bluuebunny/crossref_metadata_2025 huggingface dataset:
Extranodal Follicular Lymphoma - a Retrospective Review and Comparison with Localized Nodal Follicular Lymphoma.
Authors: Kukreti Vishal , Petersen Peter , Pintilie Melania , Tsang Richard , Crump Michael , Gospodarowicz Mary
Published: April 2020

Follicular lymphoma arising in an extranodal site is uncommon and its natural history and treatment is poorly characterized in the literature. We retrospectively reviewed a large cohort of patients with stage I and II follicular lymphoma and analyzed the outcomes of patients with extranodal (EN-FL) presentations to identify sites of involvement and treatment outcome, and compared these to patients with nodal follicular lymphoma. From 1967 to 1999, 668 cases of limited stage follicular lymphoma (stage I and II) were treated at the Princess Margaret Hospital. Of these, 157 cases (23.5%) presented in extra-nodal sites. The most common site of presentation was in the head and neck area (42%) followed by gastro-intestinal tract (14.6%) then skin (10.8%). The majority of patients had stage I disease (61.8%). Pathological type was follicular grade I: 22.9%, grade II: 33.1%, and grade III: 43.9%. Treatment consisted of involved field radiation therapy in 72%, combined modality therapy in 22.3% and chemotherapy alone in 3.8%. The treatment changed over time with increased use of combined modality treatment (CMT) [1967–77: 10.5%, vs. 1989–99: 33%] mainly due to the adoption of CMT for follicular grade III lymphoma. Overall complete response rate (CR) to primary treatment was 93%; the CR rate for radiation alone was 97.3%. The cumulative incidence of relapse (RR) was 44% at 10 years. The RR at 10 years was higher for patients age >60 (62% vs. 49%; p =0.059) but did not vary according to stage, tumour bulk, gender or histologic grade. For extranodal lymphoma, the 10-year overall survival (OS) rate was 56% and the 10-year disease free survival (DFS) was 42% and was similar for major sites of presentation.

Comparison of Stage I–II Nodal and Extra-nodal Follicular Lymphoma

Nodal Follicular Lymphoma Extra-nodal Follicular Lymphoma 10 yr Overall Survival 61% 56% (p=0.97) 10 year Disease Free Survival 41% 42% (p=0.27) 10 yr Relapse Rate 50% 44% (p=0.11)

In conclusion, a significant number of patients with localized FL present with extra-nodal disease, involving diverse sites. Patients with EN-FL were more likely to have follicular grade III histology. OS, DFS and RR were similar to nodal follicular lymphoma. These results suggest that the clinical management of stage I and II extra-nodal follicular lymphoma should be the same as for nodal, and that a significant proportion of patients have prolonged DFS with radiation-based therapy.
Non-Hodgkin’s Lymphoma in the Context of Antecedent Rheumatoid Arthritis: A Case Series from the Mayo Clinic and the Nebraska Lymphoma Study Group.
Authors: Endo Justin O. , Ooi Melissa G.M. , Black Natalie A. , Aoun Patricia , Habermann Thomas M. , Stoner Julie A. , Armitage James O. , Mikuls Ted R.
Published: April 2020

PURPOSE: Rheumatoid arthritis (RA) has been associated with an increased risk of non-Hodgkin’s lymphoma (NHL). We describe the characteristics of NHL patients with antecedent RA.

METHODS: Records of the Nebraska Lymphoma Study Group (NLSG) registry between 1982–2004 and the Mayo Clinic lymphoma registry between 1988–1998 were systematically reviewed for RA diagnosis and classified by the quality of supporting evidence. We abstracted available follow-up data through July 1, 2004. Definite cases had either a documented RA diagnosis from a trained rheumatologist or met 4 of 7 American College of Rheumatology criteria. Probable RA cases had a non-rheumatologist diagnosis of RA in addition to one of the following: 1) radiographic joint erosions; 2) disease-modifying anti-rheumatic drug (DMARD) use or; 3) RA-associated deformities (i.e., ulnar drift, swan-neck deformities). Five-year overall survival (OAS), event-free survival (EFS), and rates of treatment response were determined.

RESULTS: We identified 74 RA cases (54 definite and 20 probable) with concomitant NHL. These patients had a mean age of 67 + 8 years and were predominantly Caucasian (82%) and female (64%). Of the probable RA cases, a majority had documented DMARD use (n=17). Approximately half of the cases used methotrexate (55%) and were deceased (52%) by the common closing date. The most common NHL subtypes included B-DLCL (n=31), B-FL-1/B-FL-2 (n=12), B-FL-3 (n=6), and B-BL/B-BLL (n=4). Of the remaining 21 cases, most were of B-cell origin (n=13) and included B-EMZL (n=3), B-LPL (n=3), B-UCL (n=2), B-UCL-LG (n=2), B-DFL-2 (n=1), B-MCL (n=1), and B-SLL (n=1). The remaining NHL subtypes included NHL-NOS (n=7) or T-PTCL-LC (n=1). B-symptoms were present for 6 (19%) patients with B-DLCL and none of the patients with B-FL-1/B-FL-2. Complete response rates to NHL treatment were 48% in B-DLCL and 33% in B-FL-1/B-FL-2. For B-DLCL and B-FL-1/B-FL-2, respectively, the five-year OAS was 54% (95% CI 36%–69%) and 65% (95% CI 31%–85%) while the five-year EFS was 48% (95% CI 36%–69%) and 56% (95% CI 24%–79%). For all NHL cases the 5-year OAS was 58% (95% CI 47%–69%) and the 5-year EFS was 49% (95% CI 37%–59%).

CONCLUSIONS: Lymphomas developing in the context of RA most often include diffuse large B-cell and follicular subtypes. Complete response rates to standard treatment for these major subtypes ranged from 33 to 48%. For RA patients developing NHL, five-year overall- and event-free survival rates are approximately 50%.
Phase I/II Study of Rituxan (R) in Combination with Doxil (D) in Patients (pts) with Relapsing or Refractory B-Cell Lymphoma: Promising Early Results.
Authors: Czuczman Myron S. , Skipper Marion , Mohr Alice , Bernstein Zale P. , McCarthy Philip , Hernandez-Ilizaliturri Francisco , Chanan-Khan Asher Alban
Published: April 2020

In the past, the most common initial combination chemotherapy regimen used by most oncologists to treat B-cell lymphoma was the CHOP regimen. Although doxorubicin (DOX) is a major component of CHOP, it is associated with myelosuppression, alopecia, and potential cardiotoxicity. In vitro data has demonstrated synergistic activity between rituximab and certain drugs, including DOX. Because of D’s unique liposomal encapsulation, delivery, and toxicity profile, it may prove to be a more effective, less toxic anthracycline to combine with R. A formal Phase I/II trial to evaluate the safety and efficacy of R+D in pts with relapsing B-cell lymphoma was undertaken. R (375 mg/m2/dose) was given on day 1 and D (30 mg/m2/dose) on day 3 on q 21 day cycles for 6 cycles. Thirteen pts have completed therapy to date: 9F, 4M; Follicular lymphoma, grade 1 (n=6), grade 2 (n=1), grade 3 (n=3), DLBCL (n=3); med age = 63 (38–78); median number of prior Rx’s = 3 (range 1–8); prior anthracycline exposure (n=8). Overall, the combination of R + D was well-tolerated. Transient grade 3 cytopenias were noted in 3 pts with limited bone marrow reserve and grade 3 palmar/plantar erythrodysesthesia seen in 2 pts. All 13 pts completed the planned 6 courses of Rx. Objective responses were seen in 85% (11 of 13 pts), including 54% CR, 31% PR. Median time-to-progression has not been reached at 7+ months (range 4.5+ to 19+ months). No cardiac toxicity was seen and comparison of pre-Rx to post-Rx LVEF remained unchanged in 9, increased in 2, and decreased in 2 (yet still remaining > 50%). D + R immunochemotherapy is a well-tolerated, unique, non-cardiotoxic, and apparently effective salvage therapy for relapsed B-cell lymphoma. The study will continue until accrual of 42 pts has been reached.
Maintenance Rituximab after Autologous Stem Cell Transplant for High-Risk B-Cell Lymphoma Induces Prolonged and Severe Hypogammaglobulinemia.
Authors: Lim Seah H. , Zhang Yana , Wang Zhiqing , Esler William V. , Beggs David , Pruitt Brian , Hancock Paul , Townsend Mary
Published: April 2020

Rituximab, a chimeric CD20 antibody, has been incorporated in various chemotherapy regimen for B-cell lymphoma. It has also been used as maintenance therapy in some of these patients. However, the effects of its long-term use on the immune system has not been previously documented. Here, we present our preliminary analysis of the effect of maintenance rituximab therapy on the B-cell immune repertoire in nine consecutive patients with B-cell lymphoma who were treated with maintenance rituximab after autologous stem cell transplant (ASCT) for high-risk disease.

Nine patients (seven men and two women) with high-risk B-cell lymphoma were treated. Their diagnosis were: advanced mantle cell lymphoma in first CR (6), Stage IV DLCL in CR1 (1) and high grade NHL in CR2 (2). Median age was 66 years (range 38–72 years). CR was achieved using R-CHOP (8) or R-DHAP (1). Autologous stem cells were harvested during hematopoietic recovery from the last course of chemo-immunotherapy and G-CSF. Within two weeks after stem cell harvest, each patient received intravenous melphalan (200 mg/m2) following by the infusion of at least 2 x 106/kg CD34+ cells. All patients received a rituximab infusion (375 mg/m2) every three months starting D+100 after ASCT for a total of 2 years or until disease relapse. Unlike patients who underwent ASCT without rituximab, in whom B-cell recovery occured between 3–6 months, we observed severe delays in the immunoglobulin recoveries in these patients. At 9 months after transplant, all patients were IgM and IgG deficient, with only 1/9 patient acheived a normal IgA level. Even at 24 months after transplant, all patients were still IgM deficient and only 20% achieved normal IgA and IgG levels. The severity of immuno-deficiencies was next examined. We observed severe immunoglobulin deficiency (defined by < 50% normal levels) in IgM in all patients and in IgG and IgA in more than 20% patients at 24 months, suggesting that the immunoglobulin recovery was both delayed and severely affected.

The severe immunoglobulin deficiencies may be clinically relevant. One patient developed two episodes (follow-up of 12+ months), one seven episodes (follow-up 28+ months) and another two episodes of chest infection and a vancomycin-sensitive chronic diarrhea (follow-up 18+ months). All three patient had severe and prolonged immunoglobulin deficiencies. With a median follow-up of 27 months (range 12–31 months), all patients have remained lymphoma-free.

These preliminary results, therefore, suggest that rituximab administration every three months after autologous transplant for high-risk B-cell NHL delays immunoglobulin recovery and may be associated with increased risks of infection. Although this approach may reduce lymphoma relapse, careful monitoring of the immunoglobulin recovery and interventional as appropriate should be done routinely in these patients.
Genetic Abnormalities and Patterns of Antigenic Expression in Multiple Myeloma.
Authors: Mateo G. , Castellanos M. , Rasillo A. , Gutierrez N. C. , Montalban M. A. , Martin M. L. , Hernandez J. M. , Lopez-Berges M. C. , Monteiano L. , Blade Joan , Mateos M. V. , Sureda A. , Rubia J. , Diaz-Mediavilla J. , Lahuerta J. J. , Orfao A. , Miguel J.F. San
Published: April 2020

Myelomatous plasma cells (PC) show a high heterogeneity both in their immunophenotypic characteristics as well as in their cytogenetic features. So far, no extensive studies have been carried out to explore whether such antigenic diversity is associated with specific genetic characteristics. We have investigated the relationship between the immunophenotypic profile at PC and both their DNA ploidy status (evaluated by flow cytometry), and specific genetic features (ascertained by FISH) in a large series of 915 patients with newly diagnosed MM. The non-hyperdiploid MM group (n=454, 52%) was associated with a significantly higher frequency of positivity for CD28 and CD20 as well as a higher incidence of CD56− and CD117− cases (p<0.001). Remarkably, 13q deletion and IGH gen rearrangements, which were significantly more common in non-hyperdiploid MM, showed a strong association with CD117- cases. IGH translocation to 11q13 was associated with reactivity for CD20 (p<0.001), down-regulation of CD56 (p<0.001) and lack of expression of CD117 (p=0.001). By contrast, IGH translocations to other chromosome partners were almost exclusively found among CD20− and CD117− cases (p<0.001). These results suggest that genetic categories in MM exhibit a particular immunophenotypic profiles which in turn are strongly associated with the DNA ploidy status.
Annexin II Mediates Plasminogen-Dependant Matrix Invasion by Adult Human Mesenchymal Stem Cells.
Authors: Bolno Paul B. , Morgan Doris A. , Sharma Mahesh , Lazorik Martin , Wechsler Andrew S. , Kresh J. Yasha
Published: April 2020

Background: Annexin II (ANX2) is a fibrinolytic receptor that serves as a binding site for plasminogen and tissue plasminogen activator, facilitating the generation of plasmin. ANX2 is present on a wide variety of cells including vascular endothelial cells as well as macrophages. ANX2 has been shown to play a key role in extracellular matrix degradation, cellular migration, and invasion. This degradation of extracellular matrix may also cause the release of matrix-bound angiogenic factors such as VEGF and FGF. We hypothesized that adult human mesenchymal stem cells (hMSCs) express ANX2 and utilize this receptor for plasmin generation to facilitate basement membrane invasion.

Methods: Primary hMSCs were isolated from the sternal bone marrow of patients undergoing median sternotomy. Stem cell surface markers were characterized via immuno-fluorescence. The presence of ANX2 protein by hMSCs was established via western blot. ANX2 mediated plasminogen activation and plasmin generation was quantified using chromozyme-P as a colorimetric substrate. Invasion assays were performed in dual-chamber culture wells containing matrigel inserts. hMSCs were plated into upper chambers containing: serum-free medium (SFM), SFM + Plasminogen, or SFM + Plasminogen + epsilon-aminocaproic acid (e-ACA inhibits binding of plasminogen to ANX2). After 24 hours, invasive cells were isolated and counted.

Results: Sternal bone marrow derived hMSCs expressed the membrane phenotype CD34 (−), CD14 (−), CD44 (+), CD105 (+), CD106 (+). The presence of ANX2 was confirmed by western blot analysis. hMSCs generated 1.95 units of plasmin per milligram of protein. There was a 20% (p 0 .004) increase in hMSC invasion in the wells containing plasminogen as compared to SFM alone. When e-ACA was introduced there was a decrease in hMSC invasion back to control values.

Conclusion: Our observations establish for the first time the presence and functional activity of ANX2 in hMSCs. These data suggest that mesenchymal stem cell expression of ANX2 facilitates plasminogen-mediated hMSC trans-endothelial invasion, migration and the release of pro-angiogenic factors from within the extracellular matrix, promoting stem cell directed repair and angiogenesis.
Culture Conditions for Generating Human Bone Marrow Stromal Cells Influence Cell Immunophenotype and In Vivo Biodistribution in Immune Deficient Mice.
Authors: Vergidis Joanna , Suck Garnet , Wang Xing-Hua , Zandstra Peter W. , Keating Armand
Published: April 2020

Bone marrow stromal cells (MSCs) show promise for cell and gene therapies but their utility is currently limited by low levels of engraftment. A better understanding of factors underlying cell trafficking in vivo is likely to lead to enhanced MSC engraftment and tissue targeting. To address this issue, we studied the immunophenotype and transplantation potential of MSCs derived from two different culture systems, serially passaged adherent layers from standard long-term bone marrow cultures (LTBMC) and a novel stirred suspension bioreactor culture (SSBC) supplemented with IL-3 and SCF. MSCs were characterized as CD45 (−), VCAM-1 (+), CD44 (+), SH-3 (+), and CD49e (+) by flow cytometry. At earlier time points, the SSBC system generated 1.8x more MSCs compared with the LTBMC system when analyzing the CD45-negative fraction by flow cytometry. By the end of culture, however, the LTBMC system generated 7.3x more cells. Interestingly, 66% of the CD45 (−) cells from the bioreactor system were negative for both HLA Class I and II antigens. Furthermore, flow cytometry revealed that the bioreactor-derived cells expressed very low levels of the cell adhesion markers, VCAM-1 and CD44. These data suggest that the SSBC system generates cells that may have greater migratory freedom in vivo. Biodistribution patterns of the human MSCs derived from the two sources were examined in 11 completely unconditioned six-week old SCID mice. Six weeks after intravenous infusion of MSCs, femoral and tibial bone marrow, lung, liver, bone, spleen, brain, heart, and blood were analyzed for donor human cell engraftment by PCR and fluorescence in situ hybridization (FISH) against the murine background. Engraftment was determined by PCR with primers from the human alpha-satellite region (chromosome 17) that amplify a specific 850 bp fragment. One tibial bone marrow sample, two femoral bone marrow samples, one bone sample, and four lung samples from nine mice receiving LTBMC MSC were positive by PCR. In contrast, all of the same tissues from the mouse receiving SSBC MSC were negative for human donor cells, including the lung, with the intriguing exception of a positive PCR signal in heart tissue. The presence of donor MSC was confirmed in PCR+ specimens by FISH using a human Cy3- and a murine FITC-pan-centromeric probe. The frequencies of donor LTBMC MSC in the tibial bone marrow, femoral bone marrow, bone, and lung were 0.79%, 1.60%, 0.58%, and 1.99%, respectively. Donor bioreactor-derived MSCs accounted for 0.41% of the cardiomyocytes from one recipient, despite the absence of cardiac injury. Compared with LTBMC MSCs, SSBC-derived cells appear to display an unusual biodistribution pattern which may be attributable to the altered immunophenotype. The present study underscores the importance of culture conditions in influencing the immunophenotype of MSCs and holds promise for developing targeted cell therapy.
Human Myeloma Cells Express the Bone Regulating Gene Runx2/Cbfa1 and Produce Osteopontin That It Is Involved in Myeloma-Induced Angiogenesis.
Authors: Giuliani Nicola , Colla Simona , Morandi Francesca , Bonomini Sabrina , Crugnola Monica , Rizzoli Vittorio
Published: April 2020

Osteopontin (OPN) is a multifunctional bone matrix glycoprotein that it is involved in angiogenesis, cell survival and tumor progression. OPN gene expression by human cells is mainly regulated by the bone specific trascription factor Runx2 namely also CBFA1 (Runx2/Cbfa1) even if other transcription factors may be also involved in the production of OPN by cancer cells as the CCAAT/enhancer-binding protein alpha (C/EBPα) and AML-1.

In this study we show that human myeloma cell lines (HMCLs): RPMI-8226, U266, XG-1 and XG-6 but not ARH-77 and normal CD19+ cells directly produce OPN and express its major regulating gene Runx2/Cbfa1. The activity of Runx2/Cbfa1 in human myeloma cells has been also demonstrated by a gel mobility shift assay. On the other hand we found that all HMCLs tested were negative for C/EBPα mRNA, whereas AML-1A and AML-1B mRNA were expressed in all HMCLs even if any correlation has not been found between OPN expression and AML-1A/AML-1B ratio. In addition we found that OPN production by myeloma was up-regulated at both mRNA and protein level by IL-6 and IGF-I through a Runx2/Cbfa1 mediated mechanism; in turn recombinant human OPN was able to increase myeloma cells proliferation. The potential role of OPN in MM-induced angiogenesis has been also investigated in an experimental model of angiogenesis. rhOPN treatment stimulated vessel formation as compared to control and the conditioned medium (CM) of HMCLs, significantly increased vessel formation in comparison either with control or with VEGF treatment. On the contrary OPN-immunodepleted CM of HMCLs had not a stimulatory effect on vessel formation and the presence of anti OPN Ab inhibited vessel formation induced by HMCLs.

The expression of OPN by purified bone marrow (BM) CD138+ cells has been also investigated in 60 newly diagnosed multiple myeloma (MM) patients, finding that 40% of MM patients tested expressed OPN. Higher OPN levels have been detected in the BM plasma of MM patients positive for OPN as compared to control subjects.

A significant higher MVD was observed in the group of patients positive for OPN, (mean±SE: 29.1±0.7 vs. 17.55±0.37; p<0.01) and similarly, the number of microvessels per field was higher in OPN positive patients in comparison with OPN negative ones (mean±SE: 6.7±0.15 vs. 4.28±0.04; p=0.05).

In conclusion our data highlight the direct ectopic production of OPN by human myeloma cells with a Runx2/CBFA1 mediated mechanism and the capacity of myeloma-derived OPN to stimulate angiogenesis in vitro. In addition OPN has been detected in a subset of MM patients with higher BM angiogenesis suggesting its potential involvement in pathophysiology of MM-induced angiogenesis.
Constitutive Expression of CD40L on Bone Marrow Mast Cells Supports the Growth of Lymphoplasmacytic Cells in Patients with Waldenstrom’s Macroglobulinemia.
Authors: Tournilhac Olivier , Santos Daniel Ditzel , Xu Lian , Kutok Jeffery , Tai Yu Tsu , Le Gouill Steven , Hatjiharissi Evdoxia , Catley Laurie , Hunter Zachary , Branagan Andrew , Anderson Kenneth C. , Treon Steven P.
Published: April 2020

CD40 ligand (CD40L) is a potent inducer of normal and malignant B-cell proliferation through interaction with CD40. We and others have observed excess mast cells (MC) in bone marrow (BM) biopsies of WM patients, which are commonly found admixed with tumor aggregates. (Tournilhac et al, JCO 2004, 22:571S). We therefore sought to clarify the role of MC in WM. Co-culture of 0.5% paraformaldehyde fixed, or sublethally irradiated HMC-1, LAD, and KU mast or basophilic cell lines and sorted BM lymphoplasmacytic cells (LPC) from 10 WM patients resulted in MC dose-dependent tumor colony formation and/or proliferation as assessed by 3H-thymidine uptake studies. As demonstrated by immunohistochemical, multicolor flow cytometric (CD117+FceRI+) and/or RT-PCR analysis, CD40L was expressed on BM MC from 29 of 31 (94%), 11 of 13 (85%), and 7 of 9 (78%) of WM patients, respectively. In contrast, cell surface CD40L expression was not detected by immunohistochemistry (p=0.00005) and flow cytometry (p=0.003) in 5 normal donors, and only faint expression for 1 of 5 normal donors by RT-PCR (p=0.09). Moreover, by multicolor flow cytometry, CD40 was expressed on BM tumor cells from 14/17 (83%) patients. CD40 functionality was confirmed either by the G28.5 CD40 agonistic antibody which induced dose dependent proliferation or by the rh-CD40L which partly prevented serum starvation-induced-apoptosis of WM LPC from 4/4 and 3/4 patients respectively. Importantly, expansion of tumor cells from 3 of 4 patients in mixed cultures with paraformaldehyde fixed MC was blocked in a dose dependent manner by use of a CD40L blocking protein (CD40:Fc). These studies demonstrate that CD40L is constitutively expressed on the cell surface of BM MC in WM and support the growth of WM tumor cells, and therefore provide the framework for therapeutic targeting of MC and MC-WM cell interactions in WM.
Evidence for Clonality in Circulating Endothelial Progenitor Cells in Multiple Myeloma.
Authors: Ozcelik Tayfun , Vakil Varsha , Smith Eric , Braunstein Marc , Maroney Justin , Klueppelberg Uwe , Bagislar Sevgi , Akyerli Cemaliye , Batuman Olcay
Published: April 2020

A governing role for neoangiogenesis in the progression of multiple myeloma (MM) is indicated by the finding in patients of increased bone marrow microvascular density that is positively correlated with disease activity and is attenuated by treatment with thalidomide. Bone marrow angiogenesis in MM is largely driven by autocrine and paracrine effects of vascular endothelial growth factor (VEGF) via VEGF receptor-2 (KDR). Elucidation of the role of neovascularization in MM growth and dissemination is likely to result in identification of better therapeutic targets than those currently available as well as provide insight into the pathogenesis of MM. Circulating endothelial cells (CECs) contribute to angiogenesis and comprise mature ECs and endothelial progenitor cells (EPCs). The present study sought to characterize CECs and their relation to disease severity in MM. CECs, identified as CD34+/CD146+/CD105+/CD11− cells, were 6-fold higher in patients compared to controls, and correlated positively with serum M protein and β2-microglobulin (P<.001 for both). In addition, circulating EPCs displayed late colony formation/outgrowth and capillary-like network formation on Matrigel. Effective thalidomide treatment inhibited these characteristics (P<.001). Co-expression of KDR and early vascular antigen CD133 characterized EPCs in MM, and KDR mRNA elevations correlated positively with M protein levels (P<.01). To evaluate the clonality of circulating EPCs, X-chromosome inactivation patterns were quantitated in female patients by human androgen receptor gene (HUMARA) assay. Radioactive (α-[33P] -dCTP) polymerase chain reaction amplification of DNA extracted from peripheral blood mononuclear cells showed that X-chromosome inactivation status was informative in 9 of the 11 MM patients. Distribution of the X-inactivation profiles according to age did not show a shift towards the skewed range in older patients or controls. DNA obtained from confluent EPCs outgrown from 4 informative patients showed an extremely skewed pattern of X-chromosome inactivation, with allele ratios of 90/10% in one patient and 97/3% in another patient, whereas a random pattern of X-chromosome inactivation was observed in two other patients displaying clonal restriction (allele ratios of 55/45% and 54/46%). Collectively, these data underscore the angiogenic aspect of MM and suggest that angioblast-like EPCs are a pathogenic biomarker and a rational treatment target in MM. Furthermore, the clonality of EPCs in MM suggests that the pathogenesis of MM may be tightly related to vascular endothelial cells both at the functional/angiogenic and at the genetic and ontogenic levels.
Pulsed Low Dose Intravenous Melphalan in Patients with AL Amyloidosis, Ineligible for Aggressive Treatment with High-Dose Melphalan and Stem Cell Transplantation.
Authors: Sanchorawala Vaishali , Seldin David C. , Wright Daniel G. , Skinner Martha , Finn Kathleen T. , Falk Rodney H.
Published: April 2020

AL amyloidosis is caused by a clonal plasma cell dyscrasia and characterized by widespread, progressive deposition of Amyloid fibrils derived from monoclonal Ig light chains, leading to multisystem organ failure and death. Aggressive treatment of AL amyloidosis with high-dose melphalan followed by autologous stem cell transplant (HDM/SCT) can induce hematologic and clinical remissions and extend survival. However, not all patients are able to tolerate HDM/SCT, particularly those with severe multisystem disease. Consequently, we investigated an alternative treatment regimen consisting of pulsed low-dose intravenous melphalan (LDM) for a series of patients considered ineligible for HDM/SCT because of severe cardiac involvement and poor performance status. LDM was administered at 17.5–25 mg/m2 every 4–6 weeks for 3–4 cycles depending upon patients’ clinical condition, renal function and performance status. Growth factor support was used with each cycle, and the dose of melphalan was reduced by 20% for white blood cell counts <500/mm3 and/or platelet count <20,000/mm3 during any treatment cycle. Changes in serum free light chain (FLC) levels, as well as toxicity and survival were evaluated. Fifteen patients with AL amyloidosis (11 males, 4 females; median age 55, range 47–69) were treated with this regimen between July 2002 and January 2004. Light chain isotypes were l in 11 cases and k in 4. All patients had clinically significant cardiac involvement with NYHA class III/IV congestive heart failure and/or left ventricular ejection fraction ≤ 45%, and/or ≥ 2 organ systems involved. The median number of LDM cycles given was 3 (maximum; 4), and 3 patients received only 1 cycle. Serum FLC levels were available before and after treatment in 10 patients. Eight of these patients (80%) achieved a > 50% reduction in FLC levels, and 2 (20%) achieved normal FLC levels. One patient achieved a 20–25% reduction in the FLC, and 3 experienced no reduction. Of the15 patients treated, 2 patients survive 6 and 24 months after treatment, while 13 have died (median survival, 2 months; range, 0–10 months). Ten patients (67%) died within 35 days of initiating treatment. Transient myelosuppression was the major treatment-related toxicity with neutrophil and platelet nadirs occurring 12–16 days after melphalan infusions. There was 1 death from sepsis during treatment-induced myelosuppression. In summary, pulsed low-dose intravenous melphalan (LDM) was found to induce hematologic responses in most patients who could be evaluated. However, extended survival ≥ 2 years was observed in only 1 of the 15 patients with severe amyloidosis who were studied. Nonetheless, this regimen may benefit healthier patients, particularly those with less severe cardiac involvement, who are otherwise ineligible or unable to receive HDM/SCT.
Phosphatidylserine-Positive Erythrocytes Modulate Tissue Factor Activity In Vivo and In Vitro in Sickle Cell Disease.
Authors: Setty Yamaja B.N , Key Nigel S. , Stuart Marie J.
Published: April 2020

Evaluation of fluid phase markers reveals activated blood coagulation in sickle cell disease (SCD), with enhanced in vivo thrombin generation. Studies have shown that whole blood tissue factor (WTBF) activity is elevated, and that both monocytes and endothelial cells (ECs) contribute to WBTF. In a recent preliminary study, we have found an unexpected and interesting positive correlation between WBTF and the levels of both reticulocytes (R=0.6) and phosphatidylserine (PS)-positive red cells (R=0.4) in SCD and an inverse correlation with hemoglobin (R=-0.4). We have therefore evaluated potential mechanisms for erythroid modulation of WBTF. Reticulocytes being PS-positive, we initially evaluated the effects of PS-positive red cells on endothelial TF using human lung microvascular ECs and ionophore-treated PS-exposing control erythrocytes, since sickle erythrocytes also express other adhesion markers. Following incubation with appropriate treatments for 24 hours, ECs were analyzed for TF expression (with E-selectin as a positive control for EC activation). Co-incubation of PS-positive red cells (15% ±) with ECs increased endothelial expression of TF in a hematocrit-dependent manner. PS-positive cells increased TF expression by 140, 200, 260 and 320% at hematocrits of 2, 5, 10 and 20%, respectively, compared to media controls. Results similar to those observed with TF were also noted with E-selectin. PS-positive sickle erythrocytes have been classified as either type I or type II cells. Type I express low levels of PS (reticulocytes), while type II are highly PS-positive and include sickle dense cells. We next evaluated whether both type I and type II PS-positive red cells triggered endothelial TF expression. Type II cells were prepared by incubating control erythrocytes with calcium ionophore, and type I cells were generated by incubating type II cells with predetermined amounts of annexin V (100 μg annexin V per 25X106 PS-positive cells). FACS analyses demonstrated appropriate PS levels in each PS positive category. We show that type I cells had no effect. TF expression occurred following incubation of ECs with type II erythrocytes (0.21±0.06 units TF with type II vs 0.10±0.06 with PS-negative cells at a 2% hematocrit, n=5, P<0.01). In final patient related studies we have observed a marked correlation between WBTF and type II PS-positive red cells (R=0.5, P<0.00001, n=81). No such correlation was noted with type I. We conclude that while the reticulocyte component of the hemolytic anemia does not appear to affect WBTF, type II PS cells (including potentially dense cells) modulate WBTF activity in SCD with implications for both the thrombophilic and adhesive pathophysiologic complications of this disease entity. These studies in addition have potential implications for other disease states where erythrocyte PS exposure occurs.
Mechanisms of Activation of the FIP1L1-PDGFRα Fusion Kinase.
Authors: Stover Elizabeth H. , Cools Jan , Gilliland D. Gary
Published: April 2020

We recently identified the FIP1L1-PDGFRα fusion protein as a frequent cause of hypereosinophilic syndrome (HES), a disorder characterized by persistent eosinophilia and organ dysfunction. ~60% of HES patients who are sensitive to imatinib, a selective tyrosine kinase inhibitor, harbor the FIP1L1-PDGFRα fusion. The fusion is expressed as a consequence of an interstitial chromosomal deletion of human chromosome 4 that fuses a novel protein FIP1L1 to the cytoplasmic domain of the tyrosine kinase PDGFRα, a type III receptor tyrosine kinase (RTK). The fusion protein is a constitutively active kinase and has transforming properties in vitro and in vivo that are inhibited by imatinib. In order to understand the mechanism of activation of the FIP1L1-PDGFRα fusion kinase, a series of deletions of FIP1L1 were fused to PDGFRα and the fusions were tested for transforming activity. The data suggested that the FIP1L1 moiety was in fact dispensable for PDGFRα activation. For example, substitution of FIP1L1 with the Myc-epitope fused to PDGFRA still resulted in PDGFRα autophosphorylation; transformation of Ba/F3 cells to IL-3 independent growth; and induction of a myeloproliferative disease in a murine bone marrow transplant assay. Structural analyses of other type III RTKs, such as FLT3, have identified an autoinhibitory function of the juxtamembrane (JM) domain. Thus, the lack of requirement for FIP1L1 suggests that activation of the FIP1L1-PDGFRa fusion kinase might be caused by disruption of an autoinhibitory JM domain in PDGFRα. Indeed, in patients with the FIP1L1-PDGFRα fusion, the breakpoints in PDGFRA are tightly clustered within exon 12, which encompasses the JM domain. Whereas activation of most known fusion kinases relies on enforced dimerization by a fusion partner, disruption of an autoinhibitory JM domain may be an alternative mode of fusion kinase activation.
Clonal Origin of Circulating Endothelial Progenitor Cells in Patients with Myelofibrosis with Myeloid Metaplasia.
Authors: Bergamaschi Gaetano , Pramparo Tiziano , Rosti Vittorio , Ramajoli Isabella , Gregato Giuliana , Massa Margherita , Marchetti Monia , Zuffardi Orsetta , Cazzola Mario , Barosi Giovanni
Published: April 2020

Chronic myeloproliferative diseases are clonal disorders of the hematopoietic stem cell. Among these conditions myelofibrosis with myeloid metaplasia (MMM) is characterized by increased numbers of circulating CD34+ cells compared to other Ph-negative myeloproliferative disorders and normal controls. Increased numbers of circulating CD34+ cells are likely due to mobilization of primitive hematopoietic stem cells/progenitor cells (HSC/HPC) from the bone marrow of patients with MMM following activation of proteases and disruption of adhesion interactions, which normally result in the retention of HSC/HPC within the marrow. A more detailed characterization of circulating CD34+ cells in MMM suggested that a cell fraction which included VEGFR-2+, CD34+VEGFR-2+, CD133+, and CD34+CD133+ cells contained endothelial progenitor cells.

In the present work we cultured peripheral blood mononuclear cells (PBMNC) from three patients with MMM (one male and two females), from one female patient with essential thrombocythemia (ET) and from three normal females in the presence of fibronectin and vascular endothelial growth factor (VEGF) for 7 days, in order to obtain endothelial cell colonies. The endothelial origin of colonies was assessed by VE-cadherin and CD31 staining. Clonality of endothelial cell colonies was evaluated in female subjects by X-chromosome inactivation studies based on determination of differential DNA methylation at the two alleles of X-linked polymorphic loci (Humara or PGK). Such evaluation was performed on at least 8 single colonies and on a pool of 7–8 colonies from each subject. The male patient with MMM had a 7q34 deletion in 50 % of metaphases. The deletion resulted in loss of heterozygosity at the polymorphic marker D7S684 which was therefore evaluated on both endothelial and hematopoietic colonies grown from PBMNC. Normal subjects showed a polyclonal pattern of X-chromosome inactivation in endothelial colonies, whereas MMM and the ET female patients showed selective inactivation of the same chromosome X which was inactivated in clonal peripheral blood polymorphonuclear cells. Loss of heterozygosity at the polymorphic marker D7S684 was observed in 6/10 and 4/9 endothelial and hematopoietic colonies, respectively, from the male patient with the 7q34 deletion. Our results suggest that in MMM, and possibly in other chronic myeloproliferative diseases, circulating endothelial progenitor cells are clonal and the disease is due to transformation of a hematopoietic stem cell which also retains endothelial differentiation potential.
Aberrant DNA Methylation Patterns in Chronic Idiopathic Myelofibrosis.
Authors: Jost Edgar , Gehbauer Gerald , Maintz Christoph E. , Jousten Peter , Habets Leo , Herman James G. , Osieka Rainhardt , Galm Oliver
Published: April 2020

Chronic idiopathic myelofibrosis (CIMF) is a clonal myeloproliferative disorder characterized by bone marrow fibrosis, angiogenesis and extramedullary hematopoiesis. No specific genetic defect underlying the disease has been identified so far. The spectrum of cytogenetic abnormalities in CIMF includes del (13q), del (20q) and partial trisomy 1q as well as abnormalities of chromosomes 1, 7 and 9. Hypermethylation of CpG islands within gene promoter regions is associated with transcriptional inactivation and represents an important mechanism of gene silencing in the pathogenesis of hematopoietic malignancies. This epigenetic phenomenon acts as an alternative to mutations and deletions to disrupt tumor suppressor gene function in cancerogenesis. In order to investigate the role of DNA methylation changes in the pathogenesis of CIMF, we have analyzed the methylation status of the promoter-associated CpG islands of 13 well-characterized tumor suppressor genes by methylation-specific polymerase chain reaction in peripheral blood cells from 20 adult patients with CIMF. The frequency of aberrant methylation among the patient samples was 25.0 % (5/20) for SOCS-1 and 5.0 % (1/20) for E-cadherin, MGMT, p73 as well as TIMP-2. There was no hypermethylation of p15, p16, RARbeta, DAP kinase 1, SOCS-3, hMLH1, TIMP-3 and RASSF1A. We detected at least one hypermethylated gene promoter region in 35.0 % (7/20) of the primary patient samples. Our data indicate that hypermethylation of tumor suppressor genes is a common event in CIMF. Epigenetic modification of genes regulating growth factor signaling, cell adhesion and DNA repair may, in addition to genetic aberrations, contribute to the malignant phenotype in CIMF. The exploration of our growing knowledge about epigenetic aberrations in tumorigenesis may help develop novel strategies in diagnosis and treatment of CIMF for the future.
PKC412 Effectively Inhibits Constitutively Activated FGFR3 Mutants in Murine Leukemia Models and t(4;14) Myeloma Cell Lines.
Authors: Chen Jing , Lee Benjamin H. , Williams Ifor R. , Kutok Jeffery L. , Duclos Nicole , Cohen Sarah , Adelsperger Jennifer , Okabe Rachel , Coburn Allison , Moore Sandra , Huntly Brian J.P. , Mitsiades Constantine S. , Anderson Kenneth C. , Griffin James D. , Fabbro Doriano , Gilliland D. Gary
Published: April 2020

Reccurent translocation t(4;14) associated ectopic expression of FGFR3, sometimes containing the activation mutation K650E (TDII), has been identified in 25% of human multiple myeloma (MM) patients and cell lines. However, current empirically-derived cytotoxic chemotherapy does not effectively treat this disease. One potential therapeutic strategy of treating MM is to inhibit the tyrosine kinase activity of FGFR3. In this report, we evaluated the efficacy of PKC412 (N-benzoyl-staurosporine), a small molecule tyrosine kinase inhibitor, for the treatment of FGFR3 mutants induced diseases. PKC412 effectively inhibits the tyrosine kinase activity and activation of downstream effector pathways of FGFR3 TDII or the constitutively activated TEL-FGFR3 fusion that was reported in a subtype of human peripheral T-cell lymphoma (PTCL), as well as proliferation of hematopoietic Ba/F3 cells transformed by the FGFR3 mutants. Furthermore, PKC412 drastically inhibits proliferation of four different multiple myeloma-derived primary cell lines that are associated with t(4;14) and expression of dysregulated FGFR3. Moreover, oral-gavage treatment with PKC412 resulted in statistically significant prolongation of survival in the murine bone marrow transplant (BMT) models of FGFR3 TDII-induced pre-B cell lymphoma or TEL-FGFR3 fusion-induced myeloproliferative disease, which suggests suitable pharmacokinetic and toxicity profiles of PKC412 for clinical use. Together, our data establish the small molecule inhibitor PKC412 as a molecularly targeted therapy for multiple myeloma and other human malignancies expressing activated FGFR3.
Effects of the Indazolylpyrimidine GW786034 on Angiogenesis and MM Cell Growth: Therapeutic Implications.
Authors: Podar Klaus , Simoncini Melissa , Le Gouill Steven , Tai Yu-Tzu , Kumar Shaji , Tassone Pierfrancesco , Catley Laurence P. , Pandite Lini N. , Kumar Rakesh , Hideshima Teru , Chauhan Dharminder , Anderson Kenneth C.
Published: April 2020

Vascular endothelial growth factor (VEGF) and its receptors play an important role in the pathogenesis of multiple myeloma (MM). Specifically, VEGF, which is present in the MM bone marrow (BM) microenvironment, induces neovascularization; triggers tumor cell growth, survival, and migration; inhibits dendritic cell maturation; and induces osteoclastogenesis. Therefore, the VEGF receptor provides a potential novel therapeutic target in MM. Previous studies showed that GW654652, a pan VEGFR inhibitor, inhibits growth and migration of MM tumor cells (Podar et al. Blood. 2004; 103:3473–3479.). Here we describe the effects of GW786034, a derivative of GW654652, on MM cells to support its potential clinical evaluation in MM patients. GW786034 inhibits VEGF- triggered Flt-1 phosphorylation and activation of downstream signaling molecules, induces concentration-dependent MM cell and HUVEC apoptosis, and inhibits VEGF- triggered MM cell migration. Specifically, GW786034 induces cleavage of caspase-8 and PARP, but not caspase-9, and induces downregulation of the pro-apoptotic molecules survivin, cIAP1, 2, and Mcl-1. Furthermore, GW786034 decreases proliferation of MM cells induced by their adhesion to BMSCs, and inhibits VEGF-induced upregulation of ICAM-1 and VCAM-1, thereby abrogating their adhesion to HUVECs. Consistent with these results, GW786034 inhibited both tubuli formation and accumulation of tumor cells at vascular branching points in a Matrigel tube forming assay. Finally, GW786034 sensitizes both MM cells alone and tumor cells bound to BMSCs or HUVECs to melphalan and bortezomib. Taken together, these studies support clinical evaluations of GW786034 either alone or in combination with other agents in MM patients.
Effect of Thalidomide and Revlimid on the Gene Expression That Reveals Molecular Circuitries Involved in T Cell Co-Activation.
Authors: Prabhala Rao , Cheema Puneet , Shammas Masood A. , Song Weihua , Blanchard Elizabeth , Chauhan Dharminder , Neri Paola , Tassone Pierfrancesco , Stirling David , Anderson Kenneth , Munshi Nikhil C.
Published: April 2020

Thalidomide (Thal) and its immunomodulatory analog, Revlimid (Rev) have demonstrated anti-myeloma activity in phase II and III studies and part of their activity is immune-mediated. We have previously demonstrated co-stimulatory effects of Thal and Rev on T cells mediated via B7-CD28 activation pathway. To understand the molecular events involved in T cell co-stimulation, we investigated the effects of Thal and Rev on the genome-wide gene expression profile of T cells with or without anti-CD3 activation. Normal PBMC were incubated with Thal or Rev in the presence or absence of anti-CD3 antibodies (200ng/ml) for 3 or 24 hours at 37oC and total RNA was analyzed using U133 GeneChip (Affymetrix). Although Thal and Rev do not show significant effect on T cell proliferation without CD3 stimulation, we observed up-regulation (>2-folds) of 56 genes and down-regulation of 42 genes at 3 hours, and up-regulation of 41genes and down-regulation of 35 genes at 24 hours with Rev. Significant up-regulation of IFN-α (10-folds) was observed at 3 hours. Up-regulation of chemokine CXC5 (9 fold); IFN-β (5 fold), IL-7 (5 fold) and IL-2 (3 fold); and signaling genes like JK 3 (10 folds) and MAPKK5 (5 folds) was observed at 24 hours with Rev. Following anti-CD3 activation, Rev induced significant upregulation of 59 and 48 genes and down-regulation of 126 and 61 genes at 3 and 24 hours respectively. These include down-regulation of Th2 cytokine genes IL-5(5 folds) and TGF-β (2 folds), and apoptotsis related genes, TNFSF12 (4 folds) and TNFRSF 6b (4 folds). Genes involved in B7-CD28 activation pathway and up-regulated include CXC 5 (9 folds), IL-8 (5 folds) and IL-3 (4 folds). Interestingly, p52, which is a key molecule in CD28 signaling, was elevated 18 folds. Additionally less than 2 fold increase was observed in other CD28 signaling genes like IKK, Vov, and RelA (p65). Rev also down-regulated, at 24 hours, the expression of IL-10 (6 folds), IL-17 (3 folds) and TGF-β (3 folds) which allows sustained co-stimulation. Similar changes with lesser magnitude were observed with Thal. These results provide insight into early molecular changes induced by Thal and Rev that is important for co-stimulation and suggest new molecular targets to generate effective immune responses. Moreover, these observations facilitate the pre-clinical rationale for the use of these immunomodulatory compounds to improve clinical outcome in myeloma.