bluuebunny/crossref_metadata_2025 huggingface dataset:
Absence of Cytogenetic Abnormalities of PRV-1, TPO and C-MPL Genes Detected by Fluorescence In Situ Hybridization in Essential Thrombocythemia.
Authors: Puigdecanet Eulalia , Espinet Blanca , Villa Olaya , Zamora Lurdes , Besses Carles , Bellosillo Beatriz , Salido Marta , Woessner Soledad , Serrano Sergi , Florensa Lourdes , Sole Francesc
Published: April 2020

Introduction. Essential thrombocythemia (ET) is a chronic myeloproliferative disorder (CMPD) with heterogeneous features and no specific diagnostic markers. Consequently, its diagnosis is based on exclusion of other CMPD and secondary thrombocytosis. The role of PRV-1 (Polycythemia rubra vera-1), TPO (Thrombopoietin) and c-Mpl (Myeloproliferative leukemia virus oncogene) in ET pathogenesis has been studied in order to find new molecular targets which would help in ET diagnosis. PRV-1 gene is overexpressed in granulocytes from polycythemia vera (PV) and in some ET patients. TPO serum levels are not diagnostically useful and c-Mpl expression in megakaryocytes and platelets are generally decreased in ET. Mutations in TPO and c-MPL genes have been detected in familial thrombocythemia, but not in patients with acquired ET. The aim of the present study was to analyse PRV-1, TPO and c-MPL genes status by fluorescence in situ hybridization (FISH) technique in order to find new molecular markers in ET patients.

Patients and Methods. Thirty bone marrow samples of ET patients (7M/23F) diagnozed by PVSG criteria with a normal karyotype and 10 bone marrow samples of normal healthy donors were included in the study. All samples were studied by three locus-specific probes for PRV-1, TPO and c-MPL genes as follows: 1. PRV-1 gene (BAC RP11-160A19, 157 Kb, located at 19q13.12-2) labeled in red cohybridized with the 19p telomeric probe (D19S238E, Vysis) labeled in green. 2. TPO gene (BAC RP11-45NP16, 183 Kb, located at 3q27) labeled in green cohybridized with the centromeric probe for chromosome 3 (D3Z1, Vysis) labeled in red. 3. c-MPL gene (BAC RP11-297L5, 190 Kb, located at 1p34) labeled in green cohybridized with the centromeric probe for chromosome 1 (D1Z5, Vysis) labeled in orange. A minimum of 100 interphase nuclei were analyzed.

Results. FISH study showed no PRV-1, TPO and c-MPL cytogenetic abnormalities in any of the analyzed cases, except for one patient in which 21% of interphase nuclei presented a trisomy for the TPO gene region. The monosomy and trisomy thresholds were 6.1% and 4.7% for PRV-1, 4.9% and 3.4% for TPO, and 5.4% and 3.71% for c-MPL, respectively.

Conclusions. Our results suggest a lack of structural and numerical rearrangements of PRV-1, TPO and c-MPL genes in ET patients. The PRV-1 gene FISH results are in line with the previously reported by Najfeld et al (Exp Hematol2003;31:118–21); regarding TPO and c-MPL results, this is the first FISH study reported in the literature in ET.
Dissecting Growth Factors Production in Patients with Agnogenic Myeloid Metaplasia (AMM).
Authors: Wang Jen-Chin , Hemavathy Kirugaval , Goldberg Amit , Chang Tsong S. , Novetsky Allan D. , Lipton Jeffrey
Published: April 2020

Growth factors including Transforming Growth Factor-β 1 ( TGF- β1), Platelet Derived Growth Factor (PDGF) and Fibroblast Growth Factor (FGF) have been implicated as responsible for bone marrow fibrosis in AMM. Although TGF-β1 and FGF have been demonstrated to be increased in blood hematopoietic progenitor cells, a direct measurement of the production of these growth factors by megakaryocytes has not been performed. The current study was devised to study the production of these growth factors production directly in megakaryocytes and monocytes of AMM patients and correlate these with the clinical features. Twelve patients with AMM and 11 normal healthy volunteers used as controls, were studied. CD 34 + cells and CD 14+ cells were obtained from blood mononuclear cells by MACS Progenitor cell isolation kits ( Miltenyi Biotec, CA). The megakaryocytic (CD41+) cells were then obtained by growing the isolated blood CD34 + for 10 days in Iscove Modified Dulbecco Medium with Stem Cell Growth Factor and Thrombopoietin. The mRNA levels of TGF-β1, FGF and PDGF in the isolated megakaryocytes and blood monocytes were assayed by Real-Time RT-PCR. The results were as shown in Table 1.

Among the AMM patients, a patient with prior history of Polycythemia Vera (PV) and a patient with Essential Throbocythemia (ET) were found to have elevated PDGF and FGF expression in their monocytes but the expression of growth factors was not elevated in their megakaryocytes.. These results demonstrate that megakaryocytes are the main source of growth factors responsible for marrow fibrosis. The study also suggests that growth factors produced by monocytes may be responsible for fibrosis in AMM patients with a prior history of PV or ET.

Table-1

Megakaryocytes Monocytes * Denotes number of patients with elevated growth factor levels as compared with controls. ** Denotes numbers of patients tested. TGF- β 1 8*/10* 0/10 PDGF 8/10 3/7 FGF 6/10 2/7
Enumeration and Immunologic Characterization of Basophils in Normal Bone Marrow and Patients with Myeloproliferative Disorders.
Authors: Agis Hermine , Krauth Maria T. , Muellauer Leonhard , Schwartz Lawrence B. , Horny Hans P. , Valent Peter
Published: April 2020

Basophils are highly specialized granulocytes that express a unique profile of antigens and increase in myeloproliferative disorders. In chronic myeloid leukemia (CML), basophilia is an independent prognostic variable. So far, however, no reliable immunohistochemical approach for routine-detection and enumeration of bone marrow (bm) basophils has become available. To overcome this disadvantage, we have applied the anti-basophil antibody 2D7 on formalin-fixed, paraffin-embedded sections of normal bm and bm from patients (pts) with chronic myeloid leukemia (CML; chronic phase, n=21; accelerated phase, n=9), other myeloproliferative disorders (idiopathic myelofibrosis [IMF], n=3; polycythemia vera [PV], n=7; essential thrombocythemia [ET], n=7), and normal / reactive bm (n=32). As assessed by serial section-staining of bm specimens, the 2D7 antibody was found to be a basophil-specific immunohistochemical reagent. In serial bm sections, 2D7+ basophils co-expressed histidine decarboxylase, CD15, and CD43, but did not express B- or T-cell restricted antigens corresponding to the phenotype of normal blood basophils. Bm basophils were found to increase in number in pts with CML and other myeloproliferative disorders compared to normal bm (median 2D7+ cells/mm2 bm: normal bm: 7; CML: 46; IMF: 26; PV: 21; ET: 21, p<.05). The highest numbers of bm basophils were recorded in pts with accelerated phase CML (111 2D7+ cells/mm2). Together, we have established a useful immunohistochemical staining procedure for basophil detection in normal bm and pts with myeloid neoplasms. This approach should enable the quantification of basophils in these pts and the monitoring of bm basophil counts during follow up examinations and anti-leukemic therapies.
Monocyte Activation Studies in Patients with Essential Thrombocythemia.
Authors: Goette Nora P. , Marta Rosana F. , Chazarreta Daniel C. , Molinas Felisa C.
Published: April 2020

Essential thrombocythemia (ET), polycythemia vera and mielofibrosis with mieloid metaplasia (MMM) are myeloproliferative diseases that differ in their potential for developing bone marrow fibrosis. In addition to the role of megakaryocytes, the participation of monocytes in collagen deposits and fibroblast proliferation in the pathophysiology of MMM has already been described. We studied here monocyte activation in ET patients by measuring IL-2 receptor expression (CD25) in ex vivo monocytes and after its adhesion to polyestirene. Besides, we evaluated IL-1-b released by mononuclear cells after 24 hs culture. We evaluated 14 untreated ET patients. Blood samples were obtained under sterility conditions and mononuclear cells were separated by Ficoll-Hypaque gradient. CD25 positivity was evaluated by flow cytometry identifying monocytes as the CD14 + population before and after 18 hs cell culture in IMDM and 2% albumin. IL-1b was measured by ELISA technique in the supernatant of cells cultured in IMDM supplemented with 10% FCS during 18 hs. There were no statistical differences in CD25 expression in ET patients (n=8) neither before (3.1%, 1–11.4%) (median and range) nor after (15.2%, 4.7–39.3%) cell culture of CD14 population compared to normal controls (n=12, 2.0%, 0.2–10.3% and 20.3%, 1.4–34.3%, respectively). Levels of IL-1b were found decreased in patients with ET (n=10), 481 pg/ml, 53–1290 pg/ml, compared to normal controls (n=11), 779 pg/ml, 68.7–2166 pg/ml, although not reaching statistical difference (p=0.057). In conclusion, we did not find monocyte activation in ET patients as demonstrated by CD25 expression and IL-1b release. These results suggest that there is not a constitutive or induced activation of monocytes in ET but a trend towards a decreased capacity of activation. We believe this different monocyte behaviour from ET patients compared to that described in MMM patients could account for the dissimilar natural course of these diseases.
Demonstration of Biclonal Chronic Lymphocytic Leukemia with Mutated and Unmutated Clones and Concurrent but Clonally Unrelated Myeloma in the Same Patient by Single Cell Immunoglobulin Heavy and Light Chain Gene Analysis.
Authors: Yavuz Selim A. , Hartmann Dan-Paul , Baidas Said , Lipsky Peter E. , Ozdemirli Metin
Published: April 2020

Patients with chronic lymphocytic leukemia (CLL) may develop other B cell malignancies in their clinical course including aggressive diffuse large B-cell lymphomas and rarely myelomas. In a large proportion of cases, the secondary B cell malignancies reflected the emergence of immunophenotypically and genetically different clones. An immature type plasma cell myeloma developed in a 73-year-old female patient in whom CLL was diagnosed four years previously. The CLL expressed CD5, CD19, CD23, CD38 and surface kappa light chain, but were negative for ZAP-70. Trisomy 12 was detected by FISH analysis. PCR analysis of the peripheral blood for immunoglobulin heavy chain genes demonstrated two sharp bands that were initially interpreted as biallelic heavy chain gene rearrangements. Myeloma cells were CD38 and CD138 positive, CD19 negative and expressed cytoplasmic kappa light chain, but not heavy chains. In order to investigate the clonal relationship between these B cell malignancies, a detailed analysis of VHDJH and VκJκ gene rearrangements in individually sorted CD5 and CD19 double-positive CLL cells and also in CD38-positive and CD19-negative myeloma cells by single cell PCR of genomic DNA and direct sequencing was carried out. This technique permitted identification and pairing of both the heavy and light chain immunoglobulin genes from the same individually sorted cell. A total of 17 individual CLL and 23 myeloma cells were successfully analyzed. Our analysis demonstrated (a) the presence of two discrete clones of CLL, one with usage of [VH1-2*04/D3-3*01/J3*02]-[Vκ2-28*01/J1*01] without VH and Vκ hypermutation and the other with usage of [VH1-2*04/D4-11*01/J6*02]-[Vκ1-5*03/J1*01] with VH and Vκhypermutation; (b) no clonal relationship between the CLL and myeloma cells that utilized different VHDJH and VκJκ rearrangements [VH3-66*02/3-10*01/J4*03]-[Vκ1-33*01/J2*02] with VH and Vκ hypermutation. To our knowledge, this is the first demonstration of a biclonal CLL with mutated and unmutated clones in the same patient along with a third clonally unrelated B cell malignancy. This result suggests that single cell analysis may be necessary to detect subtle biclonality of CLL that might be associated with a more aggressive phenotype.
Activation and Proliferation of B Cell Chronic Lymphocytic Leukaemia (B-CLL) Cells through Anti-CD180, Anti-CD40 and IL-4.
Authors: Porakishvili Nino , Manoussaka Maria , Kulikova Nino , Walton James , Nathwani Amit , Jewell Andrew P. , Stevenson Freda , Clark Ed , Lydyard Peter M.
Published: April 2020

Introduction: We have previously shown that Toll-like receptor RP105 (CD180) is heterogeneously expressed on B-CLL cells and that the ligation of CD180 by monoclonal antibodies (mAb) on CD180+ B-CLL cells resulted in delineation of responder and non-responder B-CLL clones [1]. In this study we have examined the role of IL-4 together with CD180 and CD40 as activation signals.

Methods: Blood mononuclear cells were separated from 7 responder B-CLL patients with both mutated and unmutated Ig Vh genes and 11 controls and were cultured for 72 hours in optimum concentrations of anti-CD180 (G28-8) or anti-CD40 mAb or both in presence and absence of 15 ng/ml of IL-4. CD19+ B cells were stained with mAb to the activation marker CD86 or cell cycle protein Ki-67, measured by flow cytometry and expressed as Mean Fluorescence Intensity (MFI) or % of Ki-67+ cells.

Results: B-CLL cells and normal control B cells responded to CD180-ligation by activation and proliferation (Table). Higher levels of CD86 and Ki67 were detected when both anti CD40 mAb and anti CD180 mAb were added (p<0.05) compared with either alone. IL4 alone induced both activation and proliferation of control cells and this was even higher with the leukemic cells (p<0.01) confirming that IL-4 also provides a strong survival/activatory stimulus for B-CLL cells. Addition of IL-4 had no significant enhancing effect on normal B-cells stimulated with both anti-CD180 and anti-CD40, although IL-4 synergised with anti-CD40 in B cell activation (p=0.026) and with CD180 in B cell proliferation (p=0.044).

Conclusion: CD180 had an additive effect with CD40 ligation in activation and proliferation of both B-CLL cells and normal control B cells. IL-4 provides a strong additional stimulus for B-CLL cells.

CD86 and Ki-67 expression by CD19+ cells

CD86 Ki67 B-CLL Control B-CLL control Spontaneous −IL-4 7.2±4.1 4.0±1.0 8.1±2.7 17.4±2.3 +IL-4 22.4±11.8 11.2±4.2 17.0±12.8 23.6±14.8 CD180 −IL-4 14.7±5.8 26.9±13.0 17.1±10.9 28.1±12.0 +IL-4 35.4±14.5 30.0±14.6 26.9±25.6 48.3±9.7 CD40 −IL-4 19.4±8.8 14.8±5.2 14.9±9.7 34.0±5.1 +IL-4 286±145.5 44.0±19.4 52.7±22.8 41.0±19.8 CD180+CD40 −IL-4 32.5±12.6 97.0±26.2 28.0±10.4 65.5±26.6 +IL-4 299±163.2 63.5±27.4 49.3±14.6 55.5±26.4
CD38 Expression Has an Important Value among Prognostic Factors in Chronic Lymphocytic Leukemia (CLL): A Multivariate Analysis in 160 Patients.
Authors: Pavlovsky Carolina , Pavlovsky Miguel A. , Pardo Maria L. , Sapia Sandra , Mountford Pablo , Fernandez Isolda I. , Corrado Claudia S. , Statistics Mariana Juni , Pavlovsky Santiago
Published: April 2020

Objetives: Analyze the prognostic significance of CD38 expression compared to other prognostic factors.

Patients and methods: We studied retrospectively and prospectively at our institution 160 patients (pts) with diagnostic of CLL who met the diagnostic criteria of National Cancer Institute-Working Group (NCI-WG), 97 men and 63 female with an age range of 33 – 90 years (median 62) diagnosed between January 1989 and December 2003 and evaluated up to June 2004. Sixty nine pts showed CD38 positive cells and 91 were CD38 negative. CD38 expression was determined by flow cytometry defining as a higher expression > 7% CD38 positive cells. (Krober A et al. Blood15: 1410–1416.2002). Event Free Survival (EFS) and Overall Survival (OS) were analized according to different characteristics at diagnosis.

No. Patients %EFS 72 mo p= %OS 72 mo p= Binet Stage A /B-C 129/31 65/30 0.000 90/92 0.245 Rai Stage 0 /I – II – III – IV 92/68 72/37 0.000 94/94 0.339 TDLI < / ≥ 12 mo 23/126 41/63 0.007 65/100 0.001 Light Chains kappa / lambda 94/66 68/50 0.473 90/95 0.702 CD38 Positive / Negative 69/91 32/82 0.000 81/100 0.000

Statistical significant difference was observed in Binet stage, Rai stage, TDLI and CD38 expression for EFS and in TDLI and CD38 expression for OS. Considering these variables with p value of <0,05, a multivariable analysis was performed (Cox) in order to asses the adverse effect of each dichotomize group in EFS.

Beta S.E. H.R. 95% C.I. p CD38 Positive (1) /CD38 Negative(0) 1.70 0.32 5.5 2.89–10.53 0.000 Binet B-C /Binet A(0) (1) 1.46 0.29 4.33 2.42–7.7 0.000 RAI I-II-III-IV /RAI 0 (0) (1) 1.45 0.30 4.29 2.37–7.78 0.000 TDLI ≥ 12 (1) /TDLI<12 (0) 0.03 0.00 1.03 1.01–1.04 0.000

This analysis shows that the presence of CD38 positive cells at diagnosis conferred an adverse effect on EFS with a HR 5.5 (95% confidence interval 2.89–10.53) over the other variables included.

Conclusion:In our population, patients with CD38 positive expression showed a EFS and OS at 72 months of 32% and 82% ( p=0.000) versus 81% and 100% (p=0.000) in patients with CD38 negative expression. We can conlude that CD38 positive cells will identify a subgroup of patients with aggresive clinical outcome and worse prognosis.
ZAP-70 Versus CD38 Expression as the Prognostic Factors in B-Cell Chronic Lymphocytic Leukemia.
Authors: Hus Iwona , Bojarska-Junak Agnieszka , Podhorecka Monika , Wasik-Szczepanek Ewa , Sieklucka Malgorzata , Skorzynska Katarzyna , Rolinski Jacek , Dmoszynska Anna
Published: April 2020

B-CLL is a heterogeneous disease with a highly variable clinical course. The mutational status of immunoglobulin heavy-chain variable region (IgVH) genes in the leukemic cells seems to be of great importance in assessing the prognosis in B-CLL. Recent studies have shown that both CD38 and ZAP-70 might be surrogate markers for IgVH mutation identifying patients with a more aggressive clinical course. The aim of our study was to estimate the clinical value of both ZAP-70 and CD38 as B-CLL prognostic factors.

The analysis with use of flow cytometry technique was performed at diagnosis in the group of 100 B-CLL patients. 39/100 persons were defined as ZAP-70 positive and 61/100 persons as ZAP-70 negative (based on 20% cut off value in the CD19+/CD5+ population). ZAP-70+ patients had significantly higher lymphocytosis (p=0.003) and LDH level (p=0.009), while hemoglobin (p=0.049) and platelet levels (p=0.002) were significantly lower than in ZAP-70− patients. The proportion of CD19+/CD5+ cells expressing ZAP-70 correlated significantly with stage of disease (p=0.035). Patients with Rai stage III–IV (32/100 patients) had higher percentage of CD19+/CD5+/ZAP-70+ cells than patients with stage 0-II (68/100 patients) (24.81±17.40% and 14.89±13.54%, respectively, p=0.008). The mean treatment-free interval was longer in the ZAP-70 negative (8.46±4.63 months) than in the ZAP-70 positive patients (1.16±0.28 months). Furthermore, patients requiring treatment (57/100) had significantly higher percentage of CD19+/CD5+ cells expressing ZAP-70 protein than untreated patients (20.18±15.09 and 13.44±11.84, respectively, p=0.034). In the midst of patients requiring treatment, percentage of CD19+/CD5+/ZAP-70+ cells was significantly lower in patients achieving remission after first line therapy (26/57) than in patients with progressive disease (31/57) (p=0.004).

According to CD38 analysis the patients with ≥ 20% of CD38 expression were considered positive (CD38+) and those < 20% were considered negative (CD38−). Patients CD38 positive, like the ZAP-70 positive patients, had more advanced Rai stage of disease, significantly higher lymphocytosis and LDH serum level. Patients requiring treatment had higher percentage of CD19+/CD5+CD38+ cells than untreated patients, however no statistical significance was detected. Percentage of CD19+/CD5+CD38+ was not a dicriminating factor between patients with good response to the first line therapy and those with progressive disease.

The obtained results indicate that both ZAP-70 and CD38 expression may be prognostic markers identifying a subgroup of B-CLL patients with aggressive clinical presentation. However, the ZAP-70 expression seems to have more prognostic power, especially in predicting response to the first line therapy.
Increased Stability of Bcl-2 mRNA in B-Cell Chronic Lymphocytic Leukemia (CLL).
Authors: Kio Ebenezer A. , Pineda-Roman Mauricio , Otake Yoko , Stuart Robert K. , Fernandes Daniel J.
Published: April 2020

B-cell CLL is characterized by the accumulation of mononuclear B cells that are resistant to apoptosis as a result of bcl-2 oncogene overexpression. Nucleolin has recently been identified as a bcl-2 mRNA stabilizing protein that binds specifically to a 139 base AU-rich instability element (ARE) (Sengupta et al., J. Biol. Chem.279:10855–10863, 2004). Thus, studies were done to address the question whether the increased levels of bcl-2 mRNA in CLL are related to stabilization of bcl-2 mRNA by nucleolin. B cells were isolated from the blood of 9 patients with CLL and 5 normal volunteers by density gradient centrifugation followed by positive selection with CD19 immuno-magnetic microbeads. Flow cytometric analysis indicated that greater than 90% of the CLL and normal B cells were CD19 positive and CD3 negative. Western blotting revealed that cytoplasmic nucleolin and total cellular bcl-2 protein levels were elevated 18-fold (p<0.001) and 5-fold (p<0.001) respectively, in CLL compared to normal B cells. To directly examine the ability of nucleolin to stabilize bcl-2 mRNA, in vitro RNA decay assays were carried out using capped and polyadenylated bcl-2 mRNA transcripts. The average half-life of the bcl-2 mRNA transcript containing the ARE instability element was 12.5 min in S100 extracts of normal B cells, but was increased to 39.7 min in CLL cell extracts. Purified recombinant nucleolin (280 nM) stabilized bcl-2 mRNA when added to S100 extracts of normal B cells, but had no significant effect on bcl-2 mRNA stability in CLL cell extracts. The results are consistent with the hypothesis that resistance of CLL cells to apoptosis is related to overexpression of cytoplasmic nucleolin and increased stability of bcl-2 mRNA. These findings warrant further investigation of nucleolin as a potential therapeutic target in CLL.
Assessment of Nuclear Homogenicity in Chronic Lymphocytic Leukemia by Image Analysis.
Authors: Stein Gideon Y. , Djaldetti Meir , Zeidman Aliza , Merkel Drorit , Cohen Amos M.
Published: April 2020

Maturation of progenitor cells involves gradual chromatin condensation. We have shown that B-CLL cells represnet specific specrtral patterns by multipixel spectral imaging (J. Histocem. Cytohem. 46:113,1998). We further used image analysis techniques to quantify the alteration of chromatin homogenicity in lymphocytes from healthy subjects and B-CLL patients, as well as during maturation of myeloid and erythroid series, using light microscopic digital images of May-Grünwald Giemsa-stained (MGG) cells (B-CLL n=60, control n=22) and transmission electron microscopy (TEM). Nuclei were outlined by mouse cursor. Light microscopy images were converted to grayscale and designated as vector of the red, green and blue channels (sqrt(R*R+G*G+B*B)). Average color intensity (0–255), entropy (0–8), standard deviation, area diversity (0–100) and positive percent area (PPA) measurements were obtained from each nucleus. The entropy and area diversity of B-CLL lymphocyte nuclei was significantly lower than control lymphocytes (entropy 5.6±0.19 vs. 5.44±0.2, respectively, p<0.0001 and area diversity 33.2±4.3 vs. 30.5±2, respectively, p=0.006). In addition, we analyzed MGG-stained and TEM myeloid and erythroid cells at various stages of differentiation (n=155). Both the myeloid and erythroid cell lines showed gradual reduced color intensity, increased entropy and area diversity. Furthermore, MGG-stained megaloblastic proerythroblasts showed significantly higher levels of entropy and area diversity as compared with control proerythroblasts (n=73). Thus, assessment of nuclear entropy and area diversity may help to in the differentiation between normal lymphocytes and B-CLL cells.
R-Flurbiprofen, a Non-Steroidal Anti-Inflammatory Agent (NSAID) with Anti-Tumor Activity, Inhibits the Stromal-Induced Upregulation of Interleukin-8 in Chronic Lymphocytic Leukemia Cells.
Authors: Lilly Michael B. , Berdeja Jesus , Chen C.S. , Bashkyrova Svetlana , Zemskova Marina
Published: April 2020

NSAIDs have documented activity in the prevention or treatment of human epithelial cancers. Little information exists, however, to determine if similar anti-tumor effects can occur with hematologic malignancies. We have evaluated R-flurbiprofen (RFB), an enantiomer of an arylpropionic acid NSAID with known anti-tumor effects, as a potential treatment for CLL. PBMNCs from 7 CLL patients (WBC 30,000 –104,000/mcL; all CD5+/CD19+/CD38−) were exposed in short term culture to RFB. All samples showed caspase-dependent cell death following addition of RFB, with a median LD50 of approximately 90mcg/mL, a clinically achievable concentration. Addition of PGE2 or caffeic acid did not prevent RFB-induced cell death, demonstrating that the effect was not due to cyclooxygenase or lipooxygenase modulation. To identify molecular events resulting from RFB treatment, we performed microarray anaylsis (with Affymetrix U74v2A chips) on drug-treated CLL cells. PBMNCs were initially cultured in medium alone, or co-cultured with allogeneic primary marrow stromal cells (MSC). MSC co-culture increased or decreased (by >2-fold) approximately 13% of the 12,000 genes/probe sets interrogated by these microarrays (2 independent experiments), but had little effect on RFB cell kill. Approximately 35 genes (fusion of 2 independent experiments) were reproducibly up- or down-regulated by RFB in the non-adherent CLL cells cultured with MSC, but not in CLL cultured alone. Among the down-regulated genes were several chemokines and adhesion molecules. These included the gene for interleukin-8 (IL8). The expression of IL8 was markedly increased in CLL cells by co-cultivation with MSC. Furthermore, IL8 expression showed reproducible and dose-dependent down-regulation in CLL cells by RFB treatment. In contrast RFB did not affect expression of IL8 in CLL cells cultured without MSC. Intracellular IL8 was measured in CLL cells by flow cytometry; RFB markedly decreased expression of IL8 protein in addition to its effects on IL8 mRNA. IL8 has been identified as a growth/survival factor for CLL cells, and is a potent tumor-derived stimulator of angiogenesis. Furthermore, increased blood levels of IL8 have been identified as a poor prognostic sign in patients with CLL. RFB may modulate leukemia cell/MSC interactions by blocking the expression of chemokines and other pro-angiogenic mediators. RFB may play a supporting role as part of a multi-modality therapy regimen for CLL.
Deletion of 13q14 Is Independent of CD38 Expression in Chronic Lymphocytic Leukemia (CLL).
Authors: Mavromatis Blanche H. , Cheson Bruce D. , Ozdemirli Metin , Hartmann Dan-Paul , Abbasi Salah , Zafer Emre , Zantek Nicole , Cooksey Jessica , Meck Jeanne
Published: April 2020

Cytogenetic abnormalities detected by fluorescent in situ hybridization (FISH) can predict differences in survival in patients with CLL. Patients (pts) with the 13q14 deletion experience a longer median survival that those with the 17p13.1 deletion. Overexpression of ZAP-70 and CD38 correlate with a poor prognosis. Data on 44 pts currently enrolled on a prospective CLL prognostic study were analyzed for cytogenetic abnormalities by FISH and CD38 by flow. Pt age ranged from 25 to 85 (median 59). 25/44 patients (57%) were followed without any therapy. Zap-70 expression was measured by immunohistochemistry using a monoclonal antibody to ZAP-70 (clone 2F3.2).

Results: 22/44 (50%) pts were found to have abnormal cytogenetics. Of the 22 abnormalities, 17 had a 13q14 deletion (including one with an additional 11q22.31 deletion, and two with nullisomy 13q), five patients had trisomy 12, one patient a 17 p13.1 deletion. 8/44 patients overexpressed CD38. Of the pts with an isolated 13q deletion, 12/16 (75%) were CD38 negative and 10/17 (59%) were followed without any therapy, compared to 20/27 (74%) and 14/27 (52%) in those with other cytogenetic features respectively. Of those with other abnormal FISH findings, 4/5 pts (90%) with trisomy 12 were CD38 negative, a patient with 17p13.1 deletion and another patient with both 13q14 and 11q deletion were also CD38 positive. Of the patients with normal FISH results 19/21 (90%) were CD38 negative. ZAP-70 overexpression was noted in 22/26 pts with available data including 8/10 (80%) patients with and 13/26 (50%) patients without the 13q del.

Conclusion: The presence of the 13q14 deletion appeared to be independent of CD38 expression. Further studies to determine the association of 13q14 deletion with ZAP-70 expression are warranted, as is the correlation with clinical data.
ZAP-70 Expression in Chronic Lymphocytic Leukemia (CLL) Is Independent of Rai Stage: A Retrospective Review of 38 Patients (pts).
Authors: Mavromatis Blanche H. , Cheson Bruce D. , Zantek Nicole D. , Hartmann Dan-Paul , Kessler Craig , Cohen Philip , Gehan Edmund , Ozdemirli Metin
Published: April 2020

ZAP-70 is a tyrosine kinase protein involved in T cell signaling, overexpression of which is associated with a poor prognosis in CLL. Clinical information on 32 pts with CLL, and 6 pts with prolymphocytic transformation (PLT) followed at our institution was retrospectively reviewed for Rai stage, ZAP-70, CD38, FISH and response to treatment to determine the prognostic association among these factors. Ages ranged from 37 to 78 (median 59). 5/32 pts with CLL had received prior initial therapy, while 17/19 pts had not. They eventually were all treated with fludarabine based regimens either as salvage or upfront. The remaining 8/32 had not required therapy. Response to therapy was correlated with ZAP-70 expression as measured by immunohistochemistry using a monoclonal antibody to ZAP-70 (clone 2F3.2).17/32 patients (53%) were ZAP-70 positive, as were 3/6 (50%) of the patients with PLT. Of the patients with CLL who were ZAP-70 positive, 5 (30%) were stage 0-II, and 12 (70%) were stage III/IV, while18% did not require therapy. Of the 13 who were treated there were 4 (31%) CR, and 5 (38%) PR. Of the patients who were ZAP-70 negative, 50% (7/14) had stages 0-II and 50% had stage III/IV disease. 5/14(36%) were not treated. Of the 9 who were treated there were 2 CR 22%, and 7 PR 78% (p=0.01). 3 PLT patients were previously treated for CLL with fludarabine-based combinations. When they required therapy for PLT, two achieved a CR (one to rituximab/fludarabine/cytoxan_ZAP-70 positive patient, and one to R-CHOP combination_ZAP-70 negative), and another a PR (to R-EPOCH combination_ZAP-70 negative patient). Of the previously untreated PLT patients, two ZAP-70 positive patients achieved a CR (one to rituximab/fludarabine/cyclophosphamide, and one to R-CHOP). One ZAP-70 negative patient was refractory to fludarabine/rituximab combination.

Conclusion: ZAP-70 positive CLL is more likely to be refractory to treatment, while ZAP-70 negative pts achieved a higher ORR (p=0.01). Although pts who were ZAP-70 positive were more likely to have advanced stage, 30% of these pts had early stage disease. As such, ZAP-70 did not closely correlate with stage, and may be an independent prognostic factor in these pts. Response to treatment could also not be predicted by ZAP-70 expression.

ZAP-70 Number of Pts Previous Treatment Number of Pts Response Positive 16 No 10 3CR, 4PR Yes 3 1CR, 1PR Negative 14 No 7 2CR, 5PR Yes 2 2PR
Hepatitis B Virus Reactivation and Alemtuzumab Therapy.
Authors: Iannitto Emilio , Minardi Viviana , Calvaruso Giuseppina , Mulè Antonino , Ferraro Donatella , Ammatuna Emanuele , Craxì Antonio , Abbadessa Vincenzo , Di Stefano Rosellina
Published: April 2020

Reactivation of hepatitis B virus infection in subjects receiving cytotoxic treatment for haematological malignancies occurs in 21–53% of chronic HBsAg carriers and in an unknown number of HBsAg negative subjects harbouring occult HBV infection. Immunotherapy with alemtuzumab, a humanized monoclonal antibody against CD52 on lymphoid cells, produces deep immunosuppression. We describe two subjects with chronic lymphocytic leukaemia and occult HBV infection who developed a virological and biochemical flare of hepatitis B following immunotherapy with alemtuzumab. One of them developed a full blown hepatitis with seroreversion from anti-HBs to HBsAg after four weeks of alemtuzumab. Lamivudine (100 mg die) achieved a complete clinical recovery and HBV-DNA clearance from blood within 8 weeks. The second patient (HBsAg and HBV-DNA seronegative, anti-HBs and anti-HBc positive before treatment) was kept under prophylaxis with lamivudine up to three months after alemtuzumab. Two months after withdrawal of lamivudine, clinical and laboratory features of acute hepatitis B developed. Lamivudine therapy was restarted and obtained a prompt recovery with HBsAg and HBV-DNA clearance.

These cases suggest that alemtuzumab, as all other immune response modifiers, should be used cautiously when HBV infection is present. While a recommendation for universal pre-emptive treatment with lamivudine of all HBsAg positive patients undergoing effective immunosuppressive treatment can be made it is more difficult to establish a pattern for patients who have serological markers of previous HBV infection (anti-HBs and/or anti-HBc) and even more for those with no HBV markers. Testing for HBV-DNA in serum by highly sensitive PCR may discover up to 50% of these cases, allowing preemptive lamivudine therapy. A substantial proportion would however go undiscovered in the absence of a liver biopsy, which is clearly an unfeasible proposition as a screening procedure. Once a reactivation has been documented, nucleoside analog therapy should last for a few months after HBV-DNA clearance.
A Phase II Study of Denileukin Diftitox (ONTAKR) in Patients with Fludarabine Refractory B-Cell Chronic Lymphocytic Leukemia.
Authors: Kuriakose Philip , Turturro Francesco , Berdeja Jesus G. , Kerr Robert , Surendranathan Asha , Black Jennifer , Frankel Arthur E.
Published: April 2020

Therapeutic options for chronic lymphocytic leukemia (CLL) at relapse are limited because of myelosuppressive toxicity. Denileukin diftitox (ONTAK®, Ligand Pharmaceuticals) is a genetically engineered fusion protein comprising the enzymatically active domain of diphtheria toxin and the full length sequence of interleukin-2 (IL-2) targeting malignancies expressing the medium and high affinity IL-2 receptors. We designed a phase II study to evaluate the efficacy of ONTAK® in patients with fludarabine-refractory CLL, which is a follow-up to the previously published study (Frankel, et al, Clin. Cancer Res.2003; 9:3555). Denileukin diftitox was administered at a dose of 18μg/kg IV daily for 5 days every 3 weeks, for a maximum of 8 cycles. Thirteen patients have been treated so far, with 10 patients being evaluable for response (completed ≥ 3 cycles). Median age was 59 years (range 44–84), and 62% (8/13) were Rai stage III-IV, with a median of 3 prior therapies (range 1–6). The overall response was 40%, with 1 CR (10%, duration of response 5+ months) and 3 PR (30%, duration of response 3+, 3+ and 4+ months). Two responding patients (both PR) are still on study, while two (1 CR, 1 PR) were removed from study because of toxicities after 7 and 5 cycles, respectively. Four patients (40%) had progressive disease after cycles 3, 4, 4, and 7, respectively. One patient has completed four cycles and restaging studies are pending. Of the 3 patients not evaluable for response, two are still on study (having not completed 3 cycles), while one refused further treatment after 4 doses of cycle one. The grade 3/4 toxicities encountered were: neutropenia 4/13, thrombocytopenia 4/13, vascular leak syndrome 3/13, left ventricular cardiac dysfunction 1/13, hypotension 2/13, tachyarrhythmia 3/13, elevated PT 1/13, fatigue 1/13, rash 1/13, SIADH 1/13, constipation 1/13, vomiting 2/13, petechiae 1/13, transient elevation of GGT 1/13, transient elevation of AST/ALT 7/13, hyperglycemia 4/13, electrolyte imbalance 8/13, infection and/or febrile neutropenia 4/13, insomnia 1/13, visual disturbance 1/13, dyspnea 2/13, hypoxia 2/13. We conclude that denileukin diftitox has activity in CLL, with toxicities that can be managed with adequate premedication and close monitoring.
Anti-Tumour Effect of Zoledronate in Cells from B Chronic Lymphoytic Leukemia and Low-Grade Lymphoma Patients.
Authors: Vasconcellos Deborah Vidal , Silva Karina Lani , de Paula Castro Eric Delfraro , Moellman Arthur Coelho , Pombo-de-Oliveira Maria do Socorro , Maia Raquel Ciuvalschi
Published: April 2020

Recent studies demonstrate that bisphosphonates - anti-resorptive drugs - have direct anti-tumour effect in many tumour cell lines, including hematopoietic ones. The aim of this study was to evaluate the apoptotic effect of Zoledronate in cells from B chronic lymphocytic leukemia (B-CLL) and low-grade lymphoma (LGL) patients. Samples of 19 B-CLL or LGL patients were incubated with Zoledronate 100 μM in RPMI medium supplemented with fetal bovine serum 10% at 370C for 12h. Apoptosis using Annexin V assay, by flow cytometry (FC), was observed in 8 out of 19 (42,1%) patients despite previous treatment. Multidrug resistance (MDR) phenotype was performed using rhodamine-123 efflux assay by FC. Our results demonstrate that Zoledronate 100 m M can induce apoptosis in B malignant lymphocytes despite previous treatment and MDR phenotype. So, patients previously treated with distinct therapeutic agents or not can potentially benefit from the anti-tumour effect of Zoledronate. This is the first work demonstrating the anti-tumour effect of Zoledronate in newly obtained cells from patients with B-CLL and LGL. These results in association with evidences from recent studies suggest that Zoledronate may have an important and complementary role in hematological malignant diseases.
SPAN-Xb Expression in Myeloma Cells Can Be Upregulated by Pharmacologics.
Authors: Wang Zhiqing , Zhang Yana , Zhang Jian , Lim Seah H.
Published: April 2020

SPAN-Xb is a spermatid-specific protein. We recently identified SPAN-Xb as a novel Cancer-Testis (CT) antigen in multiple myeloma (MM) (Blood2003; 101: 955–960) and that it frequently elicited immune responses in vivo in these patients, suggesting that SPAN-Xb may be a suitable target for immunotherapy of MM. SPAN-Xb, however, was only detected in the tumor cells from 20% of MM patients. To increase the applicability of a SPAN-Xb tumor vaccine for MM, we have set out to determine if SPAN-Xb expression could be upregulated by pharmacologics in 8 MM cell lines.

Since most CT antigen expression occurs due to promoter demethylation, we first determined the ability of a hypomethylating agent, 5-azacytidine, to upregulate SPAN-Xb expression. Prior to exposure to 5-azacytidine, SPAN-Xb transcripts were only detected by RT-PCR in ARP-1 and RPMI 8226 cells. Following incubation in 5-azacytidine (2 uM) for 96 hours, the levels of SPAN-Xb transcripts were upregulated, as determined by RT-PCR and real time PCR, in all MM cell lines (ARK-B, ARP-1, H929, IM9, MM1-R, RPMI 8226 and U266) except KMS-11 (Table 1). This was associated with a corresponding increase in the level of SPAN-Xb protein expression, as shown by immunocytochemistry using a murine monoclonal antibody directed at SPAN-Xb.

We next determined if SPAN-Xb gene expression could be increased by cytokine pre-incubation of these cells. IL-2 (100 IU/ml) and IL-4 (1000 IU/ml) did not affect SPAN-Xb gene expression. On their own, IL-7 (0.1 ng/ml) and GM-CSF (10 U/ml) did not affect SPAN-Xb gene expression significantly. However, when combined, especially with 5-azacytidine, a summative effect of these pharmacologics on SPAN-Xb gene expression, as determined by real time PCR, was observed in IM9 cells (Table 2). Our results indicate the potential to upregulate SPAN-Xb expression in MM for therapeutic purpose using 5-azacytidine, GM-CSF and IL-7 and provide insight into the possible mechanisms of regulation of SPAN-Xb gene expression.

Table 1

Cells pre-5-azacytidine post-5-azacytidine SPAN-Xb mRNA copy numbers before and after 5-azacytidine exposure ARK-B 0 456 ARP-1 260 1376 H929 0 116 IM9 0 532 KMS-11 0 0 MM1-R 0 428 RPMI 8226 272 1348 U266 0 984

Table 2

Pharmacologics SPAN-Xb mRNA SPAN-Xb mRNA copy numbers after exposure to combination pharmacologics 5-azacytidine 532 IL-7 4 GM-CSF 9 IL-7 + GM-CSF 36 5-azacytidine + IL-7 1088 5-azacytidine + GM-CSF 952 5-azacytidine + IL-7 + GM-CSF 1632 medium only 0
Phase 2 Study of Three Doses of Single Agent Bortezomib in Patients with Fludarabine-Refractory B-Cell CLL.
Authors: Faderl Stefan , Rai Kanti R. , Gribben John , Flinn Ian , Byrd John C. , McConkey David , Schenkein David , Esseltine Dixie , Browning Mary L. , Keating Michael J.
Published: April 2020

Bortezomib (formerly PS-341, VELCADE®) is a selective inhibitor of the ubiquitin-proteasome pathway, which plays an important role in degrading regulatory proteins that control cell cycle functions, neoplastic growth and tumor spread. It has been approved by the FDA for the treatment of relapsed and refractory myeloma patients (pts). Phase I and II trials have suggested activity in B-cell indolent and aggressive lymphomas including mantle cell lymphoma. Preclinical studies have also shown that B-CLL cells undergo apoptotic cell death following inhibition of the proteasome pathway. We therefore conducted a multicenter phase II study to assess the safety and efficacy of 3 doses of bortezomib in pts with B cell CLL who were refractory to or intolerant to fludarabine. Pts with an ANC ≥ 1 x 109/L and platelets ≥ 30 x 109/L were eligible. Bortezomib was given as i.v. push on days 1, 4, 8, and 11 of a 21 day course for a maximum of 9 courses. Pts were initially randomized to 1.0 (n=5) versus 1.3mg/m2/dose (n=9) and later, after review of preliminary data, to 1.3 versus 1.5mg/m2/dose (n=8). Twenty-two pts were enrolled. Median age 62 yrs (range 46-83). Median number of prior therapies 4 (2–11). Median number of yrs from diagnosis to therapy 5.5 (0.8–14.2). b2-microglobulin > 5.5 mg/L in 10 pts (45%). Fifteen pts (68%) had ≥ Rai stage 3. Of 19 evaluable pts no responses according to NCIWG criteria were demonstrated [stable disease 8 pts (42%), progressive disease 11 pts (58%)] based on investigator assessment. When analyzed by specific sites of disease, five pts (23%) experienced ≥ 50% decrease in ALC and 6 pts (27%) showed ≥ 50% decrease in lymphadenopathy. Bortezomib was well tolerated throughout all 3 dosing groups. Most common toxicities were ≤ grade 2 and included nausea (n=9;42%), headache (6;27%), diarrhea (5;23%), vomiting (5;23%), dyspnea (5;23%), anorexia (5;23%), and peripheral neuropathy (3;14%/ grade 3 in 1 pt). Pharmacodynamic evaluation of 20S proteasome inhibition at 1 hour postdose was similar to that reported for these doses in other studies, and was highest in the 1.5mg/m2 dosing group. We conclude that bortezomib as single agent is not effective in pts with fludarabine-refractory B-cell CLL at these doses. However, in view of the evidence of clinical activity, further exploration of bortezomib in combination with other agents might be the appropriate next step to define the optimal role of this new agent in the management of advanced CLL.
Induction of Apoptosis by Didox Via Down-Regulation of Bcl-2 Family Proteins in Multiple Myeloma (MM).
Authors: Raje Noopur , Kumar Shaji , Hideshima Teru , Ishitsuka Kenji , Roccaro Aldo , Yasui Hiroshi , Hamasaki Makoto , Shiraisi Norihiko , Munshi Nikhil C. , Elford Howard , Anderson Kenneth C.
Published: April 2020

Ribonucleotide reductase is the rate-limiting enzyme of deoxynucleoside triphosphate synthesis and is therefore an excellent target for cancer chemotherapy. Inhibition of ribonucleotide reductase results in inhibition of DNA synthesis and has anti-neoplastic effects. Here we examine the anti-MM activity of the novel ribonucleotide reductase inhibitors (RRIs) Didox, Imidate, and Trimidox. All three RRIs showed potent cytotoxicity against MM cells in 48-hour cultures. Didox was studied further due to its potential for clinical development. Apoptosis was induced in MM cell lines exposed to Didox (200mmol/l), evidenced by increased sub-G1 cell fraction. Didox-induced cytotoxicity was associated with caspase-8 and poly ADP-ribosepolymerase (PARP) cleavage, as demonstrated by western blot analysis. Didox induced apoptosis even in cell lines resistant to conventional chemotherapy (doxorubicin, melphalan, dexamethasone) and overcame the proliferation of MM cells triggered by interleukin-6 (IL-6), insulin like growth factor-1 (IGF-1) and tumor cell binding to bone marrow stromal cells (BMSCs). Importantly, apoptosis of MM cells triggered by Didox was accompanied by a down-regulation of anti-apoptotic proteins Bcl-2, Bclxl, and XIAP. Cell signaling data indicated that Didox induced a time- and dose-dependent inhibition of pSTAT3. Although, Didox has been reported to inhibit radiation-induced NF-KB activation in prostate cancer cell lines, here we found that it triggered activation of NF-KB, associated with a degradation of IKBa, in MM. Ongoing pre-clinical studies are providing the rationale for a clinical trial of Didox either as a single agent or in combination with specific NF-KB/ IKB inhibitors, to improve patient outcome in MM.
Roles of CD19 Molecules That Suppress Proliferation and Induce Apoptosis in Human Myeloma Cells.
Authors: Zheng Xu , Ishikawa Hideaki , Tsuyama Naohiro , Abroun Saeid , Liu Shangqin , Li Fu-Jun , Otsuyama Ken-ichiro , Ma Zi , Maki Yasuko , Obata Msanori , Kawano Michio M.
Published: April 2020

CD19 is a B cell-specific membrane protein belonging to immunoglobulin superfamily, and acts as a positive or negative regulator in B cell signaling. Although plasma cells still express CD19, CD19 expression is lost in malignant plasma cells with the progression of multiple myeloma (MM) including monoclonal gammopathy of undermined significance (MGUS). To understand the pathological significance of loss of CD19 in human myelomas, we examined the biological effect of CD19 expression on myeloma cells by generating CD19 transfectants of a myeloma cell line, U266. CD19 transfectants showed slower growth rate in vitro than controls, such as mock and ΔCD19 transected with a truncated form of CD19. The growth-inhibitory effect was CD19-specific and presumably due to the intracellular signaling events via CD19 because this effect was not observed in ΔCD19. Furthermore, serum withdrawal rapidly induced the apoptosis of the CD19-transfectants that was rescued by insulin-like growth factor (IGF-I) as compared with CD19− controls. These may suggest that the CD19 transfectants are likely to be dependent on the growth factors, such as IGF-I, to survive and proliferate, and thus, susceptible to the serum deprivation-induced apoptosis. IGF-I activates extracellular signal-regulated kinase (ERK) 1/2 and phosphatidylinositol-3-kinase (PI-3K) that are important for proliferation of myeloma cells. Western bolt analysis showed that IGF-1-induced activation of ERK1/2 was reduced in the CD19 transfectants, although the similar levels of IGF-I receptors were expressed in the CD19 transfectants and CD19− controls. In contract, CD19 failed to inhibit the IGF-I-mediated PI-3K/AKT activation, whereas a downstream target in the PI-3K pathway, p70S6 kinase activation was decreased in the CD19 transfectants. However, the p70S6 kinase activity could be influenced by both ERK1/2 and PI-3K pathways after IGF-1 stimulation, and the MAPK pathway seems to be a major regulator for myeloma cell growth. Our results, therefore, suggest that the recovering expression of CD19 molecules and genes in human myeloma cells may be an important consideration in therapeutic approaches in human MM.