bluuebunny/crossref_metadata_2025 huggingface dataset:
Interaction of the Leukemogenic CALM/AF10 Fusion Protein with the Hematopoietic Key Regulator Ikaros.
Authors: Greif Philipp A. , Tizazu Belay , Kremmer Elisabeth , Krause Alexandre , Bohlander Stefan K.
Published: October 2019

The focus of our research group is the study of the t(10;11)(p13;q14) translocation that leads to the fusion of the proteins CALM and AF10. This translocation can be found in acute lymphoblastic leukemia (ALL), acute myeloid leukemia (ALL) and also in malignant lymphomas. In some patients the t(10;11) is the only cytogenetic abnormality which indicates that the CALM/AF10 fusion is a causal event during leukemogenesis. Previous studies of our group have shown that the expression of CALM/AF10 in hematopoietic stem cells triggers the development of an aggressive leukemia in a murine bone marrow transplantation model. CALM (Clathrin Assembly Lymphoid Myeloid leukemia gene) has a function in Clathrin mediated endocytosis. AF10, a putative transcription factor with a PHD-Motive (Plant Homeo Domain) and a Leucine Zipper domain, was initially identified as fusion partner of MLL. The underlying mechanism of CALM/AF10 dependent leukemogenesis, however, remains mostly unknown. Recently we could show that AF10 interacts with the transcription factor Ikaros (ZNFN1A1) in yeast-two-hybrid assays. Interestingly, Ikaros is a key regulator of hematopoesis, required for normal differentiation and proliferation of B- and T-lymphocytes. The structure of the protein is characterized by a DNA-binding and an oligomerisation domain. Through interaction with many factors in the cell nucleus, Ikaros can act both as activator and repressor of transcription. In various forms of ALL as well as chronic myeloid leukemia (CML) an aberrant expression pattern of Ikaros has been found. In a murine model the expression of a dominant negative isoform of Ikaros causes leukemias and lymphomas. Using various AF10 deletion mutants in the yeast, the Ikaros interaction domain of AF10 was mapped to the Leucine Zipper domain of AF10 which has also been shown to be required for malignant transformation by the MLL/AF10 fusion protein. Overexpression of fluorescently labelled proteins reveals a similar distribution pattern of AF10 and Ikaros in the nucleus, whereas in the presence of CALM/AF10 Ikaros appears to be localized predominately in the cytoplasm. The interaction between AF10 and Ikaros has been confirmed by GST-pull-down assays. In order to further study this interaction and its role in leukemogenesis we have raised monoclonal antibodies against the C-terminus of AF10. These antibodies are currently established for Western Blot analysis and Immunoprecipitation experiments. Reporter gene assays are carried out to measure the impact of CALM/AF10 on Ikaros’ function as repressor or activator of transcription. These studies may provide new insights into the mechanism of CALM/AF10 induced leukemia and thereby facilitate the development of new therapies.
Malignant Cells in the Marrow and Blood of Patients with Hematopoietic Malignancies Have Higher Mitochondrial Membrane Potential as Measured with the Potentiometric Dye JC-1.
Authors: Gardner Jeffrey R. , Zhang Rong H. , Rose Ruth , McKenzie Sue , Maslak Peter , Heaney Mark L.
Published: October 2019

Background: Immunophenotyping is an important method to define hematopoietic malignancies, but used alone, its ability to provide functional information about the malignant cells is limited. JC-1 is a fluorescent dye used to measure mitochondrial membrane potential in cells. We have developed a flow cytometric assay using JC-1 in conjunction with standard cell surface markers to characterize clinical samples from patients with hematologic malignancies.

Methods: Ficoll-purified cells derived from the blood or marrow were stained with JC-1, and analyzed at 530 nm and 590nm to assess mitochondrial mass and mitochondrial membrane potential, respectively. Malignant and normal cell populations were assigned and gated by analysis of forward scatter and side scatter. Gating in this manner identified energetically discrete cell populations confirmed by back gating analysis.

Results: To date we have measured the mitochondrial membrane potential from the bone marrow or peripheral blood of 3 normal individuals and 22 patients with acute and chronic leukemias and lymphoma involving the marrow. Samples have been analyzed from AML (6), ALL (3), CML- chronic phase (2), CLL (5), mantle cell lymphoma (3), diffuse large B-cell lymphoma (1) and acute biphenotypic leukemia (2). Compared with samples derived from normal individuals, malignant cells have a different energetic signature, and in nearly all instances, a substantially higher mitochondrial membrane potential. The mitochondrial membrane potential of malignant cells was 5.5 times higher (range 2.15–12) than normal cells within the same samples in 19 of 20 specimens (given the overlap in cell populations, CML samples were excluded and 1 CLL sample had a mitochondrial membrane potential that was the same as the normal population). Mitochondrial membrane potential is also higher in malignant cells compared with peripheral blood and marrow cells from normal individuals. While cells derived from patients with acute leukemias appear to be energetically homogeneous, cells from both patients with CML and 4 of 5 patients with CLL had at least 2 energetically discrete subpopulations despite being cytogenetically homogeneous (in the case of the CML samples) or immunophenotypically homogenous (in the case of the CLL samples).

Conclusions: JC-1 staining can be performed reliably in concert with immunocytochemistry and provides insight into the metabolism of malignant hematopoietic cells. Preliminary data indicate that malignant cells from patients with leukemia and lymphoma have a high mitochondrial membrane potential which may reflect altered oxidative metabolism. This may serve as a metabolic signature for monitoring minimal residual disease or assessing the effect of therapeutic agents on functional status of malignant populations.
Rituximab-Mediated Upregulation of PTEN Expression and Inhibition of Both the PI3K/AKT Pathway and Pleitrophin (PTN) Expression in B-NHL: Roles in Cell Growth Inhibition and Chemosensitization.
Authors: Suzuki Eriko , Chen Haiming , Baritaki Stavroula , Vega Mario I. , Gordon Melinda , Schiller Gary J. , Berenson James , Bonavida Benjamin
Published: October 2019

We have recently reported that rituximab treatment of B-NHL cell lines, like Ramos, inhibited the PI3K/AKT signaling pathway and downregulated Bcl-xL expression. The role of the AKT pathway in chemosensitization was corroborated by treating Ramos cells with the AKT inhibitor, LY294002, and resulted in sensitization of the cells to drug-induced apoptosis. We have investigated a potential underlying mechanism responsible for the rituximab-mediated inhibition of the AKT pathway. PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a tumor suppressor phosphatase and functions as a negative regulator of the PI3K pathway through its phosphatase activity. We hypothesized that rituximab may upregulate PTEN expression and thus, inhibiting the AKT pathway. Treatment of Ramos cells with rituximab (20μg/ml for 20h) resulted in significant upregulation of PTEN expression (as assessed by both Western and RT-PCR). Time kinetic analysis showed that PTEN is upregulated as early as 6–9h post rituximab treatment. In addition, since rituximab inhibits cell proliferation and cell growth, we have examined the effect of rituximab on the expression of the growth factor pleitrophin (PTN). PTN is a heparin-binding and secreted growth differentiation factor that mediates various functions such as cell motility and migration, survival, growth and differentiation. Treatment of Ramos cells with rituximab inhibited PTN expression as early as 12h post treatment. The present findings suggested that rituximab-mediated induction of PTEN expression and inhibition of both the AKT pathway and PTN epxression may be interrelated and play an important role in rituximab-mediated cell growth inhibition and chemosensitization. A recent report by Li et al., (JBC, 281:10663,2006) demonstrated that PTEN null cells exhibited upregulation of PTN expression and activation of the PI3K/AKT pathway. Further, inhibition of PTN resulted in inhibition of the AKT pathway, thus establishing a feedback mechanism. Our findings with rituximab are consistent with the Li’s findings’. The mechanism by which rituximab inhibits PTN expression is not clear. Reported studies have implicated the role of AP-1 in PTN transcription and in agreement, our findings have also demonstrated that rituximab inhibits AP-1. Overall, the present studies suggest novel targets modified by rituximab namely, PTN and PTEN, which can be considered for therapeutic intervention in the treatment of both rituximab-sensitive and rituximab-resistant tumors.
Polymorphisms of Thymidylate Synthase in the 5′- and 3′-Untranslated Regions Associated with Risk of Non-Hodgkin’s Lymphoma.
Authors: Kim Hee Nam , Tran Huong Thi Thanh , Lee Il-Kwon , Kim Yeo-Kyeoung , Yang Deok-Hwan , Lee Je-Jung , Shin Myung-Geun , Kook Hoon , Hwang Tai Ju , Park Moo Rim , Jo Deog Yeon , Won Jong Ho , Kim HyeoungJoon
Published: October 2019

Thymidylate synthase (TS) catalyzes the conversion of dUMP by 5,10-methylenetetrahydrofolate to dTMP in DNA synthesis. Polymorphisms in the untranslated regions(UTRs) of TS, which may modulate TS transcription and expression, have been associated with susceptibility to several malignancies. In this study, to evaluate the association with TS the 28-bp tandem repeat(2R→3R) and the 6-bp deletion(6 bp-) and susceptibility to non-Hodgkin’s lymphoma(NHL), large-scale population-based case-control study was conducted in Chonnam National University Hospital between Mar 1997 and Feb 2006. 553 patients with histologically comfirmed lymphoma and 1,324 controls were evaluated. The cases consisted of 275 diffuse large B-cell lymphomas (DLBCL), 109 T-cell lymphomas and 169 unclassifiable lymphomas. TS 2R2R genotype was significantly associated with increased risk for NHL and T-cell lymphoma, but not for DLBCL. Using subjects with the TS 3R3R as a reference group, the OR of TS 2R2R was 2.00 (95% CI 1.21–3.31, p=0.007) for NHL and 3.30 (95% CI 1.52–7.17, p=0.003) for T-cell lymphoma. The association was 1.65 fold higher and more evident for T-cell lymphoma than NHL. However, there was no significant association of TS 6bp- with NHL.

In conclusion, theses results suggest that TS (2R→3R) may play an important role in the pathogenesis of NHL, and that DNA synthesis may play a crucial roles in the pathogenesis of specific NHL subtypes.
Majority of Transformed Lymphomas Have High SUVs on PET Scanning Similar To Diffuse Large B Cell Lymphoma (DLBCL).
Authors: Weissler Matthew , Noy Ariela , Schöder Heiko , Gönen Mithat , Yeung Henry
Published: October 2019

Background: In our previous work, we correlated non-Hodgkin’s (NHL) histology with intensity of FDG uptake (SUVmax) on positron emission tomography in untreated patients: an SUVmax greater than 10 predicted an aggressive lymphoma with >80% certainty and an SUVmax greater than 13 predicted an aggressive lymphoma with >90% certainty. We sought to evaluate SUVmax in a series of patients with transformed lymphoma.

Methods: All PET scans for lymphoma indications at our institution 1999–2005 were evaluated. Patients were included if they had biopsy proven transformation. FDG-PET scans were included if they were no more than sixty days prior to or 90 days after biopsy proven transformation. Patients had no therapy for sixty days prior to PET scan unless there was documented progression of disease after the last treatment.

Results: Among the indolent diagnoses (n= 28), the following specific histologies were identified: 7 follicular lymphoma grade I (FL I), 5 follicular lymphoma grade II (FL II), 5 small lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL), 6 marginal zone lymphoma (MZL) including one of mucosa associated lymphoid tumor (MALT) type, one mycoses fungoides, and one indolent lymphoma otherwise not specified. Three follicular lymphomas grade III (FL III), including one specified as grade IIIa (FL IIIa) were considered indolent if they preceded a diagnosis of DLBCL. Among the aggressive diagnoses (n= 28), the following specific histologies were identified: 22 DLBCL, one follicular lymphoma grade IIIa included as it was preceded by FLI, one large peripheral T-cell lymphoma, and 4 “Large cell lymphoma” (LCL), not otherwise characterized on account of biopsy size. Standard uptake values (SUV) were measured at the biopsy site when possible (n=25). In 3 patients the biopsy site was completely excised before the PET scan. The SUV of biopsy site ranged from 2.9–26.7, with a median of 9 and mean of 11. Excluding the three patients who had an excisional biopsy, 10/25 (40%) biopsies had an SUV above 10; and 8/25 (32%), above 13. The SUVmax for a transformed aggressive lymphoma ranged from 3.2–30.2, with a median of 10.8 and mean of 14. 16/28 (57%) patients had an SUVmax above 10; and 12/28 (43%), above 13.

Conclusions: Similar to patients with de-novo aggressive lymphoma, the majority of patients with transformed lymphomas have high SUVmax for a given pre-treatment staging study. Therefore, transformation to aggressive lymphoma should be suspected in patients with indolent lymphoma found to have high SUVs on FDG PET, and biopsies should be directed to the site of greatest PET avidity whenever feasible.
Achievement of FDG-PET Negativity Does Not Predict Durable Responses in T-Cell Lymphoma.
Authors: Pro Barbara , Nunez Rodolfo F. , Romaguera Jorge , Rodriguez Maria A. , Hagemeister Fredrick , Fayad Luis
Published: October 2019

Background: T-cell non-Hodgkin’s lymphomas represent approximately 12% of all lymphomas and in general are associated with a worse prognosis compared to their B-cell counterparts. In patients with Hodgkin’s disease and aggressive B-cell lymphoma (18F)fluoro-2-deoxy-D-glucose positron emission tomography (FDG-PET) represents an important early prognostic tool for predicting outcome. Very little information is available regarding the role of FDG-PET in T-cell lymphoma.

Patients and Methods: A total of 20 patients with newly diagnosed and histologically proven T/NK cell lymphomas where staged with both conventional methods and whole-body PET scanning. Patients underwent FDG-PET, concurrently with CT of neck, chest, abdomen, and pelvis within 3 weeks prior to induction treatment, after 2–4 cycles, and after the end of induction. All patients but three received HCVIDDoxil, using pegylated liposomal doxorubicin as a substitute for doxorubicin in the HyerCVAD regimen, alternating with methotrexate and cytarabine. Three patients received the standard CHOP (cyclophosphamide,hydroxydaunomycin, vincristine, prednisone) regimen. According to the International Prognostic Index (IPI), 35% of patients were stratified as low risk, 25% as low/intermediate, 35% as high/intermediate, and 5% as high risk. Baseline scans were strongly positive in all patients.

Results: During induction therapy (at 2–4 cycles) FDG-PET became negative in 16 (80%) patients and remained positive in 4 (20%) patients. Twelve out of 16 (75%) patients who were PET negative during induction therapy relapsed, and four out of four (100%) in the PET positive group. Two of the “ early” PET negative patients became positive after 4 cycles. Median progression-free survival was 240 days in the PET negative group and 85 days in the PET positive group (Log-rank p=0.07).

Conclusions: In this limited number of patients homogeneously treated with newly diagnosed T cell lymphomas the assessment of metabolic activity by FDG-PET during induction therapy did not predict long-term prognosis.
Human Mesenchymal Stem Cells Isolated from Bone Marrow and Lymphoid Organs Support Tumor B-Cell Growth: Implications in Follicular Lymphoma Pathogenesis.
Authors: Tarte Karin , Ame-Thomas Patricia , Hajjami Hélène Maby-El , Monvoisin Céline , Jean Rachel , Lamy Thierry , Fest Thierry
Published: October 2019

There is accumulating evidence that cellular microenvironment plays a key role in follicular lymphoma (FL) pathogenesis, both within tumor lymph nodes (LN) and in infiltrated bone marrow (BM) where ectopic LN-like reticular cells are integrated within malignant B-cell nodular aggregates. In normal secondary lymphoid organs, specific stromal cell subsets provide a highly specialized microenvironment that supports immune response. In particular, fibroblastic reticular cells (FRC) mediate immune cell migration, adhesion, and reciprocal interactions. The role of FRC and their postulated progenitors, i.e. bone marrow mesenchymal stem cells (MSC), in FL remains unexplored. In this study, we have investigated the relationships between FRC and MSC and their capacity to sustain malignant B-cell growth. Our findings strongly suggest that secondary lymphoid organs contain bona-fide MSC able to give rise at single-cell level to adipocytes, chondrocytes, and osteoblasts. These LN-derived MSC could also differentiate, in response to a combination of tumor necrosis factor-α (TNF) and lymphotoxin-α1β2 (LT), into fully functional FRC, able to construct a dense extracellular reticular meshwork positive for transglutaminase and fibronectin staining, to produce inflammatory (CXCL9, CXCL10, CCL5, CCL2) and LN-specific (CCL19) chemokines, and to favour lymphoma B-cell growth. Bone marrow-derived MSC (BM-MSC) acquire in vitro a complete FRC phenotype in the same culture conditions. As an exemple, BM-MSC had a strong, although not complete, protective effect on serum deprivation-induced apoptosis of BL2 cell line (mean percentage of CD20posCaspase-3pos cells: 24.8 +/− 17.5% in coculture with BM-MSC versus 80.7 +/- 10.4% in medium alone; P < .05; n =5) and pretreatment with TNF/LT fully restored BL2 viability (mean percentage of CD20posCaspase-3pos cells: 7.4 +/− 4.7%; P < .05; n = 5). Moreover, stimulation of stromal cells by TNF/LT before coculture enhanced the number of viable CD19pos primary FL B cells by 2.4-fold for BM-MSC and 2.3 fold for LN-MSC compared with the culture without stromal cells (P < .05; n = 6). Interestingly, cell contact with lymphoma B-cell lines or purified FL B cells trigger the differentiation of BM-MSC into FRC that, in turn, support malignant B-cell migration, adhesion and survival. Altogether, these new insights into the interactions between lymphoma cells and their microenvironment could offer original therapeutic strategies.
CNOP (Mitoxantrone) Chemotherapy Is Inferior to CHOP (Doxorubicin) in the Treatment of Patients with Aggressive Non-Hodgkin Lymphoma (Review).
Authors: Bjorkholm Magnus , Osby Eva , Andersson Tomas , Ahlbom Anders
Published: October 2019

Introduction: Mitoxantrone (M), an anthracenedion, was introduced in the early/mid 1980s as a more tolerable alternative to anthracyclines. This agent has a broad anti-tumour activity including lymphoma with potentially less cardiotoxicity than doxorubicin (D), which may be of particular importance in the elderly patient population. However, an important issue is whether M is as efficacious as D in the treatment of NHL patients.

Methods: Through search of several relevant databases and direct contacts with lymphoma investigators worldwide, we identified seven randomized studies of previously untreated patients comparing CHOP and CNOP chemotherapy in aggressive NHL. In this analysis we included five trials where (D;50mg/m2) was compared with (M;10–12mg/m2; table) and the interval between chemotherapy courses was 3–4 weeks. Patients reported in the Pavlovsky article were included in the Bezwoda report, why analyses were performed separately with either but not both of these reports. Odds ratios of complete remission (CR) and overall survival (OS) were pooled using a fixed effects model.

Results: CHOP was significantly superior to CNOP with regard to both CR rate and OS (figure a–b). No formal testing of side effects could be made. However, myelosuppression was similar with both regimens as was the incidence of infections and neutropenic fever episodes. In addition, clinical evidence of symptomatic congestive heart failure was not more frequent among patients treated with CHOP. In contrast, the incidence of gastrointestinal toxicity and alopecia was significantly higher in this patient group.

Conclusion: CHOP chemotherapy is more efficacious than CNOP at equitoxic (myelosuppression) doses leading to higher CR rates and improved survival. This regimen is, however, also associated with more alopecia and gastrointestinal toxicity.

Table Randomized trials comparing doxorubicin (CHOP) with mitoxantrone (CNOP) in untreated patients with aggressive NHL

Figure Figure
Efficacy and Safety of DepoCyte® (Liposomal Cytarabine) in Patients with CNS Involvement from Non-Hodgkin’s Lymphoma (NHL): A Report on 32 Patients Treated in Spain.
Authors: García-Marco José A. , Navarro B. , Ruiz Sanz E. , Palomera L. , Capote F.J. , de Sevilla A. Fernández , Sánchez E.G. , Cantalapiedra A. , Pérez R. , Casal J.R. , Mediavilla J.D. , García F.R. , Martín Y. , Panizo C. , Alvarez A.L. , García T. , Pérez G.
Published: October 2019

Lymphomatous meningitis (LM) occurs in approximately 7–15% of patients with lymphoma and carries an extremely poor prognosis (Chamberlain et al. CNS Drugs1998;10:25; Chowdary & Chamberlain J Natl Compr Canc Netw2005;3:693). Intrathecal (IT) chemotherapy with standard agents (cytarabine, methotrexate and thiotepa) is limited by the need for multiple injections per week via lumbar puncture or an Ommaya reservoir. Liposomal cytarabine (DepoCyte®) has an extended half-life in cerebrospinal fluid that permits fortnightly administration, improving convenience and reducing the potential for injection-related trauma and infections. Thirty-two Spanish patients (median age 43.5 years [range 19–78]; 22 male) with NHL received IT liposomal cytarabine for the treatment of LM between 2004 and 2006 at 21 treatment centers. Half of the patients had diffuse large B-cell lymphoma (DLBCL; n = 16); the remainder had Burkitt’s lymphoma (n = 4), T-cell NHL (n = 3), mucosa-associated lymphoid tissue lymphoma (n = 3), lymphoblastic lymphoma (n = 2), follicular lymphoma (n = 2) or primary CNS lymphoma (n = 1). A full histological diagnosis was not available for 1 patient. The dosage of liposomal cytarabine was 50 mg per cycle, with a median of 4 cycles (range 1–10). All patients received oral dexamethasone (4 mg 2–4 × daily for 4–7 days per cycle) as prophylaxis for chemical arachnoiditis. Neurological and cytological responses were obtained in 20 (62%; 16 complete responses, 4 partial responses; Figure 1) and 25 (78%) patients, respectively. Neurological progression was subsequently reported in 23 (72%) patients, with a median time to progression of 45 days (range 7–570). Twelve patients were still alive at the time of reporting, including 5 of 16 patients with DLBCL and 2 of 3 patients with T-cell NHL. Eighteen patients reported no adverse effects from treatment. The most commonly reported adverse effects were headache (n = 11), nausea (n = 4) and vomiting (n = 4). Data from this case series show that IT liposomal cytarabine is effective and well tolerated in the treatment of LM; the less intensive administration schedule of the agent may offer additional benefits to patients and their carers during the final months of life.

Figure Figure
Necessity of Intraventricular Chemotherapy with the Modified Bonn Protocol in Primary CNS Lymphoma (PCNSL) Patients.
Authors: Schmidt-Wolf Ingo G.H. , Pels Hendrik , Juergens Annika , Glasmacher Axel , Schulz Holger , Kroschinsky Frank , Egerer Gertlinde , Lamprecht Monika , Wismeth Caecilia , Schlegel Uwe
Published: October 2019

Background: Treatment of primary CNS lymphoma (PCNSL) with a combined systemic and intraventricular chemotherapy (Bonn protocol) has achieved an overall response rate (ORR) of 84% and long term complete remissions in a substantial fraction of patients younger than 60 years.

Purpose: Due to a high infection rate of the Ommaya reservoir the question was addressed if intraventricular treatment is dispensable in this polychemotherapy protocol.

Patients and Methods: Fifty patients with histologically confirmed PCNSL were enrolled onto a phase II-study evaluating chemotherapy without radiotherapy and without intraventricular treatment. A high-dose methotrexate (MTX) (cycles 1,2,4,5) and cytarabine (ara-C) (cycles 3,6) based systemic therapy (including dexamethasone, vinca-alkaloids, ifosfamide and cyclophosphamide) was administered.

Results: In an ongoing trial thirty-five of 50 patients (18 pat. < 60 years, 17 pat. over 60 years) are yet assessable for response after a median follow up of nine months (range: 1 to 26 months). In 18 patients < 60 years, the ORR was 78%. However, median time to treatment failure (TTF) was eight months, and median progression free survival (PFS) only 7 months according to frequent early relapses.

Conclusions: Early relapses are frequent in younger patients treated with the modified Bonn protocol without intraventricular treatment despite a high ORR. These preliminary results support the assumption that intraventricular treatment is essential to achieve sustained remissions after successful treatment of PCNSL.
Phase II Study of Proteasome Inhibitor Bortezomib (Velcade®) in Patients with Relapsed/Refractory T-Cell Lymphoma: Preliminary Results.
Authors: Zinzani Pier Luigi , Tani Monica , Musuraca Gerardo , Stefoni Vittorio , Marchi Enrica , Fina Mariapaola , Baccarani Michele
Published: October 2019

The mechanisms by which Bortezomib elicits its antitumor activity may vary among tumor types, and the extent to which each affected pathway is critical to the inhibition of tumor growth could also differ. The aim of this study was to determine the efficacy and toxicity of Bortezomib in previously pretreated patients with peripheral T-cell lymphoma unspecified (PTCLU) with only skin involvement and cutaneous T-cell lymphomas (CTCL). Eligibility criteria included PTCLU or CTCL (according to REAL/WHO classification) with measurable disease; any stage, any IPI, any bone marrow status; second or more relapse or refractory disease; age ≥18; ECOG performance status ≤ 2; Hb ≥10 g/dL, ANC ≥ 1.5x109/L and platelets ≥ 100 × 109/L; normal hepatic, renal and cardiac functions; and voluntary written informed consent. Bortezomib was given at 1.3 mg/m2 IV push on days 1, 4, 8 and 11 every 21 days. Restaging was done every 2 cycles. Patients were treated for up to a total of 6 cycles unless removed from study for failure to respond or toxicity. The response criteria were those recommended by NCI sponsored Working Group. At data reporting for this abstract, 15 patients were enrolled and 12 were evaluable for response. Of these 12 patients, 10 had CTCL and 2 PTCL; 11 were male and 1 female; 6 were aged >60 years (range 48–80). Overall response rate was 67% (2 CR + 6 PR) and the remaining 4 patients had PD. Histologically the responder patients were 7 CTCL and 1 PTCL. Time to event analyses, including TTP and duration of response are pending further follow-up. Treatment was well tolerated; grade ≥ 3 AEs were seen in 5 patients. The most common grade ≥ 3 AEs were neutropenia, thrombocytopenia and peripheral neuropathy. These preliminary data indicate that Bortezomib is active as a single agent for patients with relapsed/refractory CTCL and PTCL with skin involvement. Final efficacy and safety data will be presented.
New and Comprehensive Cytogenetic Prognostication and Categorization in MDS.
Authors: Haase Detlef , Germing Ulrich , Schanz Julie , Pfeilstoecker Michael , Noesslinger Thomas , Hildebrandt Barbara , Kuendgen Andrea , Luebbert Michael , Giagounidis Aristoteles , Aul Carlo , Truemper Lorenz , Mueller Thomas , Valent Peter , Fonatsch Christa , Steidl Christian
Published: October 2019

The International Prognostic Scorig System (IPSS) was established on the basis of a multicentric analysis of 816 pts. with MDS with 327 individuals (40%) showing clonal cytogenetic abnormalities. Analyses of survival and clinical courses were performed for 11 cytogenetic subgroups. Although the IPSS represents a main and meritorious progress in prognostication in MDS it has shortcomings resulting from the limitated number of pts. Thus, little is known about the prognostic impact of rare and combined anomalies and a lot of abnormalities are assigned to the intermediate prognostic group without knowing their actual prognosis. It was our aim to refine the cytogenetic prognostic categorization in MDS. For this purpose we created a multicentric German-Austrian database of 2072 pts. with MDS and successful cytogenetics of whom 1080 individuals (52%) had clonal abnormalities. Of this group 1202 individuals were treated with supportive care only, and had a well-documented follow-up and blast count. For this group we defined 23 cytogenetic prognostic subgroups (at least 5 pts. with the same abnormality) also considering the number of accompanying abnormalities. Every subgroup was categorized in relation to the IPSS-categorization according to blast counts and a comparison of survival times. The following subgroups (all either isolated or with only one additional abnormality except the complex cases) were thus established: 9q- (median survival: not reached (nr), categorization: good); 15q- (nr, good); t(15q) (nr, good); 12p- (108, good); +21 (108, good); 5q- (77.2, good); −X (56.4, good); normal (53.4, good); −Y (39.0, good); t(1q) (34.7, good); t(7q) (34.7, good); t(11q) (32.1, good); −21 (32.0, good); 11q- (26.1, intermediate I); +8 (int I); +19 (19.8, int II); 7q- (19.0, int II); any 3 abnorm. (17.1, int II); complex with 3 abnormalties (17.0, int II); −7 (14.0, int II); complex with more than 3 abnormalities (8.7, poor); t(5q) (4.4, poor). Applying our new categorization the following results were obtained: good risk (n=802 pts., ms: 55.4 mo); int I (n=170, 29.4 mo); int II (n=103, 14.6 mo); poor (n=127, 8.1 mo). The differences between the respective Kaplan-Meier curves were highly significant (p=0.0000). Our findings could be the basis for a new comprehensive cytogenetic scoring system covering most pts. with MDS by a now well-known prognosis.
Randomized Comparison of Cladribine Single (C) or in Combination with Cyclophosphamide (CC), and COP in Previously Untreated Low Grade B-Cell Non-Hodgkin Lymphoma Patients.
Authors: Kalinka E. , Wajs J. , Sulek K.S. , Blasinska-Morawiec M. , Centkowski P. , Ceglarek B. , Robak T. , Holowiecki J. , Mazur G. , Federowicz I. , Jedrzejczak W. , Watek M. , Warzocha K.
Published: October 2019

To comparatively assess first-line treatment with cladribine single (C) or in combination with cyclophosphamide (CC), and COP (cyclophosphamide, vincristine, prednisone) in low grade B-cell non-Hodgkin lymphoma (NHL), previously untreated patients (pts) with Ann Arbor stage II-IV were randomly allocated to receive 6 monthly courses of either C, CC, or COP. End points were treatment response, freedom from progression (FFP) and overall survival (OS), and tolerance. From June 1, 2000 to June 30, 2005, 196 pts were randomized in 17 centers. Of 153 pts for whom data is available, 55 (36%) were diagnosed as small lymphocytic, 11 lymphoplasmocytoid (7%), 37 marginal-zone (24%), 42 follicular (27.5%), and 8 not otherwise specified low grade B-cell NHL (5.5%). Randomization constituted comparable groups, including International Prognostic Index variables. Compared to C and CC, COP induced lower overall response rates (75%, 90%, 50%, χ2 =7.9 p<.005), including lower complete remission rates (38%, 62%, 9.5%, χ2=19.2 p<.0001). With a median follow-up of 15 months, FFP was superior in patients receiving cladribine-containing regimens (χ2 = 21.8, log-rank p<.0001). No difference in median OS was observed. Incidences of infections (9% versus 3.5% versus 7%) and non-hematological side effects (7.5% versus 3.5% versus 7%) were similar in the randomized groups, whereas CC but not C induced more frequent peripheral cytopenias compared to COP (30% versus 11%, p=.034). This resulted in higher frequency of prolongation of intervals between CC versus COP treated pts (respectively 45% and 21%, χ2=6.04 p=.014) and C versus CC treated pts (respectively 26% and 45%, χ2=4.24, p=.039). Dose reductions because of hematological or other toxicity were comparable in C (9.5%), CC (20%), and COP (21%) groups. Although final results warrant completed data for all randomised pts with longer follow-up, similar tolerance and higher efficacy of cladribine-based regimens over COP provide rationale to combine C or CC with rituximab in future clinical trials.
Cladribine in Weekly Versus Daily Schedule for Untreated Active Hairy Cell Leukemia: Final Report of Polish Adult Leukemia Group (PALG) Prospective, Randomized, Multicenter Trial.
Authors: Robak Tadeusz , Jamroziak Krzysztof , Gora-Tybor Joanna , Blonski Jerzy Z. , Kasznicki Marek , Dwilewicz-Trojaczek Jadwiga , Dmoszynska Anna , Wojtaszko Maria , Zdziarska Barbara , Calbecka Malgorzata , Hellmann Andrzej , Stella-Holowiecka Beata , Sulek Kazimierz , Skotnicki Aleksander B. , Zawilska Krystyna , Kloczko Janusz , Seferynska Ilona , Konopka Lech , Piszcz Jaroslaw , Ceglarek Berandetta , Lewandowski Krzysztof , Gawronski Krzysztof , Kostyra Aleksandra , Nowak Wieslaw , Wiater Elzbieta , Dybowicz Jacek , Wojtaszko Maria , Molendowicz-Portala Lucyna
Published: October 2019

Cladribine (2-chlorodeoxyadenosine, 2-CdA) has dramatically improved prognosis of hairy cell leukemia (HCL), though the 2-CdA treatment-associated life-threatening infections may shorten potentially long-term survival of HCL patients. In search of optimal mode of 2-CdA administration in HCL, we compared the standard 5-day 2-CdA protocol with a novel weekly 2-CdA schedule that was suggested to be equally effective, but less toxic. Between the 1st January 1998 and the 31st September 2005, 132 patients with previously untreated, active HCL from 14 hematology centers were included to the study. Patients were randomly assigned to receive 2-CdA at 0.12 mg/kg in 2-hour intravenous infusion for 5 days (64 patients), or 2-CdA at 0.12 mg/kg in 2-hour intravenous infusion once a week for 6 weeks (62 patients). The response to chemotherapy was evaluated according to the NCI guidelines and treatment-related toxicity was recorded using standard WHO criteria. One hundred and sixteen patients were included in the final analysis of the trial. Comparison of the treatment response between daily and weekly 2-CdA protocols showed similar complete remission rate (76% and 72%, p=0.86) as well as similar overall response rate (95% and 91%, p=0.41). Survival analysis did not show significant difference between the study arms. Median progression-free survival equaled 4.3 years (95%CI = 3.3 – 5.2) and 5.1 years (95%CI = 4.7 – 5.6) (p=0.28) for patients treated with daily and weekly schedule, respectively. The estimated 6.5 year overall survival was 91% for daily and 88% for weekly 2-CdA protocol, p = 0.40. Interestingly, the analysis of adverse events did not confirm lower toxicity of the weekly 2-CdA schedule. Of special interest, there were no significant differences between daily and weekly protocol in the rate of treatment-related neutropenia (47% vs. 47%, p=0.97), the rate of grade 3/4 infections (18% vs. 26%, p=0.28), or the rate of 2-CdA-related septic deaths (2% vs. 1%, p=0.64). In conclusion, the results of our randomized study indicate that HCL treatment based on weekly 2-CdA infusions for 6 weeks is equally effective, but not safer than standard 5-day 2-CdA protocol.
Nutlin-3A, an MDM2 Antagonist, Induces p53-Dependent Cell Cycle Arrest and Apoptosis in Hodgkin Lymphoma (HL).
Authors: Drakos Elias , Thomaides Athanasios , Li Jiang , Konopleva Marina , Medeiros L. Jeffrey , Andreeff Michael , Rassidakis George Z.
Published: October 2019

p53 is the most frequently mutated tumor suppressor gene in human cancer. However, in Hodgkin lymphoma (HL) p53 is mutated only in a small subset of cases suggesting that modulation of wild-type-p53 (wt-p53) levels in Hodgkin and Reed-Sternberg (HRS) cells may have therapeutic implications in these patients. MDM2 (HDM2 in humans) is a physiologic negative regulator of p53 levels through a well-established auto-regulatory feedback loop. Nutlin-3A is a recently developed small molecule, which antagonizes mdm2 through disruption of p53-MDM2 interaction resulting in p53 stabilization. We hypothesized that nutlin 3A may stabilize p53 in HRS cells carrying wt-p53 gene, thus leading to p53-dependent apoptosis and G1-S cell cycle arrest. We used two novel classical HL cell lines recently established in our Institution, MDA-V and MDA-E, which have been shown to carry wt-p53 gene. As a control, we used a HL cell line L-428 harboring a mutant p53 (mt-p53) gene product (deletion at exon 4). We investigated effects on apoptosis and cell cycle arrest after treatment of cultured HRS cells with nutlin-3A or a 150-fold less active enantiomere, nutlin-3B. Treatment with nutlin-3A resulted in substantial cell death (up to 65%) in a concentration-dependent manner associated with increased apoptosis as shown by apoptotic morphology (DAPI immunofluorescence), annexin V binding (flow cytometry) and caspase activation (Western blot analysis) in MDA-V and MDA-E cells, but not in L-428 cells. Nutlin-3A-induced apoptotic cell death was accompanied by stabilization of p53 protein as detected by western blot analysis and immunofluorescence and up-regulation of pro-apoptotic Bax, a known target of p53. Inhibition of nuclear export by leptomycin B stabilized p53 at a similar level as compared to nutlin-3A treatment in these cells, suggesting that nutlin-3A stabilized p53 through inhibition of MDM2-mediated degradation of the protein. By contrast, no changes in cell viability, growth or apoptosis were seen after treatment with the inactive nutlin-3B small molecule. Treatment with nutlin-3A also resulted in a significant decrease (up to 85%) of cells in S-phase and a dose-dependent increase of cells in G1 phase of cell cycle as detected by flow cytometry, in MDA-V and MDA-E cells, but not in L-428 cells. Cell cycle arrest was associated with up-regulation of the cyclin-dependent kinase inhibitor p21, a transcriptional target of p53. In contrast, treatment of HRS cells with nutlin-3B had no effects on the cell cycle irrespective of p53 mutation status. Furthermore, combined treatment with nutlin-3A and doxorubicin revealed synergistic effects and enhanced cytotoxicity in HRS cells with wt-p53 gene. Targeting MDM2 with the specific antagonist nutlin-3A that leads to non-genotoxic p53 activation, apoptosis induction and cell cycle inhibition may provide a new therapeutic approach for patients with HL.
Recombinant Bispecific Monoclonal Antibody (bsMAb) Against CD20 and CD22 Active In Vitro and In Vivo Against B-Cell Lymphomas.
Authors: Qu Zhengxing , Cardillo Thomas M. , Shi Victoria , Delaney Diana L. , Hansen Hans J. , Chang Chien-Hsing , Goldenberg David M.
Published: October 2019

Background: Preclinical and clinical results suggest improved anti-lymphoma activity of combining anti-CD20 and anti-CD22 MAbs. Our aim was to develop a recombinant bispecific MAb against CD20 and CD22 antigens, and to evaluate its anti-tumor potency compared to the parental MAbs.

Methods: Tetravalent anti-CD20/CD22 bsMAb in the form of anti-CD20 IgG linked to two anti-CD22 scFv’s was prepared recombinantly. The ability of the bsMAb to inhibit cell growth and mediate CDC and ADCC was evaluated by cell-based assays. Phosphorylation and distribution of CD22 in B-lymphoma cells treated with the bsMAb were studied by immunoblotting. The extension of survival of disseminated Daudi lymphoma cells in SCID mice also was evalauted.

Results: In contrast to the parental anti-CD22 MAb, epratuzumab, the bsMAb did not internalize in Ramos cells. In CDC tests, the bsMAb showed no cytotoxic effects, similar to epratuzumab, although it did bind C1q complement protein, unlike epratuzumab. In ADCC, the bsMAb was as potent as the parental humanized anti-CD20 Mab, hA20, in inducing target-cell lysis. The anti-CD20/CD22 bsMAb has distinct effects on NHL B-cell lines compared to each parental monospecific MAb or in combination:

<10 nM bsMAb causes cells to aggregate; in the soluble, non-crosslinked form, while none of the parental MAbs alone or mixed had significant anti-proliferative activity, the recombinant bsMAb was strongly anti-proliferative; the anti-proliferation activities induced by an anti-IgM Ab and the bsMAb were synergistic; and the bsMAb resulted in rapid redistribution of CD22 into detergent-resistant membrane microdomains on the cell surface.

Initial animal studies show that the bsMab significantly extended survival over control SCID mice.

Conclusion: The recombinantly fused anti-CD20/CD22 bsMAb has new properties compared to the parental monospecific MAbs, and may represent a new class of potential therapeutic agents that could replace binary combinations of antibodies.
Histone Deacetylase Inhibitor Promotes Rituximab-Induced Apoptosis in Non-Hodgkin’s B-Lymphoma Cells by NF-kB-Mediated Bcl-2/Bcl-XL Downregulation and c-Myc Degradation.
Authors: Zhao Weili , Wang Lan , Liu Yuanhua , Yan Jinsong , Leboeuf Christophe , Janin Anne , Chen Zhu , Chen Saijuan
Published: October 2019

The anti-CD20 monoclonal antibody rituximab has shown promising results in the clinical treatment of patients with B-cell non-Hodgkin’s lymphoma (B-NHL). However, its therapeutic effect could still be improved. This study examined the anti-tumor activity of rituximab combined with histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) in CD20-positivie B-NHL cell lines, as well as in primary B-NHL cells and a murine B-NHL model. The combination treatment sensitized B-NHL cells to apoptosis in a synergistic manner, concomitant with Bcl-2/Bcl-XL downregulation, mitochondrial instability, and caspase activation. Particularly in Daudi cells relatively insensitive to rituximab, these events were associated with nuclear factor-kB (NF-kB) inactivation. SAHA presented functional complementation with rituximab, through decreasing IKKa/b and IkBa phosphorylation, thus preventing NF-kB nuclear translocation. In addition, SAHA induced IkBa cleavage to a stable inhibitory form and caused NF-kB degradation in response to caspase-3 activation. As an independent approach, co-administration of rituximab and SAHA resulted in a clear decline in levels of ERK cascade members, including extracellular-signal-regulated kinase (erk) itself, upstream Raf-1, mitogen-activated protein kinase/ERK Kinase Kinase-1 (mekk1), and mekk2, as well as their downstream transcription factor target c-myc. By western blot, c-Myc protein was subsequently downregulated when treated with rituximab or SAHA, and the degradation was most significant in combination group. More importantly, rituximab-SAHA combination significantly promoted primary B-NHL cells apoptosis and improved the survival time of a SCID mouse lymphoma model established with intravenous injection of Daudi cells. Terminal deoxytransferase-catalyzed DNA nick-end labeling and ultrastructural study revealed increased apoptotic lymphoma cells on mice spleen sections of combination group. Taken together, these findings emphasized the value of targeting apoptosis signaling pathway in lymphoma therapy. Rituximab in conjunction with histone deacetylase inhibitor may represent a novel strategy in treating patients with B-NHL.
Molecular and Functional Effects of the Novel MEK Inhibitor PD0325901 in Preclinical Models of Human Leukemias.
Authors: Milella Michele , Ricciardi Maria Rosaria , Gregorj Chiara , De Cave Fabiana , Abrams Steven L. , Steelman Linda S. , Chiaretti Sabina , Konopleva Marina , Petrucci Maria Teresa , Cognetti Francesco , Foa Robert , Andreeff Michael , McCubrey James , Tafuri Agostino
Published: October 2019

The Raf/MEK/ERK signaling module plays a pivotal role in the regulation of cell proliferation, survival, and differentiation. Our group, among others, has recently demonstrated that this pathway is frequently dysregulated in hematological malignancies and may constitute an attractive therapeutic target, particularly in AML. Here we investigated the effects of PD0325901, a novel MEK inhibitor, on phospho-protein expression, gene expression profiles, cell proliferation, and apoptosis in cell line models of AML, ALL, multiple myeloma (MM), ex vivo-cultured primary AML blasts, and oncogene-transformed hematopoietic cells. AML cell lines (OCI-AML2, OCI-AML3, HL-60) were strikingly sensitive to PD0325901 (IC50: 5–19 nM), NB4 (APL) and U266 (MM) showed intermediate sensitivity (IC50: 822 and 724 nM), while all the lymphoid cell lines tested and the myeloid cell lines U937 and KG1 were resistant (IC50 > 1000 nM). Cell growth inhibition was due to inhibition of cell cycle progression and induction of apoptosis. A statistically significant reduction in the proportion of S-phase cells (p=0.01) and increase in the percentage of apoptotic cells (p=0.019) was also observed in 18 primary AML samples in response to 100 nM PD0325901. Analysis of the correlation between sensitivity/resistance to PD0325901 and Ras/Raf mutation status is currently ongoing. PD0325901 effects were also examined in a panel of IL-3-dependent murine myeloid FDC-P1 cell lines transformed to grow in response to 11 different oncogenes in the absence of IL-3. Fms-, Ras-, Raf-1-, B-Raf-, MEK1-, IGF-1R-, and STAT5a-transformed FDC-P1 cells were very sensitive to PD0325901 (IC50: ~ 1 nM), while A-Raf-, BCR-ABL-, EGFR- or Src-transformed cells were 10 to 100 fold less sensitive (IC50: 10 to 100 nM); the parental, IL-3 dependent FDC-P1 cell line had an IC50 > 1000 nM. Analysis of the phosphorylation levels of 18 different target proteins after treatment with 10 nM PD0325901 showed a 5- to 8-fold reduction in ERK-1/2, observed only in sensitive cell lines, and a 2-fold reduction in JNK and STAT3 phosphorylation. PD0325901 (10 nM) treatment also profoundly altered the gene expression profile of the sensitive cell line OCI-AML3: 96 genes were modulated after 24 h (37 up- and 59 down-regulated), most of which involved in cell cycle regulation. Changes in cyclin D1 and D3, cyclin E, and cdc 25A were also validated at the protein level. Overall, PD0325901 shows potent growth-inhibitory and pro-apoptotic activity, indicating that MEK may be an appropriate therapeutic target in an array of different hematological malignancies. Further preclinical/clinical development of this compound is warranted, particularly in myeloid leukemias.