bluuebunny/crossref_metadata_2025 huggingface dataset:
Chemokine Receptor Expression Profile in the Model of Parotid MALT Lymphomagenesis: De Novo Expression of CXCR6.
Authors: Deutsch Alexander J.A. , Aigelsreiter Ariane , Steinbauer Elisabeth , Linkesch Werner , Beham-Schmid Christine , Schaider Helmut , Neumeister Peter
Published: April 2020

Chemokine receptors mediate migration and activation of lymphocytes through binding of their ligands. Several recent studies have revealed important contributions of chemokine receptors and their ligands to the development and progression/dissemination of hematopoietic neoplasms. Strong expression of CXCR5 and its ligand BCA-1 was detected in transformed B cells in H. pylori positive gastric MALT lymphoma. Because the knowledge of chemokine receptor expression in parotid MALT lymphoma is limited, we performed a comprehensive study on tissue samples of parotid glands (P), parotid glands affected by Sjoegren Syndrome (SS), parotid MALT lymphoma (MALT) and extranodal diffuse large B cell lymphoma (eDLBCL). By investigating the expression of all 19 known chemokine receptor at mRNA levels by real time PCR using a semi quantitative approach and of 3 chemokine receptors (CCR1, CCR5 and CXCR6) at protein levels we propose a model of MALT lymphoma that the development from a non neoplastic event to Sjoegren Syndrome or malignant transformation is accompanied by significant deregulations in the chemokine receptor expression profile: in the development of SS CCR5 was 783 times up regulated and CCR6, CCR8 and CCR10 mRNAs were de novo expressed; in the progression process of SS to parotid MALT CCR7, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1 and XCR1 were de novo expressed and CCR10 lost its expression; during the transformation of MALT to eDLBCL, CCR1, CCR8 and CXCR3 were 13 times down regulated and CX3CR1 and XCR1 lost their expression. Expression of CCR1, CCR5 and CXCR6 were confirmed by immunohistochemistry. The present results support a model of a stepwise progression of parotid MALT lymphoma from a non neoplastic event to Sjoegren Syndrome and finally overt MALT lymphoma guided by differentially expressed chemokine receptors and their ligands.
Ocular Adnexal Marginal Zone B Cell Lymphoma Has Characteristic Deletion at Chromosome Band 6q23.3-24.1 Whose Target Is TNFAIP3.
Authors: Honma Keiichiro , Tsuzuki Shinobu , Nakagawa Masao , Karnan Sivasundaram , Kim Won-Seog , Kim Yoon-Duk , Ko Young-Hyeh , Seto Masao
Published: April 2020

Marginal zone B cell lymphoma (MZBCL) occupies a distinct disease entity in WHO classification, and it has been suggested that recurrent chromosomal aberrations, such as t(11;18)(q21;q21), t(14;18)(q32;q21), and t(1;14)(p22;q32) are associated with its lymphomagenesis. However, the frequency of chromosomal alteration in marginal zone B cell lymphomas varies according to the anatomical region, and genetic abnormalities other than recurrent translocations involved in the pathogenesis of MZBCL have not yet been fully identified. In this study, we aimed to investigate genomic aberrations in ocular MZBCL and to compare them with those of tumors from other anatomical sites by using genome-wide array comparative genomic hybridization (CGH). The study population comprised 24 cases of primary ocular MZBCL, 11 of pulmonary MZBCL, and seven of nodal MZBCL. FISH analysis for MALT1 gene alteration was performed for ocular and nodal MZBCL and RT-PCR for the detection of API2-MALT1 transcript was performed for pulmonary MZBCL. The recurrent genomic alterations of ocular MZBCL were losses of chromosomes 6q23.3(9/24, 38%), and gains of 3(9/24, 38%), 15(4/24, 16%), 18q(4/24, 16%). T(11;18)(q21;q21) was not detected. The genomic alteration of pulmonary MZBCL included recurrent loss of 13q34 (2/11, 19%). Fusion transcript of t(11;18)(q21;q21) was detected in five out of eight cases (63%). Nodal MZBCL showed neither recurrent genome alteration nor any change in MALT1 gene copy number. In conclusion of genome-wide array CGH analysis, the array CGH profile of ocular MZBCL is distinct from those of pulmonary and nodal MZBCL. The novel finding was recurrent deletion in the 2.9 Mb region at chromosome band 6q23.3–q24.1 including homozygous loss in ocular adnexal marginal zone B cell lymphoma. These findings suggest that deletion of chromosome band 6q23.3–24.1 may constitute a crucial genetic alteration in the lymphomagenesis of ocular MZBCL. For a further examination, we used contig bacterial artificial chromosome (BAC) array CGH, containing the 24 BAC clone to cover the 2.9 Mb region, to analyze nine cases with chromosome band 6q23.3–q24.1 loss. We narrowed the minimal common region down to a length of 586 kb with two genes and four expressed sequence tags (ESTs). All of these genes and ESTs were subjected to RT-PCR and real-time quantitative RT-PCR, and correlation between genomic loss and expression level was found only in TNFAIP3. We thus concluded that the TNFAIP3 gene is the candidate gene for this deleted region. TNFAIP3 is an inhibitor of NF-kB signaling so that loss of this gene may result constitutive activation of NF-kB which was also implicated in MALT lymphomas with t(11;18)(q21;q21) or t(14;18)(q32;q21). Thus, TNFAIP3 may act as a tumor suppressor gene in ocular adnexal marginal zone B cell lymphoma.
Decreased Hepcidin Level in Iron Deficiency Anemia after Successful Helicobacter pylori Eradication Therapy: A Preliminary Data.
Authors: Cho Yo-Han , Lee Sun-Young , Song Eun Young , Yun Yeo Min , Yoon So Young , Lee Mark H.
Published: April 2020

Background/Aim: Hepcidin, a hepatic antimicrobial protein, is usually over-expressed in iron deficiency anemia. However, whether gastric Helicobacter pylori (H. pylori) infection effects a change in hepcidin level in iron deficiency anemia is still uncertain. We evaluated whether H. pylori eradication decreased hepcidin level and increased hemoglobin and ferritin levels in iron deficiency anemia.

Method: From October 2006 to April 2007, nine females (mean age of 32.2 year-old) who were diagnosed as iron deficiency anemia without definite blood loss, were included in the study. All the subjects underwent upper gastrointestinal endoscopy and colonoscopy to diagnose gastric H. pylori infection and to exclude any source of gastrointestinal bleeding. Blood samplings for hemoglobin, hematocrit, iron (Fe), total iron binding capacity (TIBC), ferritin and hepcidin tests were taken just before H. pylori eradication and four weeks after H. pylori eradication and oral iron supplement therapy, respectively. Serum prohepcidin level was measured by Hepcidin Prohormone ELISA (Solid Phase Enzyme-Linked Immunosorbent Assay) kits. Statistical analysis was done by Wilcoxon signed rank test.

Result: H. pylori eradication was successful in all the subjects who revealed negative in urea breath test. Hemoglobin, hematocrit, Fe, TIBC and ferritin levels improved in all the subjects after H. pylori eradication (Table 1). Mean serum hepcidin level was 224.8 ± 23.2 ng/ml initially, but decreased to 179.7 ± 40.8 ng/ml after H. pylori eradication therapy (p = 0.015).

Conclusion: Our result provides evidence that hepcidin level decreases after successful H. pylori eradication with the improvement of iron deficiency anemia. A fall in serum hepcidin level resulting from successful H. pylori eradication reflects that hepcidin is an important mediator of iron absorption in iron deficiency anemia associated with gastric H. pylori infection.

Table 1. Results of blood tests before and after Helicobacter pylori eradication Before H. pylori eradication After H. pylori eradication p-value* *Wicoxon signed ranks test Fe, iron; TIBC, total iron binding capacity Hemoglobin (g/dl, mean ± SD) 7.67 ± 2.01 10.76 ± 2.43 0.008 Hematocrit (%, mean ± SD) 26.36 ± 5.76 34.26 ± 5.76 0.008 Fe (ug/dl, mean ± SD) 11.78 ± 4.21 45.00 ± 54.39 0.038 TIBC (ug/dl, mean ± SD) 480.78 ± 51.39 427.00 ± 66.30 0.008 Ferritin (ng/ml, mean ± SD) 2.67 ± 0.99 7.39 ± 6.38 0.021 Hepcidin (ng/ml, mean ± SD) 224.80 ± 23.23 179.70 ± 40.84 0.015
Erythropoietin Therapy Does Not Benefit Transfusion-Dependent Primary Myelofibrosis Patients and Treatment Response Is Infrequent with a Baseline Hemoglobin Level ≥ 10 g/dL.
Authors: Huang Jocelin , Lasho Terra L. , Li Chin Yang , Pardanani Animesh D. , Mesa Ruben A. , Tefferi Ayalew
Published: April 2020

BACKGROUND: The potential risks of tumor growth promotion and thromboembolism associated with erythropoietin (Epo) therapy warrant cautious use of erythropoiesis stimulating agents (ESA). Recent data suggest a surprising and significant association between ESA use and increased incidence of leukemic transformation in primary myelofibrosis (PMF) (data from an analysis separately submitted for American Society of Hematology meeting, 2007). It is prudent, therefore, to avoid unnecessary ESA exposure in PMF, especially in patients with low likelihood of treatment response.

METHODS: Diagnosis of PMF was based on World Health Organization criteria. Study inclusion required availability of treatment history with ESA and adequate follow-up. We excluded patients who were receiving hemoglobin-improving agents concomitantly with an ESA, such as prednisone, androgens, danazol, or thalidomide. Transfusion dependency was defined by a history of minimum 2 units of red blood cell transfusions in a 1-month period for a hemoglobin < 8.5 g/dL, not associated with bleeding. An adequate treatment response was defined as a minimum 2.0 g/dL increase in hemoglobin level or becoming transfusion-independent over a minimum 1-month period.

RESULTS: A master database of PMF patients seen at the Mayo Clinic from 1976 through 2006 was queried to identify 43 patients (median age 67 years; 35% females) treated with ESAs and in whom adequate follow-up was available to determine response. Prior to ESA treatment, median hemoglobin was 9.2 g/dL (range 7.0–11.9), and 16 (37%) were red blood cell transfusion-dependent. In 25 patients, pre-treatment serum Epo level was available (median 34 mU/mL; range 2–323). Among the 43 study patients, 16 (37%) were previously treated with other PMF-specific therapies, and 18 (42%) were receiving cytoreductive treatment concurrent with an ESA. Overall, 23% of patients (10 of 43) responded to treatment with ESA. The response rate was 0% (0 of 16) in the transfusion-dependent group compared to 37% (10 of 27) in the transfusion-independent group (p=0.007). Among the transfusion-independent patients, the proportion of ESA responders was significantly higher in the presence of a baseline hemoglobin level < 10 g/dL prior to treatment (p=0.009). ESA response was not correlated with baseline serum Epo level in the context of either all 43 study patients (p=0.68) or the 27 transfusion-independent patients (p=0.39). ESA response was not affected by PMF-specific treatment history (p=0.46), use of concurrent cytoreductive therapy (p=0.28), cytogenetic findings (p=0.62), JAK2V617F presence (p=0.69) or its allele burden at time of initial PMF diagnosis (p=0.16).

CONCLUSIONS: Clear benefit of ESAs in PMF is limited to red blood cell transfusion-independent patients and those with baseline hemoglobin levels less than 10 g/dL.
Amplification at 7q22 Targets CDK6 in T-Cell Lymphoma.
Authors: Nagel Stefan , Leich Ellen , Quentmeier Hilmar , Meyer Corinna , Zettl Andreas , Drexler Hans G. , Rosenwald Andreas , MacLeod Roderick A.F.
Published: April 2020

Genomic amplification is microscopically visible extrachromosomally as double-minute chromosomes (DMIN) or intrachromosomally as homogeneously stained regions (HSR). Both DMIN and HSR comprise iterated copies of discrete genomic regions (amplicons) effecting copy number increases associated with upregulation of target genes. In cancer cells, these usually comprise oncogenes such as MYC. Cytogenetic analysis of the anaplastic large cell lymphoma (ALCL) cell line SU-DHL-1 revealed an HSR on the long arm region of chromosome 7 (7q). Detailled analysis of this region by fluorescence in situ hybridization (FISH) using tilepath BAC clones identified an amplicon corresponding to 86–95 Mb (NCBI Build 36.1) at 7q22. Expression analysis using reverse transcription (RT)-PCR of candidate oncogenes mapped within the amplicon, comprising DBF4, SRI, AKAP9, GATAD1, CDK6 and PPP1R9A, highlighted cyclin-dependent kinase 6 (CDK6) as a plausible amplification target. With reference to other ALCL cell lines, including SR-786, lacking this amplicon, SU-DHL-1 displays upregulation of CDK6 at both the RNA and protein levels as indicated by semiquantitative RT-PCR, real-time quantitative (RQ)-PCR and immuno-cytochemistry. Another ALCL cell line, L-82, containing a larger amplicon (7q21-31), also showed enhanced CDK6 expression as analyzed by RQ-PCR. For a functional test both SU-DHL-1 and SR-786 cells were treated with rapamycin, an inhibitor of the AKT-pathway which is involved in degradation of CDK-inhibitor p27. In contrast to SR-786, SU-DHL-1 cells are resistant to the effects of rapamycin on both proliferation and G1 cell cycle arrest, implying that CDK6 overexpression may confer proliferative advantage. Copy number analysis of several tumor-relevant genes using a commercial PCR-based approach (MLPA) determined a nearly 5-fold amplification of the CDK6 gene in SU-DHL-1. Interphase FISH confirmed copy number increase in SU-DHL-1 and identified CDK6 amplification in 1/77 ALCL and 4/38 peripheral T-cell lymphoma patient samples, indicating that this genetic event is relatively rare among T-cell lymphomas. Taken together, we have identified an amplicon at 7q22 in T-cell lymphoma cells targeting CDK6 which is an important cell cycle regulator and probably connected to the increased proliferative capacity of these malignant cells. Although CDK6 amplification seems to be infrequent overall, it strengthens the importance of this gene in regulating proliferation among T-cell lymphomas and may, therefore, represent a potential molecular target for therapeutic intervention in these disease entities.
A Long-Term Study on Indolent Adult T-Cell Leukemia/Lymphoma (ATLL).
Authors: Takasaki Yumi , Tsukasaki Kunihiro , Iwanaga Masako , Imaizumi Yoshitaka , Tawara Masayuki , Joh Tatsuro , Kohno Tomoko , Yamada Yasuaki , Kamihira Shimeru , Ikeda Schuichi , Tomonaga Masao
Published: April 2020

Background: Previous report on the subclassification and the clinical course of ATLL with maximum follow-up duration of 7 years revealed that the prognosis of indolent (smouldering or chronic) type was much better than that of aggressive (lymphoma or acute) types. (Shimoyama M, et al. Brit J Haematol, 1991). The median survival time (MST) was 6.2, 10.2 and 24.3 months for acute, lymphoma and chronic types, respectively. A MST has not been established for the smouldering type. However, long-term prognosis of the indolent types has not been elucidated.

Method: The diagnosis of ATLL was made based on seropositivity for HTLV-1 and histologically- and/or cytogically-proven peripheral T-cell malignancy. Subclassification of ATLL was based on the Shimoyama’s criteria. A total of 90 patients with indolent ATLL (65 chronic type and 25 smouldering type) with a median age of 61 years, newly diagnosed from 1974 to 2003 at Nagasaki University, were analyzed for the clinical course and prognostic factors.

Results: MST for the patients was 4.5 years with 12 patients alive for more than 10 years. Projected 5-, 10- and 15-yr survival rates were 47%, 23% and 13%, respectively, with first steep slope and sequential slow slope without plateau phase of the survival curve. Sixty-three of the patients died; 46 died of ATLL, 11 of other disease, 2 of transplantation related death and 4 of unknown. Unfavorable prognostic factor for survival included advanced performance status (PS) (p=0.005), high LDH value (p=0.004), more than 4 total involved lesions (p=0.03), more than 3 extranodal lesions (p=0.004), the presence of neutrophilia (p=0.02) by univariate analyses. In multivariate analysis, advanced PS was significant. There was no significant survival-difference in relation to age, subtypes of ATLL or lymphocyte count. Thirty-five patients with complications at diagnosis tended to be poor prognostic, especially those with active multiple cancers (n=7) or opportunistic infections (n=9). Most patients were treated as a subtype of chronic lymphoid leukemia (CLL) with watchful waiting until disease progression to acute ATLL. Twelve patients, who were treated with chemotherapy immediately after diagnosis of indolent ATLL, showed poorer prognosis as compared to the others. Five patients with at least one of the following 4 factors: alteration in tumor suppressor genes, p14ARF, p16 INK4a or p53, or aneuploidy >1 chromosomal locus by Comparative Genomic Hybridization analysis in ATLL cells, showed extremely poor prognosis.

Conclusions: The long-term prognosis of indolent ATLL was not good without plateau phase in the survival curve, similar to the findings in B-CLL. Advanced PS and genetic alterations in ATLL cells were poor prognostic factors.
Folicular and Diffuse Large B Cell Lymphoma Can Be Distinguished by 1H -Magnetic Resonance Spectroscopy.
Authors: Sanz Carolina , Barba Ignasi , Tapia Gustavo , Mate Jose-Luis , Barbera Angels , Ribera Josep-Maria , Ariza Aurelio , Garcia-Dorado David , Oriol Albert
Published: April 2020

Proton nuclear magnetic resonance spectroscopy (1H MRS) has proven useful in the metabolic characterization of neoplastic tissues both in vitro and in vivo. In vivo 1H MRS can be easily performed in conjunction with conventional MR imaging. Although 1H MRS studies of lymphoma have been performed both in cell lines and animal models, its usefulness as a differential diagnostic tool in lymphoma remains to be explored. T2-filtered high resolution proton magic-angle spinning (HRMAS) MRS of small tissue samples provides sufficient spectral resolution for the identification of chemical cell changes associated with malignancy. HRMAS data from 16 fresh frozen lymph nodes were studied. Histologically, 7 lymph nodes revealed grade I or II follicular lymphoma (FL) and 9 showed diffuse large B cell lymphoma (DLBCL). Each spectrum consisted of the accumulation of 64 scans with a CPMG sequence with an effective T2 delay of 32 ms and an overall experimental time of 6 minutes. Pattern recognition analysis was feasible in all cases except in one FL sample consisting almost exclusively of adipose tissue. Compared with DLBCL, FL cases showed increases in the signal intensity of resonances associted to taurine (3.43 ppm) and decreases in the signal intensity of resonances associated to alanine (1.47 ppm). The mean taurine/alanine ratio was 4.63 (95%SD 1.67) for FL and 1.41 (95%SD 0.69) for DLBCL (P = 0.004). The taurine/alanine ratio proved to be a powerful discriminator between FL and DLBCL at a 2.5 cutpoint, yelding a 90% specificity and an 83% sensitivity to identify DLBCL. Overlapping cases included a case of FL with a diffuse pattern and two cases of DLBCL evolving from indolent lymphomas. The 1H MRS assay proved that FL and DLBCL can be differentiated from a metabolomic standpoint and offers a unique way to explore the underlying biochemistry of malignant transformations. Unlike other techniques, MRS is potentially applicable in vivo and non-invasively in the diagnostic setting. The rapid assessment of nodes or masses before tissue sampling would allow preoperative decisions to be made more efficiently.
Follicular Lymphomas with and without Translocation t(14;18) Differ in Gene Expression Profiles and Genetic Alterations.
Authors: Leich Ellen , Salaverria Itziar , Bea Silvia , Zettl Andreas , Gascoyne Randy D. , Chan Wing-Chung , Braziel Rita M. , Rimsza Lisa M. , Weisenburger Dennis D. , Delabie Jan , Jaffe Elaine S. , Lister Thomas A. , Norton Andrew J. , Staudt Louis M. , Hartmann Elena M. , Mueller-Hermelink Hans-Konrad , Campo Elias , Ott German , Rosenwald Andreas
Published: April 2020

Follicular lymphoma (FL) is a B-cell non-Hodgkin lymphoma that is characterized in approximately 85% of cases by the chromosomal translocation t(14;18) involving BCL2. While FL3b generally lack the t(14;18), this translocation is also absent in 15% of FL grades 1, 2 and 3a. The current study was designed to identify the frequency of t(14;18)-negative FL in a series of 166 cases of FL1, 2 and 3a in which global gene expression profiles had been established previously (Dave et al., NEJM351:2159–69, 2004). Furthermore, we sought to compare genetic alterations and gene expression profiles between FL with and without the t(14;18). Combined polymerase chain reaction (PCR) and tissue microarray-based fluorescence in situ hybridization (FISH) identified 17 t(14;18)-negative FL cases in this series (9%). Virtually all FL cases carrying the t(14;18) showed BCL2 expression by immunohistochemistry (Dako, clone 124), whereas 11 of the FL cases without a t(14;18) were BCL2-negative at the protein level. Clinically, there was no difference between the t(14;18)-negative and -positive FL subgroups regarding age and gender distribution as well as in median survival times. Comparative genomic hybridization (CGH) in the 166 FL cases revealed a characteristic pattern of chromosomal gains and losses, as previously described. However, significant differences were observed between the t(14;18)-negative and -positive FL subgroups. Specifically, the t(14;18)-positive FL subgroup showed gains of chromosomes 18q (18%), 8q (12%) and X (13%), as well as losses of 13q (16%) and 10q (16%), whereas none of these aberrations were observed in the t(14;18)-negative FL cases. To compare gene expression between the two groups, we used gene set enrichment analysis (GSEA), BRB array tools and a two-sided t-test. Cell cycle-associated genes were found to be enriched in the t(14;18)-negative FL subset. These differences were even more pronounced in FL cases that lacked both the t(14;18) and BCL2 expression at the protein level. Importantly, genes expressed in non-malignant bystander cells appeared also differentially enriched and a cytotoxic gene expression signature was found to be more prominent in t(14;18)-negative FL. These findings point to a different composition of the non-neoplastic cells in t(14;18)-positive and -negative FL and could indicate subtle differences in the immunological microenvironment of t(14;18)-negative FL.
Bortezomib, Thalidomide, and Dexamethasone as Induction Therapy for Patients with Symptomatic Multiple Myeloma.
Authors: Kaufman Jonathan L. , Gleason Charise , Heffner Leonard , Lonial Sagar
Published: April 2020

The optimal induction regimen for patients with symptomatic myeloma who are eligible for transplantation is currently unknown. While thalidomide and dexamethasone is an effective regimen, it only has a 60 to 65% response rate and few complete responses (CR). Bortezomib based inductions have demonstrated a high response rate and an improved CR as well. Recently the IFM reported the initial results of the randomized bortezomib plus dexamethasone versus VAD induction followed by transplant, which demonstrated that fewer patients treated with bortezomib required tandem transplants. Wang et al reported a high induction response rate with the combination of BTD for only 2 cycles given over a 28 day cycle. Here we report our experience with the combination of BTD as induction therapy. 38 patients with symptomatic myeloma were treated with BTD as induction therapy. Patients received standard dose and schedule bortezomib at 1.3 mg/m2 on days 1, 4, 8, and 11 with thalidomide at 100 mg/day, and 8 days of 40 mg dexamethasone every 21 days. The median age was 58 years (38–70) with 19 males. This was first line therapy for 29 patients, second line for 7 patients and 3rd line for 2. 12 patients had ISS stage 2 and 8 had ISS stage 3. The median β2M was 3.4 (1.66–41.89). Median creatinine was 1.1 (0.6–21.0). Nineteen patients had an IgG paraprotein, 6 an IgA, and 16 patients had light chain disease. The median number of cycles administered was 4 (2–8). Fifteen patients developed neuropathy of any grade. One patient developed grade 3 neuropathy. The overall response rate (CR, VGPR, plus PR) was 92%, with 58% of patients achieving a VGPR or better, and 21% of patients achieving an immunofixation negative CR. 1 patient had a minimal response and 2 patients had progressive disease (both patients presented with plasma cell leukemia). These two patients were treated with the combination of BTD with PACE chemotherapy. One of the two died from progressive disease and the other patient remains in complete remission after high dose therapy and autologous transplantation. 29 patients had consolidation therapy with high dose melphalan and autologous peripheral blood stem cell transplantation. Eight patients have collected stem cells without proceeding with immediate consolidation therapy. After a median follow up of 373 days, median progression free survival and overall survival have not been reached. One year overall survival is 97%. One year progression free survival is 87%. In conclusion, we report a very high response rate with a short course of bortezomib, thalidomide and dexamethasone with an acceptable toxicity profile. Follow up of patients in CR treated without high dose therapy and autologous transplant is in progress. Further studies of this active regimen are warranted.
Decreased Incidence of Osteonecrosis of the Jaw (ONJ) in Patients with Multiple Myeloma (MM) Treated with Zoledronic Acid (ZA) after Application of Preventive Measures.
Authors: Dimopoulos Meletios Athanasios , Kastritis Efstathios , Bamia Christina , Melakopoulos Ioannis , Gika Dimitra , Roussou Maria , Migkou Magdalini , Eleutherakis-Papaiakovou Evangelos , Bamias Aristotle
Published: April 2020

Background. Bisphosphonates are widely used in the treatment of MM. ONJ can occur during treatment with bisphosphonates. We have shown that use of ZA and longer exposure are associated with higher frequency of ONJ in a series of patients with both MM and solid tumors. Dental problems or interventions are precipitating factors of ONJ. For this reason, since 2003, we have implemented assessment (and management if necessary) of all patients who are candidates for ZA or who are on treatment with ZA and have dental problems by specialists with particular experience on ONJ. In addition, we recommend improved oral hygiene and we avoid dental procedures during treatment with ZA. We have investigated whether the occurrence of ONJ decreased after the implementation of these measures.

Patients and Methods. According to the policy of our center, all patients with MM who demonstrate lytic lesions or osteopenia receive ZA 4 mg i.v. every 4 weeks indefinitely. In our current analysis we included patients who received only ZA wheras patients who initially received pamidronate and were later switched to ZA were excluded. Patients were stratified into two groups depending on the date of initiation of treatment in relation to the start of implementation of the preventive measures (Group A: 26/8/1998–31/12/2002, Group B: 1/1/2003–1/12/2006). Occurrence of ONJ was studied as row percentages as well as incidence rate rates (IR: number of cases of osteonecrosis /person-months). The proportions of patients with ONJ between the two groups were compared with the Fisher’s exact test whereas the respective incidence rates were compared with the score test. The 95% confidence interval of the incidence rate ratio was also estimated.

Results. One hundred twenty-eight patients with MM were included in the analysis (Group A: 35, Group B: 93; M/F: 66/62). Overall there were 10 cases of ONJ (8%): 8 cases in Group A (23%) and 2 cases in Group B (2%) (p<0.001). The IR was 0.560/100 person-month for Group A and 0.118/100 person-month for Group B. The IRR was Group A/Group B: 4.76 (p=0.029, 95% CI: 1.01–22.40). Median time of exposure among patients with ONJ was 31 months for Group A and 8 months for Group B (p=0.044).

Conclusions. The implementation of detailed assessment by experienced specialists of patients with MM and dental problems and the avoidance of dental procedures during treatment with ZA results in a significant 5-fold reduction of ONJ. Our data provide a basis for a safer use of ZA in MM.
Bortezomib in Combination with Pegylated Liposomal Doxorubicin and Thalidomide (VDT) for the Treatment of Previously Untreated Multiple Myeloma.
Authors: Chanan-Khan Asher A. , Miller Kena C. , DePaolo Dawn , Padmanabhan Swaminathan , Whitworth Amy , Ailawadhi Sikander , Lee Kelvin , Czuczman Myron S.
Published: April 2020

Introduction: Orlowski et al were the first to report that addition of PLD resulted in enhanced efficacy of bortezomib. To further optimize the clinical efficacy of these agents we included thalidomide to the regimen. In a previous report we described the clinical efficacy of this novel, non-steroidal regimen that incorporated bortezomib (V), pegylated liposomal doxorubicin (D) and thalidomide (T) in patients with relapsed or refractory multiple myeloma (MM). High clinical responses in heavily pretreated patients, even in the absence of steroids encouraged us to investigate this regimen in previously untreated MM patients. Here we report for the first time the efficacy and toxicity profile of the VDT regimen in treatment naïve MM patients enrolled on a phase II clinical study at our center.

METHOD: All previously untreated MM patients were eligible for this study. Bortezomib (1.3mg/m2) was given on days 1, 4, 15, and 18, pegylated liposomal doxorubicin was given (20mg/m2) on days 1 and 15 and thalidomide (200mg) was given everyday continuously throughout the treatment. Patients received treatment on a 4-week cycle. Acyclovir 400mg PO BID and low-dose warfarin was given for prophylaxis of herpes zoster and venous thromboembolism respectively.

RESULTS: A total of 26 patients (median age-- 60 range 40–82 years), 16 M and 10 F have so far been enrolled. Advance stage III disease was noted in ---of the patients. The median beta 2 microglobulin was 4 (range1.6–9.7), albumin of 4 (range 3.1–4.8) and LDH was 333 (range 152–1057). Patients received a median of 5 treatment cycles (range 1–8). Toxicity: The most common grade 3 or 4 hematologic toxicities included lymphopenia (27%) and neutropenia (15%), while the non-hematologic toxicity included infections 15% (n=4 patients). The incidence of grade 3 or 4 plantar palmar erythrodysthesia was 4% (n=1). Venous thromboembolism was not observed in any patient to date. Response: To date 17 patients are available for response assessment (EBMT criteria?), 7 patients are too early for response assessment. The ORR of this regimen is 65% with IFE negative CR observed in 3 patients (17.6%). Another 5 patients achieved a stable disease with only 1 progressive disease observed on the regimen so far.

Conclusion: VDT is a novel non-steroidal regimen with clinical efficacy in previously untreated myeloma patients. Overall toxicity is manageable. Several patients remain on treatment and currently not evaluable for response. Detailed and updated results of this study will be presented at the meeting.
FcγRIIa Is Required for Outside-In β3 Integrin Signaling in Human Platelets.
Authors: Boylan Brian , Gao Cunji , Rathore Vipul , Crockett Jimmy , Newman Debra K. , Newman Peter J.
Published: April 2020

ITAMs (Immunoreceptor Tyrosine-based Activation Motifs) are signaling motifs that are found within the cytoplasmic domains of T and B cell antigen receptors, and in the Fc receptor γ (FcRγ) chain that is associated with Fc receptors for IgG and IgE and with the GPVI collagen receptor on platelets. ITAMs are also an intrinsic component of the cytoplasmic domain of the platelet and leukocyte low affinity receptor for IgG, FcγRIIa. When ITAM-bearing receptors are engaged or cross-linked, ITAM tyrosines undergo Src-dependent phosphorylation, providing a docking site for the protein-tyrosine kinase, Syk. Activation of Syk results in assembly of a multi-protein signaling complex containing the adaptor molecule SLP-76, ultimately resulting in the activation of phospholipase Cγ2 (PLCγ2), which via its lipase activity initiates a multitude of cellular activation responses. Recent studies in platelets and neutrophils have found that ITAM-mediated signaling cascades also play a novel and unexpected role in outside-in integrin signaling, as cells containing mutant forms of Syk, SLP-76, or PLCγ2 fail to become activated normally following integrin engagement. Furthermore, the FcRγ chain and DAP12 have been identified as ITAM-bearing subunits required for integrin signaling in neutrophils and macrophages, and we and others have reported that the GPVI/FcRγ chain complex can function as a weak integrin amplifier in human platelets. Nevertheless, the complement of ITAM-bearing receptors in platelets that are involved in integrin signaling in platelets is not known. To identify candidate receptors that might couple integrins to ITAM signaling, we initiated fibrinogen (Fg) binding to the integrin αIIbβ3 by

activating the thrombin receptor, PAR1, on platelets in suspension, or by allowing platelets to spread directly on immobilized Fg.

Interestingly, both forms of platelet activation initiated strong, ligand binding-dependent tyrosine phosphorylation of FcγRIIa, as well as downstream phosphorylation of Syk and PLCγ2. Importantly, prior addition of Fab fragments of the FcγRIIa blocking monoclonal antibody, IV.3, inhibited platelet spreading on immobilized Fg, as well as downstream tyrosine phosphorylation of FcγRIIa, Syk, and PLCγ2. Finally, platelets from a patient with a mild bleeding disorder whose platelets express only 30% of the normal level of FcγRIIa exhibit markedly reduced spreading on immobilized Fg. Taken together, these studies identify FcγRIIa as the major ITAM-bearing receptor in human platelets that supports outside-in integrin signaling.
CBFA2/RUNX1 Regulates Human Platelet 12-Lipoxygenase: Studies in Runx1 Haplodeficiency.
Authors: Kaur Gurpreet , Jalagadugula Gauthami , Rao A. Koneti
Published: April 2020

Core binding factor 2 (CBFA2), also known as AML1 and RUNX1, is a transcription factor that regulates the expression of genes involved in hematopoiesis, through highly conserved DNA binding region, called RUNT homology domain (RHD). We have previously reported a patient with a mutation (haplodeficiency) in the conserved region of RUNX1/CBFA2 associated with mild thrombocytopenia and impaired platelet function. Expression profiling of patient platelets revealed ∼5 fold decreased mRNA expression of 12-lipoxygenase (12-LO, gene ALOX12) (Sun L. et al. J Thromb Haemost, 5:146–154, 2006). 12-LO catalyzes 12-hydroxyeicosatetraenoic acid (12-HETE) production from arachidonic acid (AA) upon platelet activation. We have performed studies to determine whether ALOX-12 is regulated by CBFA2. We studied 12-HETE production in patient platelets and the regulation of ALOX-12 by CBFA2 in human erythroleukemia (HEL) cells treated with phorbol myristate acetate (PMA) to induce megakaryocytic (MK) transformation. 12-HETE production was decreased in patient platelets in response to 10 U/ml of thrombin (19.5 ng/10 8 platelets, normal subjects: range 29–306, n=9) and 100 μM of arachidonic acid (5.4, normal subjects: 67– 442, n=10). Three CBFA2 consensus binding sites were identified (−1498/−1493, −708/−70, −526/−521 from ATG) by computer analysis within 2 kb of ALOX-12 5′ upstream region. The binding site at −1489 is a 13nt palindromic sequence with two CBFA2 motifs. The other two CBFA2 binding sites at −708 and −526 bp overlap AP2 binding sites. Luciferase reporter studies in HEL cells using a construct carrying ∼ 1600 bp of 5′ upstream region indicate a greater than 10 fold increase in activity in PMA treated HEL cells relative to untreated cells. Truncation at − 873 bp in PMA-treated HEL cells resulted in a ∼10 fold decrease in activity with only minimal decreases with truncations at −705 or −438 to delete the other CBFA2 binding sites. Mutation of each of the putative CBFA2 binding sites individually resulted in 5–10 fold decrease in activity. Gel shift studies using a 30-mer probe (−1507/−1478) and PMA-treated HEL extracts revealed specific protein binding that was eliminated by CBFA2 antibody and by mutating CBFA2 site from TGGGGT to TGCATT. Specific protein binding was observed with probes containing putative CBFA2 sites at −708 and −526, but it was not altered by the antibody. Chromatin immunoprecipitation (ChIP) analysis using HEL cells demonstrated (PCR amplification) in vivo binding of CBFA2 to ALOX-12 promoter in the region −1507/−1478 but not the other two sites.

Conclusions: CBFA2 haplodeficiency is associated with decreased platelet 12-HETE production and 12-LO activity. ALOX-12 is regulated by CBFA2 in megakaryocytes/platelets. These findings are important because of the role of 12-lipoxygenase in platelet arachidonate metabolism and function.
Prospective Assessment of Pulmonary Hypertension in Children with Chuvash Polycythemia.
Authors: Dham Niti , Sable Craig , Okhotin Daniel , Sergueeva Adelina , Miasnikova Galina , Bushuev Vladimir , Gordeuk Victor R.
Published: April 2020

Background: Chuvash Polycythemia (CP) is an autosomal recessive disorder that is endemic to the Chuvash region of Russia; however, it does occur worldwide. It is characterized by a mutation (598C>T) of the von Hippel Lindau gene, a negative regulator of hypoxia-sensing, and increased levels of hypoxia inducible factor-alpha during normoxia. CP is manifested by vertebral hemangiomas, varicose veins, hypotension, elevated concentrations of products of hypoxia-induced genes (serum vascular endothelial growth factor and endothelin-1), and premature mortality related to cerebral vascular events and peripheral thrombosis. Echocardiography (echo) studies in adults with CP suggest an increased prevalence of pulmonary hypertension (PH), but these findings have not been validated in children with CP.

Methods: This work is supported by an NIH grant evaluating the prevalence, outcomes, pathophysiology, vascular response, and gene polymorphisms of PH in sickle cell disease and CP. We traveled to Cheboksary, Russia to prospectively enroll children with CP and age and gender matched controls to undergo echo for assessment of left ventricular (LV) systolic and diastolic function, LV size and mass, estimated right ventricular (RV) systolic pressure by tricuspid regurgitation velocity (TRV), and the RV to systemic systolic blood pressure (SBP) ratio. Data was compared between CP and control children.

Results: 24 CP and 11 control patients were enrolled in the study. 20 CP and 9 control patients had measurable TRV. 4 of 20 CP and none of the control patients had TRV > 2.5 m/sec (max TRV 2.89 m/sec). There was a trend towards higher TRV and estimated RV pressure in CP patients. Systolic function, LV size, and mass were the same in both groups. Diastolic function was normal in both groups but significantly better in CP patients. SBP was the same in both groups. Systemic mean and diastolic BP, adjusted for age, was significantly lower in CP children.

Conclusions: Preliminary prospective data show a trend towards higher echo derived estimates of RV pressure in children with CP that can not be explained by hypertension or diastolic dysfunction. Rather, congenital upregulation of the hypoxic response may be responsible. Continued patient recruitment and correlation of TRV with other variables will allow for a better understanding of PH in children with CP.

ECHO PARAMETERS CP Controls p TRV (m/sec) 2.24 ± 0.32 2.08 ± 0.14 0.08 RV systolic pressure (mm Hg) 25.4 ± 5.7 22.4 ± 2.4 0.06 RV/systemic pressure ratio 0.26 ± 0.06 0.24 ± 0.03 0.17 LV diastolic dimension z score 0.11 ± 0.91 −0.13 ± 0.79 0.42 LV Mass Index: g/m^2.7 25.8 ± 8.2 24.7 ± 6.5 0.68 Ejection fraction (%) 64.9 ± 3.7 64.7 ± 3.2 0.91 Mitral E/Tissue Doppler E 5.35 ± 0.81 6.48 ± 1.31 0.02
Correction of Anemia with Hematide™, a Synthetic Peptide-Based Erythropoiesis Stimulating Agent (ESA), in Oncology Patients Receiving Chemotherapy.
Authors: Pickering Lisa M. , Cwiertka Karel , Jassem Jacek , Petera Jira , Pettengell Ruth , Ramlau Rodryg , Vorlicek Jiri , Vyzula Rostislav , Duliege Anne-Marie , Lang William , Stead Richard B. , Harper Peter G.
Published: April 2020

Introduction: Hematide™ a novel, synthetic, PEGylated peptidic compound, binds to and activates the erythropoietin receptor. It is in development for treatment of anemia associated with chronic renal failure and cancer.

Objective: To assess the subcutaneous (SC) Hematide™ dose required to increase hemoglobin (Hb) by ≥ 1 g/dL in ≥ 50% of anemic cancer patients (pts) on chemotherapy. Safety, pharmacodynamics and pharmacokinetics were also assessed.

Method: In a phase 2, open label, multi-center, dose finding study, up to four doses of Hematide™ were given SC every 3 wks (Q3W) to 4 cohorts of fifteen pts at 0.05, 0.10, 0.15 or 0.2 mg/kg each. Entry criteria included confirmed solid tumor malignancy or lymphoma, >9 weeks of chemotherapy, baseline (BL) H ≥ 8 and <11 g/dL, and adequate iron, folate and B12 stores.

Results: Sixty patients were enrolled (45% male). Preliminary data show that across cohorts 0.05, 0.10, 0.15 and 0.20 mg/kg, mean (BL) Hb values were 10.1(±1.07), 10.0(±0.89), 9.8(±0.67), 10.1(±0.69); the % of pts completing the study were approximately 79%, 50%, 60%, and 92%. An increase in mean Hb from BL of ≥ 1g/dL in the pharmacodynamic dataset (forty two pts) at Week 7 occurred in approximately 20%, 70%, 55% and 55% of patients. Three pts withdrew due to AEs and six due to SAEs (none attributed to study drug). Three deaths occurred (two disease progression, one renal insufficiency) none attributed to study drug. One SAE, thrombophlebitis, was considered to be possibly/probably related to study drug. Final safety and efficacy data will be provided at the meeting.

Conclusion: In this study, HematideTM dosed SC Q3W resulted in an increase from BL of ≥ 1 g/dL of Hgb in ≥ 50% of patients in the 0.10, 0.15 and 0.20 mg/kg cohorts in the pharmacodynamic analysis. HematideTM appeared to be well tolerated at all doses studied.
NKG2D-Mediated Marrow Injury in Paroxysmal Nocturnal Hemoglobinuria and Its Related Disorders.
Authors: Hanaoka Nobuyoshi , Kawaguchi Tatsuya , Horikawa Kentaro , Nagakura Shoichi , Ishihara Sonoko , Tsuzuki Yasuchika , Yonemura Yuji , Nakakuma Hideki
Published: April 2020

Immune mechanism is considered to exert in the pathogenesis of marrow failure in paroxysmal nocturnal hemoglobinuria (PNH), idiopathic aplastic anemia (AA) and myelodysplastic syndromes (MDS); however, the molecular events are unknown. We have currently reported the appearance of NKG2D ligands such as cytomegalovirus glycoprotein UL16 binding proteins (ULBPs) and MHC class I-related chains A and B (MICA/B) on granulocytes and CD34+ marrow cells of some patients with PNH and its related diseases (Hanaoka N, et al. Blood. 2006;107:1184–1191). ULBP and MICA/B are stress-inducible membrane proteins that appear in infection and transformation. The ligands share NKG2D receptor on lymphocytes such as NK, CD8+ T, and γδ T-cells and promote activation of the lymphocytes. Cells expressing the ligands are then deadly injured by NKG2D+ lymphocytes (Groh, PNAS 1996; Cosman, Immunity 2001). Indeed, cells expressing NKG2D ligands were killed in vitro by autologous NKG2D+ lymphocytes of our patients (Hanaoka N, et al. Blood. 2005;106:304a; Blood. 2006;108:295a). In further analysis, ligands were detected on granulocytes in 47 (53%) of 88 patients: 11 (58%) of 19 PNH, 28 (60%) of 47 AA, and 8 (36%) of 22 refractory anemia. Ligands were also detected on immature bone marrow cells in all 11 patients (3 PNH, 5 AA, and 3 refractory anemia) who permitted analysis of their marrow cells. In the patients, it is conceivable that blood cells were exposed to a certain stress to induce NKG2D ligands, leading to NKG2D-mediated marrow injury. We also observed a close association of the ligand expression with pancytopenia and favorable response to immunosuppressive therapy by prospective analysis of 5 patients (3 AA-PNH syndrome and 2 AA) for more than one year up to 5 years. Thus, we here propose that NKG2D-mediated immunity, which drives both NK and T-cells, is critically implicated in the pathogenesis of bone marrow failure of PNH and its related disorders.
Outgrowing Endothelial Cells That Are Derived from Human Umbilical Cord Blood Improve Neovascularization in Hind-Limb Ischemia.
Authors: Yoo Eun-Sun , Kwon KiHwan , Ahn Jee-Young , Oh Soo-Ah , Chang Hye-Jung , Choi Moon-Young , Mun Yeung-Chul , Lee Kyoung-Eun , Seong Chu-Myong
Published: April 2020

Backgroud: Human umbilical cord blood (UCB) contains a high number of endothelial progenitor cells (EPCs) and may be useful for the treatment of ischemic disease. Recently, we have isolated EPCs from UCB having different biologic properties for angiogenic capabilities in vitro. In this present study, the aim is to examine the usefulness of OECs in hind-limb ischemia.

Methods: Mononuclear cells from UCB cultured using EGM-2 medium with VEGF, IGF-1 and FGF for 21 days. Early spindle-shaped cells (early OECs), which were grown during the first week of culture and late cobblestone shaped cells (late OECs), which were in peak growth during the third week of culture were found. The hind-limb ischemia was established as follows: Athymic nude mice (BALB/C-nu) 18–22 g in weight were anesthetized with pentobarbital (60 mg/kg) and their left femoral arteries and main extension arteries were operatively resected. To examine the effect of the vasculogenesis of the two types of OECs, the mice were divided into three groups (PBS, early and late OECs). Twenty-four hours after operative excision 5 × 105 OECs in 200 μl and an equal volume of PBS were administered by intramuscular injection into the mice on hind-limb ischemia. To compare the effect of OECs on neovascularization in vivo, the analysis of blood flow of ischemic and healthy hind limbs was performed on days 1 and 21 after surgery using near-infrared (NIR) imaging with incocyanne Green (ICG).

Results: Late OECs expressed a high level of mRNA on endothelial marker genes and formed capillary tubes in Matrigel plates. The early spindle cells excreted more angiogenic cytokines and had more migratory ability. We divided the mice into two groups according to the degree of perfusion; good (22.5–50%/min) and poor (0–22.5%) perfusion. OECs improved the blood flow of the ischemic hind-limb in the ’good’ perfusion group but not in the ’poor’ perfusion group. Early OECs led to a more significant improvement in blood flow than that of the late OECs.

Conclusion: The different types of OECs from UCB have different biologic properties in vitro and different vasculogenic potential in vivo as well. The results might have potential application for the treatment of hind-limb ischemia.