bluuebunny/crossref_metadata_2025 huggingface dataset:
Balanced Rearrangements of Chromosome 6 in Acute Leukaemia: Clustered Breakpoints in 6q22-q23 and Possible Involvement of c-MYB/AHI1 in a New Recurrent Translocation, t(6;7)(q23;q31).
Authors: Sinclair Paul B. , Harrison Christine J. , Jarosova Marie , Foroni Letizia
Published: April 2020

While deletions of the long arm of chromosome 6 (6q) are common in patients with ALL, translocations are rare, most are considered sporadic and none have been cloned. Among myeloid malignancies, the recurrent translocations: t(6;11)(q27;q23), t(6;11)(q21;q23) and t(6;8)(q27;p11) give rise to known fusion oncogenes. Other uncharacterised balanced or unbalanced 6q abnormalities have occasionally been reported. Using fluorescence in-situ hybridisation (FISH) with HGMP mapped PAC and BAC clones, we investigated chromosomal rearrangements of 6q in acute leukemia. Previously we reported 24 cases with deletions of 6q (Sinclair et al 2004, Can Res 64; 4089), here we describe the analysis of 11 patients with novel balanced rearrangements.

Six of seven breakpoints in ALL and two in a single case of AML were localised to a 10.5 Mb ‘hotspot’ within 6q22–q23. Of these, five were analysed down to the level of a single clone. In two cases of childhood T ALL, both carrying a t(6;7)(q23;q31~36), split FISH signals were produced by adjacent PACs, RP1–32B1 and RP3–388E23, mapping the breakpoints to within a ~150Kb region. A search of published karyotypes identified five similar rearrangements, four also in pediatric T ALL, all present as a sole abnormality or represented in the major clone. Of genes in the region, two (c-MYB and AHI1) are tightly regulated during normal hemopoietic development and over-expression of both has been reported to occur in hemopoietic malignancies. Moreover, clusters of retroviral insertion sites occur within both genes and immediately proximal to c-MYB in murine and feline leukemias and lymphomas. Thus we have defined a new primary recurrent translocation in T ALL that is likely to target either c-MYB and/or AHI1.Three other non recurrent rearrangements of 6q22–q23, mapped in detail, interrupted regions without known genes.. Therefore, these abnormalities were unlikely to have promoted leukemia through classical oncogene activation, but may have influenced expression of one or more common target genes through a long-range position effect. Alternatively these clustered rearrangements may mark the position of a new fragile site on 6q. Additional 6q breakpoints in three cases of AML and one of ALL mapped to 6q15, 6q21, 6q25 and 6q26–q27, and were coincident with the positions of a known oncogene partner AF6q21, the haemopoietic transcription factor BACH2 and two cloned fragile sites FRA6E and FRA6F. Putative partner genes of four translocations were investigated by FISH and the involvement of MLL was demonstrated in one case with t(6;11)(q15;q23).
Acute Myeloid Leukemia with Deletion 9q Is Associated with CEBPA Loss-of-Function Mutations.
Authors: Frohling Stefan , Schlenk Richard F. , Krauter Jurgen , Ganser Arnold , Thiede Christian , Ehninger Gerhard , Haase Detlef , Harder Lana , Scholl Claudia , Caligiuri Michael A. , Bloomfield Clara D. , Dohner Hartmut , Dohner Konstanze
Published: April 2020

The transcription factor CCAAT/enhancer binding protein alpha (C/EBPalpha) plays an important role in the development of mature neutrophils. Two common types of mutations in the CEBPA gene encoding C/EBPalpha have been identified in approximately 10% of adults with acute myeloid leukemia (AML): N-terminal, dominant-negative frameshift mutations that result in loss of C/EBPalpha function and C-terminal in-frame mutations that result in C/EBPalpha proteins with decreased DNA-binding potential. Previous studies concluded that mutant CEBPA predicts favorable outcome in younger AML patients with intermediate-risk karyotypes or normal cytogenetics. To assess the prevalence of CEBPA mutations in specific cytogenetic subgroups, mutational analysis of the CEBPA gene was performed in 125 AML patients. No CEBPA mutations were detected in 77 patients with t(8;21), inv(16)/t(16;16), t(15;17), or balanced translocations with breakpoints in band 11q23. In 58 patients with various non-complex karyotypic abnormalities, CEBPA mutations were present in 8 (14%). Surprisingly, 5 of the 8 patients with del(9q) as the sole aberration or in combination with a single additional abnormality other than t(8;21) had CEBPA mutations associated with loss of C/EBPalpha function. Consequently, 41 additional del(9q) cases were analyzed; 9 had CEBPA loss-of-function mutations. The overall prevalence of CEBPA loss-of-function mutations in cases with del(9q) within a non-complex karyotype was 41% (14 of 34 patients), whereas none of the patients who had a del(9q) within a complex karyotype (n = 7), in combination with a t(8;21) (n = 10), or together with a t(15;17) (n = 1) demonstrated mutant CEBPA. Analysis of associated mutations indicated that alterations of the FLT3, MLL, and NRAS genes are not common cooperating events in the pathogenesis of del(9q) AML with inactivating CEBPA mutations. This is the first study to show that AML with del(9q) is strongly associated with CEBPA loss-of-function mutations. The coincidence of del(9q) with inactivating CEBPA mutations and the fact that del(9q) is a common secondary cytogenetic abnormality in t(8;21)-positive AML, that is characterized by specific down-regulation of CEBPA, raise the possibility that loss of a critical segment of 9q, most likely in 9q22, and disruption of C/EBPalpha function cooperate in the pathogenesis of these leukemias. Further refinement of the commonly deleted segment of 9q using high-resolution techniques is underway to identify the critical gene(s) involved and their role in normal hematopoiesis and leukemogenesis. A collaborative intergroup study has been initiated to define whether the relatively good prognosis associated with del(9q) is related to the presence of a CEBPA mutation.
Expansion of Bone Marrow (BM) Progenitors and Mobilization of Multilineage Progenitor Cells Associated with Once Per Cycle Administration of Long-Acting Growth Factors Neulasta and Aranesp with Chemotherapy (CT): Biology and Nature of Response.
Authors: Vadhan-Raj Saroj , Bueso-Ramos Carlos E. , Hangoc G. , Christopherson K. , Collard M. , Broxmeyer H.E.
Published: April 2020

Hematopoietic growth factors (HGF) G-CSF and GM-CSF have been utilized widely to facilitate mobilization of progenitor cells that can be used to support high dose CT. Recently long-acting HGF neulasta (pegfilgrastim) and aranesp (darbepoetin alfa) were developed to reduce the frequency of administration. While these agents are effective in reducing CT-induced neutropenia and anemia similar to their parent compounds, very little is known about their efficiency in mobilizing progenitor cells. The purpose of this study was to evaluate biologic effects of these agents, used in combination with CT, on progenitor cells. Chemo-naïve sarcoma patients receiving adriamycin and ifosfamide (AI) were treated once per cycle with aranesp (500 mcg) SC prior to initiating CT (day 0) and neulasta (6 mg) SC after completion of CT (day 4). BM and peripheral blood (PB) samples were studied at the baseline and around day 14 (at the time of WBC recovery) for progenitors and mediators of response. The treatment was associated with a significant increase in PB CD 34 + cells (median increase 36-fold, p=0.005) and marked mobilization (p=0.003) of CFU-GM (24-fold), BFU-E (22-fold), and CFU-GEMM (62-fold) (n=14). To better understand the biology and nature of response, we examined BM before and at the time of mobilization (n=12). There was an expansion of multi-lineage BM progenitors (p=0.06) and CD 34+ cells (P=0.001). BM exam at the time of recovery showed increased cellularity (2-fold) with increased granulopoietic elements. An increased expression of MMP-9 but unchanged expression of TIMP-1 was observed by Immunohistochemistry (IHC). c-kit ligand level by Elisa was decreased in the BM supernatant. In addition, phospho c-kit (phospho site 568) was increased in the myeloid and erythroid precursors by IHC but phospho site 823 was unaltered, indicating the specificity of activation of the downstream pathway in response to c-kit ligand-receptor activation, possibly leading to expansion of progenitor cells. Interestingly, expression of CXCR4 at the protein level (flow cytometry and western blot) and RNA level (RT-PCR) was decreased (p<0.05) in the BM at the time of mobilization (day 14) as compared to the baseline. These findings indicate that long-acting HGFs, administered once per cycle of CT, are a potent stimulus for mobilization of myeloid, erythroid, and multipotential progenitors. Our findings also suggest that increased myelopiesis in the regenerating BM may be associated with increased protease activity such as MMP-9 that may result in the cleavage of c-kit leading to activation of c-kit receptors and expansion of progenitors, and the cleavage of CXCR-4 leading to release of progenitors from the BM anchorage and mobilization into PB.
The Novel Role of the Third Complement Component (C3) in Megakaryopoiesis: Implications for Pathogenesis of Reactive Thrombocytosis.
Authors: Wysoczynski Marcin , Reca Ryan , Kucia Magda , Ratajczak Janina , Ratajczak Mariusz Z.
Published: April 2020

An elevated platelet count is frequently observed in several clinical conditions (e.g., chronic inflammation, acute hemorrhage) that may lead to reactive thrombocytosis (RT). Recently we found that i) complement is activated/cleaved in the bone marrow with release of C3a and des-Arg C3a fragments that sensitize responses of hematopoietic cells to SDF-1 (Blood2003;101:3784), an alpha-chemokine that plays an important role in Meg maturation/proplatelet formation, and ii) C3 deficient mice display delayed recovery of platelet counts after sublethal irradiation (Leukemia 2004, in press). Thus, since levels of C3 cleavage products (C3a and des-Arg C3a) and SDF-1 are elevated in bone marrow during inflammation and acute hemorrhage, we hypothesized that they may play a role in the pathogenesis of RT by enhancing Meg maturation/platelet production. In support of this notion we found that i) the receptor for C3a (C3aR) is highly expressed and functional on CFU-Meg progenitors and maturating Megs, ii) C3a induces a calcium flux and actin polymerization in Megs and enhances chemotactic response of human CFU-Meg and maturing Megs to an SDF-1 gradient, and iii) in the presence of C3a + SDF-1, Megs secrete more VEGF and MMP-9, which play a role in proplatelet formation. These effects were not inhibited by the small molecular antagonist of C3aR, SB290157, suggesting the involvement of the C3a derivative, des-Arg-C3a, that binds another C3a receptor called C5L2. At the molecular level we observed that C3a and des-ArgC3 promote the incorporation of CXCR4 into membrane lipid rafts in hematopoietic cells, allowing for its better interaction with small GTP-ases (Rac/Rho). We conclude that C3a and des-Arg C3a modulate the responsiveness of Megs to SDF-1 and postulate that the novel crosstalk between C3aR, C5L2 and CXCR4-G-protein coupled receptors we identified here plays an important and previously unrecognized role in the final maturation of Megs and pro-platelet formation during stress. Thus our results shed more light on the pathogenesis of RT and suggest that inhibitors of both CXCR4 and C3 could find a potential novel clinical application as drugs controlling elevated platelet counts.
Randomized, Double-Blind, Placebo-Controlled Trial of Epoetin Alfa (Procrit) in Patients with Rectal and Gastric Cancer Undergoing Chemo-Radiotherapy (CT/RT) Followed by Surgery: Early Termination of the Trial Due to Increased Incidence of Thrombo-Embolic Events (TEE).
Authors: Vadhan-Raj Saroj , Skibber John M. , Crane Christopher , Bueso-Ramos Carlos E. , Rodriguez-Bigas Miguel A. , Feig Barry W. , Lin Edward H. , Ajani Jaffer A. , Collard Mindy , Johnson Marcella M. , Hamilton Stanley R. , JanJan Nora
Published: April 2020

Patients with resectable gastric and rectal cancer undergoing CT/RT followed by surgery are at increased risk for anemia, fatigue, and need for red cell transfusions. Anemia and tumor hypoxia may contribute to adverse treatment outcome. A multi-center, placebo-controlled trial of 184 patients was designed to determine if Procrit treatment can maintain Hb (≥13 gm/dL), reduce the need for PRBC Tx and improve treatment outcome. Procrit was administered weekly (40,000 units starting dose) for 16 wks during CT/RT (5 FU or Xeloda + 45 Gy over 5 weeks) followed by surgery. After 60 pts were randomized, the trial was terminated due to increased incidence of TEE. 59 of these pts (53 rectal and 6 gastric) received at least one dose of treatment; 31 in the placebo and 28 in the Procrit group. Baseline median Hb for the study group was 13 gm/dL. Seven (11.9%) pts experienced venous TEE; 1/31 (3.2%) pts treated with placebo compared with 6/28 (21.4%) pts treated with Procrit (p= 0.045). In exploratory analyses including the multivariate modeling, it was observed that the Procrit treatment was a predictor of TEE (p = 0.060), however this was not observed for Hb (p = 0.998). Using a mixed-effects model, Hb was significantly higher in pts treated with Procrit than pts treated with placebo (p = 0.0004) during CT/RT. In addition, Hb was significantly different across weeks (p < 0.0001). During the study period, there was also a significant treatment by time interaction (p = 0.0061) with Hb maintained/increased in Procrit pts while it steadily decreased in the placebo pts. Four (14.3%) pts treated with Procrit compared with 10 (32.3%) pts treated with placebo required red cell transfusions (p = 0.133). Platelets (PLTs) decreased significantly from baseline during CT/RT (at weeks 4) for the placebo pts and for the pts treated with Procrit who did not experience TEE (p < 0.0001), but there was a trend for an increase in PLT in pts who experienced TEE. The tumor response for rectal ca at MDACC site was similar between both treatment groups with 14/22 (63.6%) in each treatment group (p = 0.777). These findings indicate that treatment with Procrit may prevent anemia during CT/RT/surgery, however there is increased risk for TEE in this setting for prevention of anemia. Correlative analysis is ongoing on biomarkers and molecular markers on biopsy specimens to examine the markers predictive of pathological complete response, TEE, and the treatment outcome.
An Oral, Non-Petide, Small Molecule Thrombopoietin Receptor Agonist Increases Platelet Counts in Healthy Subjects.
Authors: Jenkins Julian , Nicholl Richard , Williams Daphne , Baidoo Charlotte , Phillips Jennifer , Deng Yanli , Kitchen Valerie , Erickson-Miller Connie
Published: April 2020

SB497115, is an orally bioavailable, small molecule thrombopoietin receptor agonist that induces differentiation and proliferation of megakaryocytes. In a randomized, single blind, placebo-controlled, parallel group, phase I study conducted in the UK in 72 healthy male subjects, SB497115 was administered as oral capsules once daily for 1 day and, after a 1 week washout, for 10 days at doses of 5 to 75 mg. Subjects were randomized into six groups of 12 subjects to receive either active or placebo medication in a ratio of 9:3. The study was conducted according to Good Clinical Practice and all subjects gave their written informed consent to participate in the study.

SB497115 was well tolerated in the study, there were no serious adverse events, no significant changes in laboratory or cardiovascular safety parameters and there was no observed relationship between the incidence or severity of adverse events and dose. Most adverse events were mild in intensity and self-limiting. SB497115 was shown to be orally bioavailable in humans with a linear pharmacokinetic profile suitable for a once daily oral medication. When administered at oral doses of 30mg and above for 10 days a dose dependent increase in the platelet count was observed, maximum platelet count was observed on days 14 to 16 following initiation of dosing. The dose dependent increase in platelet count is shown in the table below. On the basis of these safety, pharmacokinetic and pharmacodynamic data the oral thrombopoietin receptor agonist, SB497115, will be studied in phase II trials involving thrombocytopenic patients.

Preliminary Data: Mean Platelet Count (platelets/uL)

Oral Dose Baseline (Day 1) Maximum (Day 14 or 16) Change from Baseline Placebo 234000 255000 21000 5 mg 217000 249000 32000 10 mg 251000 291000 40000 20 mg 236000 279000 43000 30 mg 249000 323000 74000 50 mg 254000 356000 102000 75 mg 239000 357000 118000
Induction of Polyploidization in Leukemic Cell Lines and Primary Bone Marrow by Src Kinase Inhibitor SU6656.
Authors: Lannutti Brian J. , Minear Jennifer N. , Blake Noel , Drachman Jonathan G.
Published: April 2020

Megakaryocytes (MKs) undergo successive rounds of endomitosis during differentiation, resulting in polyploidy (typically, 16-64N). Previous studies have demonstrated that this occurs through an interruption of normal cell cycle progression during anaphase. However, the molecular mechanism(s) controlling this unique process is undefined. Our studies of Src family kinases demonstrate that Lyn and Fyn kinases are activated by thrombopoietin (TPO) and have an inhibitory effect on cellular proliferation. In fact, adding PP1 to murine bone marrow cells in culture resulted in a higher percentage of polyploid cells. In the present report, we examined the effect of SU6656 on TPO-induced growth and differentiation. SU6656 is reported to be more specific than previous inhibitors for the Src family of tyrosine kinases. Remarkably, when SU6656 (2.5 mM) was added to a megakaryocytic cell line, UT-7/TPO, the cells underwent dramatic increase in cell size and polyploidization. By 24 h, 8N cells were present. After 48 h a 16N population was identified by FACS, and continued growth in SU6656 resulted in higher ploidy states (32N and 64N). This was accompanied by cessation of cellular proliferation (i.e. cell number remained constant), increase in CD41 and CD61 expression, and was notable for the absence of apoptosis. Similarly, polyploidization was observed when SU6656 was added to expanded human bone marrow progenitors with partial MK differentiation. The ability to induce differentiation was also seen using bone marrow from two patients with myelodysplastic syndrome and thrombocytopenia, suggesting that SU6656 might be useful as a differentiation-inducing agent for MKs. Although SU6656 is clearly a potent inhibitor of the Src family kinases, we have undertaken studies to determine if an additional target can be identified. We found no affect on Jak2, STAT3, and STAT5 tyrosine phosphorylation. However, the activity of Aurora kinase B was inhibited in vitro by as little as 50 nM SU6656. We propose that SU6656 may be an important tool for understanding the molecular basis of MK endomitosis and may also have therapeutic potential for individuals with MDS, thrombocytopenia, or other disorders resulting from incomplete megakaryocyte maturation.
A Novel Strategy for Combining Immunotherapy with High Dose Chemotherapy and Auto-Transplantation in High Risk Multiple Myeloma.
Authors: Szmania Susann , Cottler-Fox Michele , Rosen Nancy , Viswamitra Sanjaya , Moreno Amberly , Pomtree Mindy , Barlogie Bart , Tricot Guido , van Rhee Frits
Published: April 2020

Although high dose chemotherapy and autologous transplantation (AT) can induce complete remission in approximately 50% of multiple myeloma (MM) patients, the majority eventually relapse. We hypothesize that a combination of AT and vaccination may sustain remissions by inducing specific cytotoxic T cells against chemotherapy resistant plasma cells. Vaccinations are typically given after AT when the immune system is very defective. We tested the novel strategy of priming anti-myeloma T-cells before AT and protecting the primed T-cells from high-dose chemotherapy by apheresis and cryopreservation of the primed cells. After high-dose chemotherapy induced cytoreduction, the primed T-cells were returned early after AT and preferentially expanded by a series of booster vaccinations (figure 1). The vaccines are monocyte derived-dendritic cells (DC) loaded with myeloma lysate (ML) and tracer antigen keyhole limpet hemocyanin (KLH), matured with trimeric CD40-ligand, administered intranodally, and followed by 5 days of low dose subcutaneous IL-2. Four patients with poor prognosis MM (characterized by abnormal metaphase cytogenetics) have been vaccinated and three are evaluable (the 4th patient is 60 days post AT2 and only 14 days post vaccine 8). ML specific immune responses were detected by delayed type hypersensitivity reactions and proliferation in 2/3 and by ELISPOT in 3/3 patients. Strong responses to KLH were observed in 3/3 patients. The pattern of response is a dramatic and rapid increase in ML specific T cell proliferation (figure 2) and IFN-g secretion after LP2 infusion plus booster vaccinations post AT, suggesting that the primed T-cells had been protected by collection and storage during AT. A high pre-existing ML specific antibody response was observed in 2/3 patients and a low antibody response pre-vaccination was clearly boosted in a third patient. The patient receiving the lowest DC dose (50 versus 115 and 139 x106) had a response to ML only detected by IFNg secretion assays, but is in near complete remission, 719 days post AT2, while disease progression was observed in the other two patients 496 and 626 days after AT. Our immune monitoring data suggest that the problem of applying vaccination in heavily treated patients may be overcome by early vaccination, collection and subsequent reinfusion of primed cells followed by repeated booster vaccination. Optimization of the vaccine regimen by increasing the frequency of vaccination or the use of highly immunogenic, well characterized tumor-associated antigens may further improve efficacy. We have an ongoing trial for high risk MM patients utilizing NY-ESO-1 and MAGE-A3 peptide vaccines based on a similar strategy to preserve immune competence.
Imatinib in the Treatment of CML Patients ≥ 65 Years Old in Late Chronic Phase: Results of a Phase II Study of the GIMEMA CML Working Party.
Authors: Bassi Simona , Castagnetti Fausto , Amabile Marilina , Giannini Barbara , Cilloni Daniela , Izzo Barbara , De Vivo Antonio , Luatti Simona , Testoni Nicoletta , Rege-Cambrin Giovanna , Iacobucci Ilaria , Gottardi Enrico , Pane Fabrizio , Salvatore Francesco , Dorigo Alessandra , Saglio Giuseppe , Martinelli Giovanni , Rosti Gianantonio , Baccarani Michele
Published: April 2020

Older age constitutes a poor prognostic variable in CML patients: the negative effect of age on long-term survival has been consistently observed with most effective therapeutic modalities, both drug therapies (busulfan, hydroxyurea and interferon) and allogeneic transplant. In particular, older patients treated with interferon experienced much more adverse events than younger ones. In part their poorer prognosis was probably due to poor treatment compliance and few older patients have been included in prospective studies of interferon. Actually, imatinib is the first-line treatment for CML: its efficacy is very high and it seems to be well tolerated across age groups. We performed a sub-analysis of the effects of age on response and tolerance within the phase II trial of the italian GIMEMA CML Working Party (serial n.CML/002/STI571), which included 284 late chronic phase patients, treated with imatinib (400 mg daily) after interferon failure. Following the WHO, who defines “old” a patient ≥ 65 years, we analyzed the safety and efficacy of imatinib by age group: 226/284 patients (80%) were < 65 years (extr. 17–64) and 58/284 (20%) were ≥ 65 years (extr. 65–85 yrs) old. No significant differences between the two age groups were present at enrollment. Table 1 shows the responses and incidence of hematologic and non-hematologic adverse events (AEs). As expected, older patients experienced more AEs, both hematologic and non-hematologic. In part this significantly poorer tolerance probably justifies the lower response rates, both hematologic and cytogenetic. However, the overall survival was not different between the two age groups: with a median observation time of 33 months, the overall survival curves were superimposed (91%). Moreover, the rate of progression to accelerated/blastic phase was the same (10%). This study demonstrates that imatinib is greatly effective in older CML patients in late chronic phase (53% of MCR and 36% of CCR) and allows a good long-term survival. It is conceivable that treating old CML patients at the onset of the disease, as already shown by Jorge Cortes et al (Cancer2003;98:1105), could translate into response rates as the ones of younger patients.

Responses and adverse events

≥ 65 (n. 58) ≤ 65 (n226) p value Complete Hematologic response 91% 99% 0,001 Major Cytogenetic Response 53% 74% 0,003 Complete Cytogenetic Response 36% 57% 0,001 Grade III Hematologic AEs 72% 50% 0,002 Grade III+IV Hematologic AEs 86% 60% 0,0001 Grade II non Hematologic AEs 83% 68% 0,0001 Grade III + IV non Hematologic AEs 29% 10% 0,0001
High Sensitive Detection of Imatinib-Resistance Associated Mutations Based on ARMS-PCR.
Authors: Gruber Franz X.E. , Soevershaev Mikchail , Olsen Marita , Skogen Bjoern
Published: April 2020

Background: Point mutations in the Abl kinase domain are associated with resistance against imatinib. Strategies to overcome resistance include dose escalation, combination treatment using imatinib with conventional or other developmental agents or, in the future, imatinib may be replaced by other tyrosine kinase inhibitors which work effectively against mutated clones. Mutational profiling of the BCR-ABL kinase domain will in this scenario become an important analysis as a supplement to BCR-ABL quantitation and may provide the rational basis for therapy, once resistance is diagnosed. Our group reported recently a sensitive, single step PCR assay for quantitation of mutated clones based on the ARMS principle.

Aim: We describe an optimized, two step analysis for high sensitivity screening of mutated clones associated with resistance against imatinib targeting all P-Loop mutations, the T315I and M351T.

Methods: In a first conventional PCR-reaction a cDNA-region spanning the BCR-ABL breakpoint is amplified resulting in an isolation of the BCR-ABL kinase domain for further analysis. An aliquot is then analysed in a second PCR step, conducted on the real time PCR Taqman platform. Selectivity for the mutated clone is conferred by the amplification refractoriness of non complementary primer 3′-ends (ARMS principle). By introducing potent nucleotide-mismatches in position n-2, selectivity of the assay could be further increased. Even in the P-Loop region, which is known to be a difficult PCR template, misannealing could be reduced to an acceptable level.

Results: Assays targeting all P-Loop mutations inclusive the T315I and M351T were tested by analysis of patient samples diluted in normal cDNA and non-mutated BCR-ABL and plasmid dilutions, containing the targeted mutation in a background of wildtype plasmids. Generally a 1:1000 dilution of mutated templates could be detected (sensitivity 0.1%). For some mutations even higher sensitivity could be achieved (0.01%). The level of sensitivity is generally higher than reported for other methods described before. The first PCR step can be conducted in parallel to other PCR-based detection strategies. The second step can be run simultanously to Taqman based BCR-ABL quantitation. This makes the described assay the ideal supplement to general mutation detection approaches like D-HPLC or sequencing strategies. Compared to the single step assay we desribed before, the two step approach increases sensitivity with one or two log factors.

Conclusion: The described assay may be suitable for highly sensitive detection of mutated clones in resistant CML patients as a supplement to less sensitive general screening approaches and BCR-ABL quantitation.
Gene Expression Profiling in Quiescent Stem Cells from Normal and Chronic Myeloid Leukaemia Patients.
Authors: Graham Susan M. , Graham Gerry J. , Holyoake Tessa L.
Published: April 2020

Earlier studies have shown that Ph+ quiescent cells exist in chronic myeloid leukaemia (CML) (Blood (1999)94:2056) and we have previously shown that these cells are primitive in that they express the stem cell marker CD34. We have also shown that quiescent CML stem cells are insensitive to the effects of imatinib (IM Novartis Pharma) (Blood (2002) 99:319) and may present a possible source for relapse. This quiescent population therefore represents a potentially significant clinical problem and thus studies aimed at developing methods for eradicating this population are timely. In an effort to identify molecular markers of this population that may allow it to be specifically targeted during therapy, we have set out to investigate the transcriptional differences between quiescent and cycling stem cells. To this end, we have used specific stem cell enrichment and sorting protocols. Leukapheresis products from CML patients (N=5) in chronic phase at diagnosis and mobilised peripheral blood from allogeneic donors (N=3), were selected for CD34+ cells. Hoechst 33342 and Pyronin Y were used to discriminate the quiescent (G0) cells identified as Hoechstlo/Pyroninlo from the cycling cells. In combination with propidium iodide for dead cell exclusion we were able to sort 4–9x105 viable, quiescent stem cells and 4–11x106 cycling cells, which were processed for microarrays. Affymetrix gene chips (U133A) were used for the analysis and the data obtained was analysed using GeneSpring.

Number of Genes Changed in Each Comparison

3 Fold 4 Fold 5 Fold CML G0 V CML Div 37 21 10 Norm G0 V Norm Div 188 92 47 CML G0 V Norm G0 168 85 49 CML Div V Norm Div 49 27 8

Initial analysis indicates that the greatest differences in gene expression are between the normal quiescent cells (G0) and normal dividing cells (Div) and between the normal quiescent cells and CML quiescent cells. A large percentage of the genes differentially expressed between the quiescent and cycling normal cells encode regulators of the cell cycle confirming the success of the sorting strategy for quiescent and cycling cells

A selection of Genes Up-Regulated in Normal Cycling Cells Compared to G0

Gene Fold Up-regulation PCNA 3 CDC2 8 CCNB2 5 CCN1 3.5 CDC20 6 CDC25A 3.5 MCM5 3

In addition, many of the genes identified in our analysis are consistent with other published expression profiles for haemopoietic cells. Curiously, we have identified unanticipated changes in expression of cell cycle genes in the CML quiescent cells, which merit further investigation. We have also identified a number of unexpected genes as being more than 5 fold changed in the quiescent cells compared to dividing cells for both normal and CML samples. Specifically, there is a large cohort of genes preferentially expressed in quiescent normal or CML cells, which encode members of the chemokine family of proteins. Work is ongoing to establish the relevance, if any, of these genes to stem cell quiescence.
The Abl Carboxyl Terminal Region Is Required for the Induction of Acute Lymphoblastic Leukemia by V-Abl but Not for the Induction of Myeloproliferative Disease by BCR/ABL or V-Abl.
Authors: Ji Yongli , Gross Alec W. , Zhang Xiaowu , Ren Ruibao
Published: April 2020

The BCR/ABL oncogene, generated by the t(9;22)(q34;q11) chromosomal translocations known as the Philadelphia chromosome (Ph), is associated with virtually all human chronic myelogenous leukemia (CML), and is also associated with 20% of the adult and 5% of the pediatric cases of acute lymphoblastic leukemia (ALL). v-Abl, the oncogene of the Abelson murine leukemia virus (A-MuLV) originally found to cause pre-B cell leukemia/lymphoma when introduced into newborn mice by intraperitoneal or intravenous injection, was created by a recombination event that fused viral gag sequences to c-abl sequences, deleting the Abl SH3 domain and accumulating point mutations in the process. Using a mouse bone marrow transduction and transplantation model, we have previously shown that expression of BCR/ABL in mouse bone marrow cells rapidly (in approximately 3 weeks) induced a myeloproliferative disease (MPD) resembling human CML, while v-Abl rapidly (in approximately 4 weeks) induced B-ALL along with a minor expansion of v-Abl-infected myeloid cells. It has long been shown that the Abl carboxyl terminal region is required for the lymphoid cell transformation by v-Abl both in vivo and in vitro. We decided to investigate the role of the Abl carboxyl terminal region in BCR/ABL- and v-Abl-leukemogenesis using the mouse bone marrow transduction and transplantation model. We found that the Abl carboxyl terminal region is required for v-Abl-induced B-ALL, as expected. But, interestingly, v-Abl without C-terminus (v-Abl/p160/ΔC) induced a MPD in mice, similar to that induced by BCR/ABL, with a relative long latency (approximately 3 months in average). On the other hand, BCR/ABLΔC still induced MPD efficiently, similar as wild type BCR/ABL. These results demonstrated the Abl carboxyl terminal region is required for the induction of acute lymphoblastic leukemia by v-Abl but not for the induction of MPD by BCR/ABL or v-Abl. The results also suggested that v-Abl has a capacity to induce MPD, albeit much weaker than that of BCR/ABL and much weaker than v-Abl’s capacity to induce B-ALL. The role of the ABL carboxyl terminal region in BCR/ABL-induced B-ALL and the mechanism by which the Abl carboxyl terminal region contributes to the lineage-specific transformation are under investigation. Such research will shed lights into the mechanism of lineage-specific leukemogenesis and help to identify therapeutic targets for lymphoid malignancies.
Reduced Tissue Factor Expression in Rat Prostate Tumor Cells Limits Tumor Development in Vivo.
Authors: Song Hui , Liang Zhong , Wolter William , Suckow Mark A. , Rosen Elliot D.
Published: April 2020

Many tumors express procoagulant activities that contribute to the hemostatic complications associated with cancer. Tumor coagulant activities include tissue factor (TF) and cancer procoagulant (CP), a cysteine protease that activates Factor X. In current models of hemostasis, TF is critical in the initiation of coagulation following vascular injury as TF expressed on cells within the vessel wall complexes with blood-borne coagulation Factor VIIa to activate FX and FIX. In addition, TF transported to a growing thrombus by blood-borne microparticles binds FVIIa and contributes to thrombus propagation and stabilization. Recently, TF as well as other coagulation factors have been implicated in a variety of non-hemostatic processes including inflammation, angiogenesis, vascular development and cancer. To study the role of TF in tumor development we down-regulated TF expression in a prostate tumor cell line developed from the Lobund-Wistar (LW) rat. The LW rat combines histologic features of prostate cancer with the clinical features resembling clinical human disease; androgen-modulated growth, age-dependent spontaneous onset, and metastatic potential. Autochthonous tumors develop spontaneously or can be induced by treatment with MNU and testosterone. In addition, a cloned cell line from a spontaneous LW prostate tumor, PA-3, provides a transplantable tumor model. PA-3 cells were transfected with a series of plasmids expressing hairpin siRNAs designed to interfere with TF expression. The plasmids contained a U6-hairpin siRNA expression cassette, a neoR gene and EGFP. Cloned stable transformants of PA-3 cells expressing a siRNA corresponding to positions 260–278 of rat TF mRNA, PA-3[797], reliably reduced TF expression by 65% compared to control clonal PA-3 transformants, PA-3[776], expressing neoR and EGFP but no siRNA. To test the consequences of reduced TF on tumor development in vivo, 4 independently isolated PA-3[797] cell lines and 4 independently isolated PA-3[776] control lines were injected subcutaneously into LW rats (3 rats per cell line). Ten of the 12 rats injected with control cells developed detectable tumors 4 weeks post transplantation (tumor mass = 2.2g +/− 1.8g). In contrast, only 1 of the 12 rats receiving the low TF expressing PA-3[797] cells developed a small tumor (0.07 gm) suggesting TF expression is required for tumorigenesis.
No Fibrinolytic Benefit of Adjunctive Anticoagulation during Arterial Thrombolysis Induced by tPA Combined with TAFIa Inhibitor in a Rabbit Arterial Thrombosis Model.
Authors: Klement Petr , Liao Peng , Chen Frank , Weitz Jeffrey I.
Published: April 2020

Introduction: In a previous study, we reported that inhibition of activated TAFI (TAFIa) with potato carboxypeptidase inhibitor (PCI) enhanced tPA-induced clot lysis in a rabbit aortic thrombosis model. Building on these results, this study used the same model to determine whether the addition of anticoagulants to PCI would further enhance tPA-induced clot lysis.

Methods: After balloon injury and creation of a critical stenosis, a radiolabelled thrombus of known mass was generated in the distal aorta. Rabbits were then given tPA (0.25 mg/kg bolus followed by 0.25 mg/kg infusion over 60 min) in conjunction with PCI (30 μg/kg bolus followed by 30 μg/kg/hr) or saline (SAL, 1ml/kg bolus followed by 1 ml/kg/hr). Rabbits treated with tPA/PCI were also supplemented with hirudin (HIR, 0.2 mg/kg bolus followed by 0.2 mg/kg/hr) or heparin (H, 50 U/kg bolus followed by 50 U/kg/hr) or pentasaccharide (PENTA,1 mg/kg iv bolus) or SAL. To assess safety, standardized ear incisions were made 15 min after starting tPA and cumulative blood loss was measured over a 30 min period. This time point was chosen because both tPA and anticoagulant levels are at their peak. To assess efficacy, clot lysis was measured at 90 min by subtracting the radioactivity of the residual thrombus from that of the initial thrombus and expresing this value as a percentage of initial radioactivity.

Results: As illustrated in the table, PCI increases tPA-induced clot lysis from 71.3 to 89.2 % with a minimal effect on bleeding. The addition of anticoagulants produced no further increase in clot lysis, but all 3 of the anticoagulants tested increased blood loss.

Conclusions: These preliminary data support the concept that PCI enhances tPA-induced clot lysis to an extent where concomitant administration of anticoagulants provides no additional benefit and compromises safety.

Thrombolytic efficacy/safety profile of various treatments.

Treatment Clot lysis (%) Blood loss (μL) Values are expressed in Mean ± SEM, four rabbits per treatment group SAL 19.7 ± 2.2 15.6 ± 5.3 tPA 71.3 ± 7.1 66.8 ± 14.0 tPA + PCI 89.2 ± 3.3 104.2 ± 42.7 tPA + PCI + H 90.3 ± 5.5 134.0 ± 60.9 tPA + PCI + PENTA 90.6 ± 3.2 215.6 ± 41.5 tPA + PCI + HIR 92.0 ± 5.7 680.3 ± 536.6
Retrospective Comparison of Using or Not Using Donor Lymphocyte Transfusion in the Treatment of Hematological Relapse after Allogeneic Stem Cell Transplantation in 489 Adults with Acute Myeloid Leukemia.
Authors: Schmid Christoph , Labopin Myriam , Finke Juergen , Ehninger Gerhard , Ringden Olle , Niederwieser Dietger , Sakellari Ioanna , Urbano-Ispizua Alvaro , Ruutu Tapani , Gurman Gunham , Clarke Richard E. , Gorin Norbert , Polge Emmanuelle , Fouillard Loic , Frassoni Francesco , Kolb Hans-Jochem ,   Vanderson Rocha for the EMBT ALWP
Published: April 2020

Relapsed AML after allogeneic SCT has a poor prognosis. So far, no standard therapy could be defined. Donor lymphocyte transfusion (DLT) has been effective in a minority, however, no data is available to identify patients who will benefit from the procedure. Neither, the outcome of patients treated with or without DLT have been compared. We retrospectively evaluated overall survival (OS) of 489 adults with de novo AML in hematological relapse after SCT, receiving DLT (n=190) or not (n=299). DLT and noDLTgroups were well balanced in terms of patient age (median:37y in both groups), donor age, cytogenetics (good:5vs7%, intermediate:83vs79%, poor:12%vs14%), WBC at diagnosis, donor type (geno-id:71vs72%, MUD:18% both, mismatched:11vs10%), status at transplantation (CR1:38vs41%, CR2:13vs15%, advanced:49vs44%), conditioning, source of stem cells, and time from transplant to relapse (5vs4.5 months). However, DLT patients had a median of 39% BM blasts, as compared to 54% for the noDLT group (p=0.03). Follow-up was 32 and 30 months. Within the DLT group, chemotherapy was additionally given in 130 cases. Nevertheless, only 33% of patients received DLT in CR or aplasia, 67% had measurable disease. AGvHD developed in 41% of patients following DLT. CR and PR were achieved in 31.1% and 4.8% of DLT patients. In a multivariate analysis, younger patient age (<36 years) (HR=1.53,p=0.02) and a longer interval (> 5 months) from transplant to relapse (HR=7.74,p=0.002) were associated with better OS after DLT. When comparing the outcome of patients receiving or not DLT, OS at 2 years was 10±1% for the entire cohort, 18±3% for DLT and 6±1% for noDLT (p<.0001). In a multivariate analysis, use of DLT (HR=2.11,p<0.0001); recipient’s age<36 y (HR=1.69, p<0.001); longer interval (>5 months) from transplant to relapse (HR=2.40, p<0.0001) and number of BM blasts (<48%) at relapse (HR=1.56,p=0.002) were favorable for OS. In this retrospective analysis the results suggest that DLT may be of advantage in the treatment of AML relapse post transplant, at least in younger patients with a longer post transplant remission and relapsing with smaller amounts of blasts in BM. However, patients receiving DLT might represent a positive selection among all relapsed cases, since a considerable number from the noDLT cohort had died too early to proceed to DLT. An intetion-to-treat analysis and further prospective studies should investigate the role of DLT and other approaches, such as second reduced intensity SCT.
Targeted Replacement of the Murine Thrombomodulin Gene with Human Thrombomodulin Coding Sequence Results in Decreased Protein C Activation and Enhanced Thrombotic Response to Photochemical Injury.
Authors: Raife Thomas J. , Erger Rochelle A. , Wilson Katina M. , Fernandez Jose A. , Alternburg Jennifer , Kim Hyung-Suk , Griffin John H. , Maeda Nobuyo , Lentz Steven R.
Published: April 2020

Thrombomodulin is an endothelial transmembrane glycoprotein that regulates hemostasis and inflammation. Deletion of the thrombomodulin gene in mice results in embryonic lethality. To examine the function of human thrombomodulin in a murine model, we used gene targeting to replace the entire coding sequence of the intronless murine thrombomodulin gene with a human thrombomodulin cDNA. Mice derived from successfully targeted 129/SvEv embryonic stem cells were maintained on either a 129/SvEv background or a mixed (129/B6) background. Mice homozygous for the modified allele (hthm/hthm) were viable, and the total amount of thrombomodulin mRNA (measured by real time PCR) did not differ between hthm/hthm and wild-type (+/+) mice (p>0.05). In heterozygous (hthm/+) mice, the ratio of human to murine thrombomodulin mRNA was similar in lung, heart, and kidney, and expression of murine thrombomodulin transcripts was 50% of that in wild-type (+/+) mice. Expression of human thrombomodulin protein was detected in endothelium by immunohistochemistry and in lung lysates by enzyme linked immunoassay. Thrombomodulin cofactor activity, measured in a protein C activation assay with human protein C and thrombin, was decreased in the aorta of hthm/hthm mice (1.1±0.5 nmol/mm) compared with +/+ mice (3.6±0.3 nmol/mm; p<0.001). Thrombomodulin activity also was lower in lung lysates of hthm/hthm mice (0.7±0.1 nmol/mg protein) than in +/+ mice (2.5±0.5 nmol/mg protein; p<0.01). The relative difference in lung thrombomodulin activity between hthm/hthm and +/+ mice was even greater when measured with murine protein C and thrombin (0.7±0.6 vs. 5.2±0.9 nmol/mg protein; p<0.001). Hthm/+ mice had intermediate levels of cofactor activity with either human or murine reagents. Time to thrombotic occlusion of the carotid artery after photochemical injury (rose bengal and green laser) was shorter in hthm/hthm mice than in +/+ mice (10.4±3.0 vs. 34.6±10.6 minutes; p<0.04). We conclude that the humanized thrombomodulin gene is expressed normally in mice but produces a phenotype with markedly diminished anticoagulant activity. The increased susceptibility to experimental thrombosis in hthm/hthm mice suggests that this animal model may be useful for investigation of human thrombomodulin function in vivo.
Development of a Human Acute Myeloid Leukaemia Screening Panel and Identification of Novel Gene Mutations.
Authors: Fitzgibbon Jude , Smith Matthew , Arch Rachael , Smith Lan-Lan , Bainton Nigel , Neat Michael , Bonnet Dominique , Cavenagh Jamie , Lister T. Andrew
Published: April 2020

Mutation studies in Acute Myeloid Leukaemia (AML) are complicated by the existence of distinct morphological and cytogenetic subtypes; consequently although mutations in any one gene may occur in only 5% of AML, the frequency of mutation may differ both between the different FAB-types and cytogenetic risk groups studied. It is important therefore not only to validate the screening methodology used but also the suitability of the patient panel tested. A screening panel was developed allowing detection of novel recurring gene mutations within samples derived from patients with AML. Mutation analysis of 6 previously described genes (RUNX1, FLT3, KIT, CEBPA, PTPN11, NRAS) and 2 candidate genes (CCND3, FES) were carried out in a cohort of 175 AML samples representing all FAB types (except M3) and cytogenetic risk groups using a combination of SSCP, DHPLC and sequence analysis. One hundred and fifteen mutations were identified in 97 (55%) patients comprising 81 patients (46%) with one mutation, 14 patients (8%) with 2 mutations, and 2 patients (1%) with 3 mutations. Fifty-five out of 88 (63%) patients with normal karyotype AML had at least one mutation. There was was a weak negative association between FLT3 ITD and loop mutation (p = 0.095), a positive association between KIT mutation and favourable risk cytogenetics (p = 0.001), CEBPA mutation and intermediate risk/normal cytogenetics (p = 0.045) and PTPN11 mutation and poor risk disease (p = 0.001). The frequency of individual gene mutation was in accordance with previously published studies. Three novel mutations of FLT3 (Y589D, D839G, Y842H) were detected in 4 patients that would have been overlooked by conventional gel electrophoresis techniques. A single in frame 51bp deletion of nucleotide 939 – 990, resulting in a deletion of 17 amino acids at the carboxyl-terminus of the cyclin D3 protein was identified in a single patient. Overall, both the pattern and mutation frequencies reported in this cohort are similar to those in the literature supporting its further use as an investigational tool in the evaluation of candidate genes in the genesis of myeloid malignancy.