bluuebunny/crossref_metadata_2025 huggingface dataset:
High-Sensitivity Mutational Analysis of BCR-ABL Mutations in the Kinase Domain Using Pyrosequencing Could Provides Alternative Methodology for Monitoring the Proportion of Mutant Alleles in Patient with Chronic Myelogenous Leukemia.
Authors: Lee Il-Kwon , Yun Ju Hyun , Kim Yeo-Kyeoung , Kim Hee Nam , Park Kyeong-Soo , Shin Myung Geun , Choi Chan , Kim Hyeoung Joon
Published: April 2020

Imatinib mesylate has substantially changed treatment of chronic myeloid leukemia (CML). But the resistance-conferring mutations in tyrosine KD (Kinase Domain) of BCR-ABL region constitute the leading cause of clinical resistance in CML patients on imatinib or dasatinib monotheray. Therefore early detection and accurate quantification of the common mutations in KD in post-transplantation patient could provide vital information regarding disease progression or prognosis. Here we report results of pyrosequencing to quantify the non-mutated and mutant alleles in 11 CML patients who were treated by imatinib mesylate or dasatinib. Patients were monitored over periods ranging from 24 to 67 months. 11 mutations in the region identified by direct or pyrosequencing and/or pGEMT cloning are Y215F, T253F, E255K, L323P, N336S, A337T, M351T, E355K, F359I, H396R and P480L. Out of 11 mutations, two (F359I and P480L) are found to be novel. Four mutations (L323P, N336S, A337T and E355K) were positioned between P loop and catalytic domain, one (F359I) was in the activation domain and one (P480L) was near to the carboxy terminal of the kinase. One novel mutation (Y215F) located outside (N terminal region of kinase domain) region was also identified. Y215F mutation which lies outside the kinase domain might be sporadic one without affecting kinase activity. We successfully measured proportion of KD mutant cells with greater precision by using pyrosequencing (Fig 1). The dynamics of the mutated clones obtained from pyrosequencing were correlated with RQ-PCR (Fig2) suggesting quantitative pyrosequencing method could be alternative to various methods including allele-specific oligonucleotide-polymerase chain reaction (ASO-PCR) and conventional sequencing which have limited sensitivity at low concentrations of mutant allele. Taken together, we established a novel method for early identification of KD mutations and precise monitoring of the proportion of mutant alleles in patient with CML. Most importantly, those results were in correlation with clinical response to imatinib mesylate or dasatinib. Therefore quantitative measurement of mutant cell dynamics could provide early indications of clinically significant disease progression.

T3151 mutation T3151 mutation T3151 Mutation T3151 Mutation
Functional Gfi-1 Is Important for BCR/ABL-Mediated Proliferation and Survival of Myeloid Cells.
Authors: Basu Suchitra , Qiu Yaling , Dong Fan
Published: April 2020

Chronic myelogenous leukemia (CML) is a clonal hematopoietic disorder caused by the BCR/ABL fusion oncogene. CML typically evolves in three distinct clinical stages: chronic and accelerated stages and blast crisis. The progression of CML from chronic phase (CP) to blast crisis (BC) is characterized by the increasing failure of myeloid precursors to differentiate into mature granulocytes and aggressive proliferation of immature myeloid cells. It has been shown that CML progression from CP to BC is associated with downregulation of C/EBPs including C/EBPa and C/EBPe, critical regulators of myeloid development. Forced expression of C/EBPs suppresses transformation and restores granulocytic differentiation of BCR/ABL-expressing myeloid cells, suggesting that downregulation of C/EBPs may be involved in CML transition from CP to BC. The proto-oncogene Gfi-1 encodes a nuclear zinc-finger transcriptional repressor that is required for the survival and proliferation of myeloid cells. We recently showed that expression of a dominant negative Gfi-1 mutant, N382S, led to markedly increased expression of C/EBPe in myeloid cells, suggesting that Cebpe encoding C/EBPe is a Gfi-1 target. We investigated the possibility that inhibition of Gfi-1 function in BCR/ABL-expressing cells may increase C/EBPe expression, thereby exerting a negative effect on BCR/ABL-mediated transformation. Myeloid 32D cells transfected with BCR/ABL (32D/BCR/ABL) are independent of IL-3 for proliferation and survival. Expression of the N382S mutant in 32D/BCR/ABL cells (32D/N382S) had a significant inhibitory effect on proliferation and survival in the absence of IL-3, but only a weak effect in the presence of IL-3, suggesting that the N382S mutant specifically inhibited BCR/ABL-mediated proliferation and survival. The level of C/EBPe protein was markedly augmented in 32D/N382S cells. Interestingly, when cultured in medium containing no growth factors, 32D/N382S cells exhibited certain features of macrophage differentiation including increased cell size, adherence to the surface of culture flasks, spreading and increased surface expression of macrophage differentiation marker F4/80. 32D/N382S cells also became more sensitive to c-ABL kinase inhibitor imatinib. These data indicate that Gfi-1 may play an important role in BCR/ABL-mediated transformation and thus represent a potential therapeutic target in the treatment of CML.
Low Dose Busulfan and ATG with Fludarabine as Conditioning Regimen Was Effective in Sibling and Unrelated Donor HSCT in CML Patients.
Authors: Lange Andrzej , Sedzimirska Mariola , Dlubek Dorota , Jaskula Emilia , Mlynarczewska Anna , Sok Agnieszka
Published: April 2020

Allogeneic hematopoietic stem cell transplantation offers complete remission in CML patients. However, transplant related mortality and long term consequences especially associated with cGvHD make patients reluctant to accept this treatment modality especially in the era of Imatinib. Therefore, in 1999 we started a transplant program for CML patients characterized with a reduced dose of Busulfan to 8 mg/kg of. b.w., Fludarabine (120 mg/m2 in sibiling and 150 mg/m2 in MUD setting) and ATG to facilitate engraftement. At the beginning ATG cumulative dose was 40 mg/kg of b.w. (5 patients), or 20 mg/kg of b.w (13 patients). In this high dose ATG group 6 patients had CMV or EBV reactivation what prompted us to decrease ATG dose to 2.5 mg/kg of b.w. given in three consecutive days and then on day 4 the dose was increased to 5 mg/kg of b.w. if patients lymphocyte count did not reach the target values (≤300/ul of WBC and < 0.1% of CD3+ cells in nucleated blood cells population).

With the use of the above program 52 adults and two children (F/M: 22/32, age from 11 to 55 yrs (median: 34) were transplanted from sibling (20 patients) and alternative (33 MUD and 1 related haploidentical) donors. Thirty nine and 15 patients received PBPC and marrow, respectively. Seven patients died by + 100 days post transplant (6 infectious complications including 1 case with viral encephalitis and 1 blast crisis). One patient had marrow failure, 15 patients developed ≥ IIo aGvHD, 23 patients had cGvHD and 14 of them extensive form. Thirty four patients (63%) are alive with an observation time from 1 to 81 months post transplant (median 22 months). Survival was better in sibling than in MUD transplanted patients (77% vs 50%, p=0.03, Cox analysis). In alive patients group STR technique proved complete chimerism in 26 out of 31 examined patients. Five cases lacking full chimerism include: 1 patient with hematological relapse, and 4 with molecular relapse. All other cases were bcr/abl negative.

In conclusion: low dose ATG was sufficient to secure the take and the employed reduced intensity conditioning was associated with:

low rate of transplant related mortality, aGvHD was infrequent and mild, cGvHD was the main cause of late complications, full chimerism and bcr/abl free status were observed in a vast majority (84%) of long term survivors.
Acquired Mutations in the BCR-ABL Kinase Domain in Imatinib Resistant Patients. Multicentric Experience in Argentina and Uruguay.
Authors: Gargallo Patricia , Moiraghi Beatriz , Bullorsky Eduardo , Santarelli Rosa , Riva Maria , Magarino Alicia , Giuliani Ricardo , Bianchini Michele , Milone Jorge , Rosenfeld Ester , Pavlovsky Santiago , Lanari Emilio , Arizo Adriana , Avila Celina , Miguel Castro R. , Conti Rafael , Alu Fernanda , Barreyro Paula , Custidiano Rosario , Bitton Roberto , Larripa Irene , Bengio Raquel
Published: April 2020

Imatinib induces complete cytogenetic response (CCyR) in 82% of patients with CML in chronic phase. There is a subset of patients who either failed to achieve or lose hematological/CCyR. Kinase domain mutations have emerged as a potential cause for treatment failure.

Aims: In the current study we have investigated ABL kinase domain mutations, amplifications and quantification of BCR-ABL transcripts by Q-PCR in Imatinib resistant patients.

Patients and Methods: A total of 84 patients (pts) with CML treated with Imatinib were studied and 82 were evaluable. Sixty-one (74%) were in chronic phase (CP), eleven (14%) were in accelerated phase (AP), and ten (12%) were in blast crisis (BC).We analyzed the DNA from blood samples. Mutations were screened by conformation sensitive gel electrophoresis (CSGE). The amplification products displaying an abnormal pattern were sequenced using automatic system. The amplification of BCR-ABL rearrangement was studied in interphase nuclei using Vysis extra-signaling probe. Real time quantitative PCR (RQ-PCR) of BCR-ABL transcripts was performed in a subset of 44 patients to assess molecular response, using Light Cycler (Roche®), Syber Green Method.

Results: Eleven mutations from 82 evaluable patients were detected (13%). Seven were in p- loop: M244R (1), L247A (1), G250E (2), Q252H (1), E255K (2), and four in the Imatinib binding: T315I (3), H318L (1). Two patients had BCR-ABL amplifications with 4–6 signals in interphase nucleous and one of them showed extramedullary involvement in skin nodules. Five patients had clonal evolution with double Ph chromosome. Most of mutations were found in patients in late chronic phase (6/11: 54%). The detection occurred at a median of 49 months (range 12–154) after diagnosis. Eighty four percent of cases studied with RQ-PCR, had null molecular response (<1Log Reduction). Imatinib dose in patients with mutations had been escalated to 600–800 mg/day previously.

Conclusion: This is the first multicentric study in spanish-speaking South America. T315I was the most frequent mutation detected in our study and was associated with poor outcome. We found 13% of resistant cases with point mutations, all of them located in the p-loop or imatinib binding.This is an ongoing study and further recruitment is needed to confirm these findings. Early detection of mutations can have prognostic implication and allow therapeutic intervention such as dose escalation, combination therapy or second generation tyrosine kinase inhibitors.
Front - Line Combined Immuno-Chemotherapy (R-CHOP) Significantly Improves the Time to Treatment Failure and Overall Survival in Elderly Patients with Advanced Stage Follicular Lymphoma - Results of a Prospective Randomized Trial of the German Low Grade Lymphoma Study Group (GLSG).
Authors: Buske Christian , Kneba Michael , Lengfelder Eva , Pfreundschuh Michael , Ludwig Wolf-Dieter , Graeven Ullrich , Hallek Michael , Dreyling Martin , Unterhalt Michael , Hiddemann Wolfgang
Published: April 2020

Follicular lymphoma (FL) is an indolent disease of the advanced age with more than 40 % of the patients being older than 60 years at diagnosis and an age-specific incidence peaking above 75 years. We now analyzed the treatment outcome of elderly patients in the GLSG multicenter phase III study comprising a prospective randomized comparison of R-CHOP versus CHOP alone in patients with advanced stage FL. 221 patients > 60 years with untreated FL were randomized for therapy with R-CHOP (R-CHOP: Rituximab 375 mg/m2 d0–1; cyclophosphamide 750 mg/m2 d1; doxorubicine 50 mg/m2 d1; vincristine 1.4 mg/m2 d1; prednisone 100 mg/m2 d1–5) (n=109) or CHOP alone (n=112). Patient characteristics were well balanced between the treatment groups, also with regard to the distribution of the FLIPI risk groups (≥ 3 adverse factors 73% and 66 % in the R-CHOP and CHOP arm, respectively). R-CHOP induced higher overall response rates and significantly prolonged the time to treatment failure (TTF)(median 5.0 years versus 2.1 years, respectively; logrank test: p<0.0001) compared to CHOP in the elderly patient group. Furthermore, the estimated 4-years progression free survival was 62.2% for R-CHOP versus 27.9 % after CHOP (logrank: p< 0.0001). Importantly, R-CHOP was able to prolong the overall survival in elderly patients compared to CHOP with an estimated 4-years overall survival of 90% after immunochemotherapy versus 81 % after CHOP alone (logrank test: p=0.039). In the multivariate analysis individual FLIPI risk factors such as elevated serum LDH level, a hemoglobin level below 12 g/dl, the number of nodal areas (> 4) as well as application of CHOP alone were independently associated with a shorter TTF. Treatment related side effects were similar in both patient groups and comprised predominantly myelosuppression. In summary, the addition of Rituximab to CHOP significantly improves the outcome of elderly patients with previously untreated advanced stage FL without adding major side effects.
JAK2 V617F Mutation Is Infrequent in “5q-Syndrome” and 5q-Associated MDS.
Authors: Zhang Ling , Gueller Saskia , Raynaud Sophie , Koeffler Phillip H. , Lee Stephen
Published: April 2020

Background: V617F mutation in Janus Kinase 2 (JAK2) gene has been found in chronic myeloproliferative disorders (MPD) including polycythemia vera (90%), essential thrombocythemia and chronic idiopathic myelofibrosis (30–50%), and occasionally in myelodysplastic syndromes (MDS). “5q- Syndrome” is a MDS that shares features with MPD and characterized by an atypical megakaryocytic hyperplasia in bone marrow and usually thrombocytosis in peripheral blood. The most common deleted region for this syndrome is 5q13.3q33.1. An interstitial deletion with variable proximal (5q12-14) and distal (5q31-33) breakpoints has been found in other MDS with/without additional chromosomal abnormalities beyond “5q- Syndrome”. To date, JAK2 mutation was detected in 6/97(6.2%) of patients having diagnosis of MDS with “5q- Syndrome”.

Design: In our study 21 MDS patients (10 with “5q- Syndrome” and 11 MDS with isolated or complex 5q-) whose diagnosis by both bone marrow aspiration/biopsy and conventional chromosomal analysis were confirmed.

Materials and Method: Primers were created to amplify a 460 bp fragment containing the site of JAK2 V617F mutation. Forty-five cycles of PCR were performed at an annealing temperature of 57°C. Resulting PCR product was digested with 2 U BsaXI for 16 hours and with an additional 2 U BsaXI for another 16 hours at 37°C, then analyzed on a 2% agarose gel. The mutant allele remained undigested whereas the wild-type allele was digested into 241 bp, 189 bp and 30 bp fragments. All experiments included a positive (HEL cells) and negative (K562 cells) control.

Results: PCR results showed clear wild type PCR patterns in all 21 cases.

Conclusion: No JAK2 mutations were detected in 21 patients either with “5q- Syndrome” or other 5q- associated MDS suggesting that JAK2 mutations are infrequent in these MDS patients.
Diagnostic Potential of Immunophenotyping CD34+ Cells in Myelodysplastic Syndromes.
Authors: De Waele Marc , Leus Barbara , Trullemans Fabienne , Verschraegen Inge , Urbino Montse , Jochmans Kristin , Renmans Wim
Published: April 2020

Myelodysplastic syndromes (MDS) constitute a heterogeneous group of clonal hematopoeitic stem cell disorders. They are characterized by abnormal bone marrow differentiation, peripheral blood cytopenia and a risk of transformation into acute myeloid leukemia (AML). The diagnosis of MDS depends on cytomorphology and cytogenetics and may be difficult especially in cases with normal numbers of blasts and without ringed sideroblasts in the bone marrow. Cytomorphology is subjective and dysplastic features may be present in other disorders than MDS. In this study we examined the potential of immunophenotyping CD34+ hematopoietic precursors for the diagnosis and classification of MDS. Bone marrow samples of 31 patients with low grade MDS (21 without and 10 with ringed sideroblasts), of 17 patients with refractory anemia with excess of blasts (RAEB), of 25 patients with AML and of 39 patients with cytopenia not due to MDS (controls) were examined. CD34+ cells were enumerated and the expression of B cell antigens (CD19), of myeloid antigens (CD13, CD33, CD117) and of immature antigens (CD133) was determined by flow cytometry. Statistical analysis was done with a Mann-Whitney test. A high number of CD34+ cells was found in MDS and AML. This was accompanied by an increase of the number of myeloid precursors and a decrease of the B cell precursors. CD117 appeared to be the best marker of myeloid precursors followed by CD13. A wide range of CD34+CD133+ and of CD34+CD33+ cells was found in all types of samples. Forty percent of the patients with low grade MDS showed an increased expression of CD117 on their CD34+ cells. In 25% of the cases without ringed sideroblasts a high expression of CD133 was present. Similar changes were more frequently found in RAEB and AML together with an increased expression of CD13 and CD33 and a low positivity for CD19. With a scoring system based on the expression of these antigens 57% of low grade MDS samples (score 1/6 or 2/6) could be distinguished from the controls (score 0/6). An elevated score was also found in respectively 84% and 100% of the RAEB and AML samples. 85% of them even had a score between 3/6 and 6/6. In conclusion, immunophenotyping of CD34+ cells is able to differentiate 60% of low grade MDS samples from other causes of cytopenia. Increased expression of CD117, CD133 and CD34 are the main differences. Similar changes are even more frequently found in RAEB and AML. A scoring system based on the antigen expression on the CD34+ cells is a powerful tool for the diagnosis and classification of MDS.
Erythroid Precursors from Patients with ‘Low Risk’ Myelodysplasia Demonstrate Ultrastructural Features of Autophagy.
Authors: Houwerzijl Ewout J. , Pol Henk-Willem , Blom Nel R. , Stokroos Ietse , van der Want Johannes J.L. , De Wolf Joost , Vellenga Edo
Published: April 2020

Anemia in myelodysplasia (MDS) is partially ascribed to enhanced cell death of committed erythroid cells in the bone marrow compartment. To define in more detail the underlying type of cell death in MDS, immunohistochemical staining and ultrastructural analysis were performed on MDS and normal bone marrow samples. Immunohistochemistry of MDS bone marrow biopsies (n=20) demonstrated no positive staining of the erythroid lineage for active caspase -3 or -8. Ultrastructural analysis was performed in low-risk MDS patients (Refractory anemia (RA; n=3); RA with ringed sideroblasts (RARS; n=4) on isolated bone marrow hematons, which are compact hematopoietic complexes containing adipocytes, mesenchymal and endothelial cells, and hematopoietic cells. In vitro culture assays have demonstrated that these hematons contain a high number of progenitor and primitive cells. Ultrastructural analysis of the hematons of the MDS patients as well as the healthy controls demonstrated no signs of apoptosis in erythroid cells. In contrast, abnormalities compatible with autophagy were present in >60% of the MDS cells. Especially, cytoplasmic vacuoles with double membranes were noticed in basophilic and polychromatic normoblasts. Morphometric analysis of the erythroid cells showed no differences between healthy controls and MDS patients with regard to cell volume, mitochondria content or nuclear surface, but a significant increase was shown for the ratio area of vacuoles/cytoplasm in the MDS basophilic and polychromatic normoblasts (healthy controls vs MDS: 0.003 vs 0.013 (p<.0001) and 0.004 vs 0.014 (p<.0001). In view of the reported enhanced apoptosis in MDS bone marrow cells we questioned whether the discrepancy with our results might be linked to the cell processing, especially the presence or absence of signals from the bone marrow microenvironment. Subsequently MDS (n=4) and normal (n=2) bone marrow mononuclear cells (MNC) and hematons were in vitro cultured for 24 hrs and analyzed ultrastructurally. Upon in vitro culture a significant increase in the percentage of apoptotic erythroid cells was noticed predominantly in MDS erythroid cells. In addition, after 24 hrs of culture apoptosis was more frequently demonstrated in erythroid cells of the MNC fraction than in the hematons (21 ± 4 vs 8 ± 3 %, p= 0.04). In summary, these data demonstrate that autophagy is present in MDS erythroid cells in vivo in significantly higher numbers than in healthy controls and suggest that autophagy may switch to apoptosis in the absence of an appropriate bone marrow microenvironment.
Addition of Rituximab to First-Line MCP (Mitoxantrone, Chlorambucil, Prednisolone) Chemotherapy Prolongs Survival in Advanced Follicular Lymphoma - 4 Year Follow-Up Results of a Phase III Trial of the East German Study Group Hematology and Oncology (OSHO#39).
Authors: Herold Michael , Haas Antje , Srock Stephanie , Neser Sabine , Al Ali Kathrin H. , Neubauer Andreas , Doelken Gottfried , Naumann Ralph , Fietz Thomas , Freund Mathias , Rohrberg Robert , Hoeffken Klaus , Franke Astrid , Ittel Thomas H. , Kettner Erika
Published: April 2020

Introduction: Rituximab plus chemotherapy has been proved to be the gold - standard in treating advanced follicular lymphoma. Here we report the 4-year-follow-up data of our phase III trial comparing MCP - chemotherapy vs rituximab + MCP both followed by interferon maintenance in advanced symptomatic follicular lymphoma.

Methods: Previously untreated patients with advanced stage (III + IV) symptomatic CD 20-positive indolent NHL and mantle cell lymphoma (n=358) were randomized to receive either MCP-chemotherapy (mitoxantrone 8 mg/m² d1+2, chlorambucil 3x3 mg/m² d 1–5, prednisolone 25 mg/m² d 1–5 x 8 q 4 weeks) or MCP + rituximab (375 mg/m² d −1).

Here we report the results of the ITT population of patients with follicular lymphoma (FL) (grade 1+2), who represented the majority of patients and for whom the sample size primarily was calculated, so this is not a subgroup analysis. Study endpoints included overall and complete response rate (RR + CR), progression free survival (PFS), event free survival (EFS), time to next treatment (TTNT), overall survival (OS) and toxicities.

Results: with a median follow - up of nearly 4 years (47 months) we are able to provide relatively mature data. Concerning toxicities there was no striking difference, but there was a significantly increased risk to experience a CTC grade III or IV toxicity for leukocytes in the R-MCP arm, however this did not increase the risk of infections.

For the FL - ITT population the results are given in the table.

Conclusions: Concerning all end points rituximab plus MCP is significantly superior to MCP alone in the treatment of advanced follicular lymphoma. Special attention should be drawn to the fact, that after a median follow-up of 47 months we can demonstrate a clinically and statistically significant survival advantage for the immunochemotherapy.

Results R-MCP (n=105) MCP (n=96) p-value Response rate 92,4% 75% .0004 Complete Response 49,5% 25% .0009 PFS median n.r. 29 months < .0001 PSF 4 years 71% 40% EFS median n.r. 26 months < .0001 EFS 4 years 69% 35,5% TTNT median n.r. 29,4 months .0002 OS median n.r. n.r. .0096 OS 4 years 87% 74%
Azacytidine in the Management of Myelodysplastic Syndrome: A Single Institutional Retrospective Review of Responses and Tolerability.
Authors: Grinblatt David L. , Bavisi Seema , Lawton Jessica , Kaminer Lynne S.
Published: April 2020

Twenty two patients (pts) with primary or therapy-related MDS were treated with 5-azayctidine (AZA) at the Kellogg Cancer Care Centers (KCCC) from July 2004–May 2006. We retrospectively reviewed all consecutive patients with myelodysplastic syndrome treated at the KCCC who received this agent. Charts were reviewed for response, time to response, duration of response and rate of hospitalization with complications of therapy or disease during AZA therapy. Treated patients had Refractory Anemia with Excess Blasts-type 1 (RAEB-1, n=6), RAEB type-2 (RAEB-2, n=3), Chronic Myelomonocytic Leukemia (CMMoL, n=4) or Refractory anemia (RA, n=6). Median age at study entry was 80 yrs (range, 58–95). Patients were treated with azacytidine 75mg/m2 daily for seven consecutive days or five days followed by a two day break and an additional two days of treatment. Patients were treated until best response and treatment frequency was decreased to every 6–8 weeks once a response had occurred.

Responses were as follows: Complete response (CR) with normalization of cytopenias, marrow dysplasia and blasts (as defined in IWG criteria by Cheson et al) occurred in 2 pts (9%). Complete hematologic response (CHR) meeting all of the peripheral blood criteria but without a confirmation bone marrow performed was noted in 4 pts (18%) while hematologic improvement (HE) major (>2 gm rise in Hgb) occurred in 1 pt (5%) and HE minor (1–2 gm rise in Hgb or 50% decrease in RBC requirements) occurred in 2 pts (9%). Thus, a response using the IWG criteria was seen in 9/22 pts yielding an overall response rate of 41%. The median time to response was 3 cycles (range, 1–4). The median duration of response was 8 months (range, 3–18+). Responses are ongoing in 4/9 of the responders. One of the complete response patients had a complex karyotype (5q-, 7q-, del (18p)) that was not present on follow-up marrow after response was noted. Though treatment related cytopenias did occur frequently in the first two cycles of therapy, there were no treatment related hospitalizations for neutropenic patients in this consecutive cohort of patients and no deaths due to therapy. Azacytidine is a well tolerated agent with responses occurring with a significant rate and duration in MDS.
Study of CD28 Costimulatory Super Family and CD4+Treg in Myelodysplastic Syndromes.
Authors: He Guangsheng , Zeng Hui , Wu De Pei
Published: April 2020

Objective: To explore the possible immune annormality of CD28 costimulatory super family and CD4+T regulatory (CD4+Treg) in myelodysplastic syndrome (MDS).

Methods: Gated by CD3 and SSC, the changes of CD28, CTLA-4, PD-1, and CD25, on CD3+CD4+ cells were evaluated in 38 new diagnosed cases with MDS(RA 17, RAS 1, RAEB 14, RAEB-t 6) and 11 normal controls respectively by directive immune labels.

Results: [circ1]In the group of MDS, the CD3+CD4+CD28+ was 79.82%±8.99% ( RA 78.65%±10.4%, RAEB/RAEB-t 80.10%±7.28% respectively), was lower than 89.56%±3.06% of normal controls (P<0.01); CD3+CD4+CTLA-4+, CD3+CD4+PD-1+, and CD3+CD4+CD25+ were 2.14%±1.25% (RA 1.51%±0.80%, RAEB/RAEB-t 2.65%±1.33%), 1.44%±0.88% (RA 0.93%±0.63%, RAEB/RAEB-t 2.02%±0.76%), and 3.18%±2.11%(RA 2.80%±1.38%, RAEB/ RAEB-t 3.41%±2.46%) in MDS, which were all higher than those (0.11%±0.12%, 0.09%±0.14% and 1.65%±1.14% respectively) of normal controls (P<0.01). [circ2] Along with the progress of MDS, the expression of CTLA-4, PD-1 and CD25 on CD3+CD4+ cells increased significantly in RAEB/RAEB-t than RA. [circ3] The antagonism costimulatory of CTLA-4/CD28 decreased from normal controls (0.11±0.11) to RA (2.14±0.97), then to RAEB/RAEB-t (3.58±1.55) significantly.

Conclusion: [circ1] In MDS, the immune priming factor-CD28 decreased while the suppressive factor-CTLA-4 increased. [circ2] The negative immune CD4+ regulatory increased in MDS, and was predominance in immune balance. [circ3] Along with progression of MDS, negative regulatory factors was enforced, which promoted clone of MDS expansion.
A Pharmacokinetic & Pharmacodynamic Study of Oral Lenalidomide in Patients with Low- or Intermediate-1-Risk Myelodysplastic Syndromes.
Authors: Wu Anfan , List Alan , Lush Richard , Bretz Kelly , Knight Robert , Zeldis Jerry , Lau Henry
Published: April 2020

Twelve patients diagnosed with low- or intermediate-1-risk myelodysplastic syndromes (MDS) were administered a single, 10 mg dose of lenalidomide followed by 24-hour blood and urine sampling on one occasion, and again at Day 14 of multiple dosing, followed by 5-hours of blood and urine sampling. Patients then continued into a combined treatment phase in which recombinant EPO was administered with lenalidomide. Table 1 describes the pharmacokinetic profiles of lenalidomide found for the first 12 MDS patients.

Following the single oral 10 mg dose, lenalidomide had an average renal clearance of 7.48 ± 1.74 L/hr. Greater than half of the dose (65.6% ± 6.9%) was excreted unchanged in the urine over 24 hours.

Conclusions: Among these MDS patients, the single and multiple dose pharmacokinetics of lenalidomide were similar, suggesting no accumulation of lenalidomide with multiple dosing. After 24 hours, 65.6% of the dose is eliminated in the urine as the parent compound. No relationship was apparent between the total number of adverse events normalized per day or Grade’s 3+4 adverse events and the drug exposure.

Table 1. Pharmacokinetics (Mean ± SD) in MDS Patients After a Single and Multiple 10 mg Oral Dose of Lenalidomide Parameter Single Dose Multiple Dose 1 Cmax and tmax are presented on Day 14; 2 median (min, max); NA = Not Applicable Cmax1 188 ± 59.5 168 ± 55.4 AUCt 852.3 ± 272 NA AUCss,0–5 NA 553.1 ± 205.6 T½,z 3.72 ± 0.75 NA tmax1,2 1.00 (0.5, 2.00) 0.75 (0.75, 2.50)
High Level of Pretransplant Leukemic Blasts and the Absence of Chronic Graft-Versus-Host Disease Provide Negative Effects on Clinical Outcome of Transplantation with Fludarabine-Busulfex Based Reduced Intensity Conditioning for Patients with the De Novo Myelodysplastic Syndrome According to WHO Criteria.
Authors: Cho Seok-Goo , Cho Byung-Sik , Kim Yoo-Jin , Kim Sung-Yong , Eom Ki-Seong , Kim HeeJe , Lee Seok , Min Chang-Ki , Kim Dong-Wook , Lee Jong-Wook , Min Woo-Sung , Kim Chun-Choo
Published: April 2020

Background: Allogeneic hematopoietic cell transplantation (HSCT) is the only curative potential for patients with MDS. HSCT following reduced-intensity conditioning (RIST) have enabled the extension of transplantation to include older or medically infirm patients with myeloid malignancies. However, it is very difficult to determine the value of outcomes of RIST for myelodysplastic syndrome (MDS) because of the heterogeneity of diseases included in most trials and the small number of MDS.

Method: Twenty-two patients with de novo MDS classified by WHO criteria received an allograft using a Fludarabine (30 mg/m2/day for 5 days)/Busulfex (3.2 mg/kg/day for 2 days) (14 patients) or Fludarabine (30 mg/m2/day for 5 days)/Busulfex (3.2 mg/kg/day for 2 days)/ATG (2.5 mg/kg/day for 2 days) (8 patients). The median age of patients was 43 years (range, 16–55). Donors were HLA-compatible sibling (n = 19) or matched unrelated (n = 3).

Result: Nineteen patients (86.4%) achieved engraftment. At a median follow-up of 18.9 months (range, 13.1–24.8 months), the estimated 2-year overall survival (OS), event-free survival (EFS), transplantation-related mortality and relapse incidence were 78.7%, 67.7%, 12.6%, and 22.5%, respectively. Acute GVHD greater than grade II was developed in 3 patients (15.8%). Chronic GVHD were developed in 10 patients (55.6%) and all of them did not receive ATG as conditioning. Variables influencing EFS were chronic GVHD (None/Limited [20.8%] versus Extensive [100%], P = 0.001), marrow blasts before transplantation (<10% [85.1%] versus >10% [20.8%], P = 0.006), and WHO criteria (RA [100%] versus RCMD [75%] versus RAEB-1 [80%] versus RAEB-2 [20.8%], P = 0.043).

Conclusion: These observations indicate that high level of pretransplant leukemic blasts and the absence of chronic graft-versus-host disease provide negative effects on clinical outcome of transplantation with Fludarabine-Busulfex based reduced intensity conditioning for patients with the de novo myelodysplastic syndrome according to WHO criteria.
Detection of the JAK2 V617F Mutation by Real Time PCR in Granulocytes and Platelets of ET Patients.
Authors: Bellosillo Beatriz , Gimeno Eva , Longaron Raquel , Florensa Lourdes , Salar Antonio , Espinet Blanca , Sole Francesc , Serrano Sergio , Besses Carlos
Published: April 2020

Introduction. The JAK2 V617F mutation has been detected in 23%–57% of ET patients by direct sequencing or allele-specific (AS) PCR. It remains unknown, however, if the mutation detected in the granulocyte population, may be equally detected in platelets from these patients.

Objective. To compare the detection of the JAK2V617F mutation in granulocytes and platelets from ET patients by real time AS RT-PCR.

Patients and methods. Platelets and granulocytes from 50 ET patients from a single institution were studied. Patients were diagnosed according to the WHO criteria. At the time when JAK2 mutation was analyzed 16/50 patients were receiving platelet-lowering therapy ± ASA, 14/50 patients only received ASA and 20/50 received no specific treatment. JAK2 mutation was analyzed by real-time AS RT-PCR with probes specific for the mutated and the wild type form.

Results. The V617F JAK2 mutation was detected in 18 out of 50 patients in both granulocytes and platelets by real time AS RT-PCR, and was negative in both cell populations in the remaining 32 patients. In the V617F JAK2 positive cases, the mean Ct(V617FJAK2)/Ct(wild type JAK2) ratio was 1.074±0.062 for granulocytes and 1.038±0.039 for platelets (p=0.048). These values corresponded to a 17.79 ±7.4% of mutated population when granulocytes were analyzed, whereas, a significantly higher percentage of mutated population was observed, 23.45±7.78 %, when platelets were analyzed (p=0.032).

Conclusions. The results of V617FJAK2 mutation detection by AS RT-PCR were the same in granulocytes and platelets (either positive or negative). The percentage of clonal population detected in ET patients was significantly higher in platelets than in granulocytes.
Tyk2 Mutation Homologous to V617F Jak2 Is Not Found in Essential Thrombocythaemia, Although It Induces Constitutive Signaling and Growth Factor Independence.
Authors: Shide Kotaro , Shimoda Kazuya , Kamezaki Kenjirou , Kakumitsu Haruko , Kumano Takashi , Oku Seido , Numata Akihiko , Ishikawa Fumihiko , Takenaka Katsuto , Harada Mine
Published: April 2020

A single somatic mutation, V617F, in the pseudokinase domain of the Jak2 is the cause of the majority of polycythaemia vera (PV) cases and one-third of essential thrombocythaemia (ET) cases. PV is characterized by an increase in RBC and erythropoiesis is regulated by erythropoietin (EPO). ET is characterized by the elevation of megakaryopoiesis and platelet numbers, while thrombopoietin (TPO) is a primary regulator of thrombopoiesis. EPO selectively activates Jak2, whereas TPO selectively activates Jak2 and Tyk2. As valine 617 of Jak2 is conserved as valine 678 of Tyk2, we examined the effect of a homologous mutation in Tyk2 (V678F Tyk2) on cell growth. V678F Tyk2 augumented the transcriptional activity of Stat3 and Stat5 in 293T cells. The expression of V678F Tyk2 in Ba/F3 cells induced the phosphorylation of Tyk2 and Stat5 in the absence of cytokine stimulation and induced autonomous cell growth. Ba/F3 cells harboring V678F Tyk2 showed hyper-responsiveness to IL-3. As V678F Tyk2 might cause MPD, we examined the somatic mutation of Tyk2 in 6 ET patients lacking the V617F Jak2 mutation. No cases harbored the Tyk2 mutation. It requires a two-base-pair change to substitute phenylalanine for valine in Tyk2, and the enhancing effects of V678F Tyk2 on cell growth are less dramatic than those of V617F Jak2. The lesser effect of V678F Tyk2 on cell proliferation, together with the lower incidence of VF mutations in Tyk2, might explain why mutations in Tyk2 are rare in ET patients.
Endothelial Dysfunction Is Independent of the JAK2 V617F Mutation in Polycythemia Vera.
Authors: Gisslinger Heinz , Gwechenberger Marianne , Neunteufl Thomas , Bachofner Nicol , Gisslinger Bettina , Kralovics Robert
Published: April 2020

In polycythemia vera (PV) thrombotic events are the major cause of morbidity and mortality. Although in PV vascular complications encompass the whole spectrum of thrombembolic events, arterial occlusions (stroke, TIA, myocardial infarction and peripheral arterial disease) play a major role. In a large retrospective analysis arterial thromboses accounted for about 75% of all thrombotic complications while venous thrombotic events were less frequent and accounted only for a quarter of vascular complications. As a preclinical sign of arterial thrombosis we observed in a previous study a significantly more pronounced endothelial dysfunction in PV compared to a sex and age matched control group with even a higher cardiovascular risk profile. So far the pathogenesis of this predisposition to endothelial dysfunction and arterial thrombosis in PV remains poorly understood and no clear association with any laboratory parameter could be established. The V617F mutation in the JAK2 kinase gene (JAK2-V617F) showed an association with thrombosis in essential thrombocythemia. Virtually all patients with PV acquire JAK2-V617F although the abundance of myeloid cells carrying the V671F mutation varies greatly among patients. Therefore, we quantified the presence of the mutated allele of JAK2 (%V617F) in granulocytes using a quantitative allele-specific PCR-assay and examined the endothelial function as a preclinical indicator for arterial vascular disease in 27 patients with PV. Endothelium-dependent flow-mediated vasodilatation (FMD) was measured using high-resolution ultrasound of the brachial artery. We did not observe a correlation between the FMD values and %V617F in our cohort of PV patients (P=0.48). A similar lack of correlation was observed when only patients with low and high %V617F were compared (P=0.60). To date the low FMD values in PV have not been associated with any laboratory or genetic parameters. Thus, a unique V617F-independent pathogenetic mechanism is likely to play a role in endothelial dysfunction in PV.
The Clinical Implications of JAK2V617F Mutation in 137 Chinese Patients with Myeloproliferative Diseases.
Authors: Zhang Ri , Fei Hairong , Chen Suning , Pei De
Published: April 2020

A single acquired mutation in the JAK2 gene has recently been described in the classic myeloproliferative diseases(MPDs) including 65–97% of patients with polycythemia vera (PV), 23–57% of those with essential thrombocythemia (ET) and 35–57% of those with idiopathic myelofibrosis (IMF). The understanding of the molecular mechanisms has been considered a point mutation in exon 12 of the JAK2 gene, a key hematopoietic regulator, resulting in substitution of valine for phenylalanine at amino acid position 617 in the JH2 domain. In order to clarify the frequency and some clinical implication of JAK2V617F mutation in the Chinese patients with MPDs, we investigated this mutation in 137 Chinese patients aged 15 to 65 years. There are 57 patients with PV, 68 with ET, and 12 with IMF and the diagnosis of MPDs was defined according to published criteria. Genomic DNA samples obtained from all three MPDs and twenty cases of healthy people as negative control and the HEL, an erythroleukemic cell line, as positive control was screened by allele-specific PCR and gel electrophoresis for JAK2V617F mutation described by Baxter EJ et al.

The results indicated that 65.69% of heterozygous JAK2V617F mutation was shown in 137 patients of over all MPDs, 73.68% in PV (42/57), 58.82% in ET (40/68), 66.67% in IMF (8/12). Sequence analysis of PCR products from selected patients confirmed existence of both mutant and wild-type alleles in patients with JAK2V617F mutation. A higher prevalence was observed in older patients with MPDs, and the WBC count of ET patients with JAK2V617F mutation is higher than that of ET patients without JAK2V617F mutation, and the difference has a statistical significance (P<0.05). The HGB concentration and platelet number of MPDs patients with JAK2V617F mutation were higher than those without JAK2V617F patients, but the difference did not achieve a statistical significance(P>0.05).

Cytogenetic analyses were performed in 115 of the 137 MPDs patients. The chromosomal karyotype was normal in 108 of the 115 patients. Among them, JAK2V617F mutation was detected in 74 patients (68.51%) as compared with 5 of the 7 patients with karyotypic abnormalities (71.4%). Three patients with detective bcr/abl low expression assayed by FISH and real-time PCR techniques, two have JAK2V617F mutation. Our datas suggest that JAK2V617F mutation also occurs in a significant percentage of Chinese patients with the myeloproliferative diseases.
Relationship between Autoimmune Phenomena and Disease Stage and Therapy in B-Cell Chronic Lymphocytic Leukaemia.
Authors: Barcellini Wilma , Capalbo Silvana , Agostinelli Rosa M. , Mauro Francesca R. , Ambrosetti Achille , Calori Rossella , Cortelezzi Agostino , Laurenti Luca , Pogliani Enrico M. , Pedotti Paola , Liso Vincenzo , Mandelli Franco , Zanella Alberto
Published: April 2020

Autoimmune haemolytic anaemia (AIHA) and thrombocytopenia (AITP) are known complications of B-cell chronic lymphocytic leukaemia (B-CLL), whereas there are only few reports on other autoimmune diseases and on serological markers of autoimmunity. In this multicentric GIMEMA study we investigated the presence of different autoimmune phenomena in B-CLL (haematological cytopenias, other autoimmune diseases, and serological markers of autoimmunity) and their relationship with B-CLL stage and therapy by multivariate analysis.

Among the 194 cases recorded, the more frequent complication was AIHA (66%), followed by AITP (18%) and other autoimmune diseases (16%). The occurrence of autoimmune complications was associated with advanced and multi-treated disease, as demonstrated by multivariate analysis, which identified age over the median, stage C and I and II line therapy as independent risk factors for autoimmune complications. The distribution of the autoimmune diseases was different with respect to stage and therapy, in that AIHA and AITP were typically present in advanced and multi-treated disease, whereas the non-haematological autoimmune complications were mostly observed in early B-CLL. Interestingly, fludarabine treatment was not associated with an increased risk of AIHA. In addition, 41% of B-CLL patients, mostly in stage A, showed positive serological markers of autoimmunity.

Non-haematological autoimmune complications, although usually less severe than haematological ones, should be carefully searched for, particularly in early B-CLL patients. The distribution of autoimmune diseases with respect to stage and therapy suggest the existence of different pathogenic mechanisms underlying haematological and non-haematological autoimmune phenomena in B-CLL.

Distribution of autoimmune phenomena grouped as haematological complications (AIHA and AITP, ▪), non-haematological disease (***), and serological autoantibodies only (□), χ2 test: p<0.001 Distribution of autoimmune phenomena grouped as haematological complications (AIHA and AITP, ▪), non-haematological disease (***), and serological autoantibodies only (□), χ2 test: p<0.001
CD20 Antigen Expression Is Increased in Trisomy 12 CLL as Compared to Other Cytogenetic Subtypes.
Authors: Tam Constantine S. , Otero-Palacios Joselle , Lerner Susan , Abruzzo Lynne V. , Jorgensen Jeffrey L. , Ferrajoli Alessandra , Wierda William G. , O’Brien Susan , Keating Michael J.
Published: April 2020

CD20, a B-cell specific 297-amino acid transmembrane phosphoprotein and the target of the monoclonal antibody rituximab, is expressed at lower levels in malignant cells from patients with CLL as compared to other indolent B-cell malignancies. Anecdotal observations have suggested that CLL patients with trisomy 12 may have higher cell-surface CD20 expression than other cytogenetic subtypes. In order to test this hypothesis, we analyzed bone marrow (n=454) and peripheral blood (n=56) samples from 510 chemotherapy-naïve patients using a Vysis FISH panel and a flow cytometric quantitative CD20 antigen assay. FISH studies identified 99 patients (19%) with trisomy 12. Compared to non-trisomy 12 patients, patients with trisomy 12 were older (p=0.002) and were more likely to have increased serum B2-microglobulin (p<0.001), LDH (p<0.001), splenomegaly (p=0.007), and hepatomegaly (p=0.01). Mean CD20 antigen sites were significantly higher in patients with trisomy 12 in both peripheral blood and bone marrow samples (log mean 4.64 vs. 4.03 for PB, p<0.001; log mean 4.36 vs. 3.99 for BM, p<0.001). Mean CD20 antigen expression by Döhner subtypes were (per cell): trisomy 12 24,255; 17p deletion 11,114; negative FISH 10,557; 13q deletion 10,431; and 11q deletion 6,391. A multivariate analysis confirmed trisomy 12 as the only significant predictor of increased CD20 expression. To date, 11 CLL patients with trisomy 12 have received rituximab and GMCSF as initial therapy, and partial response was achieved in 8 of 9 (89%) assessable patients. Increased CD20 antigen expression in CLL patients with trisomy 12 provides a rationale for exploration of rituximab-based regimens in these patients.
Cytogenetic Aberrations and Ig VH Gene Mutations of Clinically Benign CD5-Monoclonal B-Cell Lymphocytosis (MLUS).
Authors: Wang Chen , Amato Dominick , Oscier David G. , Davis Zadie , Mould Sarah , Kolomietz Elena , Zheng Jianing
Published: April 2020

Clonal expansion of lymphocytes is a hallmark of lymphoproliferative disorders. However, the finding of clonal lymphocytes may not be diagnostic of a defined lymphoid malignancy. In this study, we had an opportunity to identify and characterize a series of 7 patients with clinically benign clonal B-cell lymphocytosis. The clonal lymphocytes were clearly of CD5− and non-CLL phenotype. All patients were elderly and identified with a mild to moderate absolute lymphocytosis (lymphocytes range 3.6–9.4 ×109/L). The clonal population accounted for 95–99% of total B cells. For a follow-up period of 4–16 years, clonal lymphocytosis was persistent but virtually not progressing. All patients remained clinically stable and totally asymptomatic. The clonal CD5− B cells were shown to have somatic hypermutations of VH gene in 6 of the 7 patients, indicating a cell origin of germinal center B lymphocytes. Karyotypic aberrations were found in 5 of 6 patients examined. Two clones were found to have an isochromosome 17q, one as the sole abnormality and one as part of a complex karyotype. The loss of the short arm of chromosome 17 resulted in loss of p53, which was confirmed by the FISH analysis in both cases. The finding of p53 loss is unusual in such a benign condition. Two other clones were shown to have 7q abnormalities; one 7q31–34 deletion and one t(2;7)(p11;q22), the latter is impliated in dysregulation of the CDK6 gene. Even though deletion of 7q is strongly associated with, and t(2;7) is confined to splenic marginal zone lymphoma (SMZL), neither patients were hematologically or clinically diagnostic for SMZL. It remains to be determined whether additional cytogenetic and molecular changes may occur to lead to progression to a clinical malignancy. The patients in this study differ from those clonal CD5− monoclonal B-cell populations identified by sensitive flow cytometry in the normal population, by the presence of absolute lymphocytosis. In addition, the phenotypic profile is distinct from that of benign variant CLL. These clinically benign lymphocytoses may represent an intermediate between covert clonal expansion and overt malignancy.