bluuebunny/crossref_metadata_2025 huggingface dataset:
Prevalence and Prognostic Significance of c-KIT Mutations in Pediatric CBF AML Patients Enrolled on Serial CCG/COG Protocols.
Authors: Pollard Jessica , Alonzo Todd , Gerbing Robert , Zeng Rong , Hirsch Betsy , Raimondi Susana , Heerema Nyla , Woods William , Lange Beverly , Hurwitz Craig , Arceci Robert , Radich Jerald , Bernstein Irwin , Heinrich Michael , Meshinchi Soheil
Published: April 2020

Pediatric patients with core binding factor (CBF) acute myeloid leukemia (AML) [t(8;21) and inv(16)] typically have favorable outcome, yet 20–40% of patients will relapse, suggesting that additional events may impact disease response. Recently, mutations of the c-KIT receptor tyrosine kinase gene were implicated as a prognostic factor in adults with CBF AML. The prevalence and prognostic significance of c-KIT mutations in pediatric CBF AML has not been well established. We analyzed diagnostic specimens from 156 pediatric patients with CBF AML enrolled on serial AML protocols CCG 2891, 2961 and COG AAML03P1 with 3 different methodologies (direct sequencing, HPLC, and micro capillary electrophoresis) to identify mutations in c-KIT exons 8 and 17. Mutations were detected in 27 samples (17%); 13 (48%) involved exon 8, 13 (48%) involved exon 17, and 1 (4%) involved both regions. Of 15 inv(16) specimens with c-KIT mutations, 9 (60%) involved exon 8, 5 (33%) involved exon 17, and 1 (7%) affected both exons. In contrast, of 12 t(8;21) specimens with c-KIT mutations, 4 (33%) involved exon 8 whereas 8 (67%) affected exon 17. Clinical outcome data from 98 CBF AML patients enrolled on CCG 2961 was analyzed to determine the prognostic significance of c-KIT mutations in this patient population. CBF AML patients with c-KIT mutations had similar clinical characteristics (age, sex, race, presenting white blood cell count and blast percentage) to those CBF AML patients without c-KIT mutations. There was no difference in the rate of complete remission (CR) (c-KIT mutations, 88% vs. no mutation 92%; p=0.645), overall survival (OS) at 5 years from study entry (c-KIT mutation, 68% vs. no mutation 71%; p=0.733), or corresponding relapse risk (RR) from the time of CR (c-KIT mutations, 33% vs. no mutation 31%; p=0.92). The relative prognostic significance of the location of c-KIT mutation (i.e. exon 8 or exon 17) was also evaluated. No differences were found in CR rates (exon 8 mutation, 100% vs. exon 17 mutation, 75%; p= 0.467), OS from CR (exon 8 mutation, 86% vs. exon 17 mutation, 47%; p=0.088), or corresponding RR at 5 years from CR (exon 8 mutation, 29% vs. exon 17 mutation, 50%; p=0.517). This study demonstrates that the prevalence of c-KIT mutations in pediatric CBF AML patients enrolled on serial CCG/COG AML protocols is lower than that observed in adult studies. We found that c-KIT mutations lacked prognostic significance in pediatric CBF AML patients treated with intensive chemotherapy on CCG 2961, though there was a suggestion that patients with exon 17 mutations may have worse outcome. Outcome data from the entire cohort (CCG 2891, 2961 and COG AAML03P1) will be analyzed to determine whether this finding is significant with larger scale analysis.
Delayed Response to Lenalidomide in International Prognostic Scoring System (IPSS) Low Risk Myelodysplastic Syndrome (MDS).
Authors: Tubb Erev E. , Besa Emmanuel C. , Giagounidis Aristoteles , Fonseca Gustavo A.
Published: April 2020

Lenalidomide was approved by the US Food and Drug Administration in December of 2005 for the treatment (tx) of transfusion-dependent anemia (TDA) in patients (pts) with MDS and chromosome 5q deletions (del5q). In the trial that secured the approval, 67% of 148 pts achieved red blood cell (RBC) transfusion independence (TI) with a median time to TI of 44 weeks. 90% of pts who achieved TI did so by completion of 3 months of therapy. In a companion study (MDS-002) pts lacking del5q the frequency of TI was only 26%. We report three cases of IPSS Low risk myelodysplasia MDS with a delayed response to lenalidomide. The patient characteristics are outlined in Table 1.

Following a 6 to 11 month trial of lenalidomide for TDA in IPSS Low risk MDS delayed TI can occur 1 to 4 months after discontinuation of lenalidomide. Delayed TI was not associated with a cytogenetic response. The duration of response can last from 11 months to 16 months. Lenalidomide, a thalidomide analogue, is an immunomodulatory agent with anti-angiogenic and anti-neoplastic properties. Effects of lenalidomide such as increased production of interleukin-2 and interferon-γ have been linked to the appearance of lymphoid aggregates of a mixture of B and T cells in the BM specimens of responders. TI has previously been correlated with suppression of the del5q clone. In our patients, TI was achieved after discontinuation of lenalidomide following a reasonable ineffective trial and no apparent suppression of the del5 clone. A similar phenomenon was described by Raza, et al. in a single MDS patient treated with thalidomide. Interestingly, one patient had lymphoid aggregates of B and T cells throughout treatment, raising the possibility that the immune stimulatory effects of lenalidomide may be a variable against the del5q clone. The transient response may be due to immunomodulatory effects but persistence of the del5q clone in two of our patients and and dysplasia in the other, is responsible for the relapse after the immune response has waned. Re-treatment with lenalidomide in our patient did not prolong or re-induce another response. References Giagounidis AAN, Germing U, Aul C: Biologic and prognostic significance of chromosome 5q deletions in myeloid malignancies. Clin Cancer Res 2006, 12:5–10. List A, Kurtin S, Roe DJ, et al.: Efficacy of lenalidomide in myelodysplastic syndromes. N Engl J Med. 2005, 352:549–557. List A: Recent advances in the treatment of MDS. Clin Advances in Hematology. Oncology. 2007, Supp 10, Vol 5, Issue 7: 4–5 Raza A, Meyer P, Dutt D, et al. Thalidomide produces transfusion independence in long-standing refractory anemias of patients with myelodysplastic syndromes. Blood. 2001 Aug 15, 98(4):958–65.
Results of an Exploratory Study of Oral (po) and Intravenous (iv) Clofarabine in Patients with Myelodysplastic Syndrome.
Authors: Faderl Stefan , Garcia-Manero Guillermo , Gandhi Varsha , Ravandi Farhad , Borthakur Gautam , Thomas Deborah A. , Ferrajoli Alessandra , Estrov Zeev , Plunkett William , Byrd Anna , Kwari Monica , Kantarjian Hagop M.
Published: April 2020

Clofarabine (CLO) is an adenosine nucleoside analog with activity in adult acute myeloid leukemia (AML). Its activity in MDS is less well defined. To evaluate the activity and safety of CLO in MDS we designed two phase II studies with iv CLO and po CLO, respectively. Pts were eligible if they had MDS with ≥ 5% blasts, fell into IPSS intermediate-2 and high-risk groups, or had CMML. No prior intensive chemotherapy or high-dose cytarabine was allowed. Hematopoietic growth factor support was permitted. In the iv study pts were adaptively randomized to receive CLO 15 or 30 mg/m2 iv daily x 5 every 4–6 weeks. The second study used a starting dose of CLO 40 mg/m2 orally daily x 5 every 4–6 weeks (assuming oral bioavailability about 50%), which was then decreased to 30 mg/m2 orally daily x 5. Thirty pts (18 RAEB, 6 RAEB-T, 6 CMML) have been treated. Assignment by IPSS for RAEB and RAEB-T pts: 10 high-risk, 10 intermediate-2, and 4 intermediate-1. Fifteen pts received iv CLO and 15 po CLO. Median age was 68 yrs (range 57–86). Median number of prior therapies was 1 (0–4). Twenty (67%) pts received prior decitabine or azacitidine. Karyotype was abnormal in 24 (80%) pts, including 10 pts with −5/−7 abnormalities. Responses were determined according to International Working Group criteria (IWG). Twenty-seven pts were evaluable for response (2 pts too early, one refused further treatment on day 4 of first course). Eleven (41%) pts responded [7 (26%) CR, 2 (7%) HI, 2 (7%) clinical benefit (CB)] (see table).

Response (%) Route Dose (mg/m2) N CR HI CB po 40 6 - - 1 (17) 30 6 1(17) 2 (33) 1 (17) iv 30 6 2 (33) - - 15 9 4 (44) - -

Response by IPSS: 67% intermediate-1, 44% intermediate-2, 40% high. Twenty-seven pts are evaluable for toxicities. Grade ≥ 3 toxicities occurred in 8 of 13 pts on iv CLO (rash, hyperbilirubinemia, elevated transaminases, elevations of creatinine, acute renal failure) and 4 of 14 pts with po CLO (rash, hyperbilirubinemia, elevated transaminases). Myelosuppression resulting in febrile episodes and hospitalizations with both iv and po CLO was ubiquitous, but prolonged myelosuppression (> 42 days) was rare. In summary, CLO has activity in pts with higher-risk MDS. The optimal dose and schedule for po and iv CLO have not been defined yet. Lower doses of CLO are associated with responses. Correlative PK studies may help to provide further support for optimal dose and route of administration of CLO in pts with MDS.
Lenalidomide in High-Risk Myelodysplastic Syndrome and Acute Myelogenous Leukemia Associated with Chromosome 5 Abnormalities.
Authors: Borthakur Gautam , Garcia-Manero Guillermo , Faderl Stefan , Estrov Zeev , Verstovsek Srdan , Hood Mary , Kantarjian Hagop
Published: April 2020

Background: Treatment with lenalidomide produces remarkable responses in patients with myelodysplastic syndrome and deletion 5q 31 (5q-) cytogenetic abnormality; transfusion independence in 67% of patients, major cytogenetic responses in 44% and a median duration of response of 115.9 weeks (List et al. Abstract #251; Blood: 108). Responses have been reported in few patients with MDS and complex karyotype as well. Since chromosome 5 abnormality is considered to be an early genetic event, we designed a phase 2 study of lenalidomide in patients with high-risk MDS and relapsed/refractory acute myelogenous leukemia (AML) with any chromosome 5 abnormality.

Method: The dose of lenalidomide was 25 mg/day, orally for 21 days with 7 days rest (28 day cycle). For patient with platelet count < 50,000/μl, the starting dose is 15 mg/day orally for 21 days with 7 days rest (28 day cycle). Dose interruptions/reductions are planned for ≥ grade3 neutrpenia with fever or ≥ grade 4 neutropenia (for patients starting with absolute neutrophil count [ANC] >1000/μl) and for grade 3 thrombocytopenia (for patients starting with platelet count ≥ 50,000/μl).

Results: Eleven patients out of this 30 patient study have been treated so far; 6 with AML and 5 with MDS. All patients with AML and 2 patients with MDS had previous therapy. Median age is 63 years (range, 44 to 80 years). Nine patients had complex karyotypic abnormality and 2 patients had single additional chromosomal abnormality (+8 in one and 11q abnormality in another) along with 5q deletion. One patient with AML (previously treated with decitabine) and with karyotypic abnormality of trisomy 8 in addition to 5q deletion, achieved complete remission after 3 cycles of therapy. Another patient had reduction in bone marrow blasts from 21 to 5%, but could not continue lenalidomide due to profound thrombocytopenia complicated by gastrointestinal bleeding and eventually had disease progression. Four (4/6) patients with a platelet count of ≥ 30,000/μl and 5 (5/6) patients with ANC of ≥500/μl at study entry experienced worsening cytopenias. No grade 3/4 non-hematological toxicities were seen. One patient in CR currently remains on study. Reasons off study include disease progression (8 patients), stem cell transplant (1 patient) and one patient died while on study from sustained pancytopenia, infection and multiorgan failure.

Conclusion: The role of lenalidomide in the treatment of high-risk MDS and AML with chromosome 5 and additional karyotypic abnormalities needs to be defined in the context of clinical trials. Future studies are needed to examine lower doses with or without growth factor support.
Outcome of Allogeneic Stem Cell Transplantation after Hypomethylating Therapy with 2′-Deoxy-5 Azacytidine for Patients with Myelodysplastic Syndrome.
Authors: De Padua Silva Leandro , de Lima Marcos , Kantarjian Hagop , Champlin Richard , Faderl Stefan , Giralt Sergio , Kebriaei Partow , Davisson Jan , Estey Eli , Garcia-Manero Guillermo , Issa Jean-Pierre , Ravandi Farhad
Published: April 2020

Decitabine is approved for the treatment of patients with intermediate- and high-risk myelodysplastic syndrome (MDS). In vitro studies have demonstrated an increased expression of MHC class I molecules, HLA-DR and beta-2-microglobulin on the surface of MDS cells after decitabine therapy, potentially increasing their susceptibility to immune surveillance mechanisms and a graft-versus-leukemia effect. We analyzed the outcome of 12 patients with MDS with a median age of 58.5 years (range, 37 – 66) who underwent an allogeneic stem cell transplant (5 sibling, 5 unrelated, 2 cord blood) after prior therapy with decitabine. At diagnosis, 2 patients had intermediate-1, 7 intermediate-2 and 3 high risk MDS by the international prognostic scoring system. Nine had a non-myeloablative and 3 an ablative regimen. The source of stem cells was marrow in 2, peripheral blood in 8 and cord blood in 2. The patients had received decitabine for a median of 5.5 cycles (range, 1 – 20) and a median duration of treatment of 7.1 months (range, 1 – 27). Decitabine was well tolerated with reversible gastrointestinal toxicity and neutropenic infections as the main toxicity. Best response to decitabine was CR in 4 patients, PR in 6, and hematological improvement in 2. Eight had disease progression on decitabine. Five patients received additional chemotherapy and achieved a CR before transplant. The median time between completion of decitabine and transplant was 3.4 months (range, 0.2 – 11.7). At the time of transplant, 8 patients were in CR and 4 in PR. The median CD34+ cell number was 4.17 x 106 cells/kg (range, 0.58 – 10.1 x 106 cells/kg). Eleven patients engrafted and one had secondary engraftment failure. Median times to neutrophil and platelet engraftment were 13.7 days (range, 7 – 27) and 17.3 days (range, 7 – 28), respectively. No unusual toxicity was encountered. Nine patients (75%) developed acute GVHD, and 6 chronic GVHD (55% of those alive beyond 100 days). Ten patients were in CR and 1 in PR at day 100 post transplant. With a median follow 11.5 months (range, 3 – 26), 9 patients are alive (8 in CR and 1 with progressive disease) and 3 have died (2 after relapse and 1 from GVHD and sepsis). We conclude that prior therapy with hypomethylating agents may potentially improve the outcome of allogeneic transplant in MDS through enhancement of graft versus leukemia effect and should be examined prospectively.
A Clinical Test for the Identification of Amyloid Proteins in Biopsy Specimens by a Novel Method Based on Laser Microdissection and Mass Spectrometry.
Authors: Vrana Julie A. , Gamez Jeffrey D. , Theis Jason D. , Plummer Timothy B. , Bergen Robert H. , Zeldenrust Steven R. , Kurtin Paul J. , Grogg Karen L. , Dogan Ahmet
Published: April 2020

The management of systemic amyloidosis relies on the treatment of the underlying etiology and differs radically for different amyloid types. Therefore, given that at least 25 different proteins have been associated with amyloidosis, accurate identification of proteins deposited as amyloid fibrils is an important clinical problem. In this study, we describe a novel method that can characterize amyloid subtypes using laser microdissection (LMD) and mass spectrometry (MS) on routinely processed paraffin-embedded tissues. The study used 60 cases consisting of 16 transthyretin, 9 serum amyloid-associated protein, 20 immunoglobulin light chain lambda, 5 immunoglobulin light chain kappa, and 10 amyloid negative control samples. The biopsy specimens studied included heart, kidney, gastrointestinal tract, lung and decalcified bone marrow specimens. The amyloid type in all cases was previously characterized based on clinical findings, immunohistochemistry and, where indicated, by molecular testing for transthyretin mutations. Amyloid plaques were captured from an 10 micron paraffin section exhibiting positive Congo Red staining using LMD. Proteins were extracted, digested with trypsin and identified following MS/MS using the Mascot search algorithm analysis. MS correctly identified each of the 4 types of amyloidosis analyzed. Serum Amyloid P component and Apolipoprotein E were also identified as constituents of the amyloid deposition. The analysis was successful on all tissue types including decalcified bone marrow specimens and small biopsy specimens such as endomycardial biopsies and renal biopsies. The use of LMD from paraffin embedded biopsies and subsequent analysis by MS allows identification of the type of amyloid protein deposited with high specificity and sensitivity. This method promises to be a clinical test for accurate identification of amyloid proteins in routinely processed biopsy specimens and overcomes many of the specificity and sensitivity issues associated with current methods such as immunohistochemistry.
Immunoglobulin Free Light Chain Ratio Is an Independent Risk Factor for Progression of Smoldering Multiple Myeloma.
Authors: Dispenzieri Angela , Kyle Robert A. , Katzmann Jerry A. , Larson Dirk , Benson Joanne , Clark Raynell , Melton L. Joseph , Gertz Morie A. , Kumar Shaji K. , Fonseca Rafael , Jelinek Diane F. , Rajkumar S. Vincent
Published: April 2020

Background: Smoldering multiple myeloma (SMM) is an asymptomatic plasma cell proliferative disorder with a high risk of progression to symptomatic multiple myeloma. Identification of risk factors that predict progression of SMM to symptomatic MM could identify higher risk patients who might benefit from chemoprevention or more intensive surveillance. We hypothesized that increased monoclonal free kappa or lambda immunoglobulin light chains in smoldering myeloma (SMM), as detected by the serum free light chain (FLC) assay, indicates an increased the risk of progression to active myeloma.

Methods: Of 276 pathologically confirmed SMM patients seen at the Mayo Clinic from 1970 to 1995, baseline serum samples obtained within 30 days of diagnosis were available in 273.

Results: At a median follow-up of surviving patients of 12.4 years, transformation to active disease has occurred in 161 (59%) patients. An abnormal FLC ratio was present at baseline in 90% of patients. The best break-point for predicting risk of progression was a FLC ratio less than or equal to 0.125 or greater than or equal to 8 (hazard ratio, 2.3; 95% CI, 1.6–3.2) [Figure 1]. The extent of abnormality of FLC ratio was independent of SMM risk categories defined by number of plasma cells in the bone marrow and size of serum M-proteins (bone marrow plasma cells ≥ 10% and serum M protein ≥ 3 g/dL; bone marrow plasma cells ≥ 10% but serum M protein < 3 g/dL; and serum M protein ≥ 3 g/dL but bone marrow plasma cells < 10%). Incorporating the FLC ratio into the risk model, the division of patients into high-, intermediate-, and low-risk groups is 28, 42, and 30% with 5 year progression rates of 76, 51, and 25%, respectively [Figure 2].

Conclusions: The serum immunoglobulin FLC ratio is an important additional determinant of clinical outcome in patients with SMM.

Figure Figure Figure Figure
Polymorphisms of the Transforming Growth Factor Beta 1 (TGFB1) Gene Define a Subgroup of Patients with Late Onset of Disease and Poor Outcome in Multiple Myeloma.
Authors: Neben Kai , Mytilineos Joannis , Heiss Christiane , Ho Anthony D. , Benner Axel , Opelz Gerhard , Goldschmidt Hartmut
Published: April 2020

Common genetic variants in immune and inflammatory genes can affect the risk of developing multiple myeloma. The aim of this study was to determine the effect of polymorphic variants in the interleukin 10 (IL10), transforming growth factor beta (TGFB1) and tumor necrosis factor alpha (TNFa) genes on the predisposition to multiple myeloma and the effect of these polymorphisms on survival after treatment with chemotherapy. Genotype data of 7 single-nucleotide polymorphisms and clinical follow-up information were available for 239 consecutive patients with multiple myeloma who presented at our outpatient department. All patients were treated with chemotherapy, including high-dose chemotherapy and peripheral blood stem cell transplantation in 191 patients. In particular, there were 155 males and 84 females with a median age of 55 years (range, 30 – 84). The median follow-up period for the entire cohort was 75 months (range, 3 – 190 months). The TGFB1 low producer genotype 10Leu/Leu (n=78) was associated with a 3.5 years older age at start of treatment (57.5 versus 54 years, p=0.005), a higher level of beta2-microglobulin (3.4 versus 2.5 mg/l, p=0.01), a higher frequency of ISS-scores II and III versus I (OR=0.50, p=0.01) and an inferior median survival rate of 71.8 versus 91.6 months (p=0.02) as compared to other genotypes, respectively. In a multivariate analysis, the TGFB1 low producer genotype 10Leu/Leu was identified as an independent factor for survival (p=0.03), indicating that the poor prognosis of these patients is not due to the older age at the start of treatment. All other gene polymorphisms analyzed (IL10–1082A>G, IL10–819C>T, IL10–592C>A, TGFB1 Arg25Pro, TNFa -308G>A, TNFa -238G>A) showed no statistically significant effect on overall survival. Our findings suggest that patients carrying the TGFB1 low producer polymorphism show a later onset of disease and need for chemotherapy treatment as compared to other genotypes. In addition, our findings indicate that TGFB1 genetic variants influence prognosis in patients with multiple myeloma who received chemotherapy and suggest that genetically determined cytokine production affects the clinical course of the disease possibly through regulation of immune surveillance.
Combination of Bortezomib with Tipifarnib (R115777) Induces Synergistic Myeloma Cell Apoptosis Via Down-Regulation of HDAC6 and Inhibition of Aggresome Formation.
Authors: David Ebenezer , Schafer-Hales Katherine , Marcus Adam I. , Kaufman Jonathan L. , Lonial Sagar
Published: April 2020

Background: We have previously demonstrated that the combination of bortezomib and a farnesyl transferase inhibitor (FTI) induce synergistic myeloma cell apoptosis, and that maximal cell death is sequence dependent (AACR meeting-2004, ASH meeting -2005, David, E. et.al., Blood, (2005)106: 4322–4329,). The mechanism responsible for this synergy has been poorly understood. As FTI’s are known to downregulate HDAC6 expression, we further evaluated alternative mechanisms responsible for the observed synergy between bortezomib and tipifarnib.

Methods: Myeloma cell lines and primary human myeloma cells were used for the analysis. Confocal microscopy was used to assess ubiquitination in RPMI8226 cells exposed to each drug combination. Western blot analysis and flow cytometry were used to assess phospho-protein expression and apoptosis in MM.1S cell line.

Results: The use of tipifarnib and bortezomib resulted in a time dependent reduction in HDAC6 expression as measured by western blot. Confocal images revealed that bortezomib treatment, both alone and in combination with tipifarnib resulted in an accumulation of ubiquitinated proteins, suggesting a disruption of the aggresome complex. Moreover, a potential sequence-dependent antagonistic affect was observed when tipifarnib was given prior to bortezomib resulting in the reduction of ubiquitination. Additional experiments also demonstrated that apoptosis induced by the combination was only partially inhibited by the pan-caspase inhibitor ZVAD-FMK. This led us to explore caspase independent causes of apoptosis. Inhibition of cathepsins demonstrated overall apoptosis was significantly inhibited, suggesting that both caspase independent and dependent pathways are important in myeloma cell apoptosis induced by this potent combination. Although FTI alone inhibited GO/G1/S phases to some level, only the combination of bortezomib and tipifarnib resulted in the enhanced level of apoptosis which was confirmed by different biochemical methods.

Conclusion: The use of a FTI represents an important and alternative mechanism by which to inhibit HDAC6 and thus the aggresome pathway of protein degradation. Given that combinations of bortezomib and aggresome inhibition have been shown to be very active in vitro and in animal models, this approach represents an attractive method by which to target both critical pathways of protein degradation within malignant cells.
Polymorphisms of NOS3 and FCGR2A Genes Contribute to Thrombotic Risk in Essential Thrombocythemia
Authors: Olcaydu Damla , Gisslinger Heinz , Esterbauer Harald , Wiesbauer Franz , Kralovics Robert
Published: April 2020

Thrombosis is the most frequent complication in essential thrombocythemia (ET) that impairs quality of life and contributes to mortality and morbidity of patients. The molecular basis of thrombosis in ET remains unclear. Polymorphisms (SNPs) in coagulation factors (FII-G20210A, FV-Leiden) and the V617F mutation of JAK2 gene (JAK2-V617F) have previously been shown to associate with thrombosis in ET. Since thrombosis occurs frequently in JAK2-V617F negative ET and FII-G20210A and FV-Leiden polymorphisms occur only in up to 5% of patients, we examined the hypothesis that other genetic factors contribute to the thrombotic risk in ET. In addition to FII-G20210A and FV-Leiden we studied thirteen candidate SNPs of genes involved in coagulation (F7, F13A1, VWF, THSD1, ATIII), platelet aggregation (P2Y12, GPIIIA, GPIIB, FCGR2A) and endothelial function (NOS3, ADRA2B) previously implicated in thrombosis in non-MPD patients. We studied 94 patients with the diagnosis of ET retrospectively observed for clinical variables such as venous and arterial thrombosis, stroke, myocardial infarction, deep vein thrombosis (DVT), bleeding, angina pectoris, superficial venous thrombosis (SVT), and pulmonary embolism. SNP genotyping was performed using allele-specific PCR and sequencing. We examined the association of individual SNPs with the presence or absence of clinical variables (adjusted for age, sex, and BMI). In our ET cohort, JAK2-V617F showed significant correlation with the presence of DVT (OR 10.08; 95%CI 1.15–88.49; P=0.01). Both FII-G20210A and FV-Leiden were rare in this cohort (2% and 4%, respectively) and thus did not show any significant associations. Homozygosity for the 298Asp variant of NOS3 gene (encoding the endothelial nitric oxide synthase) showed significant correlation with stroke (OR 46.75, 95%CI 1.38–147, P=0.014). Similarly, homozygosity for the 131Arg variant of Fc receptor gamma 2A (FCGR2A) involved in platelet GPVI signaling correlated with the presence of SVT (OR 9.46, 95%CI 1.48–60.82, P=0.014). Our results indicate that genetic factors other than JAK2-V617F, FII-G20210A and FV-Leiden contribute to thrombotic risk in ET. Furthermore, the nitric oxide and the platelet GPVI signaling pathways are likely to play an important role in the pathogenesis of thrombosis in ET.
Microparticles (platelet, Endothelial, and Leucocyte) Are Elevated in Patients with Myeloproliferative Disorders.
Authors: Lynch Susan , Stirling David , Ludlam Christopher A.
Published: April 2020

Patients with myeloproliferative disorders (MPD) have an excess of thrombotic complications which is not fully corrected by normalisation of their elevated haematocrit or thrombocytosis. The mechanisms responsible for this are not completely understood but platelets, leucocytes and endothelial cells have all been implicated. Plasma microparticles are observed to be elevated in other diseases associated with thrombotic complications. We measured platelet (PMP), endothelial (EMP), red cell (RMP) and leucocyte (LMP) microparticles in patients with MPD compared to healthy controls. Microparticles, of less than 1μm were measured by flow cytometry on platelet poor plasma obtained from citrated peripheral blood samples. Subtypes were identified using fluorescent conjugated monoclonal antibodies; EMP CD31+CD42-, PMP CD31+42+, RMP Glycophorin A + and LMP CD45+ events. Absolute concentrations of microparticles per μl plasma were calculated using Trucount beads. Results are currently available for 27 MPD patients, 9 polycythaemia vera (PV), and 18 primary thrombocythaemia and 14 healthy controls. All patients except 1 PV were on current treatment with hydroxycarbamide and all but 3 were receiving aspirin. PMP and LMP were elevated in MPD patients compared to healthy controls, PMP 12432 (1653) v 2371 (278) per μl, mean (SEM) p<0.001, LMP 98.7 (14) v 62.6 (7.7) per μl, p=0.03. EMP were elevated in MPD patients at 202 (34) v 34 (14) per μl, p<0.001. There was no significant difference in RMP levels 646 (131) v 589 (241) per μl, p=0.84. There were no significant differences in MP numbers between the PT and PV patients or by Jak2 V617F mutation status. Platelet count (x109/l) was higher in the MPD patients, PT 503 (39) v PV 372 (43) v control 236 (12), all comparisons p<0.04. PMP/platelet count was calculated to adjust for this and the ratio remained elevated in the MPD patients, PV 28.5 (4.5) and PT 26.1 (3.7) v controls 9.9 (1.2), both p=<0.03. In conclusion, these results demonstrate that PMP, EMP and LMP are elevated in MPD despite standard treatment, with the PMP elevated disproportionately to the platelet count. This is further evidence for platelet activation, endothelial dysfunction and a role for leucocytes in the thrombotic risk of MPD and that the microparticles also play an active role in promoting the prothrombotic state.
Activating JAK3 Mutations in Transient Myeloproliferative Disorder and Acute Megakaryoblastic Leukemia Accompanying Down Syndrome.
Authors: Sato Tomohiko , Toki Tsutomu , Kanezaki Rika , Xu Gang , Terui Kiminori , Kamio Takuya , Kojima Seiji , Kiyoi Hitoshi , Mano Hiroyuki , Ito Etsuro
Published: April 2020

Children with Down syndrome (DS) have an approximately 20-fold higher incidence of leukemia than the general population. The majority of leukemia cases associated with DS are acute megakaryoblastic leukemia (AMKL). Although GATA1 mutations have been found in almost all cases of transient myeloproliferative disorder (TMD) “a preleukemia” that may be present in as many as 10% of newborn infants with DS and AMKL accompanying DS (DS-AMKL), GATA1 mutation alone may not be sufficient for development of leukemia. Following identification of acquired activating JAK3 mutations in DS-AMKL, JAK3 mutations have been reported also in TMD patients. However, the frequency and functional consequence of JAK3 mutations in TMD remain unknown. To further understand how JAK3 mutations are involved in the development and/or progression of leukemia in DS, we screened TMD patients and two DS-AMKL cell lines for JAK3 mutation, and examined the functional consequences of these JAK3 mutations. In one out of the two DS-AMKL cell lines, MGS, we identified novel JAK3 mutations (JAK3Q501H mutation in the SH2 domain and JAK3R657Q mutation in the psuedokinase domain in the same allele). JAK3Q501H and JAK3R657Q each constitutively phosphorylates STAT5 and transformes Ba/F3 cells to factor-independent growth, whereas the double mutant (JAK3Q501H and JAK3R657Q) has more potent transforming activity than each mutant. Biochemichal analysis in Ba/F3 cells revealed that the degrees of phosphorylation of STAT5 in the cells transduced with each JAK3 mutant were correlated with its transforming activity. Although we previously identified a JAK3I87T mutation in one of two TMD patients, no JAK3 mutations were detected in another 9 TMD patients. Together with the previous results, we found JAK3 mutations in each of 11 TMD and 11 DS-AMKL patients. Although the number of the patients analyzed was small, these results indicate that there are no significant differences in the frequency of JAK3 mutations between TMD and DS-AMKL. In this study, we showed for the first time that the TMD patient-derived JAK3 mutation was also an activating one. JAK3I87T transformed Ba/F3 cells to factor-independent growth. Treatment with JAK3 inhibitors (WHI-P131 and WHI-P154) resulted in a significant decrease in the growth and viability of Ba/F3 cells expressing each activating JAK3 mutant. These results suggest that the JAK3 activating mutation is an early event during the development of AMKL in DS. Furthermore these results provide a proof-of-principle that JAK3 inhibitor should have therapeutic effects on the AMKL and TMD patients carrying the activating JAK3 mutations.
Venous Thromboembolism among 1,570 Patients Undergoing Hematopoietic Stem Cell Transplantation: Incidence, Risk Factors, Treatment and Outcomes.
Authors: Gerber David E. , Segal Jodi B. , Levy M. Yair , Kane Joyce M. , Jones Richard J. , Streiff Michael B.
Published: April 2020

Introduction: Venous thromboembolism (VTE) is increasingly diagnosed among individuals with hematologic malignancies. It is unclear whether patients undergoing hematopoietic stem cell transplantation (HSCT) are at risk for VTE. We aimed to describe the incidence of VTE and risk factors among patients undergoing HSCT.

Methods: Using ICD-9 procedure codes in a discharge database, we identified all patients undergoing inpatient HSCT at Johns Hopkins between July 1, 1993, and June 30, 2005. We reviewed electronic medical records for each patient to identify VTE and bleeding. The associations between VTE and clinical characteristics were tested using parametric and non-parametric statistical tests and survival analysis methods.

Results: 1,570 patients had 3,425 person-years of follow-up. Median age was 46 years and 51% of patients were male. All patients had indwelling central venous catheters; pharmacological VTE prophylaxis was not used for any patient but ambulation was encouraged. Between admission and 180 days after HSCT, 75 VTE occurred in 70 patients (4.5%; 95% CI, 3.5–5.6%). VTE included 55 (73%) catheter-associated and 11 (15%) non-catheter-associated deep venous thromboses (DVT), and 9 (12%) pulmonary emboli (PE). Median platelet count at time of VTE was 77 K/mm3 (interquartile range [IQR] 42–159 K/mm3), and platelet count was < 50 K/mm3 at the time of VTE in 31% of cases. In multivariate analyses, VTE was associated with a history of prior VTE (OR 2.86; 95% CI, 1.25–6.55) and with graft-versus-host-disease (GVHD) (OR 2.36; 95% CI, 1.38–4.04). 86% of patients received anticoagulation for treatment. Clinically significant bleeding occurred in 238 patients after HSCT (15%), of whom 56 patients had fatal bleeding. Bleeding was associated with GVHD (OR 2.32; 95% CI, 1.71–3.16) and with initiation of anticoagulation for VTE diagnosed after HSCT (OR=3.00; 95% CI, 1.73–5.21). Bleeding was not associated with the continuation of anticoagulation initiated prior to admission (OR=1.13; 95% CI, 0.54–2.35).

Conclusions: VTE occurs relatively infrequently among patients undergoing HSCT and primarily in association with central venous catheter use. In contrast, clinically significant bleeding is a relatively common complication of HSCT, even in the absence of pharmacologic VTE prophylaxis. The unique hemostatic milieu associated with HSCT should be carefully considered when contemplating routine VTE prophylaxis in this patient population. The low risk of VTE associated with the high risk of bleeding makes pharmacologic VTE prophylaxis in this population both unnecessary and hazardous.
Clinical Trial of Therapeutic Blockade of CTLA4 with Ipilimumab in Patients with Relapse of Malignancy Following Allogeneic Hematopoietic Transplantation.
Authors: Bashey Asad , Medina Bridget , Corringham Sue , Pasek Mildred , Carrier Ewa , Vrooman Linda , Streicher Howard , Lowy Israel , Solomon Scott R. , Morris Lawrence E. , Holland Kent , Mason James R. , Soiffer Robert J. , Ball Edward D.
Published: April 2020

Relapse/progression of malignancy (RM) is an important cause of treatment failure and death following allogeneic hematopoietic stem cell transplantation (allo-HCT). CTLA-4 is an important negative regulator of effector T-cell activation. CTLA-4 inhibition has demonstrated potent anti-cancer effects in animal models, and in patients with some solid tumors. CTLA-4 blockade following allo-HCT may potentially augment graft-versus-malignancy but GVHD may also possibly be increased. We report the safety and preliminary efficacy of a dose-escalation study of a neutralizing human monoclonal antibody targeting CTLA-4 (ipilimumab) in patients with RM following allo-HCT. Eligibility criteria included allo-HCT ≥90 days previously, > 50% donor T-cell chimerism, no prior grade 3/4 GVHD, no prophylaxis/therapy for GVHD for ≥ 6 weeks. Patients received a single dose of ipilimumab over 90 min. DLI at a dose of 5 × 10e6 CD3 cells/kg was allowed 8 weeks following ipilimumab if no GVHD occurred and RM was present. A total of 29 patients were treated at four centers (23M, 6F; median age 43 (21–65); Hodgkins disease [HD] =14 Myeloma [MM]=6, CML=2, CLL=2, AML=2, NHL=1, Renal Ca =1, Breast Ca=1; 19 related donors, 10 unrelated; 6 myeloablative, 23 RICT).(4 at dose-level 1 [DL1] 0.1 mg/kg, 3 at 0.33 mg/kg [DL2], 4 at 0.66 mg/kg [DL3], 3 at 1.0 mg/kg [DL4] and 15 at 3.0 mg/kg [DL5]). Eight patients had failed prior DLI. Median time between BMT and ipilimumab was 366 d (125–2368). Dose-limiting toxicity was not encountered. No patient developed clinically significant GVHD within 90 days following ipilimumab alone. Three possible immune adverse events were documented: grade 3 polyarthropathy 14 weeks following ipilimumab, and 6 weeks post DLI, which resolved with corticosteroid therapy, (AML, DL1); grade 1 hyperthyroidism with thyroid-stimulating antibody 6 weeks post ipilimumab (CLL, DL3). Grade 2 pneumonitis responsive to corticosteroids (HD, DL5). Ten patients received DLI after ipilimumab. Three patients developed objective evidence of disease response after ipilimumab alone: PR in a patient with mantle cell NHL lasting 3m [DL4]; ongoing CR in a two patients with HD [DL5]. Two of these patients had failed prior DLI. Three additional patients demonstrated possible anti-cancer effects (MR in a HD patient, reduction of PB and BM blasts in AML, DL1; maintenance of molecular remission in a CML patient given ipilimumab alone for 3.5 yrs despite stopping imatinib, DL1). This study shows that doses of ipilimumab (up to 3.0 mg/kg) can safely be administered to patients with RM following allo-HCT without inducing/exacerbating GVHD while inducing regressions of malignancy including durable CR in some patients.
No Difference in Survival or Long-Term Disease Outcomes in Palifermin-Treated Patients with Hematologic Malignancies Undergoing Hematopoietic Stem Cell Transplantation.
Authors: Spielberger Ricardo , Territo Mary , Durrant Simon , Nimer Stephen , McCarty John , Hurd David , Berger Dietmar , Aycock Jeff , Chen Mon-Gy , Stiff Patrick
Published: April 2020

Background: Oral mucositis is an adverse effect of myeloablative therapy which has serious clinical and economic consequences as well as a negative impact upon quality of life. The duration and severity of oral mucositis can be reduced by administering palifermin to patients with hematological malignancies receiving myeloablative therapy and undergoing hematopoietic stem cell transplantation (HSCT). However, we still require additional data on the long-term disease outcomes of patients treated with palifermin. Therefore we present here the long-term, safety data for palifermin-treated HSCT patients followed up for approximately 60 months after the last palifermin dose.

Methods: The long-term safety data were collected during the follow-up phase of 4 parent trials where patients had received at least one dose of palifermin or placebo. Study assessments included overall survival (OS), progression-free survival (PFS), and secondary malignancies. Assessments were made at 6-month intervals during year 1 and annually thereafter until death or loss to follow-up. Kaplan-Meier curves for overall survival and PFS were calculated and the treatment groups were compared using stratified log-rank test.

Results: Altogether 662 patients were randomized to treatment and received either palifermin or placebo (421 palifermin, 241 placebo); 538 patients entered the follow-up study (342 palifermin, 196 placebo). The median follow-up time for patients alive at last visit was 49.8 months (palifermin N=290) and 49.5 months (placebo N=169). There were 131 (32%) and 72 (30%) deaths in the palifermin and placebo groups, respectively. The overall survival curves were similar for both groups (p=0.717). Disease progression occurred in 167 (41%) palifermin- and 87 (36%) placebo-treated patients; the difference in PFS between the two groups was non-significant (p=0.280). Secondary malignancies were observed in 8% of patients in both groups: the incidence of secondary hematologic malignancies was 4% (palifermin: 14/342) versus 5% (placebo: 10/196) while the incidence of solid tumors was 2% in both groups.

Conclusion: The results of this 60-month follow-up study indicate that long-term disease outcomes are not affected by administering palifermin to patients with hematological malignancies who are receiving myeloablative therapy and undergoing HS. There was no difference in OS and PFS between the palifermin and placebo groups. Furthermore there was no difference in the incidence of secondary malignancies between the two patient groups. The incidence of secondary hematologic malignancies and solid tumors was low, comparable between groups, and within the expected range for this patient population.
Experiment Therapy of Aplastic Anemia Using Cyclosporin A Combined with IDO Agonist.
Authors: Huang Lifang , Dai Min , Liu Wenli , Zhou Jianfeng , Sun Hanying , Zhang Yicheng
Published: April 2020

Aplastic anemia(AA) is an unusual hematologic disease and the paradigm of the human bone marrow failure syndromes. Abnormal immunological responses have been found in most aplastic patients. Activated type 1 cytotoxic T cells may be the main contributing factor of pathogenesis. IDO is a rate-limiting enzyme in Tryptophan(Trp) metabolism. The proliferation of Th1 cells is specifically inhibited by the over-expression of IDO, which can degrade the Trp in local environment, hamper the conductioin of the activating signal in T cell and induce immune tolerance. 3,4-DAA is an IDO agonist and can activate IDO. In our experiment, Balb/c mice were irradiated (5.0Gy of 60Co), and then infused with 5×106 lymph node(LN) cells from DBA/2 mice in 4 hours. Blood count was monitored and marrow damage was assessed by histogical study. Concentrations of serum IFN-γ were measured by ELISA. The levels of Tryptophan and kynurenine were evaluated by high performance liquid chromatography (HPLC). CD4+CD25+ T cells in spleen were analyzed by flow cytometry. The level of Foxp3 in CD4+CD25+ T cell was measured by RT-PCR. Irradiation and infusion of LN cells led to rapid development of severe pancytopenia and bone marrow hypoplasia. Bone marrow of affected mice showed lymphocyte infiltration. Serum IFN-γ concentration increased 3.7 fold at d6 post infusion. The recipient mice were divided into 4 different treating groups as follow:

0.9% Sodium Chloride as control; Cyclosporin A (CsA) (50ug/g/d ×5d, peritoneal injection); 3,4-DAA(5mg/d, orally daily); the combination of CsA and 3,4-DAA.

The effects of CsA, IDO agonist and CsA combined IDO agonist were analyzed at day 6,10,14,21,24 and 28 after LN cells infusion. The white cell and the platelet recovered to near normal, respectively (4.2±0.32)×109, (937±190.47)×1012 at d21 in the combination group. Early stage treatment with CsA can improve periphery blood cells and BM nucleated cells, but long term effect was not remarkable. In contrast, the 3,4-DAA group exhibited slow and gradually enhanced role. Periphery blood cells and BM nucleated cells were improved remarkably in the combination group. The number and function of CD4+CD25+T cells also increased remarkably. In the treatment of AA, abnormal immune response in bone marrow was inhibited by CsA, meanwhile immune tolerance was induced through up-regulating the regulatory T cells(Treg) by 3,4-DAA. In this way, the balance of immune in bone marrow could be reestablished quickly. CsA combined IDO agonist could provide a new strategy for the management of AA.
Monosomy 7 Detected by FISH at Disease Presentation Is a Marker for Non-Response to Immunosuppression.
Authors: Sloand Elaine , Scheinberg Phillip , Fenlon Edward , Blancato Jan , Pfannes Loretta , Sloand Elaine M. , Young Neal S.
Published: April 2020

About fifteen percent of patients with severe aplastic anemia (sAA) who undergo immunosuppression therapy (IST) develop clonal disease in the decade following treatment. Of 122 patients treated with horse ATG/CsA at NIH, 13 developed cytogenetic abnormalities (monosomy 7 in 9 patients, 7p deletion in one, and trisomy 8 in 2) (Rosenfeld et al: JAMA289:1130; 2003). Monosomy 7 usually occurs in patients with sAA who are unresponsive to IST. Factors responsible for clonal progression in bone marrow failure are still unclear. We and others have reported that monosomy 7 may be detected by fluorescent in situ hybridization (FISH) months before conventional cytogenetics and that high levels of GCSF foster preferential expansion of the monosomy 7 clone in vitro (Sloand E et al; Proc. Natl. Acad. Sci2006;103:14483). We hypothesized that a clone of monsomy 7 cells might indicate an underlying stem cell disorder unlikely to respond to immunosuppression or reflect more severe disease marked by higher endogenous GCSF levels. FISH was undertaken on 40 bone marrow samples obtained from aplastic anemia patients at presentation and before immunosuppressive treatment. Bone marrow mononuclear cells were hybridized with centromeric probes specific for chromosome 7 and chromosome 8 (to control for hybridization efficiency); samples were assessed on duplicate slides by three investigators who were unaware of the diagnoses and outcomes. Twenty-five healthy controls were tested concurrently. The upper limit of normal was set at 6% based on control data. Of twenty-one patients with > 6% monosomy 7 cells, response to IST was observed in 12 (57%), while 6 of 19 (84%) with normal FISH responded to IST (p=0.08). Thirteen patients had monosomy 7 frequencies of >10%. Of these 5 (38%) responded to IST, compared with 23/27 (85%) with ≤10% monosomy 7. The proportion of monosomy 7 cells in the bone marrow correlated with age (R2 =0.6, p<0.001). Two of the 23 non-responding patients later developed clonal progression with monosomy 7 or 7q-; both had >10% monosomy 7 by FISH at presentation, but normal cytogenetics. The two responding patients tested with >6% monosomy 7 prior to IST demonstrated <2% following successful IST, suggesting that the high endogenous levels of G-CSF present before IST response may have facilitated expansion of the clone. Significant monosmy 7 clones may reflect a stem cell disorder in patients not responding to IST.