bluuebunny/crossref_metadata_2025 huggingface dataset:
The Effect of Telomerase Antisense on the Differentiation of CD34+ Cells from Bone Marrow to Blood Cells.
Authors: Park Kyogn Hwa , Kim Byung Soo , Choi In Keun , Kim Seok Jin , Oh Sang Cheul , Seo Jae Hong , Choi Chul Won , Shin Sang Won , Kim Yeul Hong , Kim Jun Suk
Published: October 2019

Background: This study was designed to evaluate the effect of adenovirus-mediated telomerase antisense (TA) expression on cellular proliferation and apoptosis of CD34+ cells in the ex-vivo culture condition collected from normal bone marrow donors.

Methods: We used adenovirus vector containing recombinant antisense telomerase template RNA. Mononuclear cells were obtained from 21 bone marrow donors with normal marrow profile. Each samples were divided into two groups, consisted of the study group treated with telomerase antisense oligonucleotides(TA) and control group without treatment of TA. And then, CD34+ cells were purified from each sample with MACS method and were cultured with LTC-IC method. We evaluated the total and viable cell number, apoptosis degree and blood cell morphology at 2 weeks after LTC-IC culture, and compared between control and experimental groups.

Results: Total cell number of study group (2.3–3.3(median:2.6)X104) was not significantly different from that of control group(2.1–3.5(median:2.7)X104). Viable cell percentage of control and study groups were 71–83(median:76)% and 69–84(median:77)%, respectively, without significant difference. Apoptosis degree of study group (25–38%, median:32%) was significantly higher than that of control (13–27%, median 18%), Blood cell morphology of study group was more differentiated without blast cells (0%) than that of control group showing blast cells (4–9%, median 7%) at 2 weeks of culture.

Conclusion: We found that adenovirus-mediated antisense expression of telomerase RNA might accelerate the differentiation of hematopoietic stem cells with significant effect on the apoptosis degree. Further studies will be warranted to confirm these results and verify the mechanism of these phenomena.
Cell Cycle Related Stem Cell Gene Expression.
Authors: Dooner Gerri J. , Colvin Gerald A. , Dooner Mark S. , Quesenberry Peter J.
Published: October 2019

We have previously reported that marrow stem cells show changes in engraftment (Habibian, et al J Ex. Hem, 188:393–398, 1998), homing (Cerny et al., J Hematother Stem Cell Res11:913–922, 2002) and differentiation (Colvin et al., J Cell Phys199:20–31, 2004) phenotype as they transit a cytokine-driven cell cycle. mRNA and surface expression of adhesion proteins also change (Becker et al., Exp Hematol27:533–541, 1999). We have evaluated gene expression by Real-time PCR of murine lineage negative, Sca+ (Lin-Sca+) stem cells stimulated by Il-3, Il-6, Il-11 and Steel factor (at 0, 24 and 48h) and lineage negative Rhodamine low, Hoescht low (LRH) stem cells stimulated by TPO, Flt-3 and Steel at various points in cell cycle transit (0,32,40,48h). In Lin-Sca+ cells (4experiments, time 0) expression of the following genes in descending order was as follows: IKAROS, L-selectin, Pu-1, Gata-2, Pecam, Cd84, Rock-1, c-fms, FOG, Cxcr4, c-kit, Cd4. The following were either not expressed or expressed at very low levels: Il-11, Ccr4, Sdf-1, Gata-1, P-selectin and Vecam. A pattern of depressed gene expression in S-phase (24h) with subsequent recovery (48h) was seen with c-fms and c-kit. With LRH cells (2 experiments, time 0) approximate descending rank order of gene expression was Cd45r, Cd34, G-CSFR, Mac-1, GM-CSFR and Flt-3. Il7r was not detected. With cycle progression Cd34 and Sca-1 were markedly elevated while Mac-1 and c-mpl were decreased. The expression of GM-CSFR, G-CSFR, Cd45r and Cd4 showed variable fluctuation. Il-7r was negative throughout. These data show that primitive marrow stem cells express a wide variety of “hematopoietic genes”, that expression modulates with cell cycle transit and perhaps most importantly that observed changes in gene expression are reversible. This is consistent with the continuum theory of stem cell regulation.
Genome-Scale Analysis of Hematopoietic Regulatory Sequences by Digital Analysis of Chromatin Structure.
Authors: Stamatoyannopoulos John A. , Humbert Richard , Hawrylycz Michael , Wallace James , Dorschner Michael O. , Kohli Ajay , Li Qiliang , McArthur Michael , Sabo Peter J.
Published: October 2019

In vivo, cis-regulatory elements coincide with focal disruptions in chromatin structure that manifest as DNaseI hypersensitive sites (HSs). We developed a novel stochastic methodology for comprehensive, genome-wide mapping of DNaseI HSs in vivo. We show that 19–20bp genomic DNA tags can be used to effect genome-wide localization of individual DNaseI cutting events in nuclear chromatin, and that such tags can be generated and sequenced in large numbers from limited numbers of starting cells. We analyzed 257,000 tags from a hematopoietic cell line (K562) and applied a quantitative algorithm to discriminate statistically significant tag clustering events. Such tag clusters identified both known and novel functional elements of hematopoietic genes across the genome. A unique feature of this approach is that it permits unbiased evaluation of the chromatin context of regulatory sequences from disperse genomic loci. We observed surprisingly large differences in the chromatin structural configuration of a variety of active erythroid and hematopoietic regulatory sequences, suggesting a discrete hierarchy of nuclear organization that is not apparent with conventional assays. This approach can be applied readily to generate a comprehensive catalogue of cis-regulatory sequences active in hematopoietic stem cells.
Autologous Peripheral Blood Stem Cell Implantation as Therapeutic Angiogenesis for Severe Limb Ischemia Autologous Peripheral Blood Stem Cell Implantation as Therapeutic Angiogenesis for Severe Limb Ischemia.
Authors: Han Zhong Chao , Huang Pingping , Li Shangzhu
Published: October 2019

It is estimated that 100 million individuals suffer from severe limb ischemia worldwide. Here, we report a novel therapy for severe limb ischemia by transplanting autologous peripheral blood stem cells (PBSCs). Thirty patients with different limb ischemia were enrolled and randomized to either stem cell injection or standard therapy in the study. The patients in the transplant group received intramuscular injections of autologous PBSCs collected after G-CSF-induced stem cell mobilization. Lower limb pain, ulcer repair and wound healing and blood perfusion were improved in the cell treated group. All cell treated patients achieved successful limb salvage. Analysis by digital subtraction angiography demonstrated the formation of new collateral vessels in the ischemic limbs of transplanted patients after cell injection. In contrast, no significant improvement was observed in the control patients. These results provide pilot evidence indicating that transplantation of autologous PBSCs is a simple and effective therapeutic angiogenesis for limb ischemia. To determine the mechanism of action, several animal experiments were performed by transplanting human PBSCs via tail vein into nude mice. It was found that the transplanted cells survived and incorporated into capillary networks in murine ischemic limb. Laser Doppler Perfusion Image (LDPI) and histological analysis revealed that the transplantation of human PBSCs augmented blood flow and neovascularization in the ischemic hindlimb, ameliorated necrosis and improved limb salvage. Our results demonstrate that PBSCs are excellent cell population capable of augmenting neovascularization.
The Effect of Telomerase Antisense for the Differentiation of Embryonic Stem Cells to Hemopoietic Stem Cells.
Authors: Park Kyong Hwa , Kim Byung Soo , Choi In Keun , Kim Seok Jin , Oh Sang Cheul , Seo Jae Hong , Choi Chul Won , Shin Sang Won , Kim Yeul Hong , Kim Jun Suk
Published: October 2019

Background: This study was designed to evaluate the effect of adenovirus-mediated telomerase antisense(TA) expression on cellular proliferation and apoptosis of the ex-vivo culture from human embryonic stem cells to hematopoietic stem cells.

Methods: Cultured embryonic stem cell lines (SNU-3 cell line) to hemopoietic stem cells were classified to control and study groups. Control group was that of which embryonic body were cultured to LTC-IC assay not treated with telomerase antisense oligonucleotides(TA). Study group was that of which embryonic body were cultured to LTC-IC assay treated with TA. We evaluated CD34+ cell number and viable cell percentage and the degree of apoptosis of each samples at 5 days after LTC-IC assay and the results were compared between each groups. These studies were tried for 17 times.

Results: The number of CD34+ cells in study group 0.8–2.4(median: 1.5X103) showed no significant difference with that in control group 0.6–2.9(median: 1.6X103). Viable cell percentage of control and study groups were 76–89(median: 83) % and 78–90(median: 85) %, respectively, without significant difference. And there was no difference in the degree of apoptosis (absorbance, A405nm – A490nm) between control and study groups; 0.092–0.134(median: 0.129) vs 0.087–0.152(median 0.138), with reference to negative control: 0.020, positive control: 0.186. The colony count of CFU-GM in control and study group was 0.1–0.5(medium: 0.3) X103 and 0.2–0.5(medium: 0.3) X103, respectively without no significant difference.

Conclusion: We found that adenovirus-mediated antisense expression of telomerase RNA didn’t interrupt the differentiation of embryonic stem cells to hematopoietic stem cells without significant effect on the degree of apoptosis. Further studies will be warranted to confirm these results and verify the mechanism of these phenomena.
Enhanced HDAC (Histone Deacetylase) Enzyme Activities in Blood CD 34 + Cells in Patients with Agnogenic Myeloid Metaplasia.
Authors: Wang Jen-Chin , Hemavathy Kirugaval , Suppiah Kathir , Chang Tsong S. , Novetsky Allan D.
Published: October 2019

We previously reported that Blood TIMP-1, TPO levels are elevated and blood MMP-3, bone marrow SDF-1 levels are decreased in patients with Agnogenic Myeloid Metaplasia (AMM). We postulated that these finding are likely related to the defects in TGF-β 1 pathway especially the TGF-β Type II (TGF β RII) receptor defects, resulting in enhanced TGF-β 1 activity. Decreased TGF β RII mRNA has been reported in blood CD 34 + cells previously. HDAC have been found lately to play an important role in the transcriptional repression and silencing of genes in cancer and HDAC inhibitors have proven to be useful tools in the treatment of cancer by reversre the silenced genes. We therefore measured HDAC activity in Blood CD 34 + cells to further explore the mechanism of the TGF β R II defects in this disease. HDAC activity was measured using the HDAC Colorimetric Activity Assay/Drug discovery Kit (BIOMOL Research Lab. Inc. PA). Nine patients with AMM and other patients with myeloproliferative diseases and normal volunteers used as controls were studied. The HDAC activities were expressed as the amount of the released Deacetylated Products (uM) as shown in Table 1.

This preliminary study suggests that HDAC activity is enhanced in some patients with AMM, and could be responsible for the decreased TGF β RII expression. HDAC inhibitors could therefore serve as valuable tools in the treatment of this disease.

Table 1

AMM MPD Controls Mean ± SE 19 ± 9.2 1.05 ± 0.54 2.10± 0.71
Xeroderma Pigmentosum as a Model for Treatment of Bone Marrow Failure in Fanconi Anemia and Aplastic Anemia.
Authors: Lambert W. Clark , Centurion Santiago A.
Published: October 2019

We have previously shown that the primary cell cycle defect in the inherited, cancer-prone, bone marrow failure associated disease, Fanconi anemia (FA), is not in the G2 phase of the cell cycle, as had been thought for many years, but rather in the S phase. FA cells challenged with the DNA cross-linking agent, psoralen coupled with long wavelength, ultraviolet (UVA) radiation (PUVA), fail to slow their progression through the S phase of the subsequent cell cycle, as do normal cells. FA cells are extremely sensitive to the cytotoxic and clastogenic effects of DNA cross-linkers, such as PUVA, so much so that the diagnosis of FA is based on an assay, the “DEB test”, in which cells are examined for clastogenic and cytotoxic effects of diepoxybutane (DEB), a DNA cross-linking agent. More recently, we have shown that artificially slowing the cell cycle of FA cells exposed to PUVA by subsequent treatment with agents which slow their progression through S phase leads to markedly increased viability and reduced chromosome breakage in vitro. We now show that similar results can be obtained in vivo in patients with another DNA repair deficiency disease, xeroderma pigmentosum (XP), a recessively inherited disorder associated with defective repair of sunlight induced adducts in the DNA of sun-exposed tissues followed by development of numerous mutations causing large numbers of cancers in these same tissues. We treated two patients with XP, a light complected black male and a white female, both 14 years of age, in sun-exposed areas with 5-fluorouracil, an inhibitor of DNA synthesis, daily for three months. In contrast to normal patients, who only show clinical results if an inflammatory response is invoked, marked improvement in the clinical appearance of the skin was seen with no inflammation observed. This effect was confirmed histologically by examining epidermis adjacent to excised lesions in sun-exposed areas and further verified by computerized image analysis. Treatment with agents that slow progression through S phase, such as hydroxyurea, may similarly improve clinical outcomes in patients with FA or others who are developing bone marrow failure.
Ectopic Expression of Mer Receptor Tyrosine Kinase in Childhood T Cell Acute Lymphoblastic Leukemia.
Authors: Salzberg Dana B. , Keating Amy K. , Cao Jian-zhe , Sather Susan , Hill Kimberley , Anwar Adil , Graham Douglas K.
Published: October 2019

Tyrosine kinases play an important role in normal cellular growth and differentiation. Deregulation of tyrosine kinase activity can result in cellular transformation leading to the development of human cancer. The Mer receptor tyrosine kinase, initially cloned from a human B lymphoblastoid cell line, is expressed in a spectrum of hematopoetic, epithelial, and mesenchymal cell lines. Interestingly, while the RNA transcript of Mer is detected in numerous T and B lymphoblastic cell lines, Mer RNA is not found in normal human thymocytes, lymphocytes or in PMA/PHA stimulated lymphocytes. We have developed an anti-human Mer monoclonal antibody to further study the ectopic Mer expression in lymphoblasts. The antibody detects several forms of the Mer protein including a fully glycosylated 205 kD Mer protein in monocytes and a less glycosylated 170–180 kD Mer protein in acute lymphoblastic leukemia (ALL) cell lines. We analyzed lymphoblasts from 16 T cell ALL patients diagnosed between July 1995 and July 2004 at The Children’s Hospital, Denver for Mer protein surface expression. Of the T cell ALL patient samples, we found that 9/16 (56%) were positive for Mer protein surface expression. Although we were not able to statistically evaluate the clinical outcomes relative to Mer expression due to the study sample number, there was a statistically significant association between positive expression of Mer and lack of surface expression of CD3 (p = 0.035). We found that 7/9 (78%) of the Mer positive lymphoblasts lacked CD3 surface expression, while only 1/6 (17%) of the Mer negative samples lacked CD3 surface expression (see figure). Lymphoblasts that lack surface expression of CD3 represent an immature phenotype and have been associated with a decreased event free survival as compared with surface positive CD3 lymphoblasts. Further investigation of the ectopic expression of Mer in lymphoblasts may reveal the use of this novel Mer glycosylated protein as a prognostic marker and possibly a future therapeutic target in the treatment of childhood leukemia.

Figure Figure
Effect of hTERT Antisense on Cisplatin Sensitivity of Acute Lymphoblastic Leukemic Cells.
Authors: Li Wenyu , Zhang Yuan
Published: October 2019

AIM: To investigate the effect of human telomerase reverse transcriptase(hTERT) antisense phosphorothioate oligodeoxynucleotide(AS PS-ODN) on enhancing cisplatin sensitivity in acute lymphoblastic leukemic cells.

METHODS: The expression levels of hTERT protein were detected by immunofluorescence using fluoresce isothiocyanate(FITC) lable. Cell surviving fraction was determined using the trypan blue dye exclusion assay. Apoptosis was detected by morphological observation and flow cytomertric cell cycle analysis.

RESULTS: The expression of hTERT protein was inhibited after treated by hTERT AS PS-ODN. Treatment with cisplatin after 24h of exposure to AS PS-ODN had significantly reduced the number of viable ALL cells. However, there was no difference on ALL cells survival between sense oligodeoxynucleotide(S PS-ODN)/CDDP combination and CDDP treated cells alone. In morphological observation of apoptotic cells using Hoechst 33258 and PI double staining techniques, cells displayed classic apoptotic changes treated with CDDP or CDDP combined with hTERT AS PS-ODN or S PS-ODN at 48h. Apoptotic rates of cells added CDDP and AS PS-ODN were higher than that of cells added CDDP only(P<0.05). Apoptotic rates of cells added CDDP and S PS-ODN were similar with that of cells added CDDP only(P>0.05).

CONCLUSION: hTERT AS-ODN inhibited the expression of hTERT protein and increased the CDDP induced apoptosis in primer acute lymphoblastic leukemic cells.
Single Agent Gemtuzumab Ozogamycin (Mylotarg) Is Effective in Inducing Hematologic Remission in Elderly Patients with Normal Cytogenetics.
Authors: Agha Mounzer E. , Sarah Miller , Kirkland Howard , Crandall Theodore L. , Mucenski John W.
Published: October 2019

The treatment of elderly patients with acute myelogeneous leukemia is quite difficult primarily due to the treatment related morbidity and the poor response rate to induction chemotherapy. Elderly patients are often offered supportive care only. Mylotarg has been shown to be an active agent in patients with relapsed AML with a reported response rate of 31%. The role of Mylotarg in front-line therapy for AML remains to be determined.

Patients : Six patients with newly diagnosed acute myelogenous leukemia (non-M3) were treated with single agent Mylotarg. The mean age was 77 years (71–84). All patients had low white blood cell counts upon presentation with a mean WBC of 1550. Several patients had other co-morbid conditions upon diagnosis, primarily cardiac. The average ECOG performance status before treatment was 1-1.

Treatment Plan : The treatment consisted of Mylotarg 9 mg/m2 on days 1 and 14. The treatment was given either in the hospital or as an outpatient. All patients received full doses of therapy. No further consolidation therapy was given. The bone marrow was re-evaluated within 60 days of the second dose of Mylotarg.

Results : One patient died during induction of sepsis and is not evaluable. All patients developed neutropenic fevers and required hospitalizations. One patient developed clinical evidence of pulmonary fungal infection and was treated successfully with oral voriconazole. No other end organ toxicity was encountered.

Response : Four of 5 evaluable patients (80%) achieved complete remission as documented by bone marrow evaluation. All four patients became transfusion independent, although the platelet counts remained below 100x109. The remission duration is 5.5+ months with two patients remaining in remission more than 6 months following treatment. All four responders had normal bone marrow cytogenetics upon diagnosis. The patient who died of early sepsis had complex cytogenetics and the patient who did not respond also had abnormal cytogenetics. The treatment was well tolerated despite the neutropenic hospitalizations and all responders regained normal performance status.

Conclusion : These results are very encouraging despite the small patient sample and suggest that Mylotarg as a single agent may be an effective therapy for elderly patients with normal cytogenetics. This treatment is less toxic than standard induction chemotherapy that may be poorly tolerated in this patient population. We are currently investigating the role of further Mylotarg consolidation in maintaining the duration of remission in those patients responding to initial therapy.
CD123 Expression in Chronic Lymphocytic Leukaemia - Strong Correlation with ZAP70 and CD38 Expression.
Authors: Petrie James A. , Milne Tim , Ridler Colin , Cavenagh Jamie D. , Agrawal Samir G.
Published: October 2019

CD123 constitutes the alpha subchain of the human interleukin 3 receptor (IL-3R). Interleukin 3 binds to its receptor with high and low affinities, induces tyrosine phosphorylation and promotes the proliferation and differentiation of haematopoietic stem cells. CD123 expression has been associated with a poor prognosis in AML and has been shown to be a useful marker in hairy cell leukaemia (HCL). However, it has been little studied in CLL, with data in the literature suggesting few cases of positivity. Here we studied CD123 expression by flow cytometry on fresh peripheral blood in 134 unselected, consecutive cases of CLL. We utilised anti CD123 PE (BD Pharminogen) in a dual combination with anti CD19 FITC (Coulter Immunotech) to target B cells. The results were correlated with expression of CD38 and ZAP70, markers of poor prognosis in CLL. CD123 was expressed in = 20% (median 33, range 20–92%) of cells in 32/134 cases (24%). Typical of CLL, the intensity of surface CD123 expression was weak-moderate, unlike HCL cases, where the intensity is moderate-strong. CD38 positivity was seen in 20/32 CD123+ cases (63%). Of the remaining 102 cases where CD123 was negative, a CD38 count was available in 92 cases and 29/92 (32%) were positive. ZAP70 positivity was seen in 18/32 CD123+ cases (56%). ZAP70 data was available in 98/102 CD123− cases and positivity was seen in 26/98 (27%). These findings represent the first account of a significant percentage of CLL cases expressing CD123 positivity. Furthermore we have been able to show that CD123 positivity strongly correlates with two known markers of poor prognosis ZAP70 (p= 0.004) and CD38 (p=0.004) (Chi-Square with Yate’s correction). CD123 may therefore be of prognostic significance in CLL and should be further investigated.
Effect of Administration Styles of Arsenic Trioxide on the Intracellular Arsenic Concentration and the Efficiencies of Differentiation and Apoptosis.
Authors: Zhou Jin , Meng Ran , Yang Baofeng
Published: October 2019

Objective: Study on the effects of As2O3 on intracellular arsenic concentration, differentiation and apoptosis rates and sensitivities by different administration styles.

Methods: Evaluated the intracellular arsenic concentrations,the apoptosis rates, and the ratios of differentiation phenotypes on cell surface CD33 (−)/CD11b (+), when incubated cells with As2O3 in different culture media in vitro and treated leukaemia patients with As2O3 in different administration styles in vivo.

Results: When incubated cells in a constant arsenic concentration culture medium for 24 hours, the intracellular arsenic concentrations were (33.4±0.88) μg/L in NB4, (18.6±1.12) μg/L in K562, and (28.8±0.64) μg/L in APL; the apoptosis rates were NB4 56.6%, K562 27.6%, APL 52.2%; the ratios of differentiation phenotype CD33(−)/CD11b(+) were: NB4 (19.5±0.5)%, K562 (24.5±0.6)%, APL (18.5±0.6)%, whereas, when incubated cells above in a changing arsenic concentrations culture medium for 24hours, which initial arsenic level was 5μmol/L, the intracellular arsenic concentrations were: NB4(17.6±0.88) μg/L, K562(9.2±0.64)μg/L, APL(15.2±1.04)μg/L, the apoptosis rates were: NB4 23.2%, K562 11.0%, APL 21.0%; the ratios of differentiation phenotype CD33 (−)/CD11b (+) were: NB4 (51.5±0.4)%, K562 (57.5±0.8)%, APL (46.5±0.5)%. 24hours after one time continuous and slowing intravenous As2O3 infusion, the intracellular arsenic concentrations were APL (26.6±2.5) μg/L,AML-M2 (15.5±3.1) μg/L, CML (18.5±2.3) μg/L; the apoptosis rates were APL 28.5%, AML-M2 9.5%, CML 12.5%; the ratios of differentiation phenotype CD33 (−)/CD11b (+) were: APL (29.5±0.5)%, AML-M2 (28.5±0.6) %, CML (23.5±0.5)%. Whereas, when 24hours after treated with one time general speed intravenous As2O3, the intracellular arsenic concentrations were: APL(12.3±2.1) μg/L, AML-M2 (5.5±2.3) μg/L, CML (8.5±2.7) μg/L; the apoptosis rates were: APL 13.2%, AML-M2 3.9%, CML4.2%; the ratios of differentiation phenotype CD33 (−)/CD11b (+) were: APL (53.6±0.8) %, AML-M2 (48.5±0.9) %, CML (67.5±0.8) %.

Conclusions: The administration styles affect the intracellular arsenic concentrations, the differentiation rates and the ratios of apoptosis of leukaemia cells. Treated with the continuous and slowing intravenous As2O3 infusion can obtain a higher intracellular arsenic concentration, a higher efficiency of apoptosis and a lower differentiation ratio than that in general speed intravenous As2O3 infusion in clinical dosage. The continuous and slowing intravenous As2O3 infusion can promote apoptosis and inhibit from differentiation, so this As2O3 medication can relieve leukocytosis and elevate therapeutic effects.
Comparison of Pharmacokinetics and Pharmacodynamics of two different dosing regimens of Proleukin® (IL-2) in Combination with rituximab: The effect of NK cell expansion.
Authors: Milan Sandra , Hurst Deborah , Michelson Glenn , Samara Emil
Published: October 2019

Background: Published results from studies testing two different dosing regimens of IL-2 in combination with rituximab have shown that thrice-weekly dosing of IL-2 resulted in greater expression of NK cell as compared to a daily dosing (Gluck, et al. Clin Cancer Res 2004). Expanded NK cells are desired to drive effective rituximab induced ADCC. Here we report on the observed pharmacokinetic (PK) differences associated with the differences in pharmcodynamics (PD) observed with the two regimens.

Methods: Two different subcutaneous (SC) IL-2 dosing regimens in combination with rituximab (375 mg/m² weekly IV infusions weeks 1 through 4) were studied: daily IL-2 (6 MIU daily weeks 2–5) versus thrice-weekly IL-2 (14 MIU weeks 2–5) resulting in a total of 42 MIU weekly for each regimen. Serum samples for IL-2, rituximab, and soluble IL-2 receptor (sIL-2r) were analyzed using an ELISA assay. Anti-IL-2 and anti-rituximab antibodies (Ab) were also determined. IL-2 and rituximab serum concentration-time data were analyzed by model-independent methods using WinNolin.

Results: Daily dosing of IL-2 exhibited time-dependent pharmacokinetics characterized by lower concentrations of IL-2 upon repeat SC dosing. An increase in IL-2 clearance coincided with an increase in sIL-2r concentrations in the systemic circulation. By the fifth dose, sIL-2r concentrations reached steady state at approximately 2–3 times higher concentrations than the baseline value. In contrast, the thrice-weekly dosing of IL-2 demonstrates no time-dependent kinetics; there were no observed differences in IL-2 plasma concentrations after the first dose or after repeated dosing. Surprisingly, a similar increase in sIL-2r levels from baseline was observed after multiple thrice-weekly dosing of IL-2. The pharmacokinetics of rituximab was similar after daily or thrice-weekly regimens of IL-2 and were comparable to values reported in the literature. The incidences of Ab against IL-2 and rituximab were low (<7%).

Conclusion: Concomitant administration of IL-2 with rituximab is well tolerated, with no evidence of pharmacokinetic interaction. Thrice-weekly dosing regimen results in more sustained plasma IL-2 levels with repeated dosing than daily IL-2 dosing. Hence the sustained plasma levels and AUC associated with the thrice-weekly dosing potentially explains why this regimen results in greater NK cell expansion that does the daily dosing.
Mobilization of Ph-Negative Peripheral Blood Stem Cells (PBSCs) with rHu-G-CSF in Chronic Myeloid Leukemia (CML) Patients Achieving Complete Cytogenetic Response with Imatinib Mesilate (Glivec).
Authors: Pungolino Ester , Draisci Michela , Gargantini Livio , Farioli Renata , Rossi Valentina , Cozzi Paola , Alamos Sara Miqueleiz , Cafro Anna Maria , Morra Enrica
Published: October 2019

Imatinib Mesilate (Glivec) is able to induce a high rate of Complete Cytogenetic Response (CCyR) with low treatment-related toxicity. The long-term duration of this Ph-negative status is, however, still undefined. Therefore, some groups (Drummond et al, Br J Haematol123:479, 2003; Hui et al, Leukemia17:821,2003) decided to mobilize and to collect Ph-negative PBSCs in patients reaching CCyR with Glivec. In our experience we studied 12 Ph-negative, FISH-negative patients, median age 50 yrs (26–61). Three of them were also PCR-negative, being the minimal residual disease assessed using RT-PCR. All patients received Imatinib Mesilate at the daily dose of 400 mg. The median duration of treatment with Glivec was 25 months (range 11–35). Eight of 12 pts (67%) were pre-treated with α-interferon (α-IFN) for a median time of 9.5 months (range 3–54). The median duration of CML was 39 months (range: 21–152).

We discontinued Imatinib four days (range 3–5) before starting rHu-G-CSF as single agent (5–10 μg/Kg/day), which was maintained until WBC < 50x109/L. Failure of mobilization was defined as CD34+ circulating cells < 10/m L. The collection started after a median of 6 days (range 4 –10), when CD34+ cells were > 10/μ L. Eleven of 12 pts (92%) reached the threshold of 10/μ L and started harvesting. The single patient failing collection had been pretreated with IFN and was PCR negative at the time of mobilization. The remaining 11 reached the target of 2.5 x106 CD34+ cells/Kg with a median of two apheresis (range 1–3). The median harvest was 3.14 CD34+ x 106/Kg (range 2.56–4.55). Conventional cytogenetic analysis, RT-PCR and coltural tests were performed in all apheretic samples. All the 11 mobilized patients obtained a Ph-negative harvest, which was also PCR negative in 4/11 (36%).

In our experience adequate yields of PBSCs were collected with rHu-G-CSF alone in 11/12 patients achieving CCyR with Glivec, as well as in the majority of those previously treated with α-IFN (7/8).

Results of harvesting after rHu-G-CSF alone in 11 CML patients achieving CCyR after Imatinib Mesilate

Patient N. Apheresis CD34+ x 10^6/kg RT-PCR T.M 2 4.55 pos. O.I 2 3.34 neg. V.E. 1 3.33 neg. S.G. 3 3.07 neg. R.R. 1 4.45 pos. M.G. 3 2.56 pos. S.C. 1 3.2 pos. S.C. 2 2.6 pos. C.A. 1 3.75 pos. T.G. 2 3.14 neg. V.M. 2 2.65 pos.
Unlike CML Patients, Patients with Gastrointestinal Stromal Tumors (GIST) Treated with Imatinib Do Not Develop Hematopoietic Clonal Cytogenetic Abnormalities.
Authors: Abecasis Manuel M. , Lage Pedro , Salazar Maria , Freire Joao , Moreira Antonio , Jorge Margarida , Alaiz Helena
Published: October 2019

Imatinib has the ability to inhibit the dysregulated kinase activities of the BCR-ABL fusion protein in CML pts as well as the mutated KIT in GIST patients and has been successfully used to treat both diseases. Several cases of chromosomal abnormalities in Ph-negative metaphases have been reported in CML patients in chronic phase during treatment with imatinib. This is different from true clonal evolution in that the additional cytogenetic abnormalities occur in Ph-negative cells. Although this has also been rarely reported in patients receiving other therapies its frequency in patients on imatinib is relatively high (up to 17% patients) and it may occur when imatinib is given upfront or after other treatment modalities.The most common abnormalities are trisomy 8 and chromosome 7 defects, often associated with myelodysplasia. The time from the beginning of treatment until their appearance is variable, ranging from 3 months to more than a year, with the largest published series reporting a median of 6 months (Cancer2003; 98:1905). The pathogenesis and clinical significance of these findings is still unclear and an important question is whether these clones are induced or favored by the treatment itself, since imatinib is known to have several effects on important growth regulatory proteins involved in hematopoiesis. GIST patients without hematological disease and treated with imatinib offer a unique opportunity to address this question. From Aug 2001 to Aug 2003, 11 patients with GIST were treated with imatinib at our institution and 7 agreed to participate in the sutdy. Their median age was 54-y and they were on imatinib for a median of 18 months (4 to 18) at doses between 400 to 800mg daily. Complete karyotyping on metaphase spreads from peripheral blood and bone marrow aspirates were performed. PB and BM smears were examined for myelodysplastic features. A median of 19 (15 to 24) PB and 16 (13 to 22) BM metaphases were examined for each patient. No abnormal hematological clones were detected and 1 patient had a constitutional variant 46XY, (inv9)(p11;q12) on PB and BM cells. No dysplastic features were observed. These data, although based on a small number of patients suggest that in patients without hematological disease imatinib by itself does not induce hematopoietic clonal cytogenetic abnormalities.
Characterization of Oncogene Dysregulation in Multiple Myeloma by Combined FISH and DNA Microarray Analyses.
Authors: Neri Antonino , Fabris Sonia , Agnelli Luca , Mattioli Michela , Baldini Luca , Ronchetti Domenica , Morabito Fortunato , Verdelli Donata , Nobili Lucia , Intini Daniela , Callea Vincenzo , Stelitano Caterina , Lombardi Luigia
Published: October 2019

Chromosomal translocations involving the immunoglobulin heavy chain (IGH@) locus and variuos partner loci are frequently associated with multiple myeloma (MM). We investigated the expression profiles of FGFR3/MMSET, CCND1, CCND3, MAF and MAFB genes, respectively involved in t(4;14)(p16.3;q32), t(11;14)(q13;q32), t(6;14)(p21;q32), t(14;16)(q32;q23) and t(14;20)(q32;q12), in purified plasma cell populations from 39 MMs and six plasma cell leukemias (PCL) using DNA microarray analysis, and compared the results with the presence of translocations as assessed by dual-color FISH or RT-PCR. The t(4;14) was found in six MMs, t(11;14) in 9 MMs and 1 PCL, t(6;14) in one MM, t(14;16) in 2 MMs and 1 PCL, and t(14;20) in one PCL. The translocations were associated with the spiked expression of target genes in all cases. Furthermore, gene expression profiling allowed the identification of putative translocations dysregulating CCND1 (1 MM and 1 PCL) and MAFB (1 MM and 1 PCL) without any apparent involvement of immunoglobulin loci. Notably, all of the translocations were mutually exclusive. Markedly increased levels of MMSET expression were found in one MM showing associated FGFR3 and MMSET signals on an unidentified chromosome. Our data suggest the importance of using combined molecular cytogenetic and gene expression approaches to detect genetic aberrations in MM.
Renal Impairment and the Use of Bisphosphonates in Patients with Multiple Myeloma: Retrospective Analysis of 114 Patients with Respect to Age and Other Clinical Characteristics.
Authors: Mazj Sam , Lichtman Stuart M.
Published: October 2019

Backgound: The clinical course of multiple myeloma is often associated with significant bone related morbidity. This can be modified by the use of bisphosphonates. Studies have shown renal toxicity associated with different bisphosphonates especially following the exposure of zoledronic acid. Published literature suggests renal function deterioration occurs in 8.8–15.2% of patients at recommended dose of 4 mg infused intravenously over 15 minutes.

Methods: We retrospectively reviewed the records of all patients with multiple myeloma who received bisphosphonates during the period of January 2002-June 2004 at our institution. 114 patients were analyzed (male/female 63/51; age-median 69;mean 71;range 40–92). 61 (54%) were >70 years of age. They received a total of 1301 doses (mean 11.4) during this period. The type of bisphosphonate used was: zolendronate: 58; pamidronate: 23; pamidronate changed to zolendronate (both) : 33. Patients were categorized to the type and sequence of bisphosphonates [ pamidronate vs. zoledronate and pamidronate followed by zoledronate (both) ], age and sex. Renal dysfunction was defined as an increase in serum creatinine of >0.5 mg/dl over baseline.

Results: There were 19 patients (16.7%) who developed renal dysfunction. 15 of the 19 episodes (79%) occurred in the 70 years and older group. The table shows the distribution of patients, type of bisphosphonate and the distribution of patients with renal toxicity.

Conclusion: This analysis showed increase in renal dysfunction occurs in all ages with use of bisphosphonates. The elderly may be particularly susceptible to this toxicity. Although we have not analyzed the impact of associated comorbidities (including type of multiple myeloma) leading to renal insufficiency in this study, the elderly patients may need more close monitoring of renal function with the use of bisphosphonates.

Age and renal impairment with bisphosphonate use

Age Zolendronate Pamidronate Both Total Number in paranthesis indicates the renal impairment case 40–49 6 (0) 2 (0) 2 (0) 10 (0) 50–59 10 (0) 1 (0) 4 (0) 15 (0) 60–69 14 (2) 3 (0) 11 (1) 28(3) 70–79 19 (3) 9 (1) 14 (4) 42 (8) 80+ 9 (3) 8 (0) 2 (4) 19 (7) Total 58 (9) 23 (1) 33 (9) 114 (19)
Lymphoplasmacytic Cells and Mast Cells Are Targets for Imatinib Mesylate (Gleevec, Glivec) in Waldenstrom’s Macroglobulinemia.
Authors: Tournilhac Olivier , Santos Daniel Ditzel , Xu Lian , Hatjiharissi Evdoxia , Tsu Yu , Catley Laurie , Hunter Zachary , Branagan Andrew , Burger Renate , Shringarpure Reshma , Boyce Joshua , Anderson Kenneth C. , Treon Steven P.
Published: October 2019

Recently, we demonstrated that in Waldenstrom’s macroglobulinemia (WM), clonal lymphoplasmacytic cells (LPC) appear to derive growth and survival signals from excess mast cells (MC) present in the bone marrow (BM). (Tournilhac et al, JCO 2004 22:571S). We therefore have sought agents which could target both LPC and MC. One such candidate is imatinib mesylate which has shown activity in certain mast cell disorders likely on the basis of its ability to target the tyrosine kinases, CD117 (Stem Cell Factor Receptor, c-kit), and Platelet Derived Growth Factor Receptor (PDGFa-R). By flow cytometric analysis, we demonstrated CD117 expression on sorted LPC from 17/22 (76.2%) WM patients, above 20% in 13 cases, whose expressionwe confirmed by RT-PCR analysis. Moreover, we also demonstrated expression of PDGFa-R along with its ligand PDGFa in BM LPC from 16/18 (88.9%) and 7/10 (70%) WM patients.. In addition to CD117, we found by RT-PCR analysis a PDGFa expression in LAD and HMC-1 MC lines and in KU the basophilic cell lines KU as well as in sorted BM MC (FcER1+ CD117+) from 3/10 (30%) WM patients. Importantly, co-culture of sorted LPC from WM patients along with 0.5% paraformaldehyde fixed LAD cells or ex vivo expanded (EVE) BM MC induced proliferation of sorted LPC from WM patients, which was inhibited at pharmacologically achievable levels of imatinib mesylate with an IC50 of 0.5–10 μmoles/L. Moreover, imatinib mesylate inhibited the LAD, HMC-1 and KU cell lines as well as EVE cord blood MC or EVE BM MC at an IC50 of 0.01–1 μmoles/L. Lastly, imatinib mesylate also inhibited proliferation of both MC and LPC when unfixed LAD or ex vivo MC were incubated with WM LPC with an IC50 of 1–10 μmoles/L. These studies therefore demonstrate that both LPC and MC are therapeutic targets for imatinib mesylate, and provide the framework for clinical studies evaluating its use in WM.
First-Line Treatment of Multiple Myeloma with a Combination of Thalidomide, Dexamethasone, and Zoledronate (TDZ) in an Inner-City Population with High HIV Prevalence.
Authors: Klueppelberg Uwe , Smith Eric , Chen Laurie , Aloba Chona M. , Shapira Iuliana , Moezi Mehdi M. , Batuman Olcay
Published: October 2019

Background: The combination of thalidomide and dexamethasone is a rescue regimen for multiple myeloma (MM) in relapse, and for pretransplant in newly diagnosed patients. The bisphosphonate zoledronate mitigates bone resorption and possibly tumor growth and angiogenesis. The long-term utility of this combination in a newly diagnosed inner-city MM population with high prevalence of HIV-infection is addressed in this phase II trial. MM is reported with increased frequency in patients with HIV infection, and optimal treatment of MM in these patients is unknown.

Methods: Of 30 consecutive enrollees, 22 (16F/6M) were evaluable. Mean age was 61 years (range = 43–82); all had skeletal lesions, and 27% (n = 6) were HIV+ (mean age = 47, range 46–50, all female). Patients received thalidomide 100 mg QD, dexamethasone 10–40 mg PO on Days 1–4, 9–12, and 17–20 monthly for six months, then on Days 1–4 monthly; and zoledronate 4 mg IV monthly, until progression or relapse. Baseline β2-microglobulin indicated high risk for all patients (mean = 6.2 μg/ml, SD = 3.8). At enrollment, 4 patients had AIDS, and 3 were on HAART.

Results: Overall response rate (> 50% decrease in M protein) was 72% (n = 13), and 83% (n = 5) in HIV+ patients. In 27% (n = 6), M protein levels were < 0.3 g/dL, including 3 HIV+ patients on HAART. In 23% (n = 5), M protein was reduced by < 50%. Median time to response was 2 months, and mean time on TDZ was 11 months. TDZ treatment decreased serum β2-microglobulin (P <.001). Two patients relapsed, and one patient with HIV and concomitant plasmacytoma did not respond. All three eventually died. Response to treatment was unaffected by HIV status or antiretroviral treatment. Side effects of TDZ were reversible by anticoagulant therapy or dexamethasone reduction.

Conclusions: TDZ had significant activity against newly diagnosed MM, suggesting that zoledronate may facilitate response with lower doses of thalidomide. HIV status did not affect response, indicating that this regimen is compatible with concomitant antiretroviral treatment and is equally effective in HIV+ patients. These findings underscore the potential effectiveness of this combination of anti-myeloma agents as a long-term alternative, particularly in patients for whom transplant therapy is not feasible.
HOXB4 Enforces Equivalent Fates of ES-Cell-Derived and Adult Hematopoietic Cells.
Authors: Pilat Sandra , Sebastian Carotta , Bernhard Schiedlmeier , Ute Modlich , Kenji Kamino , Elke Will , Andreas Mairhofer , Peter Steinlein , Wolfram Ostertag , Hartmut Beug , Christopher Baum , Hannes Klump
Published: October 2019

In the context of somatic gene therapy of the hematopoietic system, transplantation of molecularly defined and, hence, “safe” clones would be highly desirable. However, techniques which allow gene targeting, subsequent in vitro selection and clonal expansion are only available for embryonic stem (ES−) cells. Previously, it has been shown that in vitro differentiated ES-cells engraft when ectopically expressing HOXB4, but it remained unclear whether these cells could fully resemble adult bone marrow function after transplantation1. We here demonstrate for the first time the functional equivalence of in vitro differentiated ES-cells and adult bone marrow cells mediated by HOXB4. Differentiated ES-cells expressing HOXB4 from a retroviral vector and grown in vitro for 20 days, recapitulated the growth and differentiation properties of adult bone marrow cells after transplantation into

Rag2(−/−)γC(−/−) and C57Bl/6J recipient mice. Furthermore, we show that the amount of ectopically expressed HOXB4 influences differentiation in both systems in a similar manner. HOXB4 enforced myeloid and suppressed T-lymphoid development over a wide range of expression levels, whereas only high expression levels of HOXB4 were detrimental for erythroid development (P-values for C57Bl/6J mice, Student’s t-test, 2-sided:

CD3 + : eGFPlow vs. HOXlow = 0.003; eGFPhigh vs. HOXhigh = 0.021;

Ter119 +: eGFPlow vs. HOXlow = 0.920; eGFPhigh vs. HOXhigh = 0.0122; HOXlow vs. HOXhigh = 0.005).

Incompatibility of high levels of HOXB4 expression with erythroid differentiation was also directly demonstrated using a recently described in vitro ES-cell differentiation system2. Histological analysis of the “HOXB4-transplanted” mice revealed increased granulopoiesis both in sternal bone marrow and in spleen sections. However, all stages of granulocytic differentiation were present and neither were immature cells detected in the periphery nor was leukemic infiltration detected in other organs. Hence, none of the animals became leukemic during the observation period. In summary, ES-cells should be considered a promising alternative to bone marrow stem cells carrying the potential of safe somatic gene therapy, provided that human ES-cells can be similarly manipulated. Nonetheless, advanced cell therapy will certainly require regulated expression of HOXB4 to avoid unwanted effects such as disturbed lineage differentiation. This work was supported by the German Research Foundation (KL1311/2-3 and 2-4), German Cancer Aid (10-1763-OS5) and the Austrian Industrial Research Promotion Fund (808714).
Specific Elimination of Alloreactive T Lymphocytes Using Photodynamic Therapy Prevents GVHD and Enables Rapid Immune Reconstitution.
Authors: Boumedine Radia Sidi , Krosl Gorazd , Vaillancourt Marc , Perreault Claude , Roy Denis-Claude
Published: October 2019

Graft-versus-host disease (GVHD) and impaired immune reconstitution are the primary obstacles limiting the efficacy of allogeneic stem cell transplantation (SCT).

Graft-versus-host disease (GVHD) and impaired immune reconstitution are the primary obstacles limiting the efficacy of allogeneic stem cell transplantation (SCT). The purpose of this study was to determine whether selective depletion of donor alloantigen-specific T lymphocytes using photodynamic therapy (PDT) would prevent GVHD and enable immune reconstitution in the context of MHC-mismatched SCT. This question was addressed in an MHC-incompatible mouse model of GVHD. The donor (C57BL/6; H-2b) derived spleen cells were first activated against C3H/HeJ (H-2k) host spleen cells in a one-way mixed lymphocyte culture and then exposed to photodynamic treatment, using dibromorhodamine methyl ester (TH9402) as a photosensitizer. Activated T cells showed preferential retention of this photosensitizer compared to resting lymphocytes. In addition, in vitro experiments revealed that PDT eradicated a significantly higher proportion of activated than resting T cells. When lethally irradiated H-2k mice (C3H/HeJ and B10BR) were transplanted with C57BL/6 derived T cell-depleted bone marrow cells supplemented with C57BL/6 derived spleen cells activated with C3H/HeJ targets, they rapidly succumbed to acute GVHD (within 10–47 days). In contrast, both mouse strains receiving histoincompatible C57BL/6 T cells previously exposed to PDT after activation against C3H/HeJ survived until the end of the observation period (>100 days)(p<0.0001). Additionally, transplantation of treated T cells induced lethal GVHD in C57BL/6 histoincompatible strains of mice (third party), suggesting PDT specifically eradicated activated T cells while sparing most resting T lymphocytes. Analysis of immune recovery, evaluating T and B cell populations in thymus and spleen, activated T cells components (CD44, CD62L, CD69), proliferative responses (anti-CD3 and conA), and Vβ repertoire indicated that T and B cell reconstitution in MHC-mismatched mice transplanted with treated primed cells was similar to that of mice transplanted with treated or control autologous cells indicating that immune cells were preserved and functional. These results demonstrate that PDT can selectively eliminate alloreactive T cells and prevent the development of GVHD, while sparing T cells reactive against non-host antigens, thus offering protection against infection and disease relapse.