bluuebunny/crossref_metadata_2025 huggingface dataset:
Circulating Myeloperoxidase (MPO) as a Tumor Marker in Patients with Chronic Myeloid Leukemia (CML).
Authors: Jilani Iman , Vincente Ted , Faraji Homan , Giles Francis J. , Cortes Jorge , Kantarjian Hagop M. , Albitar Maher
Published: April 2020

MPO is a hemoprotein expressed in polymorphonuclear neutrophils and their precursors. Free circulating MPO in the plasma or serum is an important indicator of inflammation in various inflammatory diseases, whereas cellular expression levels of MPO, assessed by cytochemical staining, are used extensively for the diagnosis and subtyping of various leukemias. The role of circulating MPO levels in the evaluation of CML has not been assessed. In this study, we examined the association of plasma MPO levels with hematologic characteristics and clinical outcome in 107 patients with Philadelphia chromosome-positive CML: 74 in chronic phase (CP) and 33 in accelerated/blastic (ACC/BC) phase). 100 apparently healthy individuals were included as a control group. All patients received imatinib mesylate therapy, with a median follow-up period of 18.6 months (range, 2.1–31.1 months). Overall, MPO levels did not differ significantly between CP and ACC/BC patients (Wilcoxon test, P=0.34). However, both CP (median, 38.2; range, 4.9–11421.6 ng/mL) and ACC/BC (median, 47.00; range, 3.4–1423.6 ng/mL) groups had significantly higher levels than control subjects (median, 4.9; range, 3.5–20.6 ng/mL). Response rates were 77% for CP patients and 57% for ACC/CP patients. When both groups were considered together, nonresponders had significantly higher MPO levels than responders (P = 0.03). In separate analyses, this association held true for ACC/BC patients (P = 0.04) but not for the CP group. MPO levels correlated strongly with WBC, LDH, percent basophiles, and percent Philadelphia chromosome-positive metaphases (all P <0.001). These data suggest that plasma MPO levels are elevated in CML and may be useful in predicting response to imatinib mesylate therapy for patients in ACC/BC phase; whether the association of MPO levels with response reflects an underlying association with tumor burden requires further study. Longer follow-up with larger samples sizes may be needed to accurately evaluate the relationship between plasma MPO levels with response and overall survival for patients in CP.
Increased IL-6 Receptor Proteolytic Cleavage in Mononuclear Cells from Patients with Essential Thrombocythemia.
Authors: Goette Nora P. , Marta Rosana F. , Chazarreta Daniel C. , Molinas Felisa C.
Published: April 2020

Recently we described increased IL-6 soluble receptor (IL-6sR) levels in plasma of essential thrombocythemia (ET) patients as well as elevated IL-6sR release by cultured mononuclear cells. IL-6sR can be generated by two different mechanisms: alternative splicing and proteolytic cleavage. Here we evaluated the participation of metalloproteases responsible for the proteolytic cleavage in the increase of IL-6sR levels produced by cultured cells. We studied expression changes in anchored IL-6 receptor (IL-6R) in CD3 lymphocytes as well as monocytes before and after cell culture. We also assessed the IL-6sR levels in the supernatant of cell culture in the presence and absence of a metalloprotease inhibitor, TAPI, that prevents most of the IL-6sR shedding from the anchored receptor. Seventeen patients with ET were studied, 14 without treatment and 3 under anagrelide treatment. Blood samples were obtained under sterility conditions and mononuclear cells were separated by Ficoll-Hypaque gradient. Afterwards, cells were cultured during 24 hs in IMDM supplemented with 10% FCS in the presence and absence of 150 mMol/L TAPI and IL-6sR released to culture media was measured by ELISA technique. Anchored IL-6R was evaluated before and after cell culture by flow cytometry using anti-CD126-PE. Monocyte and lymphocyte populations were selected using anti-CD14 and anti-CD3 respectively. IL-6sR levels in supernatant of mononuclear cells from 10 untreated ET patients were found increased, 122.5pg/ml (74–248 pg/ml) (median and range), compared to 9 normal controls, 83 pg/ml (22–130 pg/ml), p=0.011. In the presence of TAPI, patients and normal controls showed similar results, 54.95 pg/ml (18–137 pg/ml) and 48 pg/ml (18–96 pg/ml), respectively, p=NS. The difference between the amount of IL-6sR released in the absence and the presence of TAPI (D) would correspond to IL-6sR generated by proteolytic cleavage. This D was statistically higher in ET patients (61 pg/ml, 18–111 pg/ml) than in normal controls (28 pg/ml, 2–87 pg/ml), p=0.034. Expression of IL-6R on monocyte and lymphocytes from 5 untreated and 3 anagrelide treated ET patients before and after cell culture was similar to that of normal controls as shown by flow cytometry analysis. In conclusion, we found that proteolytic cleavage of IL-6R is increased in cultured mononuclear cells from patients with ET although the steady-state of IL-6R on monocytes and lymphocytes membrane is not altered.
Serum Insulinlike Growth Factor Is Not Elevated in Patients with Early B-Cell Chronic Lymphocytic Leukemia but Is Still a Prognostic Factor for Disease-Progression.
Authors: Molica Stefano , Vitelli Gaetano , Mirabelli Rosanna , Digiesu Giovanna , Giannarelli Diana , Iorfida Antonella
Published: April 2020

Insulin like growth factor 1 (IGF-1) is an important growth and antiapoptotic factor for the cancer cells in several malignancies and in multiple myeloma recent studies support the hypotesis of a role for IGF-1 in disease progression. Clinico-biological relevance of IGF-1 was never studied in B-cell chronic lymphocytic leukemia (CLL). Using a quantitative sandwich immunoassay technique (ELISA)(QUANTIKINE®, Human IGF-1 and IGFBP-3, R & D Systems), we measured the concentration of IGF-1 and its major binding protein IGF-binding protein 3 (IGFBP-3) in serum drawn at the time of diagnosis from 77 Binet stage A CLL patients. Either IGF-1 or IGFBP-3 were significantly decreased compared to healthy age- and sex-matched controls (P<0.0001 for both; Mann-Whitney test).

Serum levels of IGF-1 and IGFBP-3 paralleled each other (P=0.002); in contrast, no significant correlation was found between serum levels of IGF-1 and clinico-hematological variables including age (P=0.253), sex (P=0.270), Rai clinical substages (P=0.140), LDH (P=0.956), β2-microglobulin (P=0.368), lymphocyte count (P=0.703) and lymphocyte doubling time [LDT](P=0.233). When correlation were attempted with circulating levels of angiogenic cytokines such as vascular endothelial growth factor (VEGF)(P=0.971), basic fibroblastic growth factor (FGF-2)(P=0.695), angiogenin (P=0.282) or adhesion molecules such as vascular cell adhesion molecule-1 [VCAM-1] (P=0.318), intercellular adhesion molecule-1 [ICAM-1] (P=0.883) and platelet endothelial cell adhesion-1 [PECAM-1] (P=0.772) similar results were found. Serum levels of IGF-1 were further evaluated as a dichotomous variable with respect to progression-free survival (PFS), an endpoint surrogate for overall survival in early B-cell CLL. The best separation of curves was seen with the cutoff point at the 75th percentile of IGF-1 levels (i.e., 93 pg/ml). Median PFS was 63 months in the patient group with low IGF-1, compared to a median PFS of 40 months in the remaining patients (P=0.002; HR, 0.311, 95% C.I, 0.085–0.630). In the multivariate analysis performed including variables significant at univariate analysis [i.e., Rai substage (P=0.002); LDT (P=0.004), IGF-1 (P=0.01)], only Rai substage retained prognostic significance (P=0.006). However, after removing from analysis LDT (only 6 out of 77 had an LDT< 12 months), either IGF-1 or Rai substage entered the model at a significant level ((P=0.03 and P=0.01, respectively).

In conclusion, IGF-1 did not correlate with markers of tumor burden or clinical status in CLL thus suggesting that levels of this cytokine do not reflect the intrinsic malignancy of disease. Results of the present study highlight, however, its involvement in mechanisms of disease-progression in early CLL.
Cytosolic Histone H1 Releasing under Different Apoptotic Stimuli in Chronic Lymphocytic Leukemia (CLL).
Authors: Gine Eva , Crespo Marta , Montserrat Emili , Bosch Francesc
Published: April 2020

Recently, it has been demonstrated that nuclear histone H1 could be released into cytoplasm when apoptosis is induced by DNA double-strand breaks, this process being dependent of p53 functional status. In addition, cytosolic histone H1.2 was shown to induce cytochrome C release in a Bak-dependent manner. The aims of this study were: 1) to analyze the presence of histone H1.2 in the cytosol of peripheral blood purified CLL cells during apoptosis induced by different chemotherapeutic drugs or ionizing radiation in CLL cells in culture; 2) to correlate the presence of cytosolic histone H1 with the p53 functional status. FISH analysis was used to select samples with (n=2) or without (n=4) p53 deletion. Peripheral blood purified CLL cells were cultured for 24 hours with no treatment, after irradiation (25 Gy), in the presence of fludarabine and mitoxantrone (FM) (1mg/ml and 0.5 mg/ml respectively), or with etoposide (5.8 mg/ml). Cell viability and analysis of apoptosis were performed by annexin V/PI staining and FACscan analysis. Cytosolic and nuclear fractions were separated with the Nuclear/Cytosol Fractionation kit (Biovision). The presence of histone H1 in the cytosolic fraction was assessed by Western Blott using the anti-histone H1 antibody (Upstate). Nuclear contamination of this fraction was detected by using anti-ribonucleoprotein (hnRNP) antibody (Abcam). In p53 wild-type CLL cases, increased apoptosis was observed under all stimuli, being the FM combination the most effective. In contrast, CLL cases with p53 deletion displayed a lower or no response to the different treatments. In p53 wild-type CLL cases, histone H1 was only observed in cytosolic fractions at 24 hours when irradiation or etoposide treatment was applied, but not with FM treatment. Interestingly, cytosolic histone H1 was also present in the control cells at 24h of culture, indicating that H1 was also involved in the spontaneous apoptosis observed due to the microambient deprivation of CLL cells in culture. This histone H1 traffic was also confirmed using immunofluorescence analysis. Conversely, in p53 deleted CLL cases histone H1 was not detected into cytosolic fractions with any treatment. In conclusion, cytosolic histone H1 appeared in CLL cases in a p53 dependent manner when DNA double-strand breaks were induced. These results suggest that histone H1 plays an important role in the p53-dependent cytosol apoptotic signaling.
Expression of Cannabinoid Receptors on the Neoplastic Lymphocytes in Patients with Chronic Lymphocytic Leukemia.
Authors: Piszcz Jaroslaw A. , Pietruczuk Miroslawa , Kloczko Janusz S. , Dabrowska Milena , Galar Marzenna , Milewski Marek P.
Published: April 2020

Endocannabinoids take part in the physiology of neural and immune systems. The latest data showed that these compounds and their receptors play an important role in proliferation and apoptosis of various neoplastic cells. Cannabinoids were shown to increase apoptosis in human leukemia and lymphoma cell lines and culture of neoplastic cells obtained from patients with T-cell acute lymphoblastic leukemia. The aim of the study was an assessment of cannabinoid receptors expression on lymphocytes B derived from patients with CLL. The study group contains newly diagnosed, untreated adult patients with B-cell chronic lymphocytic leukemia; 13 male and 7 female, aged from 44 to 65. All of the patients were in B or C stage according to Binet. Peripheral blood samples from 10 healthy adult donors were used as the control group. The patients were hospitalized in the Department of Haematology, Medical University in Bialystok, Poland. Diagnosis of CLL in all cases was confirmed by routine immunophenotyping study. For flow cytometric analysis 1x105 – 1x106 of peripheral blood cells were incubated with 10ul of anty CB1 and anty CB2 polyclonal antibodies (American Diagnostics). Then 10ul of monoclonal antibodies IgG1-FITC and CD19-PE (Becton Dickinson) were added and the samples were incubated for 20 min in dark in 4°C. The samples were lysed, fixed and stabilized using Immuno-Prep (Coulter procedure) and assessed by flaw cytometry (Epics XL, Coulter). Statistical analysis was performed using non parametric U Mann-Whitney and Wilcoxon Tests. The conducted study revealed high expression of CB1 and CB2 receptors on the surface of neoplastic lymphocytes. The percentage of CB1/CD19 and CB2/CD19 positive cells in CLL patients were significantly higher, compared to the control group respectively (81.2±9,8% vs 12.0±9,3% p<0,05; 94,8±11,0 % vs 9,9±4,0%, p<0,05). No difference was noticed between the percentage of lymphocytes with CB1 and CB2 receptors expression in CLL and control group. Fluorescent intensity of CB2 receptors was about ten folds higher than CB1 receptors in both groups. The study demonstrated the presence of both types of cannabinoid receptors (CB1 and CB2) on neoplastic cells derived from patients with chronic lymphocytic leukemia. The higher percentage of B-lymphocytes expressing cannabinoid receptors in CLL patient suggests that the cannabinoid system may take part in CLL development. High intensity of CB2 receptor may be another target in CLL treatment.
Chronic Lymphocytic Leukemia Cells May Become Less Dependent on Nurse-Like Cells during Later Stages of Disease.
Authors: Endo Tomoyuki , Nishio Mitsufumi , Rassenti Laura , Kater Arnon P. , Kipps Thomas J.
Published: April 2020

A subset of blood mononuclear cells from patients with B-cell chronic lymphocytic leukemia (CLL) can differentiate into large, adherent cells, termed “nurse-like cells” (NLC), that can attract CLL cells and protect them from undergoing apoptosis in vitro. When removed from NLC in vitro, CLL cells often undergo spontaneous apoptosis. We observed that the relative viability of isolated CLL cells cultured without versus with NLC appeared constant for any one sample in different experiments and did not vary with the source of the NLC used in any one experiment. Furthermore, the isolated CLL cells from some patients could survive in vitro without NLC better than could the leukemia cells of other patients. We examined for characteristics associated with CLL cells that were more or less reliant on NLC for survival in vitro. For this, we defined CLL cell populations as having “low dependency” on NLC when the relative leukemia cell viability after two days culture without NLC was less than one half-log (33%) lower than that of the same CLL cells co-cultured with NLC. Using a Multiplex Ligation-dependent Probe Amplification (MLPA) assay, we examined CLL cell populations with low dependency (n = 4) versus samples with high dependency (n = 5) for expression of 40 distinct mRNA that encoded known pro-apoptotic or anti-apoptotic proteins. CLL cells with low dependency expressed significantly higher levels of BCL-2, and significantly lower levels of BAX-2 than CLL cells with high NLC dependency. There was no significant difference between the mean white blood cell counts of patients whose CLL cells had low-dependency versus those whose cells had high dependency on NLC in vitro. However, in the high dependency group there was a significantly higher proportion of cases that expressed ZAP-70 than in the group with low dependency. This association, however, could reflect the fact that many of the samples with low dependency were acquired from patients who had long-established disease, a characteristic that typically was not associated with patients who have CLL cells that expressed ZAP-70. The mean time from diagnosis to the date of sample collection was significantly longer (m = 66.6 ± 47.0 months, median = 49 months, n=30) for samples with low dependency than it was for those cases with high dependency (m = 41.2 ± 25.1 months, median = 39.5 months, n=20) (p=0.017). Furthermore, evaluation of serial samples collected over years from the same patients (n = 5) revealed that the relative dependency of CLL cells on NLC could decrease significantly over time. In none of the serial samples did we observe increased dependency of the CLL cells on NLC over time or over that observed for the CLL cells isolated from the initial sample collection. These data suggest that over the course of the disease CLL cells might acquire less dependency on NLC, and potentially also on its stromal microenvironment.
CD11b Is Useful in the Diagnosis of Chronic Lymphocytic Leukemia/Prolymphocytic Leukemia, Mixed Chronic Lymphocytic Leukemia, and Prolymphocytic Leukemia.
Authors: Fricke William
Published: April 2020

CD11b is well known as an integrin, Mac-1, is often complexed with CD18, and is found on monocytes, granulocytes, and natural killer cells. It also serves as a receptor for iC3b. However, its occurrence in B cell chronic lymphoproliferative disorders is not generally recognized and has not been fully evaluated. To address this issue, a series of B cell leukemias and lymphomas referred for primary diagnosis was evaluated for the presence of CD11b. The purpose was to determine the frequency of its expression on these tumors and to evaluate its diagnostic value.

Consecutive cases referred for flow cytometry as possible lymphoproliferative disease were analyzed. Included were bone marrow, peripheral blood, and lymph nodes. All cases were diagnosed according to the WHO classification based on immunophenotypic, morphologic, and clinical findings. The morphologic criteria of Melo (1986) and Bennett (1989) were used for classification of chronic lymphocytic leukemia (CLL), CLL/prolymphocytic leukemia (CLL/PLL), mixed CLL, and PLL. Cases identified as not related to chronic lymphocytic leukemia or prolymphocytic leukemia were recorded but not further analyzed. Similarly, lymph node and spleen-based tumors were excluded from the final analysis.

CD11b was present on cells from 32 of 123 cases, including occasional follicular lymphoma, (5/35); mantle cell lymphoma, (1/8); diffuse large B cell lymphoma, (3/9); hairy cell leukemia, (3/5); multiple myeloma, (1/2); lymphoplasmacytic lymphoma, (2/2); nodal marginal zone lymphoma, 0/1); and splenic marginal zone lymphoma, (1/1). However, it was most consistently expressed on CLL that contained increased numbers of prolymphocytes or large cells and on PLL. A total of 16 such cases were found. Morphologic assessment showed them to include 8 CLL/PLL, 3 mixed CLL, 4 PLL, and 1 typical CLL. The typical CLL case included both large cells and prolymphocytes but did not have more than 10% PLs. Five of the 16 cases (31%) were negative for CD5, CD23, and CD38 but were positive for FMC-7. In contrast, the other 11 cases were all CD5(+) and CD23(+); 3/11 were positive for CD38; and 5/11 were positive for FMC-7. Forty-five CLLs also were identified during the study, of which 27 had sufficient data for comparison. Twenty-six of the 27 CLLs were morphologically typical. The remaining case was mixed CLL. All of the CLLs were CD11b(−), CD5(+) and CD23(+); 15/43 were CD38(+), and 6/43 were FMC-7(+).

The findings show that CD11b is expressed on chronic B cell lymphoproliferative disorders. In particular, it is expressed on almost all CLL cases that contain large cells or prolymphocytes and on PLL. Inclusion of CD11b in routine screening panels of possible chronic B cell leukemiaa will improve diagnosis of these disorders.
Leukotriene B4 Plays a Pivotal Role in CD40 Dependent Activation of Chronic B Lymphocytic Leukemia Cells.
Authors: Claesson Hans-Erik , Runarsson Gudmundur , Liu Anquan , Mahshid Yilmaz , Feltenmark Stina , Klein Eva , Bjorkholm Magnus
Published: April 2020

Biosynthesis of leukotrienes occurs in human myeloid cells and B lymphocytes. However, the function of leukotrienes in B lymphocytes is unclear. Here we report that B-cell chronic lymphocytic leukemia (B-CLL) cells produce leukotriene (LT) B4 and that specific leukotriene biosynthesis inhibitors counteracted CD40-dependent activation of B-CLL cells. Studies on the expression of the high affinity receptor for LTB4 (BLT1) by flow cytometry analysis showed that the receptor was expressed, to a varying degree, in all investigated B-CLL clones. The drugs BWA4C (a specific 5-lipoxygenase inhibitor) and MK-886 (a specific 5-lipoxygenase activating protein inhibitor), at a concentration of 100 nM, markedly inhibited CD40-induced DNA synthesis (45% and 38%, respectively) and CD40-induced expression of CD23, CD54 and CD150. Addition of exogenous LTB4 (150 nM) almost completely reversed the effect of the inhibitors on DNA synthesis and antigen expression.

Taken together, the results of the present study suggest that leukotriene biosynthesis inhibitors may have a therapeutic role in B-CLL.
Cytoplasmic PML Function in TGF-β Signaling.
Authors: Lin Hui-Kuan , Bergmann Stephan , Pandolfi Pier Paolo
Published: April 2020

TGF-β is a pluripotent cytokine that controls key tumor suppressive functions such as cell growth inhibition, induction of apoptosis, cellular senescence and differentiation. Cancer cells are often unresponsive to TGF-β. Blasts from several leukemia subtypes including acute promyelocytic leukemia (APL) display resistance to TGF-β-growth inhibitory and differentiating activity. As TGF-β, the PML tumor suppressor of APL is also known to modulate key tumor suppressive functions. PML is found to accumulate in the PML-nuclear body, although cytoplasmic isoforms of unknown function have also been described. The PML-RARα fusion oncoprotein of APL is found to accumulate both in the nucleus and cytoplasm and is thought to act as a dominant negative PML mutant in view of its ability to heterodimerize with PML. Given the functional similarities between PML and TGF-β and their functional loss in APL, we asked whether PML would modulate TGF-β function. Here we show that cytoplasmic Pml is an essential modulator of TGF-β signaling. Pml-null primary cells of diverse histological origins are resistant to TGF-β-dependent growth arrest, induction of cellular senescence and apoptosis. Induction of TGF-β target genes such as p21 and p15, as well as Smad2 and Smad3 phosphorylation and nuclear translocation is also dramatically impaired in the absence of Pml. Cytoplasmic Pml is induced by TGF-β and its add-back to Pml-null cells can fully rescue these biological and biochemical defects. Furthermore, we find that cytoplasmic PML physically interacts with Smad2/3 and SARA (Smad anchor for receptor activation), colocalizes with these proteins in cytosolic regions and is required for association of Smad2/3 with SARA and for the accumulation of SARA and TGF-β receptor in the early endosome. We demonstrate that the PML-RARα oncoprotein of APL can antagonize cytoplasmic PML function and that APL blasts display defects in TGF-β signaling similar to that observed in Pml-null cells, which can be overcome by retinoic acid (RA) and As2O3, drugs that induce PML-RARα degradation and are effective in APL treatment. Our findings identify cytoplasmic PML as a critical TGF-β regulator, and further implicate deregulated TGF-β signaling in cancer pathogenesis.
Pharmacokinetics of Single-Agent Lumiliximab (Anti-CD23 Antibody) in Patients with Chronic Lymphocytic Leukemia.
Authors: Woodworth James , Leigh Bryan R. , O’Brien Susan , Flinn Ian , Kipps Thomas J. , Weiss Mark A. , Rai Kanti , Thall Aron , Wynne Dee , Byrd John C.
Published: April 2020

CD23 is highly expressed on the cell surface of certain B-cell malignancies including chronic lymphocytic leukemia (CLL). Lumiliximab (L-mab), a macaque-human chimeric anti-CD23 antibody, has been reported to have antitumor activity against CLL in preclinical studies. In this 46 patient, Phase I, multicenter study, single-agent L-mab was evaluated as a treatment for patients with relapsed or refractory CLL. Therapy consisted of intravenous L-mab given as 6 regimens: (1) 125 mg/m2/wk for 4 weeks; (2) 250 mg/m2/wk for 4 weeks; (3) 375 mg/m2/wk for 4 weeks; (4) 500 mg/m2/wk for 4 weeks; (5) 500 mg/m2 for 3 doses during Week 1, then 500 mg/m2/wk during Weeks 2 to 4; and (6) 500 mg/m2 three times a week for 4 weeks. Pharmacokinetics (PK) and pharmacodynamic measurements were evaluated in patients with adequate data. L-mab and anti-L-mab antibody concentrations in plasma were measured by ELISA. L-mab coating of CLL cells and CD23 density on CLL cells were evaluated by flow cytometry. The absence of sustained L-mab concentrations did not allow PK calculations at the 125 mg/m2/wk and 250 mg/m2/wk dose levels. Doses at 375 mg/m2/wk or higher resulted in coating of CD23 binding sites on CLL cells and saturable PK (see table). Anti-L-mab antibody formation was not detected. CD23 density on CLL cells did not change during or after L-mab treatment. In summary, L-mab exhibits saturable PK, low clearance, and low volume of distribution without an immunogenic response. No down regulation of CD23 expression was observed on CLL cells in any dose group, and complete coating of CD23 sites was observed.

Median Pharmacokinetic Parameters

Treatment Group n C max(μ g/mL) Clearance (mL/m2/day) Volume of Distribution (mL/m2) Half-life (days) 1 3 39 - - - 2 3 84 - - - 3 8 180 310 3.02 7.9 4 9 245 335 3.22 7.3 5 8 430 406 3.45 5.4 6 9 654 293 3.58 10.6
Thalidomide Is Active Alone and in Combination with Fludarabine in Fludarabine-Relapsed and Refractory Chronic Lymphocytic Leukemia.
Authors: Furman Richard R. , Allen Steven L. , Leonard John P. , Coleman Morton , Markis Jessica M. , Gabrilove Janice L.
Published: April 2020

Background: CLL is a lymphoproliferative disease characterized by an accumulation of malignant lymphocytes due to a defect in apoptosis. CLL lymphocytes demonstrate increased NF-κB activity, which may play a role in mediating resistance to fludarabine. Additionally, several cytokines have been linked to enhanced CLL survival, including TNF-α, bFGF, and VEGF. Given thalidomide’s ability to inhibit these cytokines and NF-κB activity, we investigated whether thalidomide, either alone or in combination with fludarabine, is an effective treatment for pts with CLL previously treated with fludarabine.

Methods: CLL pts requiring therapy as defined by NCI-WG criteria and who were previously treated with fludarabine were eligible. Pts were randomized to receive either thalidomide alone (T) or in combination with fludarabine (FT). Thalidomide was started at a dose of 200 mg qHS and adjusted as tolerated. Pts assigned to FT started thalidomide prior to receiving fludarabine at a dose of 25 mg/m2 each day x 5 days x 6 cycles.

Results: 13 pts have been enrolled, 8 men/5 women, median age=60, (range 48–68), 4 pts refractory to fludarabine, randomized to receive either T (6) or FT (7). The median dose of thalidomide was 200 mg qHS (range 50–450). Of the 10 evaluable pts, there have been 1 CR (FT), 3 PRs (1T, 2FT), and 2 hematologic improvements (1T/1FT). Of interest, 5 pts on T developed tumor flares, characterized by fevers, increased size and tenderness of lymph nodes, increased lymphocyte counts, and decreased hemoglobin and platelet counts. These symptoms resolved with either continued treatment (3 pts) or temporarily discontinuing the thalidomide and re-introducing it at a dose of 50 mg (2 pts). Patients on T demonstrated hematologic improvement (HI) prior to decreases in lymph node size. Figs 1 and 2 illustrate two patients with tumor flares followed by marked HI, but only minimal improvement in LAD. Pt in fig 1 continued on T, while pt in fig 2 had T held for 4 weeks. Overall, therapy was tolerated, with grade 3 toxicities being: neuropathy(2)(requiring T dose reduction), sedation(1), catheter thrombosis(1), febrile neutropenia(1), infection(3), none requiring discontinuation of therapy.

Conclusion: Thalidomide demonstrates activity alone and in combination with fludarabine as treatment for CLL. Further data are required to see if FT can increase the response rate over historical controls for patients who relapse after fludarabine. The addition of F to T appears to ameliorate the development of a tumor flare.
Etodolac Induces Apoptosis and Inhibits Cell Adhesion to Bone Marrow Stromal Cells in Human Myeloma Cells.
Authors: Nakamura Satoki , Kobayashi Miki , Shibata Kiyoshi , Sahara Naohi , Shigeno Kazuyuki , Shinjo Kaori , Naito Kensuke , Ohnishi Kazunori
Published: April 2020

Cyclooxygenase-2 (COX-2) is reported to regulate apoptosis and to be an important cellular target for therapy. In this study, we demonstrated that etodolac, a COX-2 inhibitor, inhibited proliferation and induced apoptosis in myeloma cell lines (RPMI 8226 and MC/CAR cells), expressing the COX-2 enzyme. In both cell lines, etodolac more strongly induced apoptosis compared with thalidomide or meloxicam. Etodolac induced down-regulation of bcl-2 protein and mRNA, activation of caspase-9, -7 and -3, down-regulation of caspase inhibitors, cIAP-1 and survivin, and loss of mitochondrial membrane potential in a dose-dependent manner. In addition, our data demonstrated that when myeloma cells were coincubated with 50 mM etodolac on bone marrow stromal cells (BMSC), myeloma cell adhesion to BMSC was significantly inhibited compared with thalidomide or meloxicam coincubation, and the adhesion molecules VLA-4, LFA-1 (CD11a), CXCX4, and CD44 were suppressed on myeloma cells treated with etodolac. Moreover, we found that 100 mM R-etodolac, S-etodolac, and the combination of R- and S-etodolac, which are the stereoisomers of etodolac, slightly inhibited the proliferation of myeloma cells, while 50 to 100 mM etodolac significantly inhibited the proliferation of myeloma cells. In conclusion, our findings indicate that etodolac induced apoptosis via a bcl-2 dependent pathway, suppressed the expression of adhesion molecules, and inhibited myeloma cell adhesion to BMSC compared with thalidomide or meloxicam. Thus, the activities of etodolac potentially extend to the treatment of patients with myeloma resistant to standard chemotherapy, including thalidomide.
P53 Gene Deletion Detected by Fluorescence In Situ Hybridization Is an Adverse Prognostic Factor for Patients with Multiple Myeloma Following Autologous Stem Cell Transplantation.
Authors: Qi Xiao Ying , Yi Qi Long , Reece Donna , Stewart A. Keith , Chang Hong
Published: April 2020

We investigated the relevance of p53 deletions to the clinical outcome of multiple myeloma (MM) patients treated with high-dose chemotherapy and autologous stem cell transplantation. Hemizygous p53 gene deletions were detected by cytoplasmic Ig-enhanced interphase fluorescence in situ hybridization (cIg-FISH) in 10 of 105 (9.5%) patients studied. p53 deletions were associated with higher serum calcium (p=0.0062) and creatinine (p=0.013) levels but there were no association with patient age, gender, β-2 microglobulin, C-reactive protein, hemoglobin, albumin, bone lytic lesions, or immunoglobulin isotype. There were no association of p53 deletions with chromosome 13q deletions, translocation t(11;14) or t(4;14). The overall response rates were similar in patients with and without p53 deletions (67% vs 71%). However, patients with p53 deletions had significantly shorter progression free (median 7.9 vs. 25.7 months, p=0.0324) and overall survival (median 14.7 vs. 48.1 months, p=0.0008) than patients without a p53 deletion. A multivariate analysis confirmed p53 deletion was an independent prognostic factor predicting shortened progression free (p=0.0009) or overall survival (p=0.0002) in myeloma patients after high-dose chemotherapy and autologous stem cell transplantation.
PTEN Gene Deletions in Patients with Multiple Myeloma.
Authors: Qi Xiao Ying , Stewart A. Keith , Chang Hong
Published: April 2020

PTEN, a tumor suppressor gene, negatively regulates the anti-apoptotic action of akt phosphorylation. Allelic loss or mutation of this gene has been detected in many solid tumors and more recently in human myeloma cell lines (HMCLs). Expression of PTEN has resulted in growth inhibition and apoptosis of a HMCL, suggesting that it may play a role in the pathogenesis of multiple myeloma (MM). However, the PTEN status in tumor cells from patients with MM has not been determined. Using a triple staining method combining staining for cytoplasmic light chains and fluorescence in situ hybridization (FISH) with chromosome 10-centromere and PTEN-gene specific probes, we analyzed clonal plasma cells from 71 patients with MM, 10 with plasma cell leukemia (PCL) and 10 HMCLs. Hemizygous PTEN deletions were detected in 4 of 71 (5.6%) MM patients, 2 of 10 (20%) PCLs, and 2 of 10 (20%) HMCLs. The percentages of clonal plasma cells containing PTEN deletions ranged from 21–90% (median, 56%). Three of the 4 patients with PTEN deletions were detected at diagnosis with stage III disease (Duire-Salmon) and 1 was detected at relapse. Two patients had IgG kappa, 1 IgG lambda and 1 free lambda light chain. To correlate the PTEN status with other known genetic abnormalities in MM, we investigated 4 MM and 2 PCLs with PTEN deletions using FISH for chromosome13q, p53 status, translocations t(11;14), t(4;14) and t(14;16). One MM had a 13q deletion, 1 PCL had a t(11;14), and the other PCL had a t(14;16), a 13q deletion and a p53 deletion. All 4 MM patients with hemizygous PTEN deletions received melphalan based high-dose chemotherapy and autologous stem cell support. Their median overall survival (OS) was 48.1months, and progression free survival (PFS) was 42.8 months as compared to patients without PTEN deletions (OS, not reached, PFS, 25.8 months) (p=0.51 for OS, p=0.67 for PFS). Our results indicate that PTEN deletions are uncommon in MM patients and therefore unlikely represent a primary event for MM. PTEN deletions appear to occur in the advance stage of the disease, and are more frequently involved in PCL or HMCLs suggesting that deletions of PTEN may be associated with disease progression in a subset of MM.
t(11;14) Is Infrequent in Long-Term Myeloma Survivors.
Authors: Chang Hong , Samiee Sara , Qi Xiao Ying , Yi Qi Long , Stewart A. Keith
Published: April 2020

The translocation t(11;14) involving immunoglobulin heavy chain gene switch region and cyclin D1 is the most common IgH translocation (15–20%) in multiple myeloma (MM). MM patients with t(11;14) are considered to have a better prognosis when treated with either conventional chemotherapy or autologous stem cell transplant. In contrast, patients with chromosome 13q deletions (40–50% of patients) have a poor prognosis. To address whether long-term survivors of MM more frequently harbor a t(11;14) but not 13q deletions, we used cytoplasmic Ig-enhanced interphase fluorescence in situ hybridization (cIg-FISH) to evaluate clonal plasma cells for t(11;14) and 13q status from 20 long-term survivor MM cases. These patients met the diagnostic criteria for MM before May of 1996 and have survived more than 8 years. The 11 male and 9 female had a median age of 52 (range 39–62) at diagnosis. Eleven had IgG, 6 IgA, 2 free light chains, and 1 non-secretory. Of the 20 patients, 17 received autologous stem cell transplants. The median duration between diagnosis and transplant was 24 months (range 5–149), the median progression free survival (PFS) after transplant was 32 months (range, 1–62). One patient died 3 years after transplant and 10 years after diagnosis. The 19 other patients remain alive. Translocation t(11;14) was detected in only one of 20 patients with 94% of clonal plasma cells involved. This patient had PFS of 20 months after transplant and is alive 3 years post-transplant and 10.5 years after diagnosis. Chromosome 13q deletions were detected in only 3 cases (15%). Despite its association with a better prognosis, our results indicate that t(14;14) is infrequent in long-term myeloma survivors. As anticipated, 13q deletions were infrequent.
No Influence of Previous Thalidomide Administration on Peripheral Blood Stem Cell Collection in Patients with Multiple Myeloma.
Authors: Breitkreutz Iris , Benner Axel , Cremer Friedrich W. , Herrmann Doris , Ho Anthony D. , Goldschmidt Hartmut
Published: April 2020

OBJECTIVES: In a joint study of the GMMG and HOVON groups, induction therapy with Thalidomide (Thal), doxorubicin and dexamethasone (TAD) is currently investigated in comparison with vincristin, doxorubicin and dexamethasone (VAD) followed by mobilisation therapy with cyclophosphamide, doxorubicin and dexamethasone (CAD) and peripheral blood stem cell collection (PBSC). Munshi et al. (Blood 1999, Abstract #2577) described a dampening of PBSC-mobilisation by Thal treatment. We therefore investigated a possible influence of PBSC after previous Thal administration.

METHODS: Altogether, data on 112 patients were analyzed in terms of PBSC-mobilisation. 56 patients were randomized up-front to receive 3 cycles of TAD (Thal 400mg/d orally; doxorubicin 9mg/m2/d, 4 30-min. infusions, day 1–4; dexamethasone 480mg total dose orally). 56 patients received VAD (vincristin 0,4mg/d and doxorubicin 9mg/m2/d, 4 30-min. infusions, day 1–4.; dexamethasone 480mg total dose orally) followed by mobilisation with CAD (cyclophosphamide 1g/m2/d, 1h infusion, day 1; doxorubicin 15mg/m2/d, 4 short infusions, day 1–4; dexamethasone 160mg total dose orally) and G-CSF (Neupogen 600mg/d s.c. or Granocyte 526mg/d s.c., day 5 after the end of chemotherapy until PBSC). Thal was stopped two weeks before CAD. Low dose heparine was administered to prevent deep venous thromboses in the TAD group.

RESULTS: The median time was 14 days after the first day of CAD until PBSC in patients in both the TAD (range 12–18 days) and VAD group (range 10–19 days). In the first leukapheresis, a median total PBSC yield of 8,1x106/kg CD34+ cells in the TAD/CAD (range 0,3–34x106 CD34+ cells) and 8,7x106/kg CD34+ cells in the VAD/CAD (range 0,5–30x106 CD34+ cells) group could be harvested (p=0.31). In the best leukapheresis, a median total PBSC yield of 8,1x106/kg CD34+ cells in the TAD/CAD (range 0,7–34x106 CD34+ cells) and 8,9x106/kg CD34+ cells in the VAD/CAD (range 2–30x106 CD34+ cells) group could be reached (p=0.24).

CONCLUSIONS: No difference was found in stem cell collection and yield after TAD versus VAD. Thalidomide as a part of induction therapy does not seem to have an influence of the peripheral blood stem cell collection of patients with multiple myeloma.
Symptomatic Multiple Myeloma in Young Adults ≤ 40 Years of Age.
Authors: Symeonidis Argiris , Pouli Anastasia , Repoussis Panagiotis , Zervas Konstantinos , Anagnostopoulos Athanasios , Mitsouli Chrysanthi , Gika Dimitra , Maniatis Alice , Dimopoulos Meletios Athanasios
Published: April 2020

Introduction: Multilple myeloma (MM) rarely occurs in patients ≤ 40 years of age. Thus, information regarding disease features and outcome is limited. We performed a retrospective analysis of the database of GMSG which includes 1162 patients with symptomatic MM who were diagnosed since 1987.

Patients and methods: Multiple clinical and laboratory variables were evaluated in patients ≤ 40 years of age and in older patients including the recently proposed International Staging System (ISS) for MM. Furthermore, the same variables were assessed for possible correlation with prognosis in patients ≤ 40 years of age. Patients with asymptomatic MM were not included.

Results: Forty seven (4%) patients were ≤ 40 year old when treatment was initiated. Clinical and laboratory parameters were similar among the two groups of patients with the exception of ISS which was lower in younger patients (p=0.03). Despite a similar response to primary treatment the median survival of younger patients was significantly longer than that of older patients (65 months versus 37 months; p=0.001). Among younger patients, hypercalcemia, thrombocytopenia, bone marrow plasmacytosis and elevated serum LDH were associated with impaired survival on univariate analysis. ISS was not statistically significant (p=0.1) but the median survival of patients with ISS 1,2 and 3 was 154 months, 60 months and 33 months respectively. A Cox regression analysis showed that only elevated serum LDH was significant and was associated with a median survival of 9 months.

Conclusions: Young patients with symptomatic myeloma have similar disease features with older patients but present with a lower ISS. Patients ≤ 40 years of age live twice as long when compared to older patients with symptomatic MM. Elevated serum LDH identified a subgroup of patients (23%) who had an extremely poor outcome. 40% of young patients presented with ISS 1 and survived for more than a decade. This information may be of value when designing therapeutic strategies for young patients with myeloma especially when allogeneic stem cell transplantation is considered.
A Canadian Multicenter Case-Control Study of the Risk of Venous Thrombosis in Car and Airplane Travelers.
Authors: Opatrny Lucie , Shapiro Stanley , Miron Marie-Jose , Monczak Yury , Kahn Susan R.
Published: April 2020

Background: The potential association between venous thromboembolism (VTE) and travel, particularly air travel (“economy class syndrome”), has been the subject of extensive media coverage. While it is biologically plausible that prolonged travel is an independent risk factor for VTE, epidemiological data to date are conflicting, and confounders have rarely been accounted for.

Aim: To determine whether there is a greater risk of exposure to travel in patients with confirmed DVT compared with patients in whom DVT is ruled out. To examine the influence of confounding variables on the relation between DVT and travel.

Methods: This was a Canadian multi-center case control study. Consecutive patients presenting to the vascular laboratory with clinically suspected DVT were eligible to participate. Cases were patients with objectively confirmed DVT on venous ultrasound; controls were patients in whom DVT was ruled out. Detailed recent travel history, medications and clinical characteristics were obtained via standardized, interviewer-administered questionnaires. Genetic testing for Factor V Leiden and Prothrombin gene mutations was performed. Unconditional multivariate logistic regression analyses with adjustment for confounders and testing for interactions were performed to examine the relation between DVT and (1) any travel, and (2) duration of travel. Plane and car travel were also analyzed separately.

Results: There were 359 cases and 359 controls. Mean age among cases was 56 years and 50% were male. Among controls, mean age was 64 years and 35% were male. Body mass index, smoking status and patient location (inpatient vs. out-patient) were comparable between the two groups. The crude and adjusted odds ratio (OR) for exposure to travel in cases was 1.15 (95% confidence interval (CI): 0.78, 1.69) and 1.44 (95%CI: 0.86, 2.40), respectively. Travel of ≥ 12 hours’ duration was associated with a higher OR (adjusted OR 2.92, 95%CI: 0.54, 15.73) than shorter travel durations (adjusted OR 1.29; 95%CI: 0.62, 2.66). Analyzing plane and car travel separately showed that the adjusted OR for plane travel was 2.28 (95%CI: 0.94, 5.50) but for car travel was 1.00 (95%CI: 0.54, 1.83). Increasing durations of plane travel, but not car travel, resulted in higher ORs. For plane travel ≥ 12 hours, the crude OR was 8.22 (95%CI: 1.02, 66.05) and the adjusted OR was 7.10 (95% CI: 0.70, 72.35). No statistical interactions were detected between travel and thrombophilia, hormonal therapy, or clinical VTE risk factors.

Interpretation: This is the largest case control study to date of the relation between DVT and travel that takes into account concurrent DVT risk factors. Plane travel but not car travel appears to be a mild independent risk factor for DVT. However, flights of 12 hours or longer were associated with a 7-fold increased risk of DVT. Our findings may have future implications regarding the use of thromboprophylaxis during long-haul travel.
Campath-1H in Waldenström’s Macroglobulinemia.
Authors: Hunter Zachary R. , Branagan Andrew R. , Treon Steven P.
Published: April 2020

Campath-1H is a humanized monoclonal antibody that targets CD52, a commonly expressed antigen in WM (Treon et al, Semin Oncology 30:248). We therefore initiated this two-stage Phase II study of single agent Campath-1H in patients with WM. WM patients who failed at least one first line regimen (i.e. an alkylator agent, nucleoside analogue or rituximab) were eligible and received Campath-1H by IV for one week on a gradual dose escalation schedule (Day 1, 3 mg; Day 3, 10 mg; Day 5, 30 mg) followed by up to 12 weeks of therapy (30 mg IV TIW) provided therapy was tolerated and patients did not progress. All patients received acyclovir and bactrim prophylaxis while on therapy and for 2 months thereafter. Seven subjects with a median age of 59 (range 43–68) years and 2 (range 1–4) prior therapies have been enrolled, 4 and 7 of whom were relapsed/refractory to nucleoside analogues and rituximab, respectively. Five patients completed the intended therapy. Responses to date are as follows: Partial Response (n=3); Minor Response (n=3); SD (n=1). With a median follow-up of 7 months, no responding patient has progressed. Hematological but not infectious toxicities occurred with reversible grade IV neutropenia (n=3) and thombocytopenia (n=2), and prolonged pancytopenia (n=1) in a patient with systemic amyloidosis. These studies demonstrate that Campath-1H is active in patients with relapsed/refractory WM, with sufficient activity to continue enrollment in this two-stage study.
A Conditional Fibroblast Growth Factor Receptor-1 Derivative Stimulates Extensive Self Renewal of Genetically Modified Primary Hematopoietic Cells.
Authors: Lintmaer Ingrid , Weinreich Michael A. , Blau C. Anthony
Published: April 2020

Fibroblast growth factor receptor-1 (FGFR-1) signaling has been implicated in self renewal and proliferation of hematopoietic stem cells. We have studied the effect of a conditional FGFR-1 in genetically modified primary mouse bone marrow cells. The activity of the conditional FGFR-1 is dependent on the presence of a drug called a chemical inducer of dimerization (CID). For these studies we used a bicistronic vector encoding green fluorescence protein (GFP) as a marker and a fusion protein made up of the intracellular portion of FGFR-1 and the CID binding domain. To test the construct in vitro we plated transduced mouse bone marrow cells in 10% serum with or without the CID, AP20187 (100 nM). While the cells without CID died within two weeks, in the culture with CID the transduced, GFP marked cells proliferated. These hematopoietic cells have proliferated in vitro for over 30 weeks. This makes FGFr-1 only the second CID dependent receptor, other than mpl, that is able to support long-term self renewal of primary hematopoietic cells. By day 28 of culture cells had expanded from 106 to 1.2x1012 cells. The differentiation potential of these cells was tested by transplanting 8 million cells into each of 5 lethally irradiated (1050 cGy) congenic recipients. Four mice are alive 76 days post transplantation, with mean frequencies of GFP positive red cells, platelets, granulocytes, monocytes, B cells and T cells of 55%, 5%, 85%, 98%, 42% and 2%, respectively. These findings indicate that cells expanded under the influence of FGFR-1 signaling retain both lymphoid and myeloid repopulating ability. Additionally, CID-mediated activation of FGFR-1 induced a dramatic rise in genetically modfied red cells, granulocytes and monocytes in vivo. These findings point to a clear difference between CID induced activation of FGFR-1 compared to our previous studies using the thrombopoietin receptor, where effects on neutrophils and monocytes are modest and variable. The CID controlled activation of FGFR-1 may have utility for control of genetically modified hematopoietic cells.