bluuebunny/crossref_metadata_2025 huggingface dataset:
The t(14;19)(q32;q13) Translocation in B Lymphoproliferative Disorders: A Continuum from Chronic Lymphocytic Leukemia (CLL) to Marginal Zone Lymphoma (MZL)?.
Authors: Nguyen-Khac Florence , Chapiro Elise , Radford-Weiss Isabelle , Bastard Christian , Dominique Leroux , Callet-Bauchu Evelyne , Talmant Pascaline , Luquet Isabelle , Marie-Joelle Mozziconacci , Michel Lessard , Sophie Raynaud D. , Davi Frederic , Merle-Beral Helene , Berger Francoise , Berger Roland
Published: April 2020

Translocation t(14;19)(q32;q13) juxtaposing BCL3 in 19q13 with IGH in 14q32, is a rare recurrent event found in patients with B-cell malignancies. So far, a few cases of well-documented B-cell neoplasm have been reported. We analyzed 34 patients with t(14;19) and 1 patient with a variant t(2;19)(p11;q13) collected by the Groupe Francophone de Cytogenetique Hematologique on the basis of cytogenetic abnormality. Clinico-biological data, morphological review, immunophenotyping with Matutes’ score, conventional karyotype and FISH analysis with BCL3, IGH, CEP12, 13q14, ATM, TP53, 6q21 probes, and IgVH mutational status were recorded. The sex ratio was 22M/13F, the median age at diagnosis was 61 [39–89], the lymphocyte count was > 4×109/l in 96% of patients, and spleen enlargement was found in 47%. 31% (11/35) were morphologically classified CLL, 37% (13/35) atypical CLL, 20% (7/35) MZL; 79% had features of disease progression; 3 of the 4 latter were Diffuse Large B Cell Lymphoma, possibly transformed from MZL. 87% were CD5+, 72% had a Matutes’ score < 3. The IgVH genes were unmutated in 9/11 cases. The time to treatment was < 1 year in 68% of patients. The BCL3 locus involvement was confirmed by FISH analysis in all cases. 46% of cases showed complex karyotype. The chromosomal abnormalities associated with t(14;19) were +12 (57%), 6q- (27%), +3 (15%), 11q- (15%), 13q- (13%), 17p- (12%), +18 (12%), 7q- (12%). Comparison with published cytogenetic CLL data shows that deletion 6q was frequent and deletion 13q uncommon. Trisomies 3 and 18, and 7q deletion are less common than in published MZL. The independent analysis of our series of CLL/atypical CLL and MZL gave the same tendency. The chromosomal abnormalities associated with t(14;19) are not specific, but their frequencies are between those of typical CLL and MZL suggesting an intermediary status between the 2 malignancies. The t(14;19) identifies a subgroup of B-disorders CD5+ and Matutes’ score < 3, which could be of poor prognosis, based on progressive disease and unmutated status.
Interleukin-1 Receptor Antagonist and Interleukin-1 beta: Different Roles in Patients with Chronic Lymphocytic Leukemia.
Authors: Zhang Zhong , O’Brien Susan , Keating Michael , Jilani Iman , Kantarjian Hagop , Estrov Zeev , Ferrajoli Alessandra , Giles Francis , Albitar Maher
Published: April 2020

Interleukin-1 receptor antagonist (IL-1Ra) is a 23-KDa soluble glycoprotein that blocks the activity of IL-1α and IL-1β by competing with type I and type II IL-1 receptors without initiating signal transduction. IL-1Ra is anti-inflammatory, while IL-1β is a proinflammatory molecule. IL-1β enhances the immunological and hemopoietic systems and IL-1Ra acts as an inhibitor. IL-1β induces the cell surface expression of cytokine receptors on lymphoid and hemopoietic cells, whereas IL-1Ra suppresses this activity. IL-1β augments lymphoid and hemopoietic cell growth, whereas IL-1Ra suppresses this growth. We evaluated the clinical relevance of IL-1Ra and IL-1β levels in the plasma of 92 patients with chronic lymphocytic leukemia (CLL). IL-1Ra levels were significantly higher in CLL patients (median, 389, range 52–3667 pg/mL) than in 31 normal control subjects (median, 217; range, 94–868 pg/mL) (P <0.001). In contrast, IL-1B levels were significantly lower in CLL patients (median, 2.23; range, 1.89–12 pg/mL) than in normal controls (median, 2.68; range, 2.08–5.5) (P <0.01). Overall, neither IL-1Ra nor IL-1B correlated significantly with WBC count, β2-microglobulin (β2-M) level, Rai stage, platelet count, mutation status, or treatment history. Only age correlated with IL-1Ra (R = 0.39, P <0.001). Using a univariate Cox proportional hazards model, we found direct correlation with survival when IL-1Ra was used as a continuous variable (P <0.001). This association was independent of Rai stage, β-2M, and IgVH mutation status. However, in a multivariate analysis incorporating a combination of IgVH, β2-M, and IL-1Ra, IL-1Ra was no longer a predictor of survival. When patients were dichotomized according to the median IL-1Ra level, those with higher levels had longer survival (P = 0.057). In contrast, IL-1B did not correlate with survival (P = 0.14). These data suggest that IL-1Ra, but not IL-1B, plays a role in the biology of CLL and that the stronger the anti-inflammatory process in CLL, the more aggressive the disease.
Chronic Lymphocytic Leukaemia Has a More Aggressive Phenotype in Asians Compared to Caucasians.
Authors: Austen Belinda , Gunawardana Chaminda , Pratt Guy , Wandroo Farooq , Jacobs Abe , Powell Judith , Moss Paul
Published: April 2020

Chronic lymphocytic leukaemia (CLL) is the commonest leukaemia in the western world with an incidence of 3 per 100,000. The precise aetiology remains obscure but there is clear evidence for a genetic predisposition. CLL has a seven-fold increased incidence amongst relatives of index cases compared to controls and the disease is also more common in males. These genetic subgroups can also determine phenotype as familial CLL has a younger age of onset and increased association with second malignancies compared to sporadic cases. Similarly, CLL is more aggressive and more frequently requires treatment in males in comparison to female patients. The incidence of CLL also varies according to racial origin and has been found to be up to 20-fold less common in Japanese and Asian populations compared to Caucasian populations. However, it is not known whether or not there is also phenotypic variation in CLL patients according to racial origin.

We studied 29 Asian patients with a diagnosis of CLL and compared disease characteristics with a control group of 277 Caucasians. We found that CLL in Asians was diagnosed at a younger age (median age 62yr in Asians vs 70yr Caucasians, p=0.001) and was also more common in males (M:F Asians 3.1:1; Caucasians 1.3:1, p=0.023). No association was seen between racial origin and either IGVH status or CD38 status.

Interestingly, we also found that the Asian population had a more aggressive clinical phenotype as indicated by a shorter time to the requirement of first treatment (TTFT) (p=0.019). This observation was independent of both age and gender. Although no difference was seen in overall survival between the two groups, ‘non-leukaemia related’ deaths were more common in the older Caucasian population. 27.5% of Caucasian deaths occurred in patients who had never required treatment for CLL whereas no deaths occurred among untreated Asian patients, for whom the primary cause of death was progressive leukaemia. The failure to observe a difference in overall survival was therefore partly accounted for by an increase in non-leukaemia related deaths in Caucasians.

In conclusion, we have shown that there are phenotypic differences in CLL patients according to the racial origin. CLL in Asian patients occurs at a younger age and is associated with a more aggressive phenotype. The mechanisms underlying these observations are unknown but warrant further investigation.
Impaired Peripheral Blood Dendritic Cell Counts in B-Chronic Lymphocytic Leukaemia.
Authors: Horvath Rudolf , Budinsky Vit , Pytlik Robert , Klener Pavel , Trneny Marek , Spisek Radek , Bartunkova Jana
Published: April 2020

B-cell chronic lymphocytic leukaemia (B-CLL) is the most common leukaemia of adults in Western worls accounting for about 40% of leukemias in adults over the age of 65 years. Although, B-CLL cells can, under certain conditions, act as antigen presenting cell (APCs) they fail to generate an efficient anti-leukemic immune response. Dendritic cells (DCs) represent the most potent APCs and they are the only cell type capable of initiating a primary immune response. To asses a potential role of DCs compartment in insufficient anti-leukemic and anti-infectious immunity in B-CLL, we have analysed peripheral blood DCs subsets in 17 patients with an early stage B-CLL and 8 age matched controls by multi colour flow cytometry. Dendritic cells were identified by lack of B, T, NK and monocyte markers, HLA-DR expression and expression of CD11c and CD123 for myeloid and plasmacytoid DCs, respectively. We found a significant reduction of absolute plasmacytoid DCs counts in patients with B-CLL (average count = 49,4×106/L) when compared to healthy donors (average count = 111,3 ×106/L, p<0,05). Myeloid DCs counts were not different from healthy controls (average count = 192,7×106/L vs. 196,7×106/L p=NS). Number of plasmacytoid DCs decreased with B-CLL cells number. DC counts did not depend on CD38 or ZAP70 expression or immunoglobulin mutational status. Phenotypic analysis of circulating plasmacytoid and myeloid DCs showed low co-stimulation profile comparing to isotype controls. Thus, the depletion of pDCs in B-CLL could represent a mechanism contributing to the poor anti-leukemic immune responses in B-CLL and/or to immune deficiency in CLL patients.
An International Multicentric Trial with Fludarabine Plus Cyclosphosphamide in B-Cell Chronic Lymphocytic Leukemia (CLL) “Up Front”: Second Interim Analysis.
Authors: Bezares Fernando , Jait Cecilio , Caviglia Daniel , Bhar Daniel , Rodriguez Andrea , Pilnik Norma , Prates Virginia , Lastiri Francisco , Carpio D. del , Carrasco-Yalan Antonio A. , Giralt Sergio
Published: April 2020

Introduction: On August 2002 an international multicentric trial on Fludarabine monophosphate (FAMP) plus Cyclophsphamyde (Cy) among previously untreated B-cell CLL, was activated. Our aim is to evaluate efficacy and toxicity of FAMP plus Cy in previously untreated B-cell CLL patients (pts). This is the second interim analysis after a fourth-year period.

Material and Methods: Treatment consists in three consecutive days of oral FAMP 40 mg/m2 (n=84) or i.v. FAMP 25 mg/m2 (n=13) plus i.v. Cy 600 mg/m2 on day 1 or Cy 250 mg/m2 from day 1 to 3, every 28 days × 6 cycles. Responses were assessed according to the National Cancer Institute working group criteria after cycle 3 and again after cycle 6. Since August 2002 to March 2006, 109 CLL pts from Argentina (n=95), Perú (n=11) and Uruguay (n=3) were enrolled for this protocol; eighty-nine were evaluated for response and toxicity. Median age: 64 years old (range: 44–81); male = 47, female = 42; Binet staging: A=14, B=45, C=30; median beta-2 microglobuline = 4.00 mg/dL (range: 1.3–9.2); median LDH = 341 UI/L (range: 101–762); among patients with available data the CD 38 expression more than 10% was 38% (22 of 58 pts). Blood counts at inclusion: median values (range); Lymphocytes: 32 ×109/L (2,7–137), Hb: 120 g/L (50–164), platelets: 175×109/L (10–364). Renal and hepatic parameters within normal range limits. Cytogenetic by banding was available in 27 cases: no alterations (n=17), +12 (n=1), del (6), del (12) (n=1), lost Y (n=1).

Results: At the time of this second interim analysis (March 2006), 56.2% (50 pts) had completed 6 cycles and 97.8% (87 pts) had undergone at least 3 cycles.

Overall responses: 92% = 81 pts (CR: 39% = 35 pts; PR: 52% = 46 pts); treatment failure: 9% = 7 pts. Evaluation for toxicity: 89 episodes of haematological toxicity and 7 episodes of infection grade 3–4 were reported after 436 cycles. Thirteen pts died: seven due infectious complications because of prolonged hematologic toxicity; one due to tumoral lysis syndrome, one due hemoptysis associated with lung cancer and the remaining four due disease progression. At 24 months, estimated DFS was 70% (figure 1, SE 7.6%) and estimated Overall Survival was 76% (figure 2, SE 7.4%). The median survival was not achieved in responders (PR and CR).

Conclusion: FAMP plus Cy combination as front-line treatment is effective in B-cell CLL. Haematologic toxicity is the most severe adverse events. The response rates to this therapy is quite similar to those reported for other multicentric trials and better than others GATLA protocols.

Figure 1 Figure 1. Figure 2 Figure 2.
Combination Therapy with Alemtuzumab and Pentostatin Is Effective and Has Acceptable Toxicity in Patients with T-Lymphoid Neoplasms.
Authors: Ravandi-Kashani Farhad , Kantarjian Hagop , Faderl Stefan , Verstovsek Srdan , Koller Charles , Odinga Shirley , Wierda William , Borthakur Gautam , Jones Dan , Giles Francis , O’Brien Susan , Keating Michael
Published: April 2020

The development of effective therapeutic strategies for T-lymphoid neoplasms has been difficult partly due to their rarity and paucity of clinical trials. These neoplasms are generally refractory to traditional chemotherapy regimens and novel therapeutic strategies are needed. Alemtuzumab and pentostatin have response rates of 50% to 65% when used individually to treat various T-cell leukemias and lymphomas. However, most responses are partial and of limited duration. Immunosuppression and risk of opportunistic infections are their principal overlapping toxicities. We have treated 17 patients with T-lymphoid malignancies (10 T-PLL, 1 ATL, 2 T-ALL, 2 γδ-T cell hepatosplenic lymphoma, 2 T-LGL) with a combination of alemtuzumab 30 mg IV, 3 times weekly for up to 3 months and pentostatin 4 mg/m2 weekly × 4 followed by alternate weekly administration for up to 6 months. Prophylactic antibiotics including valacyclovir, fluconazole, and trimethoprim/sulfamethoxazole (or equivalents) were administered during the treatment and for 2 months after its completion. The median age of patients was 57 years (range 22 – 79 years), median white blood count was 54.1 × 109/L (range 0.6 – 279.5 ×109/L), and median serum β2M was 4.0 mg/L (range, 1.7 – 10.8 mg/L). Four patients had splenomegaly (1 to 5 cm), and 5 lymphadenopathy. Eleven had prior therapy (median 2, range 1 to 6 regimens). Eleven patients have responded (8 CR, 3 PR) for an overall response rate of 65% (including 8 of 10 T-PLL, 1 of 1 ATL, 0 of 2 T-ALL, 1 of 2 γδ-T cell hepatosplenic lymphoma and 1 of 2 T-LGL). Monoclonal T-cell receptor chain gene rearrangements were detected by PCR in 16 patients and became negative in 5 of 7 evaluable patients in CR. Median response duration has not been reached (range 0 to 78 weeks). Three patients have proceeded to allogeneic stem cell transplant, 4 (1 with ATL, 2 with T-PLL, and 1 with T-LGL) have died from disease progression after a response, and 6 were refractory to therapy. Opportunistic infections included reactivation of CMV in 6 patients (no disease), reactivation of HSV in 1 patient, recurrence of pre-existing Serratia pneumonia in 1 patient and Aspergillus pneumonia in 2 patients. Unexplained, marked and sustained pancytopenia occurred in 2 patients, 1 with T-LGL and 1 with T-PLL. Other toxicities were mainly grade 1 and 2 and included nausea, fever, fatigue, infusion reactions, edema, and shortness of breath. The combination of alemtuzumab and pentostatin is feasible and effective in T-cell neoplasms. Although infections including CMV reactivation are a concern, they may be minimized with adequate prophylactic antibiotic therapy.

Remission duration for patients in CR Remission duration for patients in CR
The Effect of Initial Therapy Choice in Determining Long Term Survival in Patients with Chronic Lymphocytic Leukemia (CLL).
Authors: Tam Constantine S. , Wen Sijin , Do Kim-Anh , Lerner Susan , Wierda William G. , O’Brien Susan , Keating Michael J.
Published: April 2020

Successive advances in CLL therapy have led to significant gains in response quality and remission duration but have not yet shown improved survival. An inherent limitation of randomised trials is the long followup required to demonstrate survival benefit in indolent diseases. In order to examine the effect of initial therapy on survival, we retrospectively analyzed the outcomes of patients treated on frontline protocols at the MD Anderson Cancer Center. Between 10/86 to 01/04, 616 patients received initial therapy with one of three fludarabine-based chemotherapy regimens. Patients in Cohort 1 (F; n=190) received fludarabine±prednisone, Cohort 2 (FC/M; n=140) fludarabine & cyclophosphamide or mitoxantrone, and Cohort 3 (FCR; n=286) FC & rituximab. Therapy indication and response assessment were as per the NCI 1996 Guidelines.

Patient characteristics: median age 57 years; male 67%; Rai intermediate/high risk, 60%/36%; performance status 2 or more, 3.5%; abnormal cytogenetics 27%; B2m 4mg/L or greater, 38%. Median followup was 176, 108 and 56 months for F, FC/M and FCR respectively. Excluding the effect of therapy, univariate predictors of survival were (p-value all <0.05): age, hemoglobin, B2m, abnormal cytogenetics, performance status, Rai stage, albumin and absolute lymphocyte count; only age, B2m and abnormal cytogenetics remained significant following Cox multivariate analysis. Incorporating the therapeutic regimen (F v FC v FCR) into the model, significant predictors for survival were: age (RR 1.05 per year; p<0.0001), FCR regimen (RR 0.42 vs F, 0.41 vs FC/M; p<0.001), high B2m (RR 1.13 per mg/L, p=0.001) and abnormal cytogenetics (RR 1.48, p=0.05). FCR significantly improved survival in the following adverse prognostic groups: age 60 or older (p=0.03), high-risk Rai stage (p=0.002), B2m 4mg/L or greater (p=0.005), performance status 2 or more (P=0.03) and abnormal cytogenetics not involving chromosome 17 (p=0.003). Patients with deletion or abnormality of the chromosome 17 had median survival of <24 months irrespective of treatment regimen. Our analysis underscores the importance of initial therapy choice in determining the survival of patients with CLL. Novel therapeutic strategies are required for patients with chromosome 17 deletion or abnormality.

Survival By Initial Regimen Survival By Initial Regimen
In Vitro Cytotoxicity of Alemtuzumab on B-CLL Cells: Differential Effect on B and T Lymphocytes.
Authors: Berretta Salvatore , Chiarenza Annalisa , Adamo Luana , Parrinello Laura , Buglio Daniela , Stagno Fabio , Tibullo Daniele , Conticello Concetta , Palumbo Giuseppe A. , Di Raimondo Francesco
Published: April 2020

Alemtuzumab, a monoclonal anti-CD52 antibody, has shown high efficacy against lymphoproliferative disorders such as B-cell chronic lymphocytic leukemia (B-CLL). However, its use results in a profound immunosuppression with decrease of T lymphocytes leading to increased susceptibility to infections. The aim of our study was to assess the in vitro cytotoxic effect of alemtuzumab on normal T and neoplastic B lymphocytes obtained from B-CLL patients.

Peripheral blood mononuclear cells (PBMC) from 12 B-CLL patients (5 at diagnosis and 7 previously treated) were collected and treated in vitro with alemtuzumab (10 μg/ml) and autologous serum in the culture as complement source. Spontaneous and alemtuzumab-induced apoptosis were quantified in T (CD3+) and B (CD20+) lymphocytes after 24 hours using an annexin-V flow cytometry based asssay.

Alemtuzumab was able to induce apoptosis on both B and T lymphocytes of CLL patients. However, the cytotoxic activity on neoplastic B cells was comparable in all cases analyzed, irrespective of stage or previous treatments. Spontaneous apoptosis of B CLL cells was also comparable in all studied samples.

On the contrary, the activity of alemtuzumab on normal T lymphocytes varied according to stage of disease and previous treatment. In early stages (0–I) alemtuzumab induced apoptosis in 19 % of T cells vs 60% of advanced stages (II–IV) (p=0,009). Differences were also relevant when patients were split by treatment: in fact, alemtuzumab induced apoptosis in 15% of T cells of untreated patients vs 52% of T cells of previously treated patients (p=0,005). Moreover, spontaneous apoptosis of T cells was more pronounced in treated vs. untreated patients (15% vs 2%, p=0.03) while the difference was not statistically significant in early vs advanced stage (p=0.06).Our in vitro data confirm that alemtuzumab is active against B-CLL cells in all stage of disease. However, in advanced stages and in previously treated patients, T cell compartment seems to be more fragile and susceptible to both spontaneous and alemtuzumab-induced apoptosis. This observation supports the hypothesis that using Alemtuzumab earlier in the treatment of B-CLL might result in less immunosuppression. The study is still ongoing with accrual of more patients samples.
Low-Dose Prednisone Decreases the Severity but Not the Frequency of Lenalidomide Associated Tumor Flare Feaction (TFR) in Chronic Lymphocytic Leukemia (CLL) Patients.
Authors: Musial Laurie , Miller Kena C. , Tonelli Amy , Manochakian Rami , Padmanabhan Swaminathan , Lawrence David , DePaolo Dawn , Coignet Jean-Gabriel , Chanan-Khan Asher Alban
Published: April 2020

Introduction: Lenalidomide (L) is clinically active in pts with CLL. TFR is a common side effect of L, unique to CLL pts and is characterized by tender enlargement of lymph nodes (LN), spleen and/or liver with low-grade fever, rash and/or increase in white blood cell count. Onset is usually with 24 hours of first dose and it is rarely seen after the 1st cycle. The underlying etiology of TFR remains undetermined though clinical presentation is suggestive of an immune activation phenomenon. Diagnosis, management and effective prophylaxis of TFR is crucial for the pt care as it is associated with considerable morbidity and mimics disease progression. We investigated the effectiveness of low-dose prednisone prophylaxis in CLL pts treated with L.

Method & Results: Forty five pts (36 male, 9 female), median age 64 years (range 42–75) with rel/ref CLL were enrolled on a phase II clinical trial. Single agent L (max. dose 25mg/day) was given to all pts enrolled. No prophylaxis was given to the first 29 pts (group A). Subsequently 16 pts (group B) were treated with prednisone 20mg PO QD × 5 days followed by 10mg PO QD × 5 days, starting day 1 of treatment. Median age was 64 years (range 42–75) with 36M and 9F. Stage III/IV disease was noted in 64% (n=18) of the pts. Twenty four (83%) pts in group A and 13 (81%) in group B developed TFR with > grade II TFR was seen in 31% and 6%, respectively. Among pts with TFR, 4 achieved complete remission (CR) and had grade > 2 reaction vs. 19 pts with a < partial remission who had a median of grade 1 (range 1–2) TFR.

Conclusion: Although the sample size investigated is small, we observed that use of oral prednisone prophylaxis decreased the severity but not the overall incidence of TFR. We therefore recommend that low-dose prednisone be considered for lenalidomide associated TFR in patients with CLL.
Pleiotropic Role of GSK3 Kinase in Myeloma Cell Biology.
Authors: Piazza Francesco A. , Gurrieri Carmela , Chioetto Gino , Bonanni Luca , Montini Barbara , Carraro Samuela , Cabrelle Anna , Zambello Renato , Trentin Livio , Adami Fausto , Semenzato Gianpietro
Published: April 2020

GSK3 is a cellular serine-threonine kinase discovered because of its involvement in insulin, growth factor and Wnt signalling, downstream of which it is inhibited through the action of the PI3K/AKT cell survival pathway or through other Wnt signalling-dependent mechanisms. GSK3, therefore, has been included in the group of “tumor suppressors”, as it can antagonize cell proliferation triggered by these cascades. Recent findings, however, have challenged this notion in that GSK3β has been found central for cell survival and NF-κB signalling. Since growth factor, Wnt and cytokine-dependent signalling pathways have been implicated in MM pathogenesis, we decided to investigate the role of GSK3 in multiple myeloma cell biology. GSK3 kinase activity was found slightly higher in malignant plasma cells as compared to normal resting B-lymphocytes and normal in vitro generated plasmablasts. GSK3 enzymatic activity was hampered by stimulation of MM cells with IL-6 and IGF-I but, remarkably, not with TNFα. IL-6 and IGF-I driven MM cell proliferation was significantly increased by GSK3 blockade as it was MM cell survival upon serum starvation or contact with bone marrow stromal cells (BMSC). At molecular level, IL-6-dependent STAT3 phosphorylation was unaffected by GSK3 inhibition, however, ERK1, 2 phosphorylation was increased. Importantly, NF-κB activation and transcriptional activity downstream from TNFα were only slightly affected when GSK3 function was inhibited in MM cells. However, when GSK3 inhibitors were added to MM cell cultured with BMSC, IL-6 secretion in the medium was reduced and the expression of NF-κB-dependent antiapoptotic genes was altered. Lastly, the addition of GSK3 inhibitors in MM cells-BMSC cultures led to an increased expression of Wnt/β-catenin-dependent genes both in MM and in BMSC cells. Our data indicate a different involvement of GSK3 downstream from growth factors or TNFα-induced signalling pathways in MM cells; the observed effects of GSK3 inhibition on the Wnt-signalling pathway indicate that, whereas they would be desirable in the BMSC compartment (i.e. antagonisms of Wnt-inhibitors released in the MM bone marrow, such as Dikkopf-1 (DKK1) or secreted Frizzled-Related Protein (sFRP)-2 and favouring of the osteoblast maturation) on the other hand they could lead, together with the loss of a brake downstream from growth factor signals, to uncontrolled and enhanced MM cell proliferation; thus, the inhibition of this kinase for therapeutic purposes in MM is likely to be accompanied by dangerous and unwanted side effects that may promote the progression of this disease.
Association of T Cell Costimulatory and Downregulatory Gene Polymorphisms and Susceptibility to Multiple Myeloma.
Authors: Frydecka Irena , Pawlak Edyta , Karabon Lidia , Sowinska Ewa , Jonkisz Anna , Lebioda Arleta , Dobosz Tadeusz , Suwalska Katarzyna , Tutak Anna , Kuliczkowski Kazimierz
Published: April 2020

Multiple myeloma (MM) is a B-lineage malignancy characterized by an accumulation of isotype-switched, immunoglobulin-producing monoclonal plasma cells. Additionaly phenotype and functional abnormalities of T cell are observed. The implication of T cells in the development of plasmocytoma has been shown in murine model. The study was undertaken to evaluate the association between polymorphisms of the genes encoding major T cell costimulatory (CD28, ICOS) and downregulatory (CTLA-4) molecules and susceptibility to MM in a Polish population. One hundred patients with MM and 202 healthy subjects were studied. Genomic DNA was isolated from whole frozen blood using the NucleoSpinR Blood kit. Allele identification was achieved by PCR amplification. The amplified product for SNP loci was purified and minisequenced using the commercial kit SNapShot (PE Applied Biosystems). The dinucleotide repeat polymorphism was studied by PCR and fluorescence based technique. The products were analyzed on the ABI PRISM 310 Genetic Analyzer (ABI PRISM 310 capillary electrophoresis system).

We found an association of 49G in exon 1 and (AT)82 alleles in 3′untranslated region in the CTLA-4 gene with MM [p=0.018, OR=1.57, 95% CI=1.08–2.29 and p=0.0013, OR=1.85, 95% CI=1.27–2.72, respectively] while no association between T/C substitution in intron 3 of the CD28 gene, the dinucleotide (GT)n repeat polymorphism in intron 4 in ICOS gene and C/T base exchange in the promoter (position −318) in the CTLA-4 gene and MM was observed. Our results indicate that the A/G substitution in exon 1 and microsatelite polymorphism in the 3′untranslated region in the CTLA-4 gene might be a susceptibility locus for MM.

The present study is the largest and comprehensive to date to evaluate the association between genetic polymorphisms of genes encoding T cell costimulatory and downregulatory molecules and susceptibility to MM.
Monoclonal Gammopathy (MG) Associated with Gaucher Disease (GD). Report of 16 Cases from the French Observatoire on Gaucher Disease (FROG Study).
Authors: Grosbois Bernard , Leone Jean , Camou Fabrice , Dalbies Florence , Harle Jean Robert , Lamy Thierry , Luca Luminita , Plane Christophe , Robin Jean Pierre , Rose Christian , deRoux-Serratrice Christine , Solary Eric , Steiger Jean Marie
Published: April 2020

In addition to its common bone and visceral manifestations GD is frequently associated with B cell proliferation leading possibly to MG. The objective of our study was to determine the frequency and presentation of MG in GD patients.

Material and methods. FROG is a cross-sectionnal epidemiological study on adult GD involving 64 French centers. Standard clinical, biological(including serum protein electrophoresis) and imaging data performed as part of usual management within the 3 previous years were collected at the time of a routine visit. In patients with suspected MG specific additionnal data were studied:immunofixation, dosage of immunoglobulins, bone marrow aspiration.

Results. From 05/2005 to 06/2006 seventy seven adult GD type 1(38 male,39 female;mean age 47.3 years, range 18 to 78 years) were included. Sixteen cases of MG(20.8%) were observed (10 male and 6 female;mean age 61.25 years, range 47 to 79 years). Mean age of 61 cases of GD without MG(43.8 years;range 18 to 70 years)was significantly lower(p<0.0001). Thirteen cases(17%)were classified as MGUS and 3 (3.8%) as B cell malignancy:1 multiple myeloma, 1 Chronic Lymphocytic Leukemia, 1 Non Hodgkin Lymphoma. In 3 cases MG and GD were diagnosed simultaneously. In 13 cases MG was diagnosed in the course of GD(mean interval 12.2 years;range 6.7 to 16 years). MG immunochemical typing revealed 75 % Ig G, 12.5 %Ig A, 12.5 %Ig M, 75 % Kappa and 25 % Lambda. We observed one biclonal gammopathy(IgG and IgA Kappa). One case of MGUS was associated with capillary leak syndrome. In two patients MG level decreased with enzyme replacement therapy.

Conclusion. Frequency of MG in GD observed in our study (20.8%)is largely higher than in general population (1%). Furthermore this frequency is higher than in previous series of GD. However in GD patients, as observed in general population, frequency of MG seems to be related to age as mean age of patients with MG is significantly higher than mean age of patients without MG. Finally these data assess the need of specific follow-up regarding the high frequency of MG and the risk of B cell malignancy in GD patients.
Novel Etodolac Analog SDX-308 (CEP-18082) Induces Cytotoxicity in Multiple Myeloma Cells Associated with Inhibition of Wnt/β-Catenin Pathway.
Authors: Yasui Hiroshi , Hideshima Teru , Ikeda Hiroshi , Ocio Enrique M. , Kiziltepe Tanyel , Vallet Sonia , Okawa Yutaka , Chhetri Shweta , Sukhdeo Kumar , Ishitsuka Kenji , Podar Klaus , Raje Noopur , Chauhan Dharminder , Cattasco Daniel R. , Anderson Kenneth C.
Published: April 2020

In a previous study we reported that R-enantiomer of etodolac (R-etodolac) which is under investigation in Phase II clinical trials in chronic lymphocytic leukemia, induces potent cytotoxicity at clinically relevant concentrations in both drug-sensitive and drug-resistant multiple myeloma (MM) cell lines, as well as in patient MM tumor cells. In this study, we demonstrated that SDX-308 (CEP-18082), a novel analog of etodolac, has more potent cytotoxicity than R-etodolac against MM cell lines and tumor cells from patients with refractory MM. It also induces cytotoxicity against bortezomib-resistant tumor cells. The molecular mechanisms whereby SDX-308 triggers MM cell cytotoxicity were next delineated. SDX-308 induces apoptosis via caspase-8/-9/-3 activation and poly(ADP-ribose) polymerase PARP cleavage. It strongly inhibits Wnt/β-catenin pathway by blockade of nuclear translocation of β-catenin, followed by significant inhibition of transcription and expression of target proteins. These target proteins include cell cycle regulatory c-myc molecules and anti-apoptotic survivin molecules. Neither interleukin-6 nor insulin-like growth factor-1, which induce MM cell growth and abrogate Dex-induced apoptosis, protect against growth inhibition triggered by SDX-308. Importantly, growth of MM cells adherent to bone marrow (BM) stromal cells is also significantly inhibited by SDX-308. Our data therefore indicate that the novel etodolac analog SDX-308 has more potent cytotoxicity in MM cells than R-etodolac even in the context with BM microenvironment, providing the preclinical rationale to conduct clinical trials of this agent to improve patient outcome in MM.
Correlation of Serum Free Light Chain Levels with Other Parameters in Myeloma.
Authors: Stein Regina , Mehta Jayesh , Vickrey Eric , Resseguie William , Singhal Seema
Published: April 2020

Estimation of serum free light chains (SFLC; SFKLC - kappa, SFLLC - lambda, SFKLR - kappa:lambda ratio; normal 0.26–1.65.) is useful in selected patients with non-secretory myeloma, and in light chain disease with anuric renal failure. Its utility in other clinical situations is unclear. 489 SFLC levels in 135 myeloma patients were analyzed to see if there was a significant correlation between them and other, more conventional immunologic parameters such as serum total light chain (STLC; STKLC - kappa, STLLC - lambda) levels and immunoglobulin (Ig) levels. The underlying premise was that lack of significant correlation would suggest potential value of SFLC independent of standard tests, whereas significant correlation would suggest questionable independent value of SFLC. The table below shows SFLC and STLC levels and ratios.

Parameter Median (range) Correlation with SFKLC Correlation with SFLLC Correlation with free k:l ratio SFKLC 14.4 (0.59–11525) SFLLC 18.2 (0.72–10100) Free k:l ratio 0.83 (0–1211.76) STKLC 745 (11–8810) r=0.08; P=0.07 STLLC 256 (11.6–11000) r=0.11; P=0.01 Total k:l ratio 2.51 (0–245) r=0.12; P=0.008

Poor correlation between free and total results within each light chain subtype (Figure 1 - log scale) and between free and total ratios (Figure 2- log scale) suggests that SFLC values provide information that is independent of STLC values because STLC values cannot be used to predict SFLC values.

Figure Figure Figure Figure

The table below shows correlation between Ig and SFLC/STLC levels. For these analyses, the total Ig of the specific heavy chain subtype and SFLC/STLC of the specific light chain subtype were studied (IgG and SFKLC with IgG kappa monoclonal protein, IgA and STLLC with IgA lambda monoclonal protein, etc). Very strong correlation between STLC and Ig levels suggests that STLC values do not provide information that is independent of Ig levels. On the other hand, poor correlation between SFLC and Ig levels suggests that SFLC values do provide information that is independent of Ig values.

Monoclonal protein SFLC correlation STLC correlation IgG kappa r=0.04; P=0.66 r=0.89; P<0.0001 IgG lambda r=0.13; P=0.51 r=0.99; P<0.0001 IgA kappa r=0.49; P=0.004 r=0.77; P<0.0001 IgA lambda r=-0.04; P=0.89 r=0.94; P<0.0001

These data show that SFLC estimation provides information that is independent of standard quantitative serologic tests used in plasma cell dyscrasias. However, rigorous prospective evaluation of the test is needed to see if this additional information is of any clinical relevance in settings other than non-secretory disease and light chain disease with anuria.
Role and Regulation of Heat Shock Proteins 90alpha and 90beta in Multiple Myeloma.
Authors: Chatterjee Manik , Jain Sarika , Stühmer Thorsten , Andrulis Mindaugas , Ungethüm Ute , Kuban Ralf-Jurgen , Bommert Kurt , Topp Max , Krämer Doris , Müller-Hermelink Hans K. , Einsele Herrmann , Axel Greiner , Bargou Ralf C.
Published: April 2020

The combined blockade of the IL-6R/STAT3 and the MAPK signaling pathways has been shown to inhibit bone marrow microenvironment-mediated survival of multiple myeloma (MM) cells. Here, we identify the molecular chaperones heat shock protein (Hsp) 90alpha and beta as target genes of both pathways. Interestingly, in situ overexpression of both Hsp90 proteins was observed in the majority of MM, but not in MGUS or in normal plasma cells. SiRNA-mediated knockdown of Hsp90 or treatment with the novel Hsp90 inhibitor 17-DMAG attenuated the levels of phospho-STAT3 and phospho-ERK and decreased the viability of MM cells. Although knockdown of Hsp90beta unlike knockdown of Hsp90alpha was sufficient to induce apoptosis, this effect was strongly increased when both Hsp90s were targeted, indicating a cooperation of both. Given the importance of the bone marrow microenvironment for drug resistance and MM cell survival, apoptosis induced by Hsp90 inhibition was not mitigated in the presence of bone marrow stromal cells, osteoclasts or endothelial cells. These observations suggest, that a positive feedback loop consisting of Hsp90alpha/beta and major signaling pathways supports the survival of MM cells. Our results emphasize a role for Hsp90alpha and Hsp90beta in MM pathogenesis.
No Adverse Impact of 13q14 and P53 Deletion, t(4;14)(p16;q32) or Overrepresentation of CMYC(8q24) as Detected by Fluorescence In Situ Hybridization on Outcome in Patients with Multiple Myeloma Following Dose-Reduced Allogeneic Stem Cell Transplantation.
Authors: Hansen Timon , Schilling Georgia , Liebisch Peter , Schwerdtfeger Rainer , Perez-Simon Jose Antonio , Bornhaeuser Martin , Otterstetter Svenja , Dierlamm Judith , Nagler Arnon , Hossfeld Dieter Kurt , Bokemeyer Carsten , Zander Axel R. , Kroeger Nicolaus
Published: April 2020

Deletions of chromosome bands 13q14 and 17p13 (P53) as well as t(4;14)(p16;q32) or overrepresentation of CMYC are known to be adverse prognostic factors for time to progression (TTP) and overall survival (OS) in myeloma patients receiving conventional or high-dose chemotherapy followed by autologous stem cell transplantation. We investigated the impact of these chromosomal abnormalities as detected by fluorescence in situ hybridization combined with immunofluorescent cytoplasm immunoglobulin staining (cIg-FISH) on outcome after dose-reduced melphalan/fludarabin based allogeneic stem cell transplantation (SCT) in 64 patients (pts) with multiple myeloma (MM). Median age was 52 years (34 – 67 ys.), 38 patients were male, 26 female. 45 pts received stem cell graft from an unrelated donor and 19 pts from HLA-identical sibling. Deletion of 13q14 was found in 35 out of 64 pts (55%), P53 deletion in 7 out of 64 pts (11%), t(4;14)(p16;q32) in 11 out of 56 pts (20%) and trisomy of CMYC in 12 out of 55 pts (22%). The following strong correlations among the genetic changes have been seen: six out of seven pts with P53 deletion also showed deletion in chromosome band 13q14 (86%), which was also found in nine out of eleven pts with t(4;14) (82% del 13) and nine out of twelve pts with CMYC trisomy (75% del 13). The estimated event-free (EFS) and overall survival (OS) at four years for the entire study population was 48% and 57%, respectively. No significant difference of the estimated event-free survival at 3 years was seen for pts with del 13 (42% vs 54%, p=0,4), with P53 deletion (43% vs 49%, p=0,27) and for pts with t(4;14) (46% vs 62%, p=0,69) or CMYC overrepresentation (37% vs 50%, p=0,35). Comparison of pts showing both del 13 and P53 deletion, del 13 and t(4;14), del 13 and trisomy of CMYC or t(4;14) and CMYC trisomy with the rest of the study population did not show a significantly reduced EFS either. Even the comparison of pts with any chromosomal abnormalities and pts without any genetic changes detected by FISH did not show a significant difference in EFS (48% vs 50%, p=0,8). These preliminary data suggest that the negative prognostic impact of chromosomal abnormalities such as deletion of 13q14, P53 deletion, t(4;14) and overrepresentation of CMYC may be overcome by allogeneic SCT probably due to the graft versus myeloma effect.
Leukemia Associated Mutants of SHP2 Protein Tyrosine Phosphatase Prevent HoxA10 Tyrosine Phosphorylation in Differentiating Myeloid Cells, Thereby Blocking Transcription of the Gene Encoding Gp91phox.
Authors: Lindsey Stephan , Wang Hao , Eklund Elizabeth A.
Published: April 2020

Gp91phox is a component of the membrane bound, catalytic unit of the phagocyte respiratory burst oxidase. Transcription of the CYBB gene, which encodes gp91phox, is restricted to phagocytes that have differentiated beyond the promyelocyte stage. Transcription continues in mature myeloid cells until cell death and is increased by inflammatory mediators such as LPS or interferon gamma as part of the system of host defense. Previously, we determined that a negative cis element in the CYBB promoter binds a multi-protein complex in undifferentiated myeloid cells which includes the homeodomain proteins HoxA10 and Pbx1, and the histone deacetylase HDAC2. During cytokine induced differentiation, binding of this complex decreases, permitting binding of positive trans factors which activate an adjacent cis element in the CYBB promoter. In previous studies, we also determined that tyrosine phosphorylation decreases HoxA10 binding affinity for the CYBB repressor element. We found that HoxA10 is tyrosine phosphorylated in response to treatment of myeloid progenitor cells and myeloid cell lines with hematopoietic cytokines such as G-CSF, M-CSF and interferon gamma. In undifferentiated myeloid cells, HoxA10 is maintained in a non-tyrosine phosphorylated state by the protein tyrosine phosphatases SHP1 and SHP2. We find that activity of these PTPs decreases during myeloid differentiation. This decrease in PTP activity contributes to increased HoxA10 tyrosine phosphorylation, resulting in CYBB gene transcription as differentiation proceeds. Recently, leukemia associated, activating mutations of SHP2-PTP have been described in human subjects with Juvenile Chronic Myelomonocytic Leukemia (JMMoL), AML and MDS. These SHP2 mutations induce a conformational change which unmasks the PTP domain, resulting in constitutive activation. In these studies, we find that expression of one such leukemia associated SHP2-mutant impairs HoxA10 tyrosine phosphorylation during myelopoiesis. This results in sustained repression of CYBB transcription and decreased gp91phox expression in differentiating cells. Since gp91phox is one of the rate limiting oxidase component proteins during myeloid differentiation and the inflammatory response, activating mutants of SHP2-PTP would therefore be expected to impair respiratory burst competence in malignant myeloid cells. This could contribute to immuno-deficiency in some forms of myeloid malignancy where differentiation appears histologically preserved, such as JMMoL or MDS. Further investigation of the role of SHP2-mutation in phagocyte functional competence may identify rationale molecular targets to ameliorate immuno-deficiency in chronic myeloid diseases.
Role of c-Kit in Multiple Myeloma.
Authors: Montero Juan Carlos , Miguel J.F. San , Pandiella Atanasio
Published: April 2020

C-Kit is a receptor tyrosine kinase whose activation occurs by interaction with its ligand, the Stem Cell Factor (SCF). Two distinct isoforms of c-Kit have been identified, and are characterized by the presence or absence of the tetrapeptide sequence Gly-Asn-Asn-Lys (GNNK) in the juxtamembrane domain of the extracellular region. These isoforms signal with distinct properties. c-Kit has been reported to be expressed in the plasma cells of about 20% of patients with Multiple myeloma (MM), but not in normal plasma cells. Yet, its functional and biochemical signalling properties in myeloma cells have not been described. In this study we show that patients and cell lines of MM co-expressed both c-Kit isoforms (GNNK+ and GNNK−), being predominant the GNNK− isoform. c-Kit was activated by SCF in the cell line RPMI8226, and in cell lines derived from patients. To study the properties of the two c-Kit isoforms, we have expressed these isoforms in the cell line MM1S which do not express these receptors in a significantly high level. Both receptors types were expressed by retroviral infection. Addition of SCF provoked their tyrosine phosphorylation. Imatinib mesylate, that is a known inhibitor of c-Kit, inhibited the phosphorylation induced by SCF of both isoforms. Both receptors coupled to the PI3K/Akt pathway, but were unable to substantially activate the Erk1/2 MAPKs. Differences in the kinetics of phosphorylation between the isoforms GNNK+ and GNNK− were observed. The variant GNNK− was more rapidly and highly phosphorylated than the GNNK+ isoform, which was activated more slowly but this activation persisted a longer time. In MM1S-GNNK+ cells the principal substrate of Akt activated by SCF was the p70S6K, and this activation was dependent on the activity of the mammalian Target Of Rapamycin (mTOR). Finally, the expression of the c-Kit isoforms in MM1S partially prevented their death induced by dexamethasone. These data indicate that c-Kit expression in MM cells is functional and couples to survival pathways that may modulate cell death in response to therapeutic compounds used in the treatment of this disease.
Green Tea Polyphenol (−)-Epigallocatechin Gallate Inhibits IL-6 Mediated Signalling Pathways in Human Myeloma Cells.
Authors: Burger Renate , Czekalla Hanna , Richter Kathrin , Ahrens Tanja , Guenther Andreas , Gramatzki Martin
Published: April 2020

Epigallocatechin-3-gallate (EGCG) is the predominant active ingredient of green tea leaves and has been shown to possess anti-tumor, anti-inflammatory, and anti-oxidant properties. EGCG confers its effects through potentially multiple mechanisms including inhibition of growth factor receptor signalling and decrease of cellular levels of anti-apoptotic proteins of the Bcl-2 family. We examined the effects of EGCG on a panel of human myeloma cell lines using EGCG concentrations ranging from 6.25–100 μM. After three days of culturing with EGCG, basal cell growth was inhibited in most of the cell lines as measured in an MTS based assay. IC50 concentrations were between 25 μM and 50 μM. IL-6 mediated growth of the IL-6 dependent INA-6 cell line was inhibited at similar doses. In these cells, stimulation with IL-6 leads to upregulation of Mcl-1 expression (Brocke-Heidrich et al., Blood.2004;103:242–251). Another green tea catechin, (−)-catechin gallate, seemed to be less active when used at identical concentrations. When INA-6 cells were pretreated for two hours with EGCG, a dose-dependent inhibition of IL-6 induced STAT3 tyrosine phosphorylation was observed as revealed by Western blot analysis. In contrast, phosphorylation of p44/p42 MAPK, which is constitutively activated in INA-6 cells, was not affected. The precise mechanism by which EGCG inhibits STAT3 phosphorylation remains to be determined. In conclusion, our results suggest to further evaluate the effect of EGCG not only in B-cell chronic lymphocytic leukemia, but also in plasma cell tumors.