bluuebunny/crossref_metadata_2025 huggingface dataset:
Crosslineage T-Cell Receptor Genes Rearrangements and Active Receptor Editing in B-Cell Acute Lymphoblastic Leukemia.
Authors: Makuch-Lasica Hanna , Majewski Miroslaw , Nowak Grazyna , Lewandowska Monika , Solarska Iwona , Kania Iwona , Warzocha Krzysztof
Published: October 2019

B-cell acute lymphoblastic leukemia (B-ALL) results from clonal expansion of B-lymphocytes derived at different stage of differentiation. Immunoglobulin (Ig) heavy chain genes (IGH), light chain kappa (IGK) and lambda (IGL) genes rearrange during early B-lymphocyte differentiation. T-cell receptor (TCR) genes are postulated to rearrange exclusively in normal T lymphocytes, but malignant B lymphoblasts often contain crosslineage rearranged TCR genes. The clonal leukemic cell population, carrying identical copies of rearranged Ig and/or TCR genes, can be identified above 95% of B-ALL patients. In our study Ig/TCR genes rearrangements were detected by multiplex PCR with heteroduplex analysis according to BIOMED-2 protocol. DNA was isolated by column method from mononuclear cells isolated from the peripheral blood/bone marrow samples obtained at initial diagnosis from 28 B-ALL patients. Monoclonal rearrangements of Ig genes were detected in 96% (27/28) of patients. The most frequent rearrangements were observed in IGH genes (96%), including complete IGHV-IGHJ in 75% (21/28) and incomplete IGHD-IGHJ in 31% (8/28) of patients. Among complete IGH rearrangements 4 biallelic rearrangements in IGHV1-7 and IGHJ genes (FR3) were found. Ig light chain genes rearrangements were identified in 20 patients (71%) (including 25% of IGKV-IGKJ, 50% of IGKV/intron-Kde, and 25% of IGLV-IGLJ) indicating active receptor editing occurring during B lymphoblasts leukemogenesis. Cross-lineage TCR genes rearrangements were found in 77% (23/28) of patients. TCR beta genes rearrangements were detected in 46% (13/28) of patients (complete TRBV-TRBJ in 32% (9/28), TRBD-TRBJ in 5/28 patients - 18%). TRGV-TRGV were found in 46% (13/28), TRDV-TRDJ in 50% (14/28; 10 monoallelic and 4 biallelic). TCR beta genes rearrangements with presence of TCR gamma genes rearrangements were identified in 25% (7/28) of patients. The identified Ig and TCR rearrangements were stable in patients monitored for minimal residual disease (MRD) and patients with leukemia relapse. The inactivation of potentially functional IGKV-IGKJ by secondary rearrangements indicates active receptor editing. Our data describe IGK and IGL genes rearrangements incidence, present allelic exclusion and active receptor editing in B-ALL patients. B-ALL lymphoblasts undergo many rearrangements on the same IGK allele before they rearrange IGL genes. The data suggest the role of antigen in B-ALL immunopathogenesis. The results indicate also rearranged IGK, IGL and TCR genes as a possible molecular marker for monitoring MRD in B-ALL.
Experience of a Brazilian Institution with Treatment of Adult Acute Lymphoblastic Leukemia.
Authors: Pinheiro Edilson D. , Prachia Luiz F. , Velloso Elvira R.P. , Beitler Beatriz , Bucheri Valeria , Pereira Juliana , Dorlhiac-Laccer Pedro E. , Chamone Dalton A.F.
Published: October 2019

Adult Acute Lymphoblastic (ALL) is an aggressive bone marrow neoplasm, associated with a poor outcome in adult patients. The aim of this study is to compare different treatment protocols analyzing complete response after induction treatment (CR), overall survival (OS) and disease free survival (DFS). Statistical analysis was done by SPSS 10.0. The analysis of prognostic factors, protocols and response treatment was done by Pearson’s chi-square. OS and DFS curves were constructed by Kaplan-Meyer method and differences were analyzed by log-rank test.

In our institutional department, 102 patients with ALL diagnosed between 1990 and 2005 were treated with one of the follow protocols: BFM 86 modified (BFM 86M) (48/102), OPAL86 (6/102), OPAL87 (40/102) (Linker et cols; 1986Linker et cols; 1987) and CHOP (8/102). The results were analyzed retrospectively. The median follow up was 49 months. CR for BFM 86M, OPAL86, OPAL87 and CHOP was 76,7%, 100% 63,9% and 42,9%, respectively; with no statistically difference (p=0.08). OS was better with BFM 86M than with other protocols (p=0.001). BFM 86M was associated with 4-years DFS of 42,5% (median: 26,4 m) and OS of 49,5% (median: 35,4m). OPAL86, OPAL87 and CHOP had a median survival of 21, 12, and 5,5m respectively. Four-years OS of the patients treated with protocols other than BFM 86M was 16,8%.

The distribution of the clinical and laboratory data between the BFM 86M and the other protocols (OP) was compared. Age less than 18y (p=0.05) and L1 ALL (FAB) (p=0.01) were more prevalent in OP group. Myeloid expression antigens (p=0.05) and poor prognosis karyotype (e.g. Ph cromossome) (p=0.08) were more prevalent in BFM 86M group.

Age less than 35y (p=0.02), absent of fatigue at diagnosis (p=0.04), CNS not infiltrated (p=0.01) were associated to an early complete remission with OP but not with BFM 86M. Hepatomegaly, at diagnosis, was associated with poor complete remission when patients received BFM 86M protocol (p=0.02). Age less than 18y (p=0.002) and absent of hepatomegaly at diagnosis (p=0.01) were associated with a prolonged survival only in BFM 86M protocol. T-ALL (p=0.04) and infiltration of CNS (p=0.02) were related to worst survival in other protocols (OP) but not in BFM 86M. Bleeding at diagnosis for both BFM 86M and OP were related to short survival (p=0.03; p=0.01).
The Philadelphia-Chromosome Is Unmasked as the Secondary Genetic Change in Acute Myeloid Leukemia on Imatinib Treatment.
Authors: Yip Sze F. , Wan Thomas S.K. , Liu Herman S.Y. , Wong Michael L.G. , Chan Li C.
Published: October 2019

The Philadelphia chromosome (Ph) is present in less than 3% of the acute myeloid leukemia (AML) and associated with poor prognosis. There are only few reports on the use of imatinib in Ph+ AML. We describe an adult female patient presenting with AML (Hb 3.6 g/dl, Wbc 137×109/L, Plt 295,000/uL and marrow blast 74%, with no basophilia or splenomegaly). Cytogenetic studies showed 45,XX,inv(3)(q21q26),-7,t(9;22)(q34;q11.2) [12] and RT-PCR showed p190BCR-ABL transcripts. The leukemia failed to respond to 2 cycles of daunorubicin and ara-C and imatinib 600mg daily was then started. Fluorescence in-situ hybridization (FISH) analysis of 300 marrow cells using dual fusion BCR-ABL translocation probes (D-FISH, Vysis, Downers Grove, IL, USA) showed that before imatinib, 78% of the cells were positive for BCR-ABL (at diagnosis, it was 83%), which after 6 weeks of imatinib treatment, became BCR-ABL negative (sensitivity 0.3%). However, marrow biopsy still showed 15% blasts. Repeated cytogenetics showed poor growth, but one metaphase showed 45,XX,inv(3)(q21q26),-7. With FISH, 39% of the marrow cells, including neutrophils, were positive for monosomy 7. At 4 months of imatinib, when the marrow blasts count increased to 40%, BCR-ABL remained negative by FISH, whereas monosomy 7 positive cells had risen to 82%. However, by nested RT-PCR with sets of BIOMED primers (sensitivity of 10−5), the p190BCR-ABL transcript was still present, suggesting persistence of a small and quiescent population of BCR-ABL positive leukemic stem cells. In summary, our case study demonstrates the need for careful cytogenetic and molecular analysis in cases of acute leukaemia with Ph chromosome to exclude the possibility of Ph chromosome being a secondary event, in which case durable leukemia remission will be unlikely if treatment is by BCR-ABL inhibition alone.
A Case of Relapsed Variant Acute Promyelcytic Leukemia with t(11;17)(q23q21), PLZF/RARa Fusion Gene, with Durable Molecular Remission Following an Induction and Consolidation Regimen That Included ATRA with Low Dose G-CSF and Valproic Acid as a Histone Deacetylase Inhibitor.
Authors: Efiom-Ekaha Daniel N. , Sanmugarajah Jasotha , Solomon William B.
Published: October 2019

Background:

Acute promyelocytic leukemia (APL) accounts for 10% of acute myeloid leukemias. Greater than 95% of cases are associated with the t(15;17)(q22;q21) translocation with the PML-RARa fusion product which is associated with increased affinity for nuclear co-repressor protein complex and recruitment of histone deacetylase. This leads to an alteration in chromatin conformation and inhibition of transcription and differentiation in promyelocytes in the presence of physiologic levels of retinoic acid. Pharmacologic doses of retinoic acid however are usually required to overcome this repression of transcription and leads to promyelocyte differentiaton and improvement in clinical outcomes in classic APL. A variant translocation in APL, t(11;17) (q23;q21) where the 3′ end of the RARa gene is fused to the 5′ end of a gene called PLZF (promyelocytic leukemia zinc finger), accounts for about 1% of cases. This disease phenotype is associated with refractoriness to pharmacologic doses of retinoic acid.

Case Report;

52 year-old male presented with fever and rapidly progressive leukocytosis. His HCT was 24%, WBC count was 75,000/mcl and platelet count was 109,000/mcl. He required endotracheal intubation and mechanical ventilation with leukapharesis on his second day of admission due to leukostasis. He had been diagnosed with APL variant with t(11;17) (q23;q21) two years prior to this admission and had induction with Ara-c + daunorubicin (7+3), with an additional cycle for consolidation. He was then placed on maintenance with 6-MP + MTX for 24 months. Bone marrow and peripheral blood analysis confirmed disease relapse. Flow cytometric analysis revealed immature myeloid population CD34−, CD15−, CD13+, CD56+, HLA-DR-, with partial expression of CD33. FISH analyses on peripheral blood was negative for t(15;17) and positive for the variant t(11;17)(q23q21). He was started on induction therapy with Idarubicin 12mg/m2/day for three consecutive days with ATRA 45mg/m2/day (via NGT) + G-CSF 5mcg/kg/day. His Day 21 bone marrow examination revealed complete hematologic response. Consolidation/ maintanance with sodium valproate 500mg po BID (to maintain serum levels of 50–100mcg/ml), ATRA 45mg/m2/day (via NGT) + G-CSF 5mcg/kg/day (with G-CSF dose reductions for leukocytosis with a final stable dose of 30mcg/day). He subsequently achieved a complete cytogenetic remission and relapsed after 18 months on this regimen.

Conclusion:

Induction of promyelocyte differentiation and reversal of retinoic acid refractoriness in variant APL with t(11;17) (q23;q21) can be achieved with G-CSF priming and the addition of valproic acid s a HDAC inhibitor.
Endoplasmic Reticulum Stress Is a Target for Therapy in Waldenstrom’s Macroglobulinemia (WM).
Authors: Leleu Xavier , Xu Lian , Hunter Zachary R. , Jia Xiaoying , Moreau Anne-Sophie , Ho Allen W. , Hatjiharissi Evdoxia , N’Go Hai , Santos Daniel D. , O’Connor Kelly , Manning Robert , Soumerai Jacob , Patterson Christopher , Ghobrial Irene M. , Treon Steven P.
Published: October 2019

Background: WM is an incurable low-grade lymphoplasmacytic lymphoma with limited options of therapy. Proteasome inhibition has been shown to induce components of the proapoptotic/terminal unfolded protein response (UPR), a signaling pathway activated by accumulation of misfolded proteins within the endoplasmic reticulum (ER). We previously found that UPR gene expression is related to disease activity in WM with a particular role for GRP78/Bip as a prognostic factor. We therefore examined tunicamycin (Sigma, St Louis, MO), a potent inducer of ER stress, for potential anti-tumor effects in WM.

Methods: WM cell lines (BCWM.1 and WSU-WM), IgM secreting low-grade lymphoma cell lines (MEC1, RL) and primary CD19+ selected LPC cells from WM patients were incubated with tunicamycin (0.01–10 uM) for 24–72 hours and evaluated by MTT, thymidine uptake, and Apo2.7/PI staining for effects on proliferation and survival. Since bone marrow stromal cells (BMSC) confer growth and resistance to conventional treatments, we also tested the effect of tunicamycin on WM cells co-cultured with BMSC. Immunoblotting for caspases was also performed and expression of UPR genes determined using relative quantitative RT-PCR reaction following (0.5–16 hrs) culture with tunicamycin.

Results: WM cells inherently expressed the ER chaperones GRP78/Bip and GRP94/gp96. Tunicamycin rapidly induced components of the proapoptotic/terminal UPR, including PERK, the ER stress-specific eIF-2alpha kinase; ATF6, an ER stress-induced transcription factor; and its proapoptotic target, CHOP/GADD153. Tunicamycin also induced significant cytotoxicity, and inhibited DNA synthesis with an IC50 of 0.5–1 ug/mL in all cell lines, as well as primary LPC from 3/3 WM patients. Furthermore, tunicamycin induced apoptosis in WM cells, with an increase in the sub-G1 population notable at 12 hrs. Tunicamycin induced apoptosis was preceded by caspase-12 cleavage, followed then by caspase-8, -9 and PARP cleavage. Importantly, co-culture of WM cells with the survival factors IL-6, IGF-1 as well as BMSC did not inhibit tunicamycin induced cytotoxicity. Lastly, tunicamycin did not induce cytotoxicity in healthy donor peripheral blood mononuclear or hematopoietic stem cells.

Conclusion: These pre-clinical studies provide a framework for further evaluation of ER stress inducing agents as therapeutic agents in WM.
Standard CHOP-21 as First Line Treatment for Elderly Patients with Hodgkin’s Lymphoma.
Authors: Kolstad Arne , Holte Harald , Nome Ole
Published: October 2019

Background: Outcome for elderly patients with Hodgkin’s lymphoma is not as good as for younger patients, partly due to inferior capability to tolerate appropriate chemotherapy. There is no consensus on standard treatment. In 2000 we introduced CHOP-21 as standard chemotherapy for elderly Hodgkin’s lymphoma patients at our institution with the aim to improve treatment results. CHOP-21 is known to be well tolerated in older patients with non-Hodgkin’s lymphoma and contains active drugs for Hodgkin’s disease.

Patients and methods: Twenty-nine consequtive patients admitted to our institution from 2000–2004, 60 years and older with Hodgkin’s lymphoma were included in this retrospective survey. Stage I/IIA patients received 2–4 cycles of CHOP-21 followed by 30–35 Gy involved field radiotherapy. Stage IIB-IV patients were treated with 6–8 cycles of CHOP-21. Selected cases received 30–35 Gy involved field radiotherapy against residual tumors.

Results: The median age was 71 years (range 60–91). Sixty-two percent presented in stage IIB-IV and 38% in stage I-IIA. Forty-five percent of the early stage patients had bulky disease and/or elevated SR. For the advanced stage patients IPS score of 2–3 was the most common category (72%). B-symptoms occurred in 52% of the total population. Nodular sclerosis was the most frequent histology subgroup (38%), followed by mixed cellularity (17%). Fifty-five percent had co-morbidity prior to treatment, cardiac conditions were most common. Two cases of treatment-related deaths were seen (7%). The most common toxicity during therapy was febrile neutropenia (31%). Treatment modifications were necessary in 38% of cases. The complete response rate after CHOP +/− radiotherapy was as high as 93%. Median follow-up for the whole population is now three years. Five patients have relapsed, and four have died from Hodgkin’s lymphoma. Total overall survival (OS) at three year was 80%, and better for the stage I-IIA subgroup (90% versus 60%). Total Hodgkin-specific survival (HSS) at three years was 73%, and 80% versus 60% for early and advanced stages, respectively. No relapses have occurred after 2 years.

Outcome with CHOP +/− radiotherapy for Hodgkin’s lymphoma patients 60 years and older Response 29 pts (%) NE = not evaluable, patients died after one cycle of CHOP. CR 27 (93) PR 1 (3) ORR 28 (97) NE 1 (3) Relapsed 5 (18) Cause of death Hodgkin’s lymphoma 4 (14) Toxicity 2 (7) Other 1 (3)

Conclusion: The data shows that CHOP-21 is a well tolerated and highly effective first line treatment for elderly patients with Hodgkin’s lymphoma. Of particular interest were the findings that advanced stage patients did so well and that no relapses have been seen after two years.
Addition of Rituximab to CHOP Regimen Improves Clinical Outcome in Non-Germinal Center Type Diffuse Large B-Cell Lymphoma.
Authors: Yokoyama Masahiro , Ennishi Daisuke , Ueda Kyoko , Kodaira Makoto , Yamada Shuhei , Myojo Tomohiro , Sakajiri Sakura , Mishima Yuko , Takeuchi Kengo , Saotome Takashi , Terui Yasuhito , Takahashi Shunji , Ito Yoshinori , Hatake Kiyohiko
Published: October 2019

Background: In recent years, diffuse large B-cell lymphoma (DLBCL) has been classified into the germinal center B-cell (GC) type, the activated B-cell (ABC) type, and the type 3 using global gene expression profiling or immunohistochemical staining. It has been reported that the GC type DLBCL showed significantly longer survival than the non-GC (ABC and the type 3) type DLBCL treated with CHOP or CHOP like regimen not using rituximab.

Methods: We analyzed retrospectively the prognosis between the GC and non-GC types of DLBCL treated with R-CHOP regimen. All 50 patients with DLBCL, diagnosed between July 2003 and July 2005 were included in this study. The pathology was reviewed by hematopathologist and confirmed to be de novo DLBCL according to the WHO classification. Patients with primary CNS- and post-transplant lymphomas were excluded. GC type or non-GC type DLBCL was determined by immunohistochemistry such as the expression patterns of CD10, BCL-6, and IRF-4 (MUM1). All patients were initially treated with six cycles of R-CHOP regimen consisting of rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone. If we evaluated partial response after six cycles of R-CHOP, the patients have added radiation therapy.

Results: The patients consisted of 30 GC type and 20 non-GC type DLBCL with a median age of 61.0 yr (range 31–83 yr). The median follow up of surviving patients was 24 months. CR rate between GC and non-GC types were 57.0% vs. 75.0%, p=0.186, and overall response rate were 87.0% vs. 90.0%, p=0.929, respectively. The median of progression free survival was 17.3 months vs. 19.6 months, p=0.80. There is no statistical significance difference between two groups.

Conclusion: These results suggest that addition of rituximab to CHOP regimen improves clinical outcome of non-GC type DLBCL as well as GC type DLBCL.
Cyclin-Dependent Kinase Inhibitor Seliciclib Shows In Vitro Activity in Diffuse Large B Cell Lymphomas.
Authors: Bertoni Francesco , Lacrima Katia , Rinaldi Andrea , Vignati Sara , Martin Vittoria , Tibiletti Maria Grazia , Gaidano Gianluca , Catapano Carlo C. , MacCallum David E.
Published: October 2019

Background. Despite recent improvements in treatment, a significant fraction of patients with diffuse large B cell lymphoma (DLBCL) still fail therapy. Therefore, new therapeutic modalities are needed to advance the cure rate. Seliciclib (CYC202, R-roscovitine) is a purine analogue developed as an inhibitor of CDK2/cyclin E CDK7/cyclin H and CDK9/cyclin T. Seliciclib has been shown to be active in B cell neoplasms, such as mantle cell lymphoma, chronic lymphocytic leukemia and in multiple myeloma in vitro. The aim of this study was to assess the in vitro activity of seliciclib in DLBCL.

Materials and methods. The anti-proliferative activity of seliciclib was tested in nine human DLBCL cell lines and six DLBCL primary cell cultures. The effects of seliciclib on the cell cycle and on apoptosis, as well as on transcription-related proteins were assessed.

Results. The cell viability of all DLBCL cell lines and primary cells was reduced by seliciclib treatment. The IC50 for the cell lines ranged from 13 to 36 μM. The effect of seliciclib was independent of the genetic aberrations characterizing the cell lines. After seliciclib exposure cells accumulated in G2/M or in G1 phase, with most of the cells showing signs of apoptosis. Despite the clear cytotoxic effect and induction of apoptosis, we could not identify a unique mechanism of action.

Conclusions. Our in vitro data suggest that seliciclib is an active agent in DLBCL. Its efficacy is apparently independent of the underlying chromosomal translocations characteristic of DLBCL. The drug might represent a new therapeutic agent in this lymphoma subtype.
Recombinant Protein A Activity in SCID Mouse Models of Human B Cell Tumors.
Authors: Cooper Andrew , Stein Rhona , Hall Katherine , Boismenu Richard , Chen Susan , Rusche James
Published: October 2019

Protein A from Staphylococcus Aureus acts as a potent B cell superantigen through specific interaction with B cell receptors displaying variable heavy chain sequences from the VH3 clan. In humans, 30–50% of all B cells are VH3-positive, so protein A activity may serve to interfere with host immune responses to S. Aureus through deletion of normal B cell subsets. Indeed, protein A-mediated B cell deletion has been demonstrated in transgenic mice with nearly all B cells expressing VH3-positive surface Ig [Goodyear, J. Immunol176(4), 2262, 2006]. However, it is conceivable that this activity may be usurped to target clonal B cell diseases with a VH3-positive cell origin. In the current study, the superantigen function of a recombinant therapeutic form of protein A (TpA) was exploited to investigate potential anti-tumor activity of protein A in in vitro and in vivo models of B cell neoplastic disease. TpA initiated apoptotic cell death of VH3-positive B cell tumor lines in culture within 24–48 of exposure as evidenced by alteration in mitochondrial membrane potential, activation of caspases, and external exposure of phosphatidylserine in the cell membrane. By 72–96 hours, cell viability was reduced by >50%. Addition of human IgG was essential for cell killing and optimal activity was observed at a 2:1 molar ratio of IgG to TpA. In SCID mice engrafted with the non-Hodgkin’s lymphoma cell line 2F7, TpA administered along with human IgG resulted in modest but statistically significant extension of median survival. In two independent studies employing a SCID mouse KMS-12-BM tumor model, TpA along with 40mg/kg human IgG produced a dose-dependent, reproducible therapeutic effect. At the highest dose studied (30mg/kg), median survival was 45 days compared to median survival of 27 days for control animals. Increased frequency of TpA administration also positively impacted median survival. While human IgG was required for optimal response, a five-fold increase in IgG diminished TpA anti-tumor activity, further underscoring the importance of the ratio of TpA to IgG in cell killing. These results demonstrate that the capacity of protein A to act as a B cell superantigen is not limited to normal cells but can be manifest on transformed cells as well. Since the VH3 clan is highly represented in the human B cell repertoire and is over-represented in some B cell neoplastic diseases, protein A may have utility as a therapeutic agent in cases of B cell malignancy where a VH3 origin is identified.
Preliminary Results Concerning the Prognosis Value of Vascular Endothelial Growth Factor (VEGF) in Chronic Myeloid Leukemia Patients Treated with Imatinib Mesylate.
Authors: Legros Laurence , Huault Sebastien , Mahon Francois-Xavier , Hueber Anne-Odile , Touitou Irit , Cassuto Jill-Patrice , Guilhot Joelle , Peudhomme Claude
Published: October 2019

Increased angiogenesis in bone marrow (BM) is one of the characteristics of chronic myeloid leukemia (CML), a clonal myeloproliferative disorder which expresses a chimeric Bcr/Abl protein. The therapeutic strategy in CML has been totally modified with the development of Imatinib Mesylate (STI571), a specific inhibitor of Bcr/Abl tyrosine kinase activity. Previously, we studied the effect of Imatinib on the Vascular Endothelial Growth Factor (VEGF), one of the most potent regulators of angiogenesis (1). Thus, we demonstrated, for the first time, the decrease in VEGF production in CML patients treated with Imatinib. It pointed out the potential prognostic value of determining the VEGF plasma level of patients in order to follow the evolution of this hematologic malignancy. Actually, we showed that low plasma VEGF levels could be one of the characteristics of complete cytogenetic remission.

However, investigations with a larger number of patients were necessary to evaluate the potential of VEGF levels in the monitoring of CML patients treated with Imatinib. So, we analyzed VEGF plasma level in patients treated in the SPIRIT, the CML French Group phase III, multicenter, open-label, prospective trial. Patients are randomized between the 3 experimental arms [IM 400mg daily in combination with Peg-IFN-α2a (Peg-IFNα2a, 90 μg weekly) or with Ara-C (20 mg/m2/day, days 15–28 of 28-day cycles) or IM 600mg daily] and the reference arm, IM 400mg daily. The Endpoint are overall survival (primary), rate and duration of hematologic, cytogenetic and molecular responses and tolerability. This VEGF evaluation is based on a cohort of 110 pts [median age 51 yrs (19–81); Sokal distribution : 30% of pts low, 37% intermediate, and 33% high]. Males predominated (62%) and percentage of patients enrolled in IM 400mg, IM 600mg, IM 400 with Ara-C and IM 400mg with Peg-IFN arm is respectively 28%, 28%, 24% and 30%. At diagnosis, the median of VEGF plasma level is 945 pg/ml (range: 42.3–4001.5). After 3 months of treatment, the median of VEGF level decreases significantly (p<0.05) to 116.4 pg/ml (range: 6.7–2780). After 6, 9, 12 months of treatment, the median of VEGF level was stabilized at respectively 104 pg/ml (range: 17.9–2244.9), 88.3 pg/ml (range: 21.2–907.4), 99 pg/ml (range: 22.1–689,2).

To determine prognosis value of VEGF we correlated the results with the cytogenetic and molecular response.
Pharmacological Blockade of NF-kB Targets Both Imatinib-Sensitive or -Resistant Chronic Myeloid Leukemia Cells for Cell Death.
Authors: Imbert Véronique , Lounnas Nadia , Frelin Catherine , Gonthier Nadège , Griessinger Emmanuel , Sirvent Nicolas , Hummelberger Michael , Cassuto Jil-Patrice , Mahon François-Xavier , Michiels Jean-François , Rousselot Philippe , Dreano Michel , Peyron Jean-François
Published: October 2019

The Bcr-Abl inhibitor imatinib is now the first line therapy for all newly diagnosed CML patients in chronic phase. Nevertheless resistance to the drug emerges as CML progresses to an acute deadly phase. Additional cellular targets should thus be identified to develop alternative therapeutic strategies. The transcription factor NF-kB is a pro-survival factor, found abnormally active in numerous hematologic malignancies. In the present study we show that the constitutive and abnormal activation of NF-kB in Bcr-Abl transformed BaF3 cells and in the LAMA84 CML line could be downregulated after inhibition of Bcr-Abl. Pharmacological blockade of NF-kB by the IKK2 inhibitor AS602868 (Serono International S.A.) prevented proliferation of BaF3/Bcr-Abl, LAMA84 and primary CML cells. Importantly, AS602868 led to apoptosis of an imatinib resistant variant of LAMA84 and of BaF3 clones expressing mutated form of Bcr-Abl derived from imatinib resistant patients. Moreover, NF-kB inhibition affected proliferation and hematopoietic colony formation of primary imatinib resistant CML cells. Finally, the IKK2 inhibitor prolonged survival of mice intravenously injected with the imatinib resistant clone LAMA84-r. Our data strongly suggest that NF-kB mediates important survival functions in CML cells for bcr-abl and that targeting NF-kB with the IKK2 inhibitor AS602868 may represent a new promising therapeutic strategy for CML treatment.
Overexpression of the ABC-Transporters BCRP and MDR1 in CML Patients with Resistance to Imatinib and in Untreated CML Patients.
Authors: Bendit Israel , Mello Monika C. , Dorliac-Llacer Pedro E. , Nardinelli Luciana , Bendit Marina , Novaes Mafalda N.Y. , Neto Joao E.B. , Lima Walter M. , Giorgi Ricardo R. , Chamone Dalton A.F.
Published: October 2019

Imatinib Mesylate (Gleevec™ or Glivec™ from NOVARTIS) is widely used for the treatment of CML inhibiting the BCR-ABL tyrosine kinase activity. Although, 90% of the patients respond to this treatment, resistance to this drug has been seen, mainly due mutations in the tyrosine kinase domain of BCR-ABL. Recently, has been reported that imatinib is a substrate for the ABC-transporters BCRP (breast cancer resistance protein) and MDR1 (P-Glycoprotein). Based in that report, we examined the expression of those two ABC-transporter, by quantitative PCR (Q-PCR) technology, in 29 CML patients who were resistant to IM (9 CP, 17 AP, and 3 BC), in 34 untreated CML patients (23 CP, 10 AP, and 1 BC) and 6 normal blood donors. cDNA was synthesized for RQ-PCR, which was performed on peripheral blood samples drawn after loss of CHR or MCyR for those with resistance to imatinib and prior to start of treatment with imatinib and those untreated patients. All samples were analyzed using SYBER Green technology and reported as the 2−Δ Δ Ct where it assumes that the amplification efficiency of the target gene (BCRP or MDR1) and the internal control gene (ABL) are the same. Resistant patients had a significantly higher levels of BCRP expression than untreated patients, and normal blood donors with a median ratio of 2.1 (range, 0.04–34.2) vs. 0.64 (range, 0–18.6) vs. 0.6 (range, 0.07–1.41) (Wilcoxon test, P<0.0002). For the MDR1 gene expression, patients with resistance to imatinib had also a significantly higher level, median ratio of 1.65 (range, 0.09–10.8) vs. 0.68 (range, 0.01–2.63) in untreated patients and normal blood donors median ratio 0.76 (range, 0.02–1.92) (P < 0.0017). Nineteen of 29 resistant patients (65.5%) overexpressed BCRP and 41.3% (12/29) overexpressed MDR1, meantime only 26.5% (9/34) and 11.8% (4/34) of the untreated CML patients overexpressed BCRP and MDR1 respectively (X2 test P=0.004 and P=0,022). Overexpression of BCRP was more frequent in AP of the resistant group (70%) than the untreated patients (30%) (Fisher exact test P=0.05). When comparing the expression of MDR1 between the resitant group in AP (35,3%) and the untreated one in the same phase (40%) no statistic difference was seen (P=1). Patients in CP and BC do not show any difference in the expression of BCRP and MDR1 regardless the presence or not of resistance to imatinib. These data confirms that high expression of BCRP or MDR1 may contribute to imatinib resistance phenotype leading to a reduction in intracellular imatinib concentrations.Further follow-up is necessary for those untreated CML patients that overexpress, at diagnosis, those ABC-transporters BCRP and MDR1. These may reveal correlations between the expression and clinical prognosis and may allow the development of new starategies to overcome imatinib mesylate resistance.
Whole Chromosome Painting and Multiplex Fluorescence In Situ Hybridization in Detecting Complex Chromosomal Aberrations in Myelodysplastic Syndromes.
Authors: Li Jianyong , Xiao Bing , Chen Lijuan , Zhu Yu , Xu Wei , Qiu Hairong
Published: October 2019

Objective To explore the value of the technique of whole chromosome painting (WCP) and multiplex fluorescence in situ hybridization (M-FISH) in the detection of complex chromosomal aberrations (CCAs) of myelodysplastic syndromes (MDS).

Methods M-FISH was used in seven MDS patients with CCAs detected by R-banding technique to refine CCAs, and to identify cryptic translocations and characterization of marker chromosomes. Dual-color WCP procedures were further performed in 7 cases to confirm some rearrangements detected by M-FISH.

Results In all cases, M-FISH confirmed all results of R-banding.The composition and origin of 6 kinds of marker chromosomes, 9 kinds of chromosomes with additional material undetermined and 5 kinds of derivative chromosomes undefined by CC were defined after M-FISH analysis; 4 kinds of cryptic translocations overlooked by CC were found on derivative chromosomes and previously normal appearing chromosomes. In addition, M-FISH revealed some nonrandom aberrations: aberrations involving chromosome 17 (5/7) and -5/5q- (4/7) were the two most frequent aberrations. Fluorescence flaring is a main factor leading to misinterpretations. Some misclassified and missed chromosomal aberrations by M-FISH were corrected by WCP.

Conclusions M-FISH is a powerful molecular cytogenetic tool in clarification of CCAs. Complementary WCP helped us identify misclassified and missed chromosomal aberrations by M-FISH. CC in combination with molecular cytogenetic techniques M-FISH and WCP can unravel complex chromosomal aberrations more precisely.
Causes of Thromboembolism in Polycythemia Vera Patients.
Authors: Ambrus Julian L. , Ambrus Clara M. , Akhter Selina , Islam Abul B.M.A. , Islam Pierre T.
Published: October 2019

Our experience and review of the literature suggests that thromboembolism is a major cause of morbidity and death in polycythemia vera patients. Using methods developed by our group (Ambrus et al, Angiology 34 418–426, 1984) we determined membrane fluidity, deformability of red cell membranes. We found that compared to normal controls, red cell membranes in polycythemia vera patients were relatively stiff and the red cells less deformable when passed through microtubules. These abnormal red cell membranes also caused platelet aggregation. In the circulation of polycythemia vera patients we also found increased platelet aggregates using the detection method of Wu et al (Wu et. al, Lancet2: 924–926 1974, Wu et. al, Thromb. Res.7:917–924, 1975). Other factors in the thromboembolic complications included

increased hematocrit hyperviscosity syndrome increased thromboxane A2 content of platelets increased tendency to disseminated intravascular coagulation (DIC) (factors involved were also investigated).

Red cell membrane fluidity and platelet aggregation may be points of therapeutic attention to prevent thromboembolic complications. Suggestions of approaches to this problem by future research will be discussed.
Mutation JAK2 V617F in Patients with Myeloproliferative Disorders and Technical Aspects of Its Detection.
Authors: Pekova Sona , Hoffmann Rudolf , Jindra Pavel , Novakova Ludmila , Kozak Tomas , Vozobulova Vera , Schutzova Miroslava , Balasz Jozef , Tomsikova Lucie , Prucha Miroslav
Published: October 2019

Myeloproliferative disorders (MPD) are a heterogeneous group of diseases that involve chronic myeloid leukemia (CML), polycythemia vera (PV), essential trombocythemia (ET), chronic idiopatic myelofibrosis (IMF), hypereosinophilic syndrome and chronic neutrophilic leukemia. Recently, it has been found that in Ph-negative MPD, acquired somatic mutation in JAK2 gene can be identified, causing substitution of valine for phenylalanine at aminoacid position 617 (V617F). Experimental evidence indicates that JAK2 V617F might cause erythropoietin-independence of erythroid colonies and receptor hypersensitivity, thus be implicated in the pathogenesis of PV.

The incidence of V617 in PV is high, reaching 96% of cases; in ET or IMF, the mutation is less frequent. Nevertheless, the percentage of JAK2 V617F-positive patients differs greatly among studies, depending on the detection technique used. Sequencing analysis, as one of the options, can underrate the number of positive cases, as its sensitivity in authentic samples with varying proportion of mutant granulocytes is unsatisfactory. The nucleotide context of the V617F site is complicated (direct repeat TGTG/TT), and thus prone to sequencing artifacts.

For specific and sensitive detection of V617F mutation in JAK2 gene, we have developed a novel allelic discrimination assay, based on the usage of Real-Time PCR technology and LNA-modified fluorescently labeled probes (Locked Nucleic Acids). This technology is distinguished by 100% discrimination efficiency, and sensitivity detection rate reaching 10% of mutant granulocytes in an authentic sample. LNA-modified probes are extremely powerful tool for precise genotyping, since they are short (12-mers), and due to the interspersed LNA nucleotides, melting temperature (Tm) of the probe/target hybrid is high. Hence, the LNA-based genotyping technology combines the desired features of high sensitivity and absolute specificity.

Herein, we present an analysis of JAK2 V617F detection in our group of 157 consecutive patients with the diagnosis of MPD or suspected MPD. Using our novel LNA-based Real-Time PCR genotyping technique, we were able to identify varying proportions of the mutant JAK2 allele in 87 cases (55.4%); in the remaining 70 patients the mutation was not detected.

The LNA-based Real-Time PCR technique is extremely precise and efficient in identifying even subtle proportion of JAK2 V617F mutant granulocytes, which in cases of diagnostic uncertainties on the origin of the polycythemia might be of great importance.
The Jak2-Inhibitor AG409 Affects Survival of BFU-E and CFU-GM of Myeloproliferative Disorder Cells Carrying the JAK2 V617F Mutation.
Authors: Duek Adrian , Lichman Ina , Shtalrid Mordejai , Berrebi Alain
Published: October 2019

In the last few years, point mutation of the Janus Kinase 2 (JAK2) gene (V617F) was found in the majority of patient with Polycythemia Vera and two related diseases ET and MMM. This mutation initiates constitutive activation of JAK2 transduction gene, inducing growth factor- independent hemopoiesis and plays a dominant role in origin of myeloproliferative disorder. Inactivation of this mutated gene may restore the normal hemopoiesis. Based on the evidence that the inhibition of the Jak-Stat axis by AG490 induced apoptosis in leukemic compared with normal cell, we attempt to analyzed the AG490 effect on the survival of MPD cells carrying JAK2 mutation in cultures.

Peripheral blood (PB) samples were collected from 49 MPD patients (PV n=31, ET n=14, MMM n=4) at the time of diagnosis or during the follow up. The JAK2-V617F mutation tested by an allele-specific, semi-quantitative PCR was detected in 35 patients. MNPBC’s from four patients homozygous for the JAK2 mutation and from two normal volunteers were cultured in methylcellulose with 50 ng/ml SCF, 10ng/ml GM-CSF, 10ng/ml IL-3 and 3U/ml EPO, and in the same medium without growth factors. On the sixth day, 50 micromole of AG490 were added to the cultures and dishes with no addition of AG490 were kept for control. Colonies were counted on 6th day and on 14th day, and we checked the growth patterns on 14th day.

Spontaneous colonies without growth factors were found only in the four MPD patients. Although, the number of the colonies was not significantly decreased after the addition of the AG490 inhibitor, we observed an obvious change in the normal growth pattern. The appearance of the colonies became darker reflecting an apoptotic process and death.

Such a phenomenon was not seen in normal controls or in MPD cells without addition of the inhibitor. In conclusion our experiment clearly demonstrated preferential inhibition of MPD cells with JAK2 mutation by AG490, probably by induction of apoptosis. Our results suggest a therapeutic potential of inhibitors of JAK2 for the future.
LEF-1 Regulates C/EBP α Expression and Neutrophil Differentiation in Normal Myelopoiesis and Congenital Neutropenia by a β-Catenin Independent Mechanism.
Authors: Skokowa Julia , Uenalan Murat , Schambach Axel , Scherr Michaela , Eder Matthias , Zeidler Cornelia , Baum Christopher , Stanulla Martin , Cario Gunnar , Welte Karl
Published: October 2019

Lymphoid enhancer-binding factor 1 (LEF-1) belongs to the canonical Wnt signaling pathway acting in transcriptional complexes with β-catenin. LEF-1 can also act independent of β-catenin (i.e. in the TGF-β or Notch pathway). Additionally, recent studies described LEF-1 dominant negative isoform (dnLEF-1), which lack the β-catenin binding domain and functions as either a transcriptional repressor or activator. To date, analysis of the role of LEF-1 in hematopoiesis has been restricted to the lymphoid compartment.

Previously we described the crucial role of lymphoid enhancer-binding factor 1 (LEF-1) in granulopoiesis. We found that LEF-1 mediates proliferation, survival, and differentiation of granulocyte progenitor cells. Moreover, abrogated LEF-1 expression is one of the pathomechanism of severe congenital neutropenia CN (Skokowa et al., Nature Medicine, in press). Based on these findings, we aimed to characterize the molecular mechanisms of LEF-1 in the regulation of granulocytic differentiation. C/EBPα is well known as a key transcription factor in granulopoiesis and we found it to be a target gene directly regulated by LEF-1. A screen of the known 566 bp upstream promoter of C/EBPα gene revealed a putative LEF-1 binding site (− 559 bp to − 538 bp). We confirmed LEF-1 binding to C/EBPα promoter in nuclear extracts from CD34+ and CD33+ cells in the transcription factor binding NoShift and ChIP assays. Interestingly, LEF-1 binds to the C/EBPα promoter more efficiently in CD33+ myeloid progenitors than in CD34+ cells. The direct regulation of C/EBPα by LEF-1 was further confirmed in functional studies. We found that in line with down-regulation of LEF-1, expression of C/EBPα was also significantly reduced in CD33+ myeloid progenitors of CN patients. Moreover, LEF-1 rescue of these cells resulted in a marked up-regulation of C/EBPα mRNA expression and in vitro restoration of defective granulocytic differentiation. Remarkably, transduction of CN CD33+ cells with dnLEF-1 isoform, which lacks the ß-catenin-binding domain, resulted in up-regulation of C/EBPα to a similar degree as it was observed with full-length LEF-1. A direct regulatory link between LEF-1 and C/EBPα was additionally confirmed in LEF-1 inhibition experiments. C/EBPα expression was significantly down-regulated in CD34+ cells of healthy individuals, transduced with LEF-1 shRNA. Therefore, we propose that LEF-1 is a key regulator of myeloid differentiation acting in a β-catenin-independent manner, similar as it is known for LEF-1 regulation of T-lymphocyte development.
IL-6 Down-Regulates CD33 Expression in Primary Myeloma Cells as Well as Myeloma Cell Lines.
Authors: Shamsasenjan Karim , Abroun Saeid , Liu Shngqin , Amin Jakia , Islam Khademul , Otsuyama Ken-ichiro , Kawano Michio M.
Published: October 2019

Human primary myeloma cells show the marked heterogeneity of surface marker expression and morphology; possibly for myeloma cells lose the expression of PAX-5 and EBF, master genes of B cell lineage. As reported previously, we detected the expression of a myeloid marker, CD33, in the primary myeloma cells in about 10% out of 60 cases with overt myelomas as well as in 2 myeloma cell lines (Liu-01 and ILKM8 cells) by RT-PCR and flowcytometry using 4 different anti-CD33 antibodies. The expression of CD33 in these cells appeared to be well correlated to the monocytoid morphology with convoluted nuclei, and furthermore correlated to the increased expression of PU.1 and C/EBPα genes. Induction of CD33 expression was also found by the treatment with vitamin D3 in some primary myeloma cells as well as ILKM3 cell lines. This induction was not found in PAX-5(+) B cell lines (KUS and Raji cells). Interestingly, IL-6 down-regulated the expression of CD33 in Liu-01 and ILKM8 cell lines; if cultured with IL-6 (10 ng/ml) for 12 days, the expression of CD33 on Liu-01 cells completely disappeared. By IL-6 stimulation, the morphology of Liu-01 cells was also reversed to the morphology with round nuclei. Also, it is confirmed that IL-6 markedly suppressed the expression of PU.1 and C/EBPα genes in Liu-01 cells. Therefore, these data suggest that IL-6, a growth factor for myeloma cells, could reverse phenotypic conversion of non-B cell lineage such as CD33(+) myeloid/monocytoid cells to the B cell lineage.
Molecular Cytogenetic Study of Immunofluorescently Labeled Plasma Cells and Prognostic Significance of Clonal Chromosomal Aberrations in Multiple Myeloma.
Authors: Michalova Kyra , Zemanova Zuzana , Pavlistova Lenka , Tajtlova Jana , Spicka Ivan , Zounar Rene , Gregora Evzen , Smolikova Alena , Chrz Michal , Obernauerova Jarmila , Walterova Lenka
Published: October 2019

Finding of clonal chromosomal aberrations in plasma cells is considered as one of the most important and independent prognostic factor in patients with multiple myeloma (MM). The most frequent and reliable cytogenetic indicators are chromosome 13q deletions (intermediate or moderately adverse prognosis), and translocations involving the immunoglobulin heavy chain gene (IgH) at 14q32 region. IgH gene is involved in translocations with different partner genes and these rearrangements are related to a very poor prognosis. The only exception is translocation t(11;14)(q13;q32) which is often connected with longer overall survival and therefore considered to be a favorable prognostic factor. The aim of the study was to assess the frequency of the most important chromosomal aberrations in immunofluorescently labeled plasma cells of patients with MM by interphase fluorescence in situ hybridization (I-FISH), and to evaluate their prognostic significance.

During the last two years we examined 128 newly diagnosed patients with MM by conventional G-banding technique and by I-FISH. We focused on detection of aberrations of 13q, IgH gene rearrangements, and t(11;14)(q13;q32) translocation. I-FISH was done by locus-specific DNA probes (Abbott-Vysis, Des Plaines, Illinois, USA). Fifteen patients with complex karyotype were re-examined by multicolor FISH (mFISH, MetaSystems GmbH, Altlussheim, Germany). G-banding revealed abnormal karyotypes in 25% of patients, I-FISH detected chromosomal abberations in 82.8% cases. Abberations of chromosome 13 were found in plasma cells of 84 patients (65,6%), the incidence was: del(13)(q14) in 11%, monosomy 13 in 40%, combination of del(13)(q14)/monosomy 13 in 8%, and other aberrations of chromosome 13 in 7% of cases. Aberrations of IgH gene were proved in 82 patients (64%). t(11;14)(q13;q32) was seen in fourteen patients (11%) and other translocations affecting 14q32 region in 19 patients (15%). Besides translocations, different variations of total and/or partial deletions of IgH gene were detected in 28 cases (22%). In nine patients (7%) we found clones with translocation and deletion of 14q32 simultaneously, twelve patients (9%) had other IgH aberrations. Univariate statistical testing showed aberrations of 13q (p=0.042), and IgH gene rearrangments (translocations except t(11;14)(q13;q32) or deletions; p=0.007) to be associated with significantly shorter progression-free survival. The worst prognosis in our cohort was for 18 patients with combination of chromosome 13 anomalies together with rearrangements of IgH gene (p=0.049). Method of immunofluorescent labeling of plasma cells allows higher detection of chromosomal changes by I-FISH technique and therefore correct prognosis of the disease can be done.