bluuebunny/crossref_metadata_2025_split huggingface dataset:
High Resolution Amplicon Melting Curve Analysis: A Sensitive and Rapid Method To Detect the Janus Kinase 2 (JAK2) Gene Mutation.
Authors: Park Sunita I. , Winton Elliot F. , Mann Karen P. , Hill Charles E. 
Published: April 2020

Abstract
Chronic myeloproliferative disorders (CMPDs) are clonal hematopoetic stem cell disorders resulting in the increased production of cells of the myeloid lineage. The recently described G to T point mutation at nucleotide 1849 in the JAK2 gene may be a specific molecular marker to aid in the diagnosis of CMPDs, including polycythemia vera (PV), essential thrombocythemia (ET), and chronic idiopathic myelofibrosis (CIMF). Previously described methods of detection for this point mutation include sequencing, allele-specific PCR, and restriction digestion. We report here the development of a novel method to detect the JAK2 point mutation using high resolution amplicon melting curve analysis. Genomic DNA was obtained from peripheral blood samples from patients with PV, ET, CIMF, and suspected CMPD using the Qiagen DNA Blood kit (Valencia, CA). DNA was quantified using PicoGreen (Invitrogen) fluorescent dye followed by spectrophotometry and samples were normalized to a standard concentration. Real time PCR was performed on the Roche Light Cycler in the presence of the double stranded DNA binding dye LCGreen1 using primers that flank the point mutation. High Resolution Melting curve analysis was then performed on the HR-1 instrument (Idaho Technologies), and mutation status assigned by comparing the melting curve position and shape to controls of known mutation status. For comparison, the samples were also restriction digested using BsaX I and analyzed by agarose gel electrophoresis. Mutation status was confirmed by sequencing of the PCR amplicons (Lark Technologies, Houston, TX) for a subset of specimens. Twenty six patients with known CMPD as defined by the WHO criteria (2001) were included in the study. In addition, seven patients with suspected CMPD were studied. The point mutation in JAK2 was identified in 9 (100%) of 9 PV patients, 7 (58%) of 12 ET patients, 1 (50%) of 2 CIMF patients, 3 (100%) of 3 CMPD NOS patients, and 2 (29%) of 7 patients with suspected CMPD. Patients testing homozygous for the JAK2 mutation included 6 patients with PV, 1 patient with ET, and 1 patient with CMPD, NOS; all other positive patients were heterozygous for the mutation. We observed excellent correlation between the melting curve analysis and restriction digest results. High resolution melting curve analysis is a rapid, sensitive, low reagent cost method to assess for the JAK2 point mutation in cases of suspected or known CMPDs. The advantages of this technique include the ability to discriminate heterozygous from homozygous mutations, increased sensitivity as compared to sequencing, rapid turn around time, no post-amplification modification, and the ability to screen for other mutations or polymorphisms within exon 12 of the JAK2 gene.
Differences between Peripheral Blood, Lymph Nodes and Bone Marrow in Expression of Integrins, ICAMs, CD38 and CD31 Correlate with Clinical Characteristics in B-CLL.
Authors: Jaksic Branimir , Jaksic Ozren , Kardum-Paro Mirjana M. , Kardum-Skelin Ika 
Published: April 2020

Abstract
B-CLL patients have variable tumor mass distribution within major cell compartments, and as a consequence different clinical presentation. Adhesion molecules (AM) expression phenotype of integrins (CD11a, CD11b, CD11c, CD18, CD29, CD49c, CD49d and CD61), ICAMs (CD54, CD102 and CD50), CD38 and CD31 was determined in samples taken from peripheral blood (PB), bone marrow (BM) and lymph nodes (LN) by two/three color flow cytometry in 33 B-CLL patients. Differences for particular AM expression between compartments were quantified to express respective intensity and direction (gradients). We found following significant differences: stronger expression for CD11a and CD102 in PB than in BM, while CD54 was stronger in BM than in PB; CD11b and CD102 were stronger in PB than in LN, opposite to CD54 and CD38; CD102 was stronger in BM than in LN, while CD18, CD11a, CD11c and CD38 were stronger in LN then in BM. Observed gradients were compared with clinical and laboratory parameters: PB lymphocytosis, LN size, Spleen size, Total Tumor Mass score (TTM), TTM distribution (TD), BM failure (BMF), Rai and Binet stages. Peripheral blood lymphocytosis positively correlated with LN to PB gradient for CD11a, CD11b, CD18 and CD31 (p&lt;0.05). Lymph node size negatively correlated with LN to PB gradient for CD11b and with BM to PB gradient for CD54 and CD61 (p&lt;0.05). Spleen size negatively correlated with BM to PB gradient for CD11c and CD102 (p&lt;0.05). TTM negatively correlated with BM to PB gradient for CD11c, CD61 and CD102 (p&lt;0.05). TD positively correlated with LN to PB gradient for CD11b and CD102, and with BM to PB gradient for CD11c and CD102 (p&lt;0.05). Isolated BMF irrespective of tumor load, as well as clinical stages that incorporate BMF (Rai and Binet) were associated with significant PB to BM gradient for CD18, CD11a, CD11c, CD49d, CD54 and CD102, and with PB to LN gradient for CD102 (p&lt;0.05). Cluster analysis corroborated these findings. This study shows that expression of selected integrins, ICAMs, CD38 and CD31on B-CLL cells is significantly different among lymphoid compartments suggesting possible role in resulting clinical presentation. Taking together, this investigation disclosed yet unexplained interesting interactions and warrants further studies of AM role in B-CLL.
X-Linked Gray Platelet Syndrome Due to a GATA1 Arg216Gln Mutation.
Authors: Tubman Venée N. , Levine Jason E. , Campagna Dean R. , Fleming Mark D. , Neufeld Ellis J. 
Published: April 2020

Abstract
Gray Platelet Syndrome (GPS) is characterized by a variable, mild bleeding diathesis, associated with thrombocytopenia and large, agranular, functionally abnormal platelets. While the disorder has been well-described both biochemically and pathologically, to our knowledge, no genetic mutation has been associated with the disease. Most cases are sporadic, with a few sibships and apparent autosomal kindreds reported. In our investigation, the proband is a healthy 1 year-old female whose father and uncle had recently been diagnosed with GPS. The child had a platelet count of 382 x103 platelets/μl, comprised of a dimorphic population of normal and large, agranular forms. Her CBC and peripheral blood smear were otherwise unremarkable. We subsequently analyzed four generations of this family. In addition to the proband’s father and uncle, several other members had known bleeding tendencies. The symptomatic individuals were invariably males, consistent with a sex-linked pattern of inheritance. Several symptomatic and asymptomatic family members were evaluated by CBC and blood smear. Large, agranular platelets were found in symptomatic males, while obligate carrier females exhibited both normal and abnormal forms. Using a set of fifteen microsatellite markers spanning the X chromosome, a common haplotype was identified in all affected men, their mothers, and their daughters. Aided by the published sequence of the X chromosome, we examined this region for candidate genes. Although the common haplotype between markers GATA144D04 and DXS6797 (Xp11.3-Xq22.3) contains hundreds of genes, only two, GATA1 and WAS, have known associations with thrombocytopenia. PCR amplification and sequencing of a 3′ segment of the GATA-1 promoter and the five coding exons of the GATA-1 gene revealed an G759A missense mutation resulting in an Arg216Gln substitution in exon 4 (NCBI RefSeq: NM_002049) that segregated with the phenotype and was present in all obligate carrier females. This mutation has been previously associated with X-linked thrombocytopenia and beta-thalassemia (XLTT), a syndrome characterized by splenomegaly, thrombocytopenia, and imbalanced globin chain synthesis (Balduini et al, Thromb Haemost. 2004; 91:129). Comparison of the ultrastructural characteristics of the platelets in both disorders and a review of literature on both diseases suggests that XLTT, and the associated mutation in GATA1, could represent one genetic origin for GPS. As carrier females generally display a less severe phenotype than affected males, without thrombocytopenia and with subtle dimorphism on smears, it is possible that this mutation may account for some previously reported “sporadic” cases of GPS.
Combined Therapy with Alemtuzumab and Pentostatin Is Feasible and Effective in T- Lymphoprolifertive Disorders.
Authors: Ravandi-Kashani Farhad , Kantarjian Hagop , Verstovsek Srdan , Ferrajoli Alessandra , Wierda William , Odinga Shirley , Huang Xueling , Pierce Sherry , Faderl Stefan , Giles Francis , O’Brien Susan , Keating Michael 
Published: April 2020

Abstract
Mature T-cell lymphoid neoplasms are clonal proliferation of post-thymic T-cells, which are generally refractory to traditional chemotherapy regimens. The prognosis for most patients with these disorders, particularly those with relapsed disease, is poor. New treatment strategies are urgently needed. Alemtuzumab and pentostatin have been used as single agents in various T-cell neoplasms with response rates of up to 75% and 50%, respectively. Immunosuppression and opportunistic infections are the principal overlapping toxicities. We have treated 9 patients with T-lymphoid malignancies (5 T-PLL, 1 ATL, 1 γδ-T-cell hepatosplenic lymphoma, 2 T-LGL) on a phase II study of alemtuzumab 30 mg IV three times weekly for up to 3 months with concomitant pentostatin 4 mg/m2 weekly x 4 followed by alternate weekly administration for up to 6 months. Prophylactic antibiotics including valcyclovir, trimethoprim/sulfamethoxazole, and fluconazole were administered during therapy and for 2 months after completion. Median age of patients was 57 years (range 25–80 years), median WBC was 78.5 x 109/L (range 0.7 – 279.5 x109/L), median serum β2M was 4.1 mg/L (2.7 – 10.8 mg/L). Six patients had splenomegaly (1 – 6 cm), and 3 lymphadenoapthy. Six had received prior therapy (median 5, range 1 to 6 regimens). To date, 6 of 8 evaluable patients have responded (5 CR, 1PR). One patient who had failed both single agent pentostatin and single agent alemtuzumab achieved CR with the combination. Monoclonal T-cell receptor gamma chain gene rearrangements were detected by PCR in all patients and became negative in 3 of 5 evaluable responding patients. Median response duration is 5+ months (range 2 – 6+ months). Two patients proceeded to an allogeneic stem cell transplant, 2 (1 with ATL and 1 with T-LGL) have died from disease progression after a response, 2 are alive and disease-free, and 2 were refractory to therapy. Opportunistic infections have included reactivation of CMV and HSV in one patient, recurrence of pre-existing Serratia pneumonia in 1 patient and Aspergillus pneumonia in 1 patient. We conclude that the combination of alemtuzumab and pentostatin is effective in T-cell neoplasms; infections can be prevented with prophylactic antibiotic therapy and close monitoring of patients.
The Treatment of Complicated and High Risk Chronic Lymphocytic Leukemia (CLL) with Fludarabine, Cyclophosphamide and Rituximab (FCR).
Authors: Donthireddy Vijaya , Shurafa Muhammad S. , Saleh Murad , Kamboj Ginny , Wang Ding , Janakiraman Nalini 
Published: April 2020

Abstract
Between November 2000 and November 2004, we treated 11 patients with the FCR regimen for complicated CLL. The median age was 64 years (51–80 years) and the median number of prior regimens received was 1(0–4). Median number of FCR cycles delivered was 4 (2–6). Four patients were treated due to active autoimmune hemolytic anemia (AIHA), three patients due to massive lymphadenopathy, two patients for short lymphocyte doubling time, one patient for active AIHA and immune thrombocytopenia and one patient for progressive splenomegaly. Two patients received only 2 cycles of FCR due to prolonged pancytopenia secondary to hypoplastic bone marrow. Four of the patients with AIHA had prior splenectomies. Overall response rate, according to NCIWG criteria, was 100% including the patients who had received only 2 cycles. One patient achieved a complete remission, seven patients achieved nodular partial remissions and three achieved partial remissions. Among the 7 patients with nodular PR’s, 4 had no evidence of residual CLL by flow cytometry and are continuing to be in remission at a median follow up of 53 months. The other 3 patients with nPR who had evidence of disease by flow cytometry relapsed at a median of 23 months (16–33 months). The patient who had a CR by NCIWG criteria, still had evidence of disease by flow cytometry and relapsed at 35 months. All patients with AIHA and immune thrombocytopenia achieved a remission of their autoimmune processes, including those who failed splenectomy. Grade 3–4 neutropenia, thrombocytopenia and anemia occurred in 8, 2 and 2 patients respectively. The median progression free interval was 33 months (9+ to 54 m) and the overall survival was 56 months (9+ to 56m). Four patients had expired at the time of this report due to progression of CLL(1), sepsis while still in remission(1), high grade transformation and subsequent sepsis(1) and complications of allogeneic transplant(1). Cytogenetic remissions were achieved in 7/9 patients. Three of them continue to be in remission at the time of this report with a median follow up of 53 months since initiation of therapy.
Conclusion: FCR is an effective treatment regimen for CLL associated AIHA, even after failing splenectomy and can result in high response rates and prolonged remissions. Flow cytometry remission seems to correlate with prolonged disease free survival in patients achieving nodular partial remissions according to the NCIWG criteria. We suggest treatment with FCR until elimination of minimal residual disease by flow cytometry irrespective of histopathological remission.
Induction of Chronic Lymphocytic Leukemia (CLL) Apoptosis by Nitric Oxide Synthase (NOS) Inhibitors: Drug Efficacy Correlates with Lipid Solubility and NOS1 Dissociation Constant.
Authors: Levesque Marc C. , Ghosh Dipak K. , Beasley Bethany E. , Chen Youwei , Volkheimer Alicia D. , O’Loughlin Charles W. , Gockerman Jon P. , Moore Joseph O. , Weinberg J. Brice 
Published: April 2020

Abstract
The viability of CLL cells may be dependent on the autocrine production of nitric oxide because nitric oxide synthase (NOS) inhibitors induce CLL cell apoptosis and CLL cells express inducible NOS (NOS2). Our previous study indicated that the non-specific NOS inhibitor NMMA induced CLL cell apoptosis but only at high concentrations (&gt; 1 mM) (Levesque et al., Leukemia17:442, 2003). Therefore, we performed the current study to identify NOS inhibitors that induce CLL cell apoptosis at lower concentrations and to understand factors that promote NOS inhibitor-induced CLL cell toxicity. We isolated and enriched CLL cells from the blood of CLL patients and cultured the CLL cells in media containing various concentrations of 21 different NOS inhibitors. We determined CLL cell viability following culture with each NOS inhibitor. We found that NOS inhibitors with specificity for neuronal NOS (NOS1) induced CLL cell death at concentrations lower than non-specific NOS inhibitors and lower than inducible NOS (NOS2) specific inhibitors. There was a weak correlation (r2 = 0.29, p = 0.1608) of the NOS1 (but not NOS2) half-maximal inhibitory concentration (IC50) of each NOS inhibitor for purified recombinant NOS and its ability to induce CLL cell death. We confirmed the specificity of the NOS inhibitors by inhibition of purified recombinant NOS1 and NOS2 enzyme activity, and we confirmed that NOS1 specific inhibitors induced CLL cell death by apoptosis. Because there was only a weak correlation of the NOS1 IC50 with NOS inhibitor induced CLL cell death, we considered whether other factors such as the Kd and hydrophobicity of each compound correlated with CLL cell death. We found that there was a direct correlation between the NOS1 (but not NOS2) dissociation constant (Kd) of NOS inhibitors and CLL cell death (r2 = 0.77, p = 0.0041) and a direct correlation of the partitioning coefficient (a measure of hydrophobicity) of each NOS inhibitor and its ability to induce CLL cell death (r2 = 0.68, p &lt; 0.0001). Therefore, NOS inhibitors that bound tightly to NOS1 and were hydrophobic induced CLL cell death at lower concentrations. There was variable expression of CLL cell NOS1 mRNA (6 of 28 samples positive) and we were unable to demonstrate CLL cell expression of NOS1 protein by immunoblotting. This suggests that if NOS1 is present in CLL cells, it exists at very low levels. Taken together, we believe that low level NO production promotes CLL cell viability and that inhibition of CLL NOS induces CLL cell apoptosis. Importantly, our studies provide direction for the rational design and selection of NOS inhibitors that may be useful as CLL therapeutics.
Fludarabine and Alemtuzumab Versus Fludarabine and Rituximab in the Treatment of Relapsed B-Cell Chronic Lymphocytic Leukemia: Results from a Phase II Study.
Authors: Gribben John , Stephans Katherine , Marshal Blossom 
Published: April 2020

Abstract
Although single-agent fludarabine is associated with high response rates (60%–80%) in patients with previously untreated B-cell chronic lymphocytic leukemia (B-CLL), this therapy is not curative and patients will relapse from persistence of minimal residual disease. Response rates to subsequent lines of therapy drop dramatically, as does survival. Antibodies including alemtuzumab and rituximab act in synergy with fludarabine and improve responses in salvage CLL therapy. In an effort to identify an effective chemoimmunotherapy regimen for patients with relapsed CLL, a phase II, multicenter, open-label, randomized trial was initiated. B-CLL patients who had failed prior therapy were randomized to treatment with either fludarabine combined with alemtuzumab or fludarabine combined with rituximab. Four patients randomized to the cohort received fludarabine 25 mg/m2 IV and alemtuzumab 30 mg SC, on Days 1–5. Eight patients received fludarabine 25 mg/m2 IV on Days 1–5, and rituximab 375 mg/m2 IV on Days 1 and 4 of the first cycle, followed by fludarabine 25 mg/m2 IV on Days 1–5, and rituximab 375 mg/m2 IV on Day 1 in subsequent cycles. Patients were assessed monthly for response while on therapy, and interim restaging occurred at cycle 4. Those who achieved a CR received no further therapy, whereas those who achieved a PR or SD received 2 additional cycles. 12 patients (7 male and 5 female) participated in this trial and the median age was 67 years. Nine patients had Rai III/IV (2 patients in alemtuzumab arm and 7 patients in rituximab arm). All patients had failed 1 course of therapy; 9 had failed treatment with a fludarabine-based regimen, and 3 had failed treatment with alkylating agents. In the alemtuzumab arm, 2 patients developed a CMV reactivation, one of whom developed CMV viremia, which was successfully treated with gancyclovir. Of the 8 patients in the rituximab plus fludarabine arm, 6 withdrew due to adverse events and 2 patients died while on study. In the alemtuzumab plus fludarabine arm, 1 person withdrew due to adverse events and 1 patient died after the trial was closed. Overall, 3 of 4 patients in the alemtuzumab arm responded (2 complete response [CR], 1 partial response [PR]), and 3 of 7 patients in the rituximab arm (1 CR, 2 PR). Recently published data has prompted a shift in CLL therapy. Increased numbers of patients are receiving chemoimmunotherapy combinations earlier in treatment. An increased use of FR and FCR in the first-line setting made further recruitment difficult. The potential of randomizing patients to the FR arm of the study prompted low accrual and the subsequent closing of the study.
A Novel Anti CD23 Fully Human Monoclonal Antibody Potentially Useful for B-CLL Therapy.
Authors: Delcommenne Marc , Klingemann Hans-Georg , Gregory Stephanie A. 
Published: April 2020

Abstract
B-cell chronic lymphocytic leukemia (B-CLL) is one of the most common hematological malignancies and is, in most cases, characterized by an increased expression of CD23 on the cell surface. Since cross-linking CD23 induces B-CLL apoptosis, it is an attractive target for B-CLL antibody-based immunotherapy. In this study we show that an anti-CD23 human IgG1 monoclonal antibody, C6F5, may be useful in treating B-CLL. This antibody is derived from the human single chain antibody (scFv) C6F5 that was originally raised against the RPMI-8226 multiple myeloma cell line using the antibody phage display technique. While the C6F5 scFv did not bind to other myeloma cell lines, it was able to bind weakly to normal peripheral blood B lymphocytes and strongly to EBV transformed B cells and B-CLL cells. The antigen recognized by C6F5 was also upregulated on B lymphocytes that had been stimulated by CD40 ligand. Immunoprecipitations by the scFv C6F5 identified a protein of 45 kDa which co-migrated with CD23. Furthermore, this protein was recognized by an anti-CD23 mouse mAb in Western blot analyses. Immunofluorescence staining with the C6F5 scFv was inhibited if cells were preincubated with an anti-CD23 polyclonal antiserum. Taken together, these results verify that C6F5 recognizes CD23. The VH and VL regions of C6F5 antibody were then cloned into a baculovirus transfer vector encoding the human IgG1 heavy and light chains so that fully human C6F5 IgG1 antibody could be produced in baculovirus infected SF9 cells. Since C6F5 binding specificity was preserved in the IgG1 format, this antibody is ready to be tested in in vitro cytotoxic assays against B-CLL cells.