bluuebunny/crossref_metadata_2025 huggingface dataset:
Frequent Elevation of Akt Kinase Phosphorylation in Mononuclear Cells from High-Risk Myelodysplastic Syndrome Patients.
Authors: Bosi Costanza , Finelli Carlo , Martelli Alberto M. , Nyakern Maria , Martinelli Giovanni , Tazzari Pier Luigi , Cocco Lucio , Grafone Tiziana , Piccaluga Pier Paolo , Michele Baccarani
Published: April 2020

Myelodysplastic syndromes (MDS) comprise a heterogeneous group of clonal stem cell disorders characterized by ineffective and inadequate hematopoesis. Early stages of MDS display an increased apoptosis, while later stages of MDS present an excessive survival and decreased apoptosis of progressing abnormal clonal cells. The identification of the signaling pathways responsible for increased survival of MDS cells is of high importance as they might represent promising targets for novel forms of therapy aimed at preventing MDS evolution in to acute myeloid leukaemia (AML). However, to date, very limited information is available on survival pathways activated in MDS patients. The phosphatidylinositol 3-kinase (PI3K)/Akt signalling pathway has been implicated in the aggressiveness of a number of different human cancers, and constitutive activation of the PI3K/Akt pathway has been shown to be necessary for the survival of AML cells. In this study, we have performed an immunocytochemical and flow cytometric staining analysis, using an antibody to Ser473 phosphorylated-At (p-Akt) recognizing the activated form of the Akt kinase, to evaluate the levels of p-Akt in MDS mononuclear cells from either bone marrow or peripheral bllod. We observed a high Ser473 p-Akt staining in 85 % of the patients (n=22) diagnosed as high risk MDS, whereas mononuclear cells from healthy donors or low risk MDS patients showed very low or absent Ser473 p-Akt staining. Furthermore, high levels of the class I PI3K p110d isoform was consistently observed in MDS samples also demonstrating high levels of p-Akt. PI3K p110d was recently shown to have a higher expression in contrast to other class I PI3K isoforms in AML blasts. Finally, the expression of PTEN was correlated to the elevated levels of Ser473 p-AKT and PI3K p110d detected in myeloid cells. PTEN is a lipid phosphatase having the PI3K downstream product phosphatidylinositol 3,4,5 triphosphate (PIP3) as one of its primary targets, thus negatively regulating Akt activity. We have found that PTEN is expressed in mononuclear cells from high risk MDS patients, albeit with a lower staining intensity when compared with peripheral blood mononuclear cells from healthy donors. These findings identify activated Akt and PI3K p110d as potential survival factors in high risk MDS and could be important as therapeutic targets for innovative strategies to treat MDS patients.
5′-Nucleosidase and Deoxycytidine Kinase Gene Expressions in High-Risk Myelodysplastic Syndrome (MDS).
Authors: Suzuki Keijiro , Sugawara Takeshi , Ishida Yoji
Published: April 2020

Various chemotherapeutic regimens including cytarabine (ara-C) for MDS patients have been less effective than those for AML patients. Althogh alterations in the expression of genes involved in ara-C metabolism have been reported to be related to the clinical outcome in AML patients, there is no report about MDS. Deoxycytidine kinase (dCK) is the rate-limiting enzyme in this activation process being the first phosphorylation of ara-C. Cytoplasmic 5′-nucleotidase (5′-NT) dephosphorylates ara-CMP. The concentration of the intracellular ara-CTP, active form of ara-C, depends on the activities of two enzymes. To determine whether the level of expression of 5′-NT and dCK is implicated in clinical outcome in patients with high-risk MDS (RAEB/RAEB-t), we analyzed the mRNA expression of these at diagnosis in bone marrow (BM) cells of patients with RAEB/RAEB-t using real-time PCR (rt-PCR).

Materials and Methods: BM cells obtained from 22 patients (male: female=16:6) with untreated high-risk MDS. The mean age of all patients was 66.4±7.9 y. 7 patients (31.8%) had RAEB-t subtype and 15 (68.2%) patients RAEB subtype of the FAB classification. Quantitative rt-PCR of 5′-NT or dCK mRNA of BM cells in patients and 12 healthy volunteers was performed by LightCycler using β-actin as housekeeping control. For each sample, a ratio of 5′-NT or dCK value/β-actin value was calculated and considered as the level of 5′-NT or dCK mRNA expression.

Results: 15 of patients received ara-C containing regimen (7 patients had supported care only). Complete remission was obtained in 7 patients. Other 8 patients were chemoresistant. The median overall survival (OS) of all patients was 22.9 months (range 2.0–91.0), and that of patients with chemotherapy was 20.0 (7–91). The median post-chemotherapy survival (PCS) was 15.0 months (5.0–43.0). At diagnosis, the mean and median expression level of 5′-NT mRNA in all patients was 1.56 (SD 1.61) and 1.36 (range 0.77–6.77), respectively (control: mean 0.23, SD 0.06 and median 0.67, range: 0.17–0.34, p<0.01). The expression of dCK mRNA did not show any significant difference between patients and control. Patients with chemotherapy whose BM cells were higher level of 5′-NT expression (greater than mean value) had shorter OS (14.0 months vs. 26.0 months, p<0.01) and shorter PCS (10.0 months vs. 17.0 months, p=0.011).

Figure Figure

Conclusion: These data suggest that the expression level of 5′-NT mRNA might be a candidate for prognostic factor in RAEB/RAEB-t. Patients with an increase of 5′-NT mRNA at diagnosis show shorter overall survival and chemoresistance.
Abnormal PI 3-Kinase Activity Due to Increased Lyn with Decreased PTEN and Absent SHIP in Bone Marrow Cells from Patients with Myelodysplastic Syndromes.
Authors: Lee Daniel W. , Zhu Quan-Sheng , Rudra Sonali , Shpall Elizabeth J. , Kornblau Steven , Corey Seth J.
Published: April 2020

Myelodysplastic syndromes (MDS) result from a malignant stem cell clone characterized by ineffective hematopoiesis, manifested as peripheral cytopenia with a cellular bone marrow. A leading hypothesis is that MDS results from a breakdown in the control of myeloid cell proliferation and apoptosis. Through its generation of 3′-phosphoinositides and subsequent activation of effectors such as Akt, phosphatidylinositol 3-kinase (PI 3-kinase) drives cell proliferation, apoptosis, differentiation, and motility. We show here that PI 3-Kinase is profoundly deregulated in high-risk MDS. Bone marrow cells from high-risk MDS patients displayed a 3–30 fold increase in constitutive activation of the Src kinase Lyn. Constitutive serine/threonine phosphorylation of Akt, a surrogate of PI-3 kinase activity, was seen in all specimens. Protein levels of PTEN, which dephosphorylates the D3-position phosphate of the inositol ring, were variably decreased. Since PTEN is frequently silenced by hypermethylation, we treated U937, THP-1, and Mo7e cells with 5-azacytidine. However, real-time PCR showed no increase in PTEN transcripts in these cell lines. More significantly, protein expression of SHIP-1, which dephosphorylates the D5-position phosphate of the inositol ring, was markedly decreased or absent in bone marrow cells from MDS patients, whereas they were present in AML or ALL blasts. Real-time PCR showed SHIP-1 transcripts in MDS cells to be 50% of normal CD34+ stem cells. Although 5-azacytidine treatment resulted in an increase in SHIP-1 transcripts, as measured by quantitative PCR, protein levels did not increase. Because SHIP-1 contains three PEST-rich regions, we hypothesized that the low or absent level of expression of SHIP-1 may due to protein degradation. Neither the cryopreservation of cells nor lysis buffer could explain the absence of SHIP-1 protein. Instead, when U937 and HL60 leukemic cell lines were treated with the proteastome inhibitor lactacystin (1–5 uM), protein levels of SHIP-1 increased. These results suggest that constitutive activation of Akt is likely due to decreased PTEN and absent SHIP-1 protein levels in primary MDS cells. Consistent with these findings, mice deficient in PTEN and SHIP-1 suffer from anemia, thrombocytopenia, leukocytosis and impaired function of myeloid progenitors (BLOOD103:4503, 2004). These results also suggest that a combination of a Src kinase inhibitor and a proteasome inhibitor may be a therapeutic approach for the treatment of high-risk MDS.
CHIR-258 Efficacy in a Newly Developed Preclinical Bone Marrow Model of t(4;14) Multiple Myeloma.
Authors: Xin Xiaohua , Tang Yan , Oei Yoko , Ye Helen , Menezes Daniel , Rendahl Katherine , Hollenbach Paul , Pryer Nancy , Trudel Suzanne , Mendel Dirk , Jallal Bahija , Heise Carla
Published: April 2020

Multiple myeloma (MM) remains a fatal hematological malignancy due to the development of drug resistance to conventional high-dosage chemotherapy. It has been demonstrated that the bone marrow microenvironment, where MM cells preferentially home and grow, plays a crucial role in developing resistance to therapies for MM. Recent understanding of the molecular pathology of MM has provided novel therapeutic targets for treatment of this disease. The ectopic expression of FGFR3, which occurs in approximately 15–20% MM patients resulting from a t(4;14) chromosomal translocation and confers a particularly poor prognosis in clinic, has become an attractive therapeutic target for MM. CHIR-258 is a small molecule inhibitor of Class III, IV and V receptor tyrosine kinases, including FGFR, VEGFR and PDGFR (IC50s ~5–15 nM in kinase assays). It has been demonstrated that CHIR-258 inhibits FGFR3 autophosphorylation, downstream signaling and cell proliferation in FGFR3 mutant MM cells in vitro as well as induces apoptosis in FGFR3 positive primary myeloma cells (Trudel, et.al, Blood 2005). To evaluate the anti-myeloma efficacy of CHIR-258, we developed an in vivo MM model in which multi-organ MM lesions developed after tail vein injection of human KMS-11-luc cells stably transfected with luciferase. This cell line harbors the t(4;14) translocation and expresses constitutively active FGFR3 (Y373C mutation). Non-invasive bioluminescent imaging (BLI) was used to monitor the in vivo growth and dissemination of KMS-11-luc MM tumors. Early detection and serial imaging monitoring the growth of metastatic lesions was successfully captured by BLI in this model. Nearly all mice injected with KMS-11-luc tumor cells were found to develop MM lesions, which were mainly localized in spine, skull and pelvis. We examined CHIR-258 anti-myeloma efficacy in this model and found that daily oral administration of CHIR-258 at 20 mg/kg, a dose that was demonstrated to inhibit phosphorylation of ERK in KMS-11-luc tumors in vivo, resulted in a significant inhibition of tumor growth. Furthermore, this anti-tumor activity of CHIR-258 translated to a significant improvement of animal survival compared to vehicle treatment in this model. In vitro combination studies with dexamethasone and bortezomib in KMS-11 cells demonstrated synergistic and additive effects, respectively. The development of this KMS-11-luc in vivo model will allow further evaluation of CHIR-258 combination therapy with conventional or other molecularly targeted agents. These studies have provided further rationale for the ongoing clinical trials of CHIR-258 in MM.
Incidence and Clinical Course of Peripheral Neuropathy in Patients Receiving Thalidomide for the Treatment of Multiple Myeloma.
Authors: Naina Harris V.K. , Lacy Martha Q. , Dispenzieri Angela , Kumar Shaji , Greipp Philip R. , Witzig Thomas E. , Gertz Morie A. , Rajkumar S. Vincent
Published: April 2020

Background: Although thalidomide induced neuropathy has been described for decades, many features such as incidence, risk factors, severity and character of the neuropathy are not well defined. We retrospectively reviewed the incidence and clinical course of peripheral neuropathy in patients treated with thalidomide or thalidomide/dexamethasone (Thal/Dex) on a Phase II Mayo Clinic trial.

Methods: Patients who participated in a Mayo Clinic Phase II trial of thalidomide in myeloma were studied. In this trial, patients with SMM and RMM received thalidomide 200 mg per day escalated as tolerated to a maximum of 800 mg/day. Patients with NMM received thalidomide 200 mg/day plus dexamethasone 40 mg/day orally on days 1–4, 9–12, 17–20 (odd cycles) and 40 mg/day days 1–4 (even cycles) repeated monthly. The incidence and clinical course of neuropathy was assessed by chart review. The study was approved by the Mayo Clinic Institutional Review Board.

Results: All 113 patients (70 male, 43 female) treated on trial were studied; 31 patients with smoldering/indolent myeloma (SMM), 32 patients with relapsed/refractory myeloma (RMM) and 50 patients with newly diagnosed myeloma (NMM). Patients were accrued to the parent protocol from April 1999 through March 2002. The median age was 62 years (range, 36–78). The median duration of thalidomide therapy on protocol was 15 months in SMM, 6 months in RMM and 4 months in NMM. Overall, 63 patients (56 %) developed symptoms of peripheral neuropathy. The incidence of neuropathy was 77%, 50%, and 46% in patients with SMM, RMM, and NMM, respectively. Neuropathy worsened during the treatment phase with/without dose reduction or after cessation in 15%, remained stable in 52%, and improved in 27%. Overall grade of neuropathy was grade 1 in most pts; 11% of all patients had grade 2 neuropathy and 2% had grade 3 neuropathy. Predominant symptoms of neuropathy were tingling and numbness involving both upper limb and lower limbs. Small subsets of patients complained of hearing loss 6 patients), and erectile dysfunction (4 patients). Electromyographic studies and nerve conduction studies were performed in 11 patients; the most common finding was a length dependent axonal neuropathy.

Conclusion: Peripheral neuropathy is an important and common adverse event with thalidomide therapy that often limits the dose and duration of treatment. Its incidence and severity are likely related to dose and duration of therapy.
Multimodular Analysis of Angiogenic Biomarkers in the Primary Treatment of Multiple Myeloma: Correlative Studies of the BiRD Trial.
Authors: Jin David K. , Young Lauren M. , Vincent Loic , Gelbshtein Udi , Pekle Karen , Lam George , Shido Koji , Cho Hearn J. , Pearse Roger , Leonard John P. , Chen-Kiang Selina , Coleman Morton , Rafii Shahin , Niesvizky Ruben
Published: April 2020

Biomarkers that predict the angiogenic propensity of multiple myeloma and the impact of therapy on angiogenesis remain unknown. Nevertheless, many putative anti-angiogenic agents are currently being evaluated in clinical trials. We describe the results of a multimodular analysis of angiogenic biomarkers including a novel in vitro functional assay, the human umbilical vein endothelial cell (HUVEC)-based angiogenic scale (HBAS), to assess the overall angiogenic activity in the plasma of patients with multiple myeloma. Plasma fractionated from the peripheral blood of myeloma patients was added to primary HUVEC and cultured under serum-free conditions for 24 hours. Angiogenic morphology was then examined and scored using a six-point angiogenic scale. Analysis of a cohort of ten consecutive patients with multiple myeloma undergoing the BiRD protocol (Biaxin, Revlimid and dexamethasone) showed that the majority had higher HBAS scores at baseline than age-matched normal subjects. Plasma samples from stage IIIA disease consistently scored higher than those from stage II disease on the functional angiogenic scale (mean score of 2.8 versus 1.0, p<0.005), suggesting a more pro-angiogenic state for further advanced disease. The HBAS also correlated with increased levels of plasma and platelet vascular endothelial growth factor-A (VEGF-A) as quantified by ELISA. Interestingly, VEGF-A levels in platelet lysate were significantly higher than those in plasma and more closely paralleled the functional HBAS scores. In addition, two-color flow cytometric analysis of cellular biomarkers demonstrated an angiogenic switch defined by increased mobilization of circulating CD133+VEGFR2+ endothelial progenitor cells in patients with active disease as compared to normal subjects (2.2-fold increase; p<0.005). Treatment with BiRD effectively attenuated all tested angiogenic parameters and correlated with a positive clinical response. Collective assessment of angiogenic biomarkers may be a promising resource for predicting clinical outcomes and for validating the potential impact of anti-angiogenic therapy in multiple myeloma.
SNP Genotypes Show Association with Common Toxicities during both VAD Induction and High Dose Melphalan with Autologous Transplant Support in Intergroup Trial S9321 for Myeloma: From the Bank on a Cure.
Authors: Van Ness Brian G. , Crowley John C. , Ramos Christine , Grindle Suzanne , Rasmussen Erik , Hoering Antje , Greipp Philip , Barlogie Bart , Durie Brian
Published: April 2020

Patients with multiple myeloma show significant heterogeneity with respect to progression, therapeutic response and toxicities. Part of the Bank On A Cure project is to examine potential genetic polymorphisms that may be associated with common toxicities monitored in clinical trials. The SWOG/ECOG intergroup trial S9321 therapy includes VAD induction followed by randomization to high dose melphalan with autologous transplant support. Paticularly important toxicities with VAD include infection, thrombosis and hyperglycemia; and with melphalan toxicities in GI function are noted. Toxicities and genotypes were assessable during VAD induction on 569 patients; and during high dose melphalan on 161 patients. Toxicities examined included an array of symptoms: cardiovascular, flu-like, GI, neurologic, infection, lung, metabolic, and pain. Grade 3 toxicities found in at least 20 patients were compared to SNP genotypes. For VAD induced toxicities,TNF-alpha SNP genotypes showed association with fatigue, dyspnea and thrombosis; CYP3A4 SNP genotypes showed association with fatigue; DNA repair SNP genotypes in XRCC1 showed associations with infection, hyperglycemia and dyspnea; LT-alpha SNP genotypes showed association with dyspnea; and IL-6 and IL-10 SNP genotypes were marginally associated with neuropathy. After high dose melphalan, diarrhea was associated with SNP genotypes in TGF-beta, CYP3A4 and ERCC2. Interestingly, IL-6 polymorphisms were significantly associated with infection. The p values on the above associations ranged from .05 to .004. In addition, we noted ethnic distributions of some SNP genotypes were significantly different; and significant ethnic disparities in therapy induced hyperglycemia, fatigue, dyspnea and thrombosis were noted (p values range from .04 to .009). Of particular note is that the high producer IL-6 genotype is present in over 90% African Americans, compared to 35% Caucasians. Details of each association will be presented. Not all associations can easily be explained by known biologic function of the genotype, and it is important to qualify these results, as the likelihood of seeing some genotype associations by chance is quite high given the number of comparisons made. Further, we are continuing to examine additional prognostic factors that may, together with genotypes, provide collective predictors of toxicities. We consider these associations as targets for further study through the increasing sample acquisition in the Bank On A Cure project.
Maintenance Rituximab after CVP Results in Superior Clinical Outcome in Advanced Follicular Lymphoma (FL): Results of the E1496 Phase III Trial from the Eastern Cooperative Oncology Group and the Cancer and Leukemia Group B.
Authors: Hochster Howard S. , Weller Edie , Gascoyne Randy D. , Ryan Teresa S. , Habermann Thomas M. , Gordon Leo I. , Frankel Stanley R. , Horning Sandra J.
Published: April 2020

E1496 is a phase III trial designed to evaluate the ability of 2 years (yr) of maintenance rituximab (MR) to prolong progression-free survival (PFS) after CVP (cyclophosphamide 1 G/m2 day [d] 1, vincristine 1.4 mg/m2 [max = 2 mg] d 1, prednisone 100 mg/m2 d 1–5) chemotherapy in stage III–IV follicular grade 1 and 2 and small lymphocytic lymphoma. After CVP treatment to maximum response, (6–8 cycles), stable and responding patients (pt) were randomized to MR (375 mg/m2 weekly x 4) every 6 months x 4 or observation (OBS). Stratification factors included histology, response and residual disease after CVP. With 3-yr median follow-up, survivals (from time of randomization, one-sided logrank p values) for all pt (n=304) favored MR for PFS (p = 3 x 10–8; hazard rate {HR} = 0.38 [0.28;0.54, 95% confidence intervals]) and OS (p = 0.09; HR = 0.66 [0.36–1.22]). Because the large majority of pt have FL and because rituximab efficacy is notably greater in FL, we focused in this report on the 237 FL pt. Median age was 58 yr, 65% were stage IV, 64% had marrow disease, 64% had high tumor burden and 37% had high-risk disease by the follicular lymphoma prognostic index. PFS after randomization was significantly longer for MR vs OBS (p = 3 x 10-7; HR = 0.39 [0.27;0.57]). The estimated PFS at 4 yr (~4.5 yr after start of treatment) was 56% for MR vs 33% for OBS. Differences in PFS were significant within the predefined strata and the differences were most significant in favor of MR for pt with high initial tumor burden and minimal residual disease after CVP. Overall survival was superior for MR (p = 0.03; HR = 0.51 [0.25;1.04]. Estimated OS at 4 yr (~4.5 years after start of treatment) was 88% for MR vs 72% for OBS. Of 33 deaths, 21 occurred on the OBS arm. These data demonstrate that maintenance rituximab not only significantly delays disease progression in FL compared with OBS but that a substantial proportion of patients treated with MR remain disease-free at 4 years after the completion of CVP. These are the first data to strongly suggest a survival benefit with a therapy that includes rituximab and CVP and the first to strongly suggest a survival benefit with maintenance rituximab in FL.
Structural and Signaling Requirements for Transformation Mediated by Shp2 Mutants.
Authors: Mohi M. Golam , Neel Benjamin G.
Published: April 2020

The SH2-containing tyrosine phosphatase Shp2 (PTPN11) is required for normal growth factor and cytokine signaling. Germline Shp2 mutations cause ~50% of Noonan Syndrome (NS), which is associated with an increased risk of juvenile myelomonocytic leukemia (JMML). Somatic Shp2 mutations are found in ~35% of sporadic JMML, and at lower incidence in various chronic and acute leukemias. JMML patients without Shp2 mutations have either activating Ras mutations or homozygotic inactivation of Nf1. Nearly all disease-associated Shp2 mutations affect residues known to control catalytic activity. NS and leukemia mutations can involve the same residues, but when they do, the latter are less conservative, suggesting that they may be more activating. We previously showed that leukemia-associated mutants E76K or D61Y, but not wild type (WT) Shp2 could transform bone marrow (BM) or fetal liver cells and give rise to cytokine-independent myeloid colony formation. Leukemia-associated mutants yielded much higher number and larger size of colonies than NS-associated mutants (e.g., N308D, N308S, E76D) whereas, mutants associated with both NS and leukemia (T73I, E139D, Q506P) evoked intermediate numbers. Transplantation of E76K− or D61Y− (but not parental virus or WT Shp2) transduced BM into lethally irradiated recipients evoked a fatal JMML-like disorder. To understand the structural requirements of Shp2 for transformation, we made a series of second site mutations in Shp2 E76K and expressed these mutants in murine BM. Mutation of the canonical arginine residue to methionine in the FLVRES motif in either N-SH2 or C-SH2 domain of Shp2 E76K ablated myeloid transforming ability. Mutation of the conserved arginine residue within the Shp2 E76K phosphatase domain signature motif to methionine, which diminishes phosphatase activity, also eliminated myeloid transformation. Tyrosine residues 542 and 580 in the C-terminus of Shp2 are known to undergo phosphorylation in response to many growth factors and required for Grb2 binding. Mutation of tyrosine 542 to phenylalanine markedly reduced transformation, whereas tyrosine 580 mutation alone had little effect. However, mutation of both tyrosines eliminated Shp2 E76K-evoked transformation, suggesting that Grb2-binding to Shp2 could be important for transformation. We observed that Gab2, a major Shp2 SH2 domain binding protein, was absolutely required for transformation by Shp2 E76K but not activating RasV12. Gab3, another member of Gab family protein mainly expressed in hematopoietic cells, was not critical for Shp2 E76K-evoked transformation. We also found that Stat5 was important for Shp2 E76K-mediated transformation. Mast cells derived from E76K− and D61Y− transplanted mice BM exhibited increased proliferation and enhanced activation of Erk, p38MAPK, Akt and Stat5 in response to IL-3. These results provide some mechanistic insights into how Shp2 mutants could transform primary myeloid cells and result in JMML-like MPD.
Does Combined Immunochemotherapy with the Monoclonal Antibody Rituximab Improve Overall Survival in the Treatment of Patients with Indolent Non-Hodgkin Lymphoma? Preliminary Results of a Comprehensive Meta-Analysis.
Authors: Schulz Holger , Skoetz Nicole , Bohlius Julia , Trelle Sven , Kober Thilo , Greb Alexander , Engert Andreas
Published: April 2020

Background: The monoclonal anti-CD20 antibody Rituximab (R) has been shown to induce high response rates and to improve progression free survival when added to combined chemotherapy in newly diagnosed and relapsed indolent lymphoma. It remains unclear if R combined with chemotherapy may also have an impact on overall survival (OS).

Objectives: To determine the effectivness of a combination chemotherapy plus R versus chemotherapy alone with respect to overall survival.

Methods: Randomized controlled trials (RCT) comparing chemotherapy plus R vs chemotherapy alone in patients with newly diagnosed or relapsed indolent lymphoma and mantle cell lymphoma. Medical databases (Cochrane Library, MEDLINE, EMBASE) and conference proceedings were searched (1990–2005). We included full-text and abstract publications. Data extraction and quality assessment were done in duplicate. Data were pooled under a fixed-effects model. Number needed to treat were calculated to facilitate interpretation.

Main results: We identified six eligible RCTs involving patients with follicular and mantle cell lymphoma. Treatment regimen combined with R were CHOP (2x), CNOP, CVP, FCM and MCP. A total of 5 trials with 994 randomized untreated pts were included in the survival analysis. The included studies were described by the authors as randomized. In all studies, the method for allocation concealment could not be determined. Three studies were published as full text, and two were abstract publications. Overall survival was statistically significantly improved in the R-chemotherapy group compared to chemotherapy alone (HR 0.61; 95%-CI: 0.47–0.80). There was no statistical heterogeneity between the trials compared. Assuming a survival chance of 85% after 18 months for patients with low grade NHL we estimated that it would be necessary to treat 17 (95%-CI: 13–33) patients with R in addition to chemotherapy to prevent one death. For patients with mantle cell lymphoma and a survival chance of 60% after 18 months the corresponding estimate is 6 (95%-CI: 5–13).

Conclusion: This preliminary meta-analysis demonstrated evidence for improved overall survival among patients with indolent lymphoma and mantle cell lymphoma treated with a combination of rituximab plus chemotherapy compared to chemotherapy alone.
Clonal Expansion in Paroxysmal Nocturnal Hemoglobinuria (PNH): Expression of Mutant HMGA2 Suggests That PNH Is a Benign Tumor of the Bone Marrow.
Authors: Inoue Norimitsu , Izui Tomohisa , Murakmai Yoshiko , Endo Yuichi , Nishimura Jun-ichi , Kanakura Yuzuru , Meyers Gabrielle , Wittwer Carl , Chen Zhong , Babcock William , Frei-Lahr Debra , Parker Charles , Kinoshita Taroh
Published: April 2020

In PNH, a somatic mutation of PIGA in hematopoietic stem cells causes the deficiency of glycosylphosphatidylinositol-anchored proteins (GPI-AP), but the basis of clonal expansion of the PIGA-mutant cells is speculative. Because some patients with aplastic anemia develop PNH, GPI-AP deficient stem cells may have a survival advantage in the setting of immune-mediated bone marrow injury. However, in many patients with aplastic anemia, the GPI-AP deficient cell populations remain small or disappear. Therefore, we hypothesized that additional abnormalities in the PIGA-mutant stem cells account for clonal expansion. We previously reported a patient with PNH/aplastic anemia (J20) whose PIGA-mutant hematopoietic cells had a coexistent cytogenetic abnormality [46,XX,ins(12;12)(q14;q12q14)]. In this patient, the insertion disrupted the 3′ untranslated region of HMGA2, an architectural transcription factor whose aberrant expression causes benign mesenchymal tumors. Truncated HMGA2 transcripts lacking the acidic tail (the pathophysiologically relevant form) were identified in the double mutant cells. In the present study, we characterized a similar genetic abnormality in a patient with classical PNH. In this case, PIGA-mutant cells again had a concurrent der(12) [46,XX,ins(12)(p13q14q13)]. A 20 Mbp fragment from 12q13 to q14 and a 300bp fragment from 12q14 together containing exons 1–4 and part of exon 5 of HMGA2 were inserted inversely and directly, respectively, into intron 1 of TEL at 12p13. One of the breakpoints in the HMGA2 locus was at almost the same position as the HMGA2 breakpoint in patient J20. Truncated HMGA2 transcripts (lacking the acidic tail) were highly expressed in bone marrow cells. Full-length transcripts of TEL without any fusion partners were normally expressed and no other transcription units were disrupted by the breakpoints. That similar cytogenetic abnormalities were observed in these two patients suggests that aberrant expression of HMGA2, in concert with mutant PIGA, accounted for the clonal expansion and explains the benign tumor characteristics of PNH.
Hepcidin Inhibits Erythroid Colony Formation In Vitro at Decreased Erythropoietin Concentrations.
Authors: Means Robert T. , Dallalio Gail , Law Erin
Published: April 2020

The anemia of chronic disease (ACD) results from three major processes: slightly shortened red cell survival, impaired reticuloendothelial system iron mobilization, and impaired erythropoiesis. Hepcidin is an acute phase protein with specific iron regulatory properties, which, along with the anemia seen with increased hepcidin expression, have led many to consider it the major mediator of ACD. However, if hepcidin is the major factor responsible for ACD, then it should also contribute to the impaired erythropoiesis observed in this syndrome. In this study, the effects of hepcidin on erythroid colony formation in vitro are examined. At the standard recombinant human erythropoietin (rhEPO) concentration used for erythroid colony-forming unit (CFU-E) assays in vitro (1 U/mL), hepcidin had no significant effects on colony formation. However, ACD is a syndrome associated with relative reductions in EPO concentration. The effects of hepcidin 100 ng/mL on CFU-E colony formation were evaluated at lower rhEPO concentrations (0.1 _ 0.5 U/mL). Colony formation was significantly decreased in the presence of hepcidin. The regulatory pathways controlling apoptosis and proliferation of HCD57 erythroleukemia cells replicate those observed in primary human erythroid progenitors. This cell line was studied in order to more fully define the effects of hepcidin on erythropoiesis. HCD57 cells were incubated in Iscove’s modified Dulbecco’s medium with 10% fetal calf serum with 0.3 U/mL rhEPO at 37°C for 24 hrs, with or without hepcidin 100 ng/mL. No difference in cell proliferation was seen. Apoptosis in HCD57 cells is regulated by the Bad/Bcl-xL pathway. Total Bad and Bcl-xL protein expression were unchanged by exposure to hepcidin; however, the proportion of the anti-apoptotic protein pBad was decreased approximately 50%. A recent report has demonstrated that hepcidin reduces macrophage iron efflux by binding to the iron transporter ferroportin, causing its internalization and subsequent degradation. It is unlikely that the inhibition of CFU-E colony formation reported here results from this effect: although macrophages are present in the marrow cell population, and iron is required for erythropoiesis in vitro, the amount of iron-saturated transferrin contributed to the culture medium by FCS is comparable to the amount required for maximal CFU-E colony formation in serum-free medium. CONCLUSION: At reduced rhEPO concentrations, CFU-E colony formation is inhibited by hepcidin. Short-term exposure to hepcidin induces a pro-apoptotic pattern in HCD57 erythroleukemia cells. The studies reported here do not disprove the possibility that hepcidin-induced impairment of iron flow to erythroid cells contributes to ACD: rather, they suggest an additional mechanism by which hepcidin can inhibit erythropoiesis.
High Risk Pregnancies in Women with Diamond Blackfan Anaemia: A Survey of 64 Pregnancies from the French and German Registries.
Authors: Leblanc Thierry , Faivre Laurence , Meerpohl Jorg , Da Costa Lydie , Nouvel Christian , Bauters Francis , Coiffier Bertrand , Peaud Pierre-Yves , Rispal Patrick , Berrebi Alain , Berger Claire , Varet Bruno , Niemeyer Charlotte , Tchernia Gil
Published: April 2020

Diamond Blackfan anaemia (DBA) is a rare disorder characterised by pure red cell aplasia and inconstant congenital abnormalities. Few data are available concerning pregnancy in DBA affected women.

We report on 60 women (age>18 yr) included in the French and German DBA registries. Among them, 26/60 experienced one or several pregnancies. Mean age at 1st pregnancy was 24 yr and the mean number of pregnancies by woman was 2.5. A total of 64 pregnancies occurred, 22 being normal and 42 being complicated (66%). Eight women experienced only normal pregnancies and 18 experienced at least one complicated pregnancy. Complications included abortions: 26 of which 24 were spontaneous, in utero foetal deaths:5, foetal malformations:7, intrauterine growth retardation:10, pre-term deliveries:7, gestational diabetes:2, pre-eclampsia:5 and retro-placental haematomas:3 (more than 1 complication occurred in some pregnancies). A pathological examination of the placenta was performed in 4 cases and demonstrated significant infarctions in 3.

As a result of those 64 pregnancies, only 34 children were born alive (53%) from whom 14 were DBA-affected (41%). All women survived. In most cases, treatment with transfusion or steroid was increased during pregnancy.

When complicated and normal pregnancies were compared, no obvious correlation between the outcome of the pregnancy and clinical or biological features of maternal DBA, mother treatment, genetic status (RPS19 gene mutation present or not) or the status of the child were found. This suggests that at least some complications may be related to the affected mothers, irrespective of the DBA status of their offspring. Actually, many of these complications are similar to those observed in pregnancies associated with vascular-placental disease.

Among the 34 women who did not present any pregnancy, 20 did not desire children for various reasons, 4 had a follow-up for infertility and 10 had severe polymorbidity. Infertility was likely to be treatment related (iron overload or bone marrow graft side effects).

From this study together with the literature data, we conclude that pregnancies in DBA affected women must be managed as high-risk pregnancies. Such pregnancies should therefore be carefully monitored with a satisfying control of anaemia, preferably with transfusions, and ultrasound and Doppler follow-up. Early introduction of aspirin, should be discussed especially when a woman experienced a previous complicated pregnancy. An international prospective follow-up of pregnancies in DBA is now required in order to confirm the association with placenta-vascular disease features and to improve the outcome of pregnancy in women with DBA
Somatic Mosaicism and Variable Expressivity in Diamond Blackfan Anemia (DBA): A Large Deletion Involving the 19q13 Locus in a Patient with Transient Anemia.
Authors: Garelli Emanuela , Quarello Paola , Campagnoli MariaFrancesca , Brusco Alfredo , Carando Adriana , Menegatti Elisa , Crescenzio Nicoletta , Foglia Luiselda , Dianzani Irma , Ramenghi Ugo
Published: April 2020

DBA (MIM 205900) is a rare, pure red blood cell aplasia of childhood due to an intrinsic defect in erythropoietic progenitors, accompanied by somatic malformations in about 40% of cases. Anemia is corrected by steroid treatment in half of patients; non-responders need chronic transfusions or stem cell transplantation. Heterozygous mutations in the RPS19 gene, mapping in the chromosomal region 19q13.2 and encoding for a ribosomal protein of the small subunit, are found in 25% of patients. Deletions spanning the 19q13 locus and involving RPS19 gene have yet been reported in literature; these patients showed more severe manifestations of the disease with skeletal malformations, profound anemia unresponsive to steroids needing regular red blood transfusions and mental retardation.

We report the first case of DBA due to mosaicism for a deletion on chromosome 19q13 involving RPS19 gene. The large deletion involving the maternal allele, spanning an interval restricted by loci D19S220 and D19S178, was identified by segregation analysis of 19q polymorphic markers. To assess the degree of mosaicism semiquantitative SNapShot analysis was performed with detection of one RPS19 copy in 60% of peripheral blood mononuclear cells. Mosaicism was also found in oral and urinary epithelium cells. Our patient had been affected since infancy by hyporegenerative anemia, that had spontaneously remitted at the age of ten; he showed neither malformations nor overt mental retardation. His parents and a brother are healthy and do not carry the deletion.

The involvement of tissues of different embryologic origin suggests that the deletion has appeared in an early stage of embryological development. Our findings explain the milder than expected phenotype and suggest that somatic mutations that lead to tissue mosaicism might contribute to variable expressivity of DBA. The presence of blood cells without deletion might also justify the observed hematologic remission. Mosaicism must be taken into account for genetic counselling in this patient.
Maintenance Dosing with Epoetin alfa Every Three Weeks (Q3W) in Anemic Patients with Cancer Receiving Chemotherapy Every Three Weeks: Final Results.
Authors: Montoya Vernon P. , Xie John , Woodman Richard C.
Published: April 2020

Although epoetin alfa is commonly used weekly for the treatment of chemotherapy (CT)-induced anemia, most CT is given at three week intervals. Therefore, to provide increased patient convenience we examined a dosing regimen that would allow patients to receive epoetin alfa 80,000 U Q3W during maintenance therapy. This open-label, multi-center study had two phases: Initiation Phase (IP) of 60,000 U QW to achieve target hemoglobin (Hb) of 12 g/dL (maximum duration 12 weeks), followed by a Maintenance Phase (MP) of 80,000 U Q3W to maintain Hb between 11.0 and 12.5 g/dL. Only those patients achieving Hb ≥ 12 g/dL in the IP entered the MP; once target Hb was achieved, patients proceeded to MP at the beginning of their next full CT cycle. Patients were withdrawn from the MP if Hb decreased to < 11 g/dL. Doses were held for Hb > 13 g/dL and reduced for rate of rise > 1.3 g/dL/2 weeks. Maximum study duration (IP + MP) was 24 weeks. Patients ≥ 18 years of age with non-myeloid malignancy, baseline Hb ≤ 11 g/dL, and CT planned Q3W for ≥ 15 weeks were enrolled. Endpoints included proportion of patients achieving hematopoietic response in the IP (≥ 2 g/dL Hb increase from baseline or Hb ≥ 12 g/dL) and proportion maintaining mean Hb between 11.0 and 12.5 g/dL in the MP. Also analyzed was the proportion of patients maintaining mean Hb between 11.0 and 13.0 g/dL in the MP. A total of 115 patients were enrolled and received at least one dose of epoetin alfa; mean age was 62.2 ± 11.9 years, 66.1% were female, mean baseline Hb was 10.2 ± 0.8 g/dL, and 86.1% had solid tumors. During the IP, 84 of 115 (73.0%) patients achieved hematopoietic response (≥ 2 g/dL Hb increase from baseline or Hb ≥ 12 g/dL). Seventy-three patients (63.5%) who achieved Hb ≥ 12 g/dL entered the MP after a median of 6.0 weeks in IP and spent a mean of 9.8 weeks in MP. Fifty-four of 73 (74.0%) maintained an average Hb between 11.0 and 12.5 g/dL over the course of the MP; 64 (87.7%) maintained an average Hb between 11.0 and 13.0 g/dL over the course of the MP. Twenty-five (34.2%) patients were withdrawn from the MP because Hb decreased to < 11 g/dL. Of 115 patients dosed in IP, 30 (26.1%) had doses reduced and 31 (27.0%) had doses held; of 73 patients dosed in MP, 9 (7.8%) had doses reduced and 16 (13.9%) had doses held. Fifty-one (44.3%) patients experienced serious adverse events. Eleven (9.6%) patients experienced clinically relevant thrombotic vascular events. Two and 4 patients discontinued due to adverse events in the IP and MP, respectively. Eight (7.0%) patients died during the study or within 30 days after last study dose. Although 60,000 U QW was used during the IP to achieve a HR, 40,000 U QW is the recommended initiation dose of epoetin alfa. This study suggests that epoetin alfa 80,000 U administered every three weeks to patients on a Q3W CT regimen is an effective option for maintenance therapy of CT-induced anemia.
The Minimal Epo Receptor Allele, EpoR-H, Regulates ERK1,2, (But Not JNKs or p70S6K), and Induces Pim-1, SOCS3 and Oncostatin-M (But Not BcL-XL) in Primary Erythroblasts.
Authors: Menon Madhu P. , Cuetara Bethany , Houde Estelle , Fang Jing , Bogacheva Olga , Bogachev Oleg , Karur Vinit , Ufkin Melanie , Wojchowski Don
Published: April 2020

In studies of core Epo receptor signals that are necessary-and-sufficient for erythroblast development, we have shown that an EpoR-PY343-Stat5 axis is required for efficient erythropoiesis during anemia. This involved analyses of knocked-in truncated Epo receptor alleles that retain only a PY343 Stat5 binding site (EpoR-H allele), or lack all cytoplasmic PY motifs (EpoR-HM allele). Presently we have analyzed core signaling capacities of these receptor forms (and the wt-EpoR) in purified developmentally-staged marrow- derived erythroblasts. Analyses of JNKs, p70s6K and ERKs revealed that JNK and p70s6K activation is mediated by non-essential C-terminal EpoR domains. ERKs, in contrast, remained coupled to EpoR-H and EpoR-HM forms. EpoR-H/PY343/Stat5 response genes that might support EpoR-H’s wild-type activity next were analyzed. Despite evidence for Stat5 binding at a Bcl-x intron-1 element, no significant Epo-induction of Bcl-x transcripts was detected for any EpoR allele. In contrast, Pim-1, oncostatin-M and SOCS-3 were induced multi-fold in wt-EpoR and EpoR-H, but not EpoR-HM erythroblasts (and for EpoR-HM, no activation of Stat-5, -1 or-3 was detectable). In addition, receptor cross-talk analyses revealed that Kit selectively modulates EpoR-PY343-Stat5- dependent SOCS-3 and oncostatin-M transcription, and enhances Pim-1 expression several-fold via apparent post-transcriptional mechanisms. Epo action therefore is proposed to depend primarily on the regulation of ERKs, Pim-1 and possibly oncostatin-M (but not JNKs, p70S6K or Bcl-x).
The PAR-1-Stimulated Platelet Subpopulation That Binds Factor Xa Also Expresses Matrixmetalloproteinase and Calpain Activities Resulting in Population-Specific GP1bα Shedding and Platelet Vesiculation.
Authors: London Fredda S.
Published: April 2020

We previously showed that only a subpopulation of platelets responds to stimulation by strong agonists with exposure of coagulation factor binding sites. The subpopulation of SFLLRN-amide or collagen-stimulated platelets that bound coagulation factor Xa showed 50% decreased surface glycoprotein 1bα (GP1bα) and 60% reduced forward scatter relative to unstimulated platelets. While collagen-stimulated Xa-negative platelets lost no GP1b/IX/V surface density, the PAR-1-stimulated Xa-negative population lost 25% of both GP1bα and GPIX surface density signifying decreased GP1b/IX/V complex following PAR-1 stimulation. The PAR-1-stimulated Xa-positive platelets showed a discrepancy between the surface densities of GPIX and GP1bα that suggested a procoagulant subpopulation-specific shedding of GP1bα. While Xa-positive PAR-1-stimulated platelets pretreated with 10μM GM6001, a nonspecific inhibitor of matrixmetalloproteinases, recovered 30% of the lost surface GP1bα, bringing the GP1bα /GPIX ratio to ~1, collagen-stimulated loss of GP1bα on Xa-positive platelets was little affected by GM6001 pretreatment, and GP1bα on Xa-negative platelets was unaffected by GM6001 pretreatment. While inhibition of calpains by pretreatment of platelets with 100μM calpeptin had little effect on agonist-stimulated GP1bα shedding, calpeptin pretreatment resulted in 50% decreased loss of GP1bα/GPIX from PAR-1-stimulated Xa-negative platelets, in a 35–45% loss of the Xa-positive subpopulation resulting from PAR-1 stimulation, in 25% increased forward scatter from all agonist-induced Xa-positive populations, and in a 40–50% decrease in positive events occurring outside the defined platelet region. On the contrary, GM6001 inhibition of GP1bα shedding had no effect on either the size of the Xa-positive subpopulation, on the density of binding sites or on forward scatter. Platelet vesiculation would produce smaller platelets with decreased forward scatter, and microparticles. Calpain, previously implicated in platelet vesiculation, appears to mediate specific vesiculation of Xa-positive platelets. In summary, PAR-1 agonists and collagen recruit a platelet subpopulation to undergo specific membrane changes exposing binding sites for coagulation enzymes factors IXa and Xa. This procoagulant subpopulation recruited by PAR-1 signaling is also notable for specific matrixmetalloprotease activities leading to procoagulant population-specific GP1bα shedding. Calpain activity mediates membrane vesiculation of procoagulant platelets resulting from both collagen- and PAR-1-stimulation leading to smaller procoagulant platelets and to platelet microparticles.