma (PRP). We found no platelet aggregation in Fg−/− or Fg/vWF−/− PRP induced by adenosine diphosphate (ADP) in the presence of anticoagulant reagents including divalent cation chelators (ACD, sodium citrate, and EDTA), and thrombin inhibitors (heparin, hirudin, PPACK). Since no fibrin formation occurs in Fg/vWF−/− plasma, we then induced Fg/vWF−/− platelet aggregation in non-anticoagulated platelet poor plasma (PPP). Surprisingly, robust aggregation occurred after ADP treatment. We further demonstrated that directly triggering the thrombin receptor PAR4 with thrombin receptor activation peptide (TRAP, AYPGKF-NH2) induced platelet aggregation in thrombin-inhibitor treated Fg/vWF−/− PRP and gel-filtered Fg/vWF-platelets in 1mM Ca2+ PIPES buffer. Thus Fg/vWF-independent aggregation can be induced in vitro and both divalent cations and thrombin are required for this novel platelet aggregation pathway. It is likely that ADP induced thrombin generation in Fg/vWF-/- PRP. Considering platelet aggregation has been observed in type I Glanzmann Thrombasthenic patients lacking αIIbβ3 protein, we hypothesized that Fg/vWF−/− platelet aggregation may occur in either a β3 integrin-dependent or β3 integrin independent manners. To test this hypothesis, β3 integrin deficient (β3−/−) platelets were added into the same non-anticoagulated Fg/vWF−/− plasma and no aggregation was observed after ADP treatment. β3−/− platelets also did not co-aggregate with Fg/vWF−/− platelets in vitro in aggregation assays, which were analyzed by flow cytometry with an anti-CD61 (anti-β3 integrin) antibody. Furthermore, when fluorescently labeled β3−/− platelets were injected into Fg/vWF−/− mice, β3−/− platelets did not significantly incorporate into Fg/vWF−/− thrombi under confocal intravital microscopy. The inability of β3−/− platelets to aggregate is not due to a deficiency in other adhesive receptors, since no reduction of GPIbα, β1 integrins, and P-selectin was observed on β3−/− platelets. Thus, although the GPIb complex, P-selectin, and β1 integrins may be involved in platelet aggregation, β3 integrin is essential for this Fg/vWF-independent platelet aggregation pathway. This also indicates that other alternative ligands of β3 integrin from either the plasma or platelet granules are capable of mediating platelet aggregation independent of both Fg and vWF under more physiologically relevant conditions (i.e. non anti-coagulated blood) where divalent cations and thrombin are present.
Absolute Lymphocyte Count Recovery during Standard Chemotherapy Predicts Superior Survival and Is Independent of the Hasenclever Index for Hodgkin’s Disease.
Authors: Siddiqui Mustaqeem , Porata Luis F. , Ristow Kay , Habermann Thomas M. , Colgan Joseph P. , White William L. , Witzig Thomas E. , Inwards David J. , Ansell Stephen M. , Micallef Ivana N. , Johnston Patrick B. , Martin William G. , Martenson James A. , Stafford Scott L. , Markovic Svetomir N.
Published: April 2020

Absolute lymphocyte count (ALC) post-autologous stem cell transplantation is an independent predictor for survival in Hodgkin’s Disease (HD). Lymphopenia at diagnosis is associated with worse prognosis in patients with newly diagnosed HD. The role of ALC recovery during standard chemotherapy for HD is unknown. We analyzed all patients managed for HD at the Mayo Clinic from 1986 to 2003. Of the 141 consecutive patients, two patients were excluded due to missing ALC data. Of the 139 patients, 63 patients were females and 76 males. The median age of patients at diagnosis was 31 years (range 6–75 years). 118 (85%) patients presented with nodular sclerosis HD, 10 (7%) with mixed cellularity, 2 (1%) with lymphocyte predominance, 1(1%) with lymphocyte depletion, 4 (3%) with lymphocyte predominant (nodular variant), and 4 (3%) unclassified. Frontline chemotherapy included ABVD (N = 79), MOPP-ABV (N = 47), COPP-ABV (N = 8), BEACOPP (N = 3), and Stanford V (N =2). The median number of cycles was 6 (range: 2–10). The median follow-up for the cohort was 62 months (range: 4–221 months). The ALC recovery was analyzed during the first 4 cycles as 90% of the patients received at least 4 cycles of therapy. Patients with an ALC ≥ 1.3 x 109/l at any time point within the first four cycles of chemotherapy (N = 93) demonstrated improved overall survival (OS) and progression-free survival (PFS) compared to patients with an ALC < 1.3 x 109/L at all time points within the first four cycles of chemotherapy (N =46) (10 years OS and PFS: 91% vs 51%, p < 0.0001; 85% vs 52%, p < 0.0001, respectively). Both groups were balanced for the Hasenclever Index: albumin < 4g/dl (p = 0.1), hemoglobin < 10.5 g/dl (p = 0.46), male gender (p =0.21), age ≥ 45 years (p = 0.06), stage IV disease (p = 0.68), leukocytosis ≥ 15 x 109/L (p = 0.58), and lymphopenia < 600 cell/ml (p = 0.211). Multivariate analysis demonstrated ALC recovery to be independent predictor of OS and PFS when compared to the Hasenclever index. These data suggest a critical role of lymphocyte (immune) recovery during standard chemotherapy in HD.
Mortality during Treatment of Patients with Advanced Hodgkin’s Lymphoma Undergoing Dose Escalated BEACOPP Chemotherapy: An Analysis of the German Hodgkin Study Group (GHSG).
Authors: Fuchs Michael , Franklin Jeremy , Klimm Beate , Josting Andreas , Pfistner Beate , Engert Andreas , Diehl Volker
Published: April 2020

Introduction: Due to substantial clinical progress over the past decades, the outcome of patients with Hodgkin’s Lymphoma (HL) has improved with a long-term disease free survival of nearly 80%. Even patients with advanced-stage HL show a five year freedom from treatment failure (FFTF) of 87% and overall survival (OS) of 91% when treated with 8 cycles of BEACOPPescalated (bleomycin, etoposide, adriamycin, cyclophosphamide, vincristine, procarbacine, prednisone). However, BEACOPPescalated has been associated with some acute and long-term treatment related mortality (TRM). We thus analysed the incidence, clinical features and risk factors for TRM of patients treated with BEACOPPescalated in the HD12 multicenter trial of the GHSG performed between 1998 and 2002. The HD12 was conducted for advanced HL patients (Stage IIB with large mediastinal mass and/or extranodal involvement, stage III/IV). All patients received 8 cycles of chemotherapy either 8x BEACOPPescalated (Arm A/B) or 4xBEACOPPescalated + 4xBEACOPPbaseline (Arm C/D) +/− 30Gy radiation on bulk and residual tumor.

Results: In this study, 43 patients (3.1%) from a total of 1392 included died from TRM. 5 patients were excluded from this analysis because of various reasons (change of fist-line therapy due to toxicity, TRM in BEACOPPbaseline) 38 patients were eligible for this analysis. 30 patients (79%) had infectious complications, 6 (16%) cardiac events such as arrhythmia or heart failure, 1 patient died due to bleomycin-related toxicity and 1 case remained unclear. 25 patients (66%) were older than 50 years in contrast to the whole HD12 study population with only 17% of patients being older than 50. There was no statistical difference between those cases with treatment related mortality and the whole study population in terms of other clinical risk factors such as gender, B-symptoms, extranodal involvement, stage of disease, large mediastinal mass or elevated ESR. There was also no difference between the 4 study arms. Most events occurred during the first 4 courses of BEACOPPescalated (79%) with the majority during the first cycle (n = 12; 32%). 23/26 (89%) of patients who died during cycles 2 – 8 had prior WHO grade III/IV leucopenia or infection.

Conclusion: Patient age and toxicity in previous cycles are the most obvious risk factors for TRM in patients with advanced HL undergoing BEACOPPescalated chemotherapy. In the HD12 study, the use of G-CSF was mandatory and most patients received their treatment on an outpatient basis. Thus, possible measures to reduce toxicity with this treatment include the prophylactic use of antibiotics as well as treating those with risk factors at least for the first course as inpatients.
A Prospective Study of Combined m-BACOD Chemotherapy and Radiotherapy for Aggressive Nasal NK/T-Cell Lymphoma.
Authors: Lei Kenny I.K. , Yu Brian K.H. , Hui Edwin P. , Zee Benny C.Y. , Chan Anthony T.C.
Published: April 2020

Purpose: Primary nasal NK/T-cell lymphoma is a highly aggressive tumor associated with poor response to therapy, frequent relapse, and a short median survival of 7–12 months. Moreover, the optimal treatment has not yet been established as most published studies were retrospective in nature. We performed a prospective study to evaluate the efficacy of combined m-BACOD chemotherapy and involved field radiotherapy (RT) for patients with previously untreated or relapsed nasal NK/T-cell lymphoma.

Methods: Between January 1998 and December 2004, all previously untreated or relapsed patients (pts), aged > 18 years, with biopsy proven nasal NK/T-cell lymphoma, ECOG performance status < 2, and adequate marrow, cardiac, liver and renal functions were included in this study. Previously untreated pts were treated with m-BACOD (iv methotrexate 200mg/m2 D8, D15 with leucovorin rescue, iv bleomycin 4mg/m2 D1, iv doxorubicin 45mg/m2 D1, iv cyclophosphamide 600mg/m2 D1, and iv vincristine 1mg/m2, and dexamethasone 6mg/m2 D1-5 PO) every 3 weeks for 6 cycles, followed by involved field RT (54 Gy in 27 daily fractions over 5.4 weeks). Relapsed pts with prior anthacycline-based chemotherapy were treated with 3 cycles of m-BACOD, followed by RT. Stage I/II pts with static response (SR) after 3 cycles m-BACOD were treated with early RT. Young pts (<60 years) who achieved complete remission (CR) with m-BACOD were offered a course of high-dose therapy BEAM before RT. Endpoints were objective response (OR), treatment tolerability, progression-free-survival (PFS), and overall survival (OS).

Results: Seventeen pts (14 untreated, 3 relapsed) were included. The median age was 49 years (range: 34–90) with a sex ratio of 1:1.1 (M:F). Fifteen pts had stage I disease, 1 had stage II and 1 had stage IV disease. A median of 5 cycles (range: 2–6) of m-BACOD were delivered to each pt. An OR was observed in 15 pts (88%) after m-BACOD induction with CR in 13 pts (76%), PR in 1 pt (6%) and SR in 1 pt (6%). Two pts (12%) had progressive disease during m-BACOD therapy. The two pts with PR and SR were converted to CR after RT, resulting in a final CR of 88%. Grade 3/4 adverse events were leukopenia (70%), thrombocytopenia (12%), infection, (24%), pneumonitis (12%). There was 1 (6%) therapy-related death. Only 5 pts opted for BEAM high-dose therapy. With a median follow-up of 49 months (range: 3–82), the median PFS and median OS were 31.5 months (range: 1.9–68.9) and 35.6 months (range: 2.9–81.6), respectively. The 5-year PFS and OS were 55% and 63%, respectively. These results appears superior than our historic control (5-yr OS: 33%, Clin Oncol 1999) and that reported by others (5-yr OS: 37.9%, Int J Radiat Oncol Biol Phys. 2002)

Conclusion: m-BACOD chemotherapy in combination with involved field RT is a highly active and well tolerated treatment for pts with aggressive nasal NK/T-cell lymphoma.

Figure Figure
Comparison of LIC Obtained from Biopsy, BLS and R2-MRI in Iron Overloaded Patients with β-Thalassemia, Treated with Deferasirox (Exjade®, ICL670).
Authors: Piga A. , Fischer R. , Harmatz P. , St. Pierre T. G. , Longo F. , Fung E. , Engelhardt R. , Perrotta S. , Galanello R. , Capra M. , Vichinsky E. , Janka-Schaub G. , Masera G. , Cianciulli P. , Coates T. , Janssen G. , Jeng M. , Lai M. E. , Salles G. , Cole P. , Alberti D. , Rosenkranz G. , Opitz H.
Published: April 2020

Chelation therapy for patients with transfusional hemosiderosis is guided by the degree of iron overload and ongoing transfusion requirements; liver iron concentration (LIC) is one tool for measuring current iron burden. During the clinical development of the novel oral iron chelator deferasirox, LIC was measured in subcutaneous liver biopsy samples, but also non-invasively by biomagnetic liver susceptometry (SQUID-BLS) and magnetic resonance imaging (R2-MRI: FerriScan®). These methods were compared in a substudy of a large, open-label, randomized Phase III study in 586 β-thalassemia patients with transfusional hemosiderosis; the study evaluated the efficacy and safety of long-term deferasirox and deferoxamine treatment.

48 patients (aged 17–35 yrs) gave informed consent for adding BLS and MRI assessments (baseline, 6 and 12 months) to the biopsy assessments (baseline and 12 months) of the main study. At baseline, 47 patients were assessed by BLS and biopsy, 45 by R2-MRI; at 6 months, 42 by BLS and R2-MRI; at 12 months, 41 by BLS and R2-MRI, 39 by biopsy. BLS was performed and evaluated locally at 3 centers in Hamburg, Turin and Oakland. MR images were recorded at 4 centers in Hamburg, Turin, Stanford and Los Angeles and were analyzed centrally. Data from BLS and R2-MRI remained blinded until study completion. LIC from biopsies was determined by AAS from extracted paraffin blocks in a central laboratory and used as the standardized reference method. LIC was expressed as mg/g dry weight for all methods: for BLS, a wet-to-dry weight conversion factor of 3.3 was applied, while MRI was calibrated against fresh freeze-dried liver biopsies. The relationship between BLS or R2-MRI with biopsy measurements was modeled by linear regression after log-transformation of the data. To account for the correlation between baseline and post-baseline measurements of the same patient, mixed-effects models were fitted.

On average, the LIC data obtained from BLS and biopsy were related by a factor of 0.46 (95% CI: 0.32, 0.67). With R2-MRI assessment of LIC vs biopsy, the factor was 0.72 (95% CI: 0.54, 0.97). BLS and R2-MRI measurements could not be related appropriately by a factor. Overall, LIC from biopsy was generally larger than that obtained from BLS; R2-MRI measurements fell in-between biopsy and BLS. Differences are caused in part by the variability in the wet-to-dry weight ratio relating in vivo with in vitro methods.

In conclusion, LIC data from 3 independent methods were correlated but not equivalent. It is therefore important that for an individual patient, a single method is consistently used under standardized conditions.
Identification of a Transcription Factor Potentially Involved in γ-Globin Gene Expression.
Authors: Grosso Michela , Amendola Giovanni , Petruzzelli Raffaella , Puzone Stella , Sessa Raffaele , Di Concilio Rosanna , D’Urzo Giovanna , Izzo Paola
Published: April 2020

The switch from fetal to adult globin gene expression occurs around birth when fetal hemoglobin (HbF) production gradually declines within a few months. Much effort is underway to clarify the molecular basis of this mechanism, since impaired hemoglobin switching leading to persistent expression of fetal globin genes in adults (HPFH) offers therapeutic potential for hemoglobinopathies (S.L. Thein. Br. J. Haematol2004; 124: 264–74).

In order to identify and study regulatory factors putatively involved in γ-globin gene expression, we examined the reticulocyte mRNAs differently expressed in three siblings (one brother, 54 years-old and two sisters 35- and 37-years old, respectively). The eldest brother had been referred to Umberto I Hospital for evaluation of a severe condition of β-thalassemia intermedia on chronic transfusion therapy since 1990. Both of his sisters resulted clinically affected by a milder form of thalassemia intermedia, non-transfusion dependent, showing Hb values around 8.4 g/dL, Hb A2 levels from 6.0 to 7.9% and HbF ranging from 14.5 to 27.6%, values higher than those resulted in their brother (5.9% Hb A2 and 7.2% HbF). Molecular analysis was performed on DNA extracted from peripheral leucocytes and revealed the same β-globin gene cluster genotype for all these subjects who resulted homozygous for the β+ IVSI-6 (C→T) mutation associated to haplotype VI chromosomes. Different levels of HbF were thus presumably responsible of different clinical phenotypes. To investigate the possible causes of the variations in γ-globin gene expression, extensive sequence analysis was performed on putative regulatory regions within the β-globin gene cluster (Zhi-Hong Lu et al., Blood1996; 87: 1604–11). Results showed the same genetic background in all the siblings. It was thus supposed that genetic determinants external to the β-globin gene cluster were responsible of the different γ-globin gene expression. To explore this hypothesis, the reticulocyte transcriptome was analyzed by a differential mRNA display approach. Reticulocytes were isolated from peripheral blood and total RNA extracted for all family members. Our study revealed several bands differentially displayed in the sample from the more severely affected sibling respect to his sisters. Selected bands were cloned in a pGEM T-vector (Promega, WI) and sequenced. Comparative sequence searches were performed using the BLAST algorithm. Preliminary data gave, for two of the clones originated from bands with increased expression in the brother, a complete homology (greater than 95%) with the cDNA sequence of a cold shock domain protein, displaying features of a repressor factor for several hematopoietic genes (Horwitz M. et al. JBC1994; 269: 14130–39; P. Coles et al. Nucleic Acids Res1996; 12: 2311–17).
Pharmacogenomics of Deferiprone Metabolism.
Authors: Haverfield Eden V. , Weatherall David J. , Graber Andrea Yoder , Ramirez Jacqueline , Ratain Mark J.
Published: April 2020

Individuals with transfusion-dependent β-thalassemia can be affected by iron accumulation in the heart and liver, resulting in liver fibrosis, cardiomyopathy and eventual death. As a result, iron chelation treatment is a necessary concurrent therapy for many patients. Deferiprone (1,2-dimethyl-3-hydroxypyrid-4-one) is currently the only orally active iron chelator approved outside the U.S. for the treatment of iron overload. Despite the efficacy of deferiprone in reducing serum ferritin levels and total body iron burden, not all patients respond to the drug, and serious adverse events can require discontinuation of therapy. The variability in drug response, combined with a high rate of side effects, indicate that there may be genetic factors that influence how the drug is metabolized. We have identified the uridine diphosphate glucuronosyltransferase (UGT) enzyme that metabolizes deferiprone. Utilizing human liver microsomes and insect cell supersomes, we have screened UGT 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15 and 2B17 enzymes for deferiprone glucuronidation. Deferiprone glucuronide was detected solely with UGT1A6. Previous reports of linkage disequilibrium between the UGT1A1*28 and UGT1A6*2 alleles (Kohle et. al., 2003; Peters et. al., 2003), combined with the association of the UGT1A1*28 allele with an increased risk of hyperbilirubinemia and gallstone formation in the hemoglobinopathies (Haverfield et. al., 2005), substantiate the importance of elucidating the pharmacogenomics of deferiprone metabolism. From this preliminary data, it will be possible to examine UGT1A6 genetic variation that may contribute to differences in drug efficacy and toxicity, and further delineate any additional enzymes involved in deferiprone metabolism.
Phosphorylation of AML1 (RUNX1) Controls Transcription, Nuclear Localization, and Is Regulated during DNA Damage.
Authors: Wang Suiquan , Zhang Youhong , Biggs Joseph R. , Zhang Dong-Er , Kraft Andrew S.
Published: April 2020

We demonstrate that AML1 (RUNX1) is phosphorylated on S276, S293, T300, S303 and S462 after phorbol ester treatment in K562 cells, and these phosphorylations control the transcriptional activity of this protein and regulate varied promoters including TCRb, GM-CSF, M-CSF receptor, and myeloperoxidase gene (JBC279: 53116–53125, 2004). In addition, we find that phosphorylation of the AML1 protein controls the binding of AML1 to the nuclear matrix, and regulates its degradation (MCR3: 391–401, 2005). We have now derived rabbit phosphopeptide antisera to S276, S303, and S462 in the AML1 protein. Using these antibodies, we find that in unsynchronized Jurkat T cells AML1 contain low levels of phosphorylation at all three sites. To examine whether this phosphoryaltion is cell cycle regulated, we blocked cells in G1/S with aphidicolin or in G2/M with nocodazole. In contrast to G1/S block that had no effect on phosphorylation, nocodazole treatment induces marked increases in phosphorylation of S276, S303, and S462. The nocodazole increase in phosphorylation was markedly inhibited by staurosporine, a PKC inhibitor, and to a lesser extent by the JNK inhibitor SP600125, and the p38 inhibitor, SB202190. Using elutriation to examine untreated Jurkat cells, we demonstrate increases in the level of AML1 protein as cells progress through the cell cycle to G2/M. However, the actual level of phosphorylated protein as judged by phospho303 and 462 antibodies decreases. Nocodazole treatment of Jurkat cells not only induces G2/M arrest, but also induces apoptosis. We are currently investigating the hypothesis that DNA damage causes phosphorylation of AML1 that regulates both its activity and location.
PU.l Immortalizes Hematopoietic Progenitors in a GM-CSF Dependent Manner.
Authors: Houston Isaac B. , Jennings Serena , Huang Kelly J. , DeKoter Rodney P.
Published: April 2020

Leukemia arises as a result of a multistep progression of blood cells towards malignancy. Genetic blocks in cell development and uncontrolled proliferation are often key steps in leukemic transformation. PU.1 is a transcription factor that has several known roles in hematopoietic development. Knock-out studies of PU.1 demonstrate a requirement for PU.1 in the development of myeloid and lymphoid cells. In addition, PU.1 concentration can influence cell development in normal hematopoiesis. Low concentrations of PU.1 are required for lymphoid development, whereas high concentrations of PU.1 are required for myeloid development. Mice that have been genetically altered to express abnormally low concentrations of PU.1 develop acute myeloid leukemia (AML) due to a developmental block within the myeloid lineage. In contrast, abnormally high concentrations of PU.1 in mice are capable of inducing erythroleukemias. This can occur through Friend virus integration into the PU.1 gene, which leads to over-expression of PU.1, and the subsequent blocking of erythroid development due to PU.1’s ability to antagonize GATA-1 function. Here we report that ectopic expression of PU.1 is sufficient to immortalize hematopoietic progenitors in a GM-CSF-dependent manner. When fetal hematopoietic progenitors are infected with a PU.1 retrovirus and placed into culture with GM-CSF, a small fraction become immortalized. These PU.1-infected progenitors (PIP cells) are arrested in development and can grow for several months in culture. PU.1 Immortalized Progenitors (PIP) are GM-CSF restricted and dependent. In addition, PIP cells display blast-like morphology and express cell surface markers indicative of myeloid development. In contrast, gene expression analysis of clonal PIP cell lines demonstrates the expression of stem cell, myeloid, and erythroid specific genes. Finally, PIP cells display low levels of endogenous PU.1 production as shown by real-time PCR relative to wild-type cells. These observations support a model in which high levels of PU.1, in conjunction with signaling through the GM-CSF receptor, block cell differentiation at an early stage in hematopoiesis. This discovery provides new information into PU.1’s ability to control cell differentiation and its ability to induce leukemia.