bluuebunny/crossref_metadata_2025 huggingface dataset:
P53 and p21waf1— Expression by Immunohistochemistry in Diffuse Large B-Cell Lymphoma Has a Strong and Independent Impact on Survival of Patients with Germinal Center Phenotype.
Authors: Visco Carlo , Canal Fabio , Andreoli Annalisa , Parolini Claudia , Lestani Maurizio , Ambrosetti Achille , Krampera Mauro , Pizzolo Giovanni , Chilosi Marco
Published: October 2019

Over-expression of P53-related protein detected by immunohistochemistry (IHC) and absence of p21waf1— expression, the main downstream target following P53 activation, are useful surrogate markers in identifying diffuse large B-cell lymphoma (DLBCL) patients with mutated P53-gene. Following recent studies on gene expression, tissue micro-array technology has been shown to represent a reliable technique in discriminating DLBCL with germinal center B-cell like (GC), or post- germinal center (non-GC) origin.

We analyzed 80 consecutive patients with de-novo DLBCL, homogeneously treated with cure-intent (CHOP-like regimens +/− Rituximab) in a single Institution, for expression of p53/p21waf1—, with regard to the expression of GC (CD10+, bcl-6+/mum-1−/CD138−), and post-germinal center (CD10−, mum-1+, CD138+) IHC markers. Cases with 50% or more p53-positive neoplastic cells were defined as p53+++. All patients were provided with complete clinical information. Median age of our patients was 50 years, AAS was III or IV in half of patients, IPI was high or int/high in 36%. Median follow-up for survivors was 77 months. A p53+++/p21waf1— phenotype (corresponding to loss of function of the P53-gene) was detected in 19 of 80 patients (23%), while a GC phenotype characterized 42 patients (52%). Patients with P53+++/p21waf1— phenotype were more frequently resistant to induction therapies (p<0.0001) and had a significantly lower 5-year Progression Free Survival (PFS, 27% vs 48%, p=0.01). Patients with GC phenotype were associated with a better 5-year PFS than non-GC patients (57% vs 26%, p=0.02). When analysis was restricted to the 42 patients with GC phenotype, p53+++/p21waf1— phenotype could discriminate 28% of patients with PFS of 26% vs 68% of patients with GC pattern and other p53/p21waf1— phenotypes (p=0.005). In contrast, the outcome of patients with a non-GC phenotype was not modified by p53/ p21waf1— expression. At multivariate analysis, p53+++/p21waf1— phenotype, non-GC phenotype, and high IPI resulted the strongest independent factors in worsening PFS of our patients. Combining such adverse prognostic factors we could identify 60% of patients with an extremely poor prognosis (5-year PFS of 28%).

Our results strongly point to the usefulness of a combined molecular and clinical approach for prognostic considerations in DLBCL, and should be validated in larger series.
ReSETting PP2A Tumor Suppressor Activity Overcomes BCR/ABL Leukemogenic Potential in Blast Crisis CML.
Authors: Neviani Paolo , Santhanam Ramasamy , Trotta Rossana , Notari Mario , Blaser Bradley W. , Chang Ji-Suk , Liu Shujun , Mao Hsiaoyin , Oaks Joshua J. , Roy Denis C. , Valtieri Mauro , Bruner-Klisovic Rebecca , Caligiuri Michael A. , Bloomfield Clara D. , Marcucci Guido , Perrotti Danilo
Published: October 2019

A tight control of kinase and phosphatase activity is fundamental for normal cell growth, survival and differentiation. The deregulated kinase activity of the BCR/ABL oncoprotein is responsible for the emergence and maintenance of chronic myelogenous leukemia (CML). By contrast, PP2A, a serine-threonine phosphatase involved in the regulation of many cellular functions, was found genetically inactivated in many types of cancer. We show here that, in BCR/ABL-transformed cells and CD34+ CML blast crisis progenitors, the phosphatase activity of the tumor suppressor PP2A is inhibited by the physiological PP2A-inhibitor SET whose expression is enhanced by BCR/ABL and increased in blast crisis CML. In imatinib-sensitive and -resistant (T315I included) BCR/ABL+ cell lines and in CD34+ CML blast crisis cells, molecular and/or pharmacological activation of PP2A leads to dephosphorylation of important regulators of proliferation and survival of CML progenitors, suppresses BCR/ABL kinase activity and promotes BCR/ABL proteasome degradation via a mechanism that requires the SHP-1 tyrosine phosphatase activity. Furthermore, PP2A activation achieved by shRNA-mediated SET knock-down or PP2Ac overexpression or treatment with the PP2A activator forskolin results in growth suppression, enhanced apoptosis, restored differentiation, impaired clonogenic potential and decreased in vivo leukemogenesis of wild type and T315I BCR/ABL-transformed myeloid cells. Thus, functional inactivation of PP2A phosphatase activity is essential for BCR/ABL leukemogenesis and, perhaps, required for transition of CML into blast crisis.
A New Abl Kinase Inhibitor (AMN107) Has In Vitro Activity on Chronic Myeloid Leukaemia (CML) Ph+ Cells Resistant to Imatinib.
Authors: Martinelli Giovanni , Martelli Alberto M. , Grafone Tiziana , Mantovani Irina , Cappellini Alessandra , Tazzari Pier Luigi , Ottaviani Emanuela , Soverini Simona , Amabile Marilina , Renzulli Matteo , Terragna Carolina , Colarossi Sabrina , Iacobucci Ilaria , Poerio Angela , Baccarani Michele
Published: October 2019

Imatinib mesylate (Novartis Pharma), an inhibitor of the bcr/abl tyrosine kinase, has rapidly become the first-line therapy for CML. Imatinib has proved remarkably effective at reducing the number of leukaemia cells in individual CML patients and promises to prolong life substantially in comparison with earlier treatments. However, in patients in advanced phases of the disease, the development of resistance to this drug is a frequent setback. Therefore, new inhibitors of bcr/abl are needed. Very recently, a new bcr/abl inhibitor, AMN107 (Novartis Pharma), has been developed. We have tested AMN107 on human leukaemia cell lines and on blasts isolated from imatinib-resistant CML patients. After a 24 h incubation, AMN107 (10 nM) blocked K562 cells in the G1 phase of the cell cycle. To obtain the same effect with imatinib, a 200 nM concentration was required. AMN107 had no affect on cell cycle progression of bcr/abl-negative cell lines such as HL60 and NB4, even if the concentration was raised to 500 nM. After 48 h incubation, AMN107 (10 nM) was capable of inducing a massive apoptosis of K562 cells whereas, once again, 200 nM imatinib was required to obtain the same effect.

Western blot analysis with phosphospecific antibodies revealed that in K562 cells AMN107 (50 nM) markedly down-regulated autophosphorylation of bcr/abl Tyr177 and Tyr412, whereas autophosphorylation of Thr735 was unaffected. In contrast, imatinib even if used at 200 nM, did not diminish phosphorylation of either bcr/abl Tyr177 or Tyr412. This finding seems particularly important because recent evidence has demonstrated that the signalling pathway emanating from Tyr177 plays a major role in the pathogenesis of CML. Indeed, phosphorylated Tyr177 forms a high-affinity binding site for the SH2 domain of the adapter Grb2. The main effectors of Grb2 are Sos and Ras, however Grb2 also recruits the scaffolding adapter protein Gab2 to bcr/abl via a Grb2-Gab2 complex, which results in activation of phosphoinositide 3-kinase (PI3K)/Akt and Erk signalling networks. Consistently, we found by immunoprecipitation decreased levels of bcr/abl-associated Gab2, Grab2, and p85 regulatory subunit of PI3K in AMN107-treated cells. AMN107 treatment of K562 cells also caused a reduction of STAT5, cCBL, CRKL, and Akt phosphorylation levels, as well as Bcl-XL expression. AMN107 (5 μM for 24h) significantly increased the apoptosis rate of CML blasts isolated from patients resistant to imatinib. Therefore, AMN107 might represent a new bcr/abl selective inhibitor useful for overcoming imatinib resistance.
FOXO Transcription Factors Are Negatively Regulated by p38 Map Kinases Downstream of FLT3 Receptor Signaling.
Authors: Scheijen Blanca , Ngo Hai T. , Griffin James D.
Published: October 2019

The phosphoinositide 3-kinase (PI3K)-AKT/PKB signal transduction cascade is an evolutionarily conserved pathway regulating the FOXO subclass of Forkhead transcription factors (FOXO1, FOXO3a and FOXO4) downstream of insulin, cytokine and growth factor receptors. When PI3K phospholipid kinase activity is low, FOXO proteins localize to the nucleus where they induce expression of genes that promote programmed cell death (FasL, Bim, TRAIL), inhibit cell cycle progression (p27KIP1, Rb related family member p130), facilitate DNA repair (GADD45) and protect against oxidative damage (MnSOD). Following receptor stimulation, increased levels of PtdIns(3,4,5)P3 are generated at the plasma membrane resulting in PDK1-mediated activation of the serine/threonine kinase AKT/PKB. Subsequent phosphorylation of FOXO proteins by AKT/PKB results in their active sequestration in the cytoplasm through binding to 14-3-3 proteins, thereby blocking their action as transcription factors. Recently, it has become evident that FOXO proteins can also be regulated through other signaling pathways. One example is phosphorylation and activation of FOXO4 by stress kinases JNK/SAPK in response to oxidative stress (Essers et al., 2004; EMBO J. 23:4802–12).

Here, we report that p38 mitogen activated protein kinases (MAPK) negatively regulate FOXO transcription factors downstream of FLT3 receptor tyrosine kinase signaling. Stimulation of human wild-type FLT3 receptor with FLT3 ligand, or expression of constitutively active mutants FLT3-ITD or FLT3-D835Y in Ba/F3 cells resulted in activation of p38 MAPK and phosphorylation of its known substrate ATF2 on threonine residue 71 (Thr71). Transient luciferase reporter assays showed that expression of a constitutively active MKK3 or MKK6, upstream activators of p38 MAPK, inhibited FOXO-induced gene transcription. The p38 MAPK inhibitors SB202190 and SB203580 as well as dominant-negative p38 MAPK blocked FLT3-mediated inhibition of FOXO transcription activation, suggesting p38 MAPK is both necessary and sufficient for FOXO regulation. In vitro kinase assays demonstrated that FOXO proteins are directly phosphorylated by p38 MAPK, although different p38 MAPK family members (p38α, p38β 2, p38γ and p38δ) displayed distinct FOXO protein substrate specificity. In conclusion, our data demonstrate that FOXO proteins are novel substrates of p38 MAP kinases and activation of p38 MAPK through FLT3 receptor signaling represents an important pathway to attenuate FOXO target gene regulation.
Directed Donor Cord Blood Transplantation for Hematological Disorders.
Authors: Walters Mark C. , Quirolo Lynn , Edwards Sandie , Lee Joanna , Robertson Shanda , Saba Julie D. , Trachtenberg Elizabeth , Quirolo Keith C. , Lubin Bert
Published: October 2019

The Sibling Donor Cord Blood Program was initiated in 1998 as a resource to collect, characterize, and to release for transplantation cord blood units (CBU) from families affected by malignant and non-malignant disorders. Currently, 1686 CBUs from 1587 families have been collected among referrals from all 50 US States. The categories of participation include malignant disorders (50%), sickle cell disease (29%), thalassemia (6%), and other hereditary or rare hematological conditions (15%). The mean cell volume collected was 102 ml (range, 31–284) with a mean total nucleated cell count (TNC) of 9.5 x 108 (range, 0.6–53.6) and mean CD 34+ cell count of 3.6 x 106 (range, 0.1–88.1). The post-thaw viability of CBU released for transplantation was 94.4 % (SD ±8.7%) and only 4.4% of CBUs were not processed due to having inadequate volume.

To date, 54 children have been treated by sibling donor cord blood transplantation (CBT), 38 using the cord blood unit as the sole source of stem cells. There was a very high rate of CBU utilization, particularly among thalassemia families where 18 of 105 (17%) of CBUs collected have been released for CBT. CBT recipients had hematological malignancies (N=20), thalassemia major (N=18), sickle cell anemia (N=8) or other non-malignant disorders (N=8), and all but 6 received HLA-identical allografts. The median total nucleated (TNC) and CD34+ cell dose was 3.1 x 107 TNC/kg and 0.7 x 105/kg recipient weight, respectively. The median time to ANC >500 and platelet >20,000/mm3 was 22 and 45 days, respectively. One of 52 (2%) evaluable patients had graft failure accompanied by autologous reconstitution. With a median follow-up of 9.6 (range, 0.1 – 94) months, 45 of 54 (83%) patients survive, and 9 patients (17%) died of relapse (N=6) or transplant-related causes (N=3) after CBT. Among the hemoglobinopathy patients, 23 of 26 (88%) survive, and 22 (85%) survive disease-free. Overall, the Kaplan-Meier probabilities of survival and event-free survival after sibling CBT are 79% and 72%, respectively.

These results confirm the feasibility of remote site, directed donor cord blood collection and subsequent transplantation for hematological disorders. The ability to combine cord blood collections with a marrow harvest from the sibling donor effectively reduced the incidence of graft rejection when CBU cell doses were judged to be insufficient. Transplantation of sibling CBUs in lieu of bone marrow may be particularly advantageous when there is urgency for transplantation and in non-malignant disorders where graft-versus-host disease, in particular, has a negative impact upon outcome.
Outcome and Immune Reconstitution Following T Cell Depleted Nonmyeloablative Allogeneic Transplantation Using Matched Donors.
Authors: Rizzieri David A. , Koh Liang Piu , Long Gwynn D. , Gasparetto Cristina , Gong Jerald Z. , Lagoo Anand S. , Niedzwiecki Donna , Sullivan Keith M. , Chute John , Horwitz Mitchell , Ashley Morris , Chao Nelson J.
Published: October 2019

To minimize toxicity and monitor immune recovery without interference of long term use of immunosuppressive agents, we have investigated T-cell depleted, nonmyeloablative allogeneic therapy using matched family member donors.

Methods: Seventy five patients who were not candidates for ablative therapy due to age or comorbid diseases received fludarabine 30mg/sq m and cyclophosphamide 500mg/sq m IV qd x 4 with alemtuzumab 20mg IV qd x 5 followed by stem cell infusion. No other post transplant immunomodulation was provided.

Results: Patient diagnoses included lymphoma/myeloma (20), leukemias/MDS (30), myelofibrosis/aplasia (7) and metastatic solid tumours (18). The median age was 50 (range 18–17) with a median follow up of 18 months. The median CD34+ cell dose collected was 13.4 x 106/kg. Engraftment occurred in 100% of patients with a median of 97% donor cells responsible for patient hematopoiesis by 3 months. Forty six also had a DLI (range 106–108 CD3+ cells/kg). Overall, Grade III–IV acute GVHD occurred in only 5/75 (7%) and 18 (29%) had grade II–IV. Four patients developed chronic GVHD. The transplant regimen was well tolerated with 4% 100 day treatment related mortality. In those with hematologic malignancies, only 7% started in remission, though 45 (60%) attained a CR. The most common cause of death in this group was progressive disease (28%), followed by infections (5%), and GVHD (5%). A subgroup of 21 of these older, more infirm patients who had high risk AML in 1–2nd CR or PR or ≥2 chronic stable phase CML, partially responding lymphoma, or severe myelofibrosis have been followed for at least 1 year. At 1 year, 13/21 (62%) remain alive and in continuing remission. Phenotypic analysis of lymphocyte subsets (measured by flow) revealed recovery at 6 months.

Figure Figure

T cell VBeta family recovery (spectratype analysis using PCR) revealed robust recovery by 6 months as well (first figure pre and second is 6 months post transplant), despite T depletion.

Figure Figure

TRECs analysis reveals little, if any, recovery in these patients for at least 12–18 months.

Conclusions: Nonablative allogeneic transplantation using alemtuzumab for T cell depletion results in reliable engraftment with little acute GVHD or TRM. Immune recovery assays reveal that despite T cell purging, T cell subset and VBeta families have significant recovery by 6 months and TRECS results show the recovery is primarily from peripheral expansion of transplanted donor cells, not education of new T cells. These data possibly reflect the advantage of low dose donor lymphocyte boosts without planned long term use of immunosuppressive agents. The future challenge will to be to develop strategies to further improve immune recovery in this setting.
Mode of Action of Rituximab in Chronic Lymphocytic Leukaemia; Activation of Tis11b, an Inducer of mRNA Instability, and Induction of Apoptosis.
Authors: Baou Maria , Murphy John , Jewell Andrew P.
Published: October 2019

Rituximab is a chimeric anti-CD20 monoclonal antibody that has been used successfully in the treatment of Non Hodgkin’s Lymphoma or patients with Chronic Lymphocytic Leukaemia (CLL). The mechanisms of action of Rituximab are not fully understood although antibody dependent cell mediated cytotoxicity and complement dependent cytoxicity have been shown to be important. An alternative mechanism is the induction of apoptosis through activation of pathways mediated through CD20. CD20 is involved in many cellular processes including proliferation, activation, differentiation and apoptosis. We have found that treatment of CLL cells with 20 μ g/ml Rituximab cross-linked with a secondary antibody reduced cell viability from 84±8% (in unstimulated cells) to 51.50±10% after 48h of cultivation by the Annexin/PI method. Using inhibitors specific for p38, JNK and ERK pathways, we found that inhibition of p38 inhibits the induction of apoptosis by crosslinked Rituximab. Rituximab has been reported to inhibit this pathway andlead to down regulation of bcl-2 expression in AIDS related lymphoma cells. However the mechanism is unclear. One mechanism by which many genes involved in apoptosis are regulated is through induction of mRNA instability through induction of Tis11 family genes. The Tis11 family (Tis11, Tis11b/Berg36 and Tis11d) bind to AU Rich elements present in several mRNA (eg bcl-2, TNF) and cause their degradation. We found that Tis11b/Berg36 is strongly induced by crosslinked Rituximab. Tis11d was weakly induced while Tis11 remained unchanged after treatment. Furthermore we found that induction of Tis11b/Berg36 by Rituximab is partly regulated through the p38 pathway since inhibition of this pathway resulted partial or complete inhibition of Tis11b/Berg36 induction. This suggests that Tis11b/Berg36 may mediate the induction of apoptosis by Rituximab through the degradation of proteins involved in apoptosis that contain AU Rich elements, disrupting autocrine cytokine feedback mechanisms and down regulating bcl-2.
Association of a Novel Regulatory Polymorphism (-938C>A) in the BCL2 Gene with Disease Progression in Chronic Lymphocytic Leukemia.
Authors: Nueckel Holger , Frey Ulrich H. , Sellmann Ludger , Duerig Jan , Duehrsen Ulrich , Siffert Winfried
Published: October 2019

Bcl-2 plays a key role in the regulation of apoptosis. We investigated the role of a novel regulatory single nucleotide polymorphism in the BCL2 gene (−938C>A) in B-CLL. Bcl-2 protein expression in B-cells from CLL patients carrying the −938 AA genotype was significantly increased compared to AC and CC genotypes. Moreover, the −938C- and A-alleles differed with regard to the binding of nuclear proteins.

Genotype distributions between 123 CLL patients (42 AA, 55 AC, 26 CC) and in 109 age-matched healthy controls (38 AA, 55 AC, 16 CC) were not significantly different suggesting that this polymorphism does not increase the susceptibility for B-CLL. However, time from first diagnosis to initiation of chemotherapy was significantly shorter in patients with −938AA genotype (38 months) compared to AC/CC genotypes (120 months; p=0.0077), especially in CD38 and ZAP-70 negative CLL patients. Multivariate Cox regression identified the BCL2 −938AA genotype as an independent prognostic factor for progression-free survival (hazard ratio, HR, 1.9; p=0.034) together with disease stage at diagnosis (HR 1.6; p=0.018) and ZAP-70 status (HR 2.9; p=0.001). Furthermore, the BCL2 −938C>A polymorphism in combination with the two common prognostic factors CD38 and ZAP70 status provides additional prognostic information beyond that obtained from single or double marker analysis. To the best of our knowledge this is the first report demonstrating the influence of a novel regulatory polymorphism in the BCL2 gene upon the progression of B-CLL, which, of course, requires confirmation from independent studies. On the other hand, the results presented here appear plausible with regard to differences on the protein level depending on genotype and the known impact of Bcl-2 on disease progression. Thus, our report exceeds other genetic association studies in that a mechanistic link between genotype and observed phenotype can be proposed.

In conclusion, carriage of the BCL2 −938AA genotype is a novel unfavorable genetic marker in patients with B-CLL associated with increased Bcl-2 expression.
Cytosolic Histone H1.2 Releasing under Different Apoptotic Stimuli in Chronic Lymphocytic Leukemia (CLL).
Authors: Gine Eva , Crespo Marta , Muntanola Ana , Montserrat Emili , Bosch Francesc
Published: October 2019

Recently, it has been shown that nuclear histone H1.2 is released into cytoplasm when apoptosis is induced by DNA double-strand breaks (DSB’s), this process being dependent on p53 functional status. In addition, cytosolic histone H1.2 induces cytochrome C release in a Bak-dependent manner. Thus, cytosolic histone H1.2 release represents a new mechanism that links DSB’s with activation of the apoptotic mitochondrial pathway. Against this background, we analyzed the release of histone H1.2 in the cytosol of purified CLL cells during apoptosis induced by fludarabine (F), mitoxantrone (M), etoposide, or ionizing radiation. In addition, the presence of histone H1 was correlated with p53 functional status. Cell viability and analysis of apoptosis were investigated by annexin V/PI staining and FACscan analysis. The presence of histone H1 and H1.2 in the cytosolic fraction was assessed by Western Blott using the anti-histone H1 (Upstate) and anti-histone H1.2 (Abcam) antibodies. Histone H1 traffic was also evaluated by using immunofluorescence analysis in CLL cells suspensions. FISH analysis was used to select samples with (n=3) or without (n=6) p53 deletion, and activation of p53 after treatment was assessed by Western Blot. In cases without p53 deletion, increased apoptosis was observed under all stimuli, the FM combination being the most effective. In such cases, histone H1.2 release was apparent 6 hours after the onset of irradiation or pharmacologic treatments, progressively increasing up to 24 hours. In contrast, cases with p53 deletion displayed a low cytotoxic effect upon different treatments. Interestingly, no p53 activation or histone H1.2 release into cytosol was observed. These results were also confirmed by immunofluorescence analysis, in which histone H1.2 was only visible in the cytosol of non-deleted p53 cases. These results demonstrate that, upon drug or irradiation exposure nuclear histone H1.2 is released into the cytoplasm of CLL cells in a p53-dependent manner. This suggests that, in CLL, histone H1.2 traffic contributes to the apoptosis induced by DSB’s and to drug resistance in cases with p53 deletion.
Randomized Comparison of Cladribine with Cyclophosphamide and Fludarabine with Cyclophosphamide in Previously Untreated Progressive and Symptomatic Chronic Lymphocytic Leukemia: The Interim Analysis of PALG CLL3 Trial.
Authors: Robak Tadeusz , Blonski Jerzy Z. , Gora-Tybor Joanna , Najda Jacek , Stella-Holowiecka Beata , Calbecka Malgorzata , Kowal Malgorzata , Dmoszynska Anna , Ceglarek Bernadeta , Konopka Lech , Szpila Tomasz , Warzocha Krzysztof , Piszcz Jaroslaw , Kloczko Janusz , Zawilska Krystyna , Mital Andrzej , Hellmann Andrzej , Potoczek Stanislaw , Kuliczkowski Kazimierz , Sulek Kazimierz , Zdunczyk Andrzej , Kasznicki Marek
Published: October 2019

The efficacy and toxicity of cladribine (2-CdA) + cyclophsphamide (CY) - CC versus fludarabine (FA) + CY - FC were compared in previously untreated chronic lymphocytic leukemia (CLL) patients. The study was started in January 2004. Eligible patients were asigned to either 2-CdA 0,12mg/kg/d and CY 250mg/m2/d for 3 consecutive days or FA 25mg/m2/d and CY 250mg/m2/d also for 3 days. Courses were repeated at 28 days intervals or longer if myelosupression and/or infection developes for a maximum 6 courses. The response and toxicity criteria were those recommended by NCI SWG. Minimal residual disease (MRD) was evaluated by flow cytometry if complete response (CR) was achieved. As shown in the table, there were no significant differences in the rates of overall response (OR), CR, grade 3/4 thrombocytopenia, neutropenia and infections between the programmes. MRD negativity was obtained in 4 patients in CC arm and in 6 patients in FC arm (p=0.69). The death rate was similar in both groups, 2 (3.2%) and 6 (8.2%), respectively (p=0.19). The therapy related mortality was not observed. In conclusion, CC and FC programmes seem to have similar efficacy and toxicity in previously untreated CLL patients. The results of the interim analysis of PALG CLL3 trial justify continuation of this study.

Treatment Pts enrolled Pts evaluated OR (%) CR (%) Thrombocytopenia Neutropenia Infections CC 96 63 57 (90.5) 20 (31.7) 8 (12.7) 15 (23.8) 26 (41.3) FC 100 73 62 (84.9) 25 (34.3) 6 (8.2) 17 (23.3) 20 (27.4) p value 0.24 0.45 0.27 0.53 0.06
Recombinant Mini-β3 Integrin Fragments for the Detection of HPA-1 Antibodies.
Authors: Stafford Prachi , Ghevaert Cedric , Campbell Kate , Smith Graham , Williamson Lorna , Watkins Nicholas A. , Ouwehand Willem H.
Published: October 2019

The αIIbβ3 integrin (GPIIbIIIa, CD61) is the platelet receptor for fibrinogen, fibronectin and vitronectin. A single nucleotide polymorphism (dbSNP Id= rs5918) in the ITGB3 gene defines the Human Platelet Antigen (HPA)-1 system encoding either a Leucine (1a) or Proline (1b) at position 33. HPA-1a immunisation is of clinical relevance in neonatal alloimmune thrombocytopenia (NAIT), post transfusion purpura (PTP) and platelet refractoriness. Currently, the detection of HPA-1 antibodies is based on a cumbersome sandwich ELISA requiring HPA genotyped platelets as source of antigen. We set out to express soluble calmodulin (CaM) tagged β3 fragments suitable for HPA-1 antibody detection. The design of fragments was informed by domain structural information, analysis of homology between β3 and integrin-like monomeric proteins and the evolutionary β3 domain phylogeny. Combined with computer modelling, this was the basis of the successful expression in Drosophila S2 cells of eight β3 fragments with either Leu33 or Pro33 (ΔSDL, ΔβA, PSI-Hybrid and PSI alone, both alleloforms for each of the four). Murine monoclonal antibodies Y2/51, SZ21 and CRC54 reacted with the β3 fragments as expected with only CRC54 reacting with the PSI fragment. Two of 3 human HPA-1a specific phage antibodies (19–7, 23–15, both derived from a PTP patient) reacted with all 4 Leu33 fragments but CamTran007 derived from a NAIT patient only reacted with the ΔSDL-Leu33 and ΔβA-Leu33 fragments, defining an epitope split. By ELISA, reactivity of both potency (NIBSC-03/152) and sensitivity (NIBSC-93/710) standards were comparable to those seen by MAIPA. Twelve polyclonal samples from NAIT patients (5 with cerebral haemorrhage [CH], 5 with platelet counts <40x109/L but no CH and 2 with counts >40x109/L) of potency ranging from 5–193 au/ml were detected by ELISA with Leu33 forms of ΔSDL and ΔβA. In conclusion soluble mini-β3-Leu33 fragments have been successfully expressed and shown to react with all polyclonal anti-HPA-1a samples tested from NAIT patients. The CaM tagged β3 fragments are now being further validated with a repository of 150 HPA-1a antibody samples from a NAIT case series with well defined treatment and clinical outcome histories to establish the relation between HPA-1a antibody potency, epitope reactivity and clinical phenotype.
Suppression of NK Cell-Mediated Bone Marrow Cell Rejection by CD4+CD25+ Regulatory T Cells: Linkage of Adaptive to Innate Responses.
Authors: Murphy William J. , Bareo Isabel , Hanash Alan M. , Welniak Lisbeth A. , Sun Kai , Blazar Bruce R. , Levy Robert B.
Published: October 2019

While a link between the innate to adaptive immune system has been established, studies demonstrating direct effects of T cells in regulating Natural Killer (NK) cell function have been lacking. Naturally occurring CD4+CD25+ regulatory T cells (Tregs) have been shown to potently inhibit adaptive responses by T cells. We therefore investigated whether Tregs could affect NK cell function in vivo. Using a bone marrow transplantation (BMT) model of hybrid resistance, in which parental (H2d) marrow grafts are rejected by the NK cells of the F1 recipients (H2bxd), we demonstrate that the in vivo removal of host Tregs significantly enhances NK-cell mediated BM rejection. This heightened rejection was mediated by the specific NK cell Ly-49+ subset previously demonstrated to reject the BMC in this donor/host pairing. The depletion of Tregs could also further increase rejection already enhanced by treating recipients with the NK cell activator, poly I:C. Although splenic NK cell numbers were not significantly altered, increased splenic NK in vitro cytotoxic activity was observed from the recovered cells. The regulatory role of Tregs was confirmed in adoptive transfer studies in which transferred CD4+CD25+ Tregs resulted in abrogation of NK cell-mediated hybrid resistance. Thus, Tregs can potently inhibit NK cell function in vivo and their depletion may have therapeutic ramifications with NK cell function in BMT and cancer therapy.
Managing Erythropoietin Usage in a Large Veterans Healthcare System.
Authors: Karri Sirisha , Carter Gary , Safar Mazin , Kyasa Mouhammed Jameel , Mehta Paulette
Published: October 2019

Erythropoietin (EPO) is used for many hematologic and other diseases and for adjunctive, supportive care of patients with cancer undergoing chemotherapy. However the cost is high, and excessive use may cause complications and/or compromise cancer survival. EPO utilization was the singlest largest pharmacy expense in the Central Arkansas Veterans Healthcare System and averaged $780,000 in fiscal year 2002–2003, with projections of over $2 million for the following year. We therefore studied methods to manage EPO usage through education, guideline development, monitoring, and through development of EPO-clinics modeled after coumadin-clinics. We designed and disseminated guidelines and algorithms for use of EPO, using national guidelines tailored to the specific needs of veterans and the Veterans Healthcare System. Guidelines for monitoring EPO included ongoing surveillance of iron status including iron and TIBC, ferritin, and reticulocyte hemoglobin measurements. Reticulocyte hemoglobin measurements were used as an immediate parameter of iron reserve and compared to iron profile as an indicator of iron status. Other monitoring included patient symptoms, blood counts, and course of disease. We stopped automatic refills of medication and required new prescriptions for each 6-week period so as to assure re-evaluation of the continued need for EPO and to assure titration of dosage to optimal hemoglobin and patient condition. We also flagged EPO orders in the V.A. computerized patient record system (CPRS) so that we could monitor compliance to guidelines. A pharmacy subcommittee reviewed patient profiles, symptoms, laboratory parameters, compliance to guidelines, and outcomes in relation to EPO usage. We also initiated an EPO-clinic, modeled after coumadin-clinics in which pharmacists, nurses, and physicians worked together to counsel patients about EPO, to titrate dose to best response, to prevent excess dosing, and to provide ongoing outpatient, inpatient and at-home education. Prior to initiation of the program, the projected annual cost of EPO was $2 million; after initiation of the program, the actual cost was $l.l5 million, or 43% less than the total projected amount. The program also resulted in greater attention to EPO usage, better staff and patient education, and to piloting of an EPO-clinic to coordinate medical, pharmacy, and psychosocial resources into a single clinic. This model may be effective in use of other, highly effective but very expensive medications being used in patients with hematologic disorders.
Effects of STI571 on the Expression of c-kit Receptor in Mononuclear Cells from the Bone Marrow of Patients with Acute Non-Lymphocytic Leukemia.
Authors: Feng Ru , Huang Fen , Tantai Linfang
Published: October 2019

Objective: To explore the effects of STI571 on the expression of c-kit gene of the bone marrow mononuclear cells from patients with acute non-lymphocytic leukemia (ANLL) and to study the gene difference expression in original leukemia cells from the bone marrow of ANLL patients with CD117 overexpress treated by STI571, so as to explore initially the mechanism of the restraint of proliferation caused by STI571.

Methods: The expression of CD117 and C-kit mRNA level were assayed by FACS and RT-PCR before and after different concentration STI571 cultivatio. The changes of gene expression were examined with the cDNA array in original leukemia cells after treated by STI571 and control.

Results: After STI571 treatment, the expression of CD117 and the mRNA expression of c-kit were significantly lower than before (P<0.05). There were over 730 different genes in each pair of chips, in which 156 different genes expressed together. In all different genes, 66.67% (104/156) were up-regulated, 33.33% (52/156) were down-regulated. Cellular skeleton accounted to 4.49% (7/156), oncogenes and tumor supressors 6.41% (10/156), cells receptor 7.05% (11/156), extracellular signal transduction proteins 23.07% (36/156), cyclin proteins7.05% (11/156), apoptosis correlation proteins 1.92% (3/156) and other genes accounted to50% (78/156). Genes with over ten times relative difference amounted to 26.92% (42/156).

Conclusion: The restraint of c-kit on ANLL fresh cells was related to concentration of STI571. The mechanism of the restraint of proliferation caused by STI571 may relate to three ways: 1. STI571 realizes its biological function by affecting different signal conduction pathways through protein tyrosine kinase, guanine-nucleotide-binding protein and protein kinase C; 2. STI571 restrains the infinite differentiation of cells through affecting of tumor cellular mitosis; 3. Destroy cellular skeleton.
Combination of PP1/PP2 and LY294002 Induce Apoptosis Efficiently to TEL-FGFR3 Transfected Ba/F3 Cells.
Authors: Okamura Daisuke , Yagasaki Fumiharu , Maeda Tomoya , Ishikawa Maho , Jinnai Itsurou , Bessho Masami
Published: October 2019

We previously reported that TEL-FGFR3 in a patient with peripheral T-cell lymphoma and AML conferred IL-3 independency to Ba/F3 cells and activates MAPKs, p38, PI3K, Stat3 and Stat5 through its constitutive tyrosine kinase (TK) activity in TEL-FGFR3 transfected Ba/F3 cells (TF-V5). Both FGFR3 TK specific inhibitor SU5402 and PI3K inhibitor LY294002 inhibited cell growth of TF-V5 in a dose dependent manner (IC50=5μM, 10μM respectively). SU5402(25μM) resulted in G1 arrest (90%) and induced 80% of apoptosis to TF-V5 after 24hr. LY294002(50μM) decreased expression of c-Myc, Cyclin D3 and CDK4 in TF-V5 and induced G1 arrest (90%) and apoptosis about 20% to TF-V5 after 24hr. SU5402 completely suppressed expression of Bcl-xL and Bcl-2 after 24hr. LY294002 partially suppressed these expressions. As LY294002 did not affect the phosphorylation of STAT5 and STAT3, we hypothesized that these distinct apoptosis inducing abilities between SU5402 and LY294002 might be caused by the difference of Bcl-xL and Bcl-2 expression levels. Recent report suggests that FGFR3 activates Stat5 through recruitment of Pyk2 (proline-rich tyrosine kinase 2)-Src to its juxta-membrane (JM) domain. Therefore, we examined whether PP1/PP2 which are known as Src kinase inhibitors inhibit TF-V5 cell growth. PP1 and PP2 inhibited TEL-FGFR3 induced cell proliferation in a dose dependent manner (IC50=15μM, 15μM respectively). In contrast, growth of mock with IL-3 was not inhibited at a high concentration of PP1or PP2(30μM). PP1/PP2 did not affect auto-phosphorylation of TEL-FGFR3. Interestingly, PP1/PP2 inhibit Tyr phosphorylation of Pyk2 except Tyr402 and markedly suppressed activation of Stat3, Stat5 without affecting MAPKs and PLC gamma activation. PP1/PP2 suppressed expression of Bcl-xL and Bcl-2 partially but not of Bax and induced apoptosis 20% and 30% respectively. PP1/PP2 partially blocks cell cycle at G1 phase. When TF-V5 was co-treated with PP1(30μM)+LY294002(50μM) or PP2(30μM)+LY294002(50μM), apoptosis was induced in 60–70% of TF-V5 cells which remained at G1 phase after 24hr. Although PP1+LY294002 or PP2+LY294002 did not suppress Bcl-xL and Bcl-2 completely like SU5402, PP1/PP2 induced N-terminal cleavage form of Bax.

Conclusion: PP1/PP2 induces apoptosis in TF-V5 by activating Bax and by Pyk2-Src kinase inactivation that leads to the change of Bax/Bcl-2 or Bax/Bcl-XL ratio. Combination of PP1/PP2 and LY294002 induce high rate of apoptosis under G1 arrest in TF-V5 and may represent a novel approach against TEL-FGFR3 associated hematopoietic malignancies.
Trisomy 11 and Partial Tandem Duplication of the MLL Gene in a Case of Infant Acute Myeloid Leukemia M0.
Authors: Serravalle Salvatore , Purgato Stefania , Melchionda Fraia , Tonelli Roberto , Pession Andrea
Published: October 2019

Trisomy 11 is uncommon in pediatric acute myeloid leukemia (AML) and molecular studies of AML with trisomy 11 in adult patients have recently revealed high incidence of partial tandem duplication (PTD) of the MLL gene. We report the first case of trisomy 11 and MLL-PTD in a 3 months old girl with AML, the immunophenotype of bone marrow aspirate documented blast cells positive for CD34, CD33, AntiHLA DR, CD117, and MPO <3%, M0 according to FAB classification. No exposure to potential mutagenic agents during pregnancy was documented. The most frequent translocations of MLL, t(4;11) and t(9;11), were investigated by RT-PCR at diagnosis and resulted negative, as well as other genetic abnormalities common in pediatric AML as inv 16, t(8;21), t(15;17), FLT-ITD, D835 mut. Otherwise, interphase FISH analysis, performed with a specific MLL probe, revealed a MLL triple signal (Fig. 1a). Moreover trisomy 11 was confirmed by FISH analysis with a centromeric probe for chromosome 11 (Fig. 1b). RT-PCR analysis revealed PTD of an internal portion of MLL and direct sequencing confirmed the in-frame fusion of exon 6 and exon 2 (Fig. 1c). The self-fused MLL has a duplicated N-terminal region that contains AT hook DNA-binding and DNA methyltransferase motifs. MLL-PTD may involve different exons, and it is known to be associated with a poor prognosis. Even if the oncogenic mechanism of MLL-PTD is not clear yet, several hypothesis suggested a role of DNA binding motifs duplication in the increase of affinity for MLL binding to a target site, or in the separation of autoregolatory domains from their sites of action. Beside its significance as a prognostic marker at diagnosis, MLL-PTD may be a target for PCR-based detection of minimal residual disease in AML patients.

Figure 1. Interphase FISH analysis with MLL probe revealed a MLL triple signal (a) and with a centromeric probe confirmed trisomy 11 (b). Direct sequencing confirmed the MLL PTD with in-frame fusion of exons 6 and exon 2(c). Figure 1. Interphase FISH analysis with MLL probe revealed a MLL triple signal (a) and with a centromeric probe confirmed trisomy 11 (b). Direct sequencing confirmed the MLL PTD with in-frame fusion of exons 6 and exon 2(c).
Different Patterns of Cytogenetic Evolution in CML Treated with Imatinib.
Authors: Haase Detlef , Steidl Christian , Bierbaum Anne , Soeling Ulrike , Truemper Lorenz , Griesinger Frank
Published: October 2019

In CML the Ph+ cell clone is genetically unstable leading to the acquisition of typical additional abnormalities during accelerated phase and blast crisis in up to 60 to 80 % of pts. Imatinib, by selectively inhibiting bcr-abl tyrosinkinase induces complete cytogenetic remissions in up to 80% of pts. Beneath others, two main mechanisms are responsible for the development of Imatinib-resistance: On the molecular level mutations of the bcr-abl domain and on the cytogenetic level acquisition of additional abnormalities can occur. To investigate the latter phenomenon a cohort of 67 pts treated with Imatinib was karyotyped sequentially. Treatment results were as follows: CCR: 40 (60%), MaCR: 4 (6%), MinCR: 9 (13.4%), NR: 7 (10.5%), cytogenetic progression: 5 (7.5%). Five different patterns of cytogenetic progression under Imatinib were observed. I (occurrence of an additional abnormality): One case with initial t(3;9;22) and additional 17p-/P53 allelic loss 36 after months under Imatinib. II (expansion of an initially present additional abnormality): Initial t(9;22) and a subclone with additional t(2;3), followed by expansion of the sublone with additional t(2;3). III (occurrence of multiple additional, partly atypical abnormalities and expansion of this clone): One case with initial t(9;22) and additional inv(3q), 7q-, +19, +Ph. IV (occurrence of typical additional abnormalities and expansion of the clone with the highest number of abnormalities): Initial t(9;22) and additional +8,+Ph. V (discordant response): Initial t(9:22), subsequent MaCR with parallel occurrence of a clone with additional +8,+8,+Ph and further progression. Of the five cases with cytogenetic progression two pts (I,V) had been treated with Imatinib only, one pt had a combination of Imatinib and IFN (II) and two pts (III, IV) had low-dose AraC before Imatinib. It can be concluded that pretreatment with AraC might have modulated the genetic response to Imatinib possibly leading to genetic evolution via selection of cytogenetically resistant clones in 2/5 cases. In 3/5 cases intrinsic mechanisms must have contributed to Imatinib-resistance. As demonstrated by case V our observations furthermore draw the attention to the underestimated problem of discordant genetic responses only traceable by classical cytogenetics. Thus the therapeutic consequences of these findings have not been clearly defined as yet.
Multiplex Fluorescence In Situ Hybridization (M-FISH) in the Detection of Complex Karyotypic Abnormalities of Acute Myeloid Leukemia and Myelodysplastic Syndromes.
Authors: Li Jianyong , Pan Jinlan , Xiao Bing , Ma Li , Qiu Hairong , Li Li , Xu Wei , Xue Yongquan , Ruan Changgeng
Published: October 2019

The complex chromosome abnormalities (CCAs) were one of the most important poor prognostic risk factors in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). Chromosome analysis using classical cytogenetic banding techniques fails to completely resolve complex karyotypes and cryptic translocations. The technique of multiplex fluorescence in situ hybridization (M-FISH) allow for the simultaneous visualization of all chromosomes of a metaphase in a single hybridization step and thereby enable to comprehensively analyze complex karyotypes and the identification of new and cryptic translocations. To investigate the value of M-FISH in the detection of complex karyotypic abnormalities of AML and MDS. M-FISH was used in combination with interphase-FISH to study 24 cases of AML and MDS with CCAs showed by R-banding of conventional cytogenetics (CC). In 14 cases of AML with CCAs, 4 gains of whole chromosome and 4 losses of whole chromosome were confirmed by M-FISH, while 12 losses of whole chromosome were revised as derivative chromosomes resulted from various structural aberrations. 26 derivative chromosomes and 19 marker chromosomes were characterized precisely by M-FISH. Most of them were unbalanced translocations, including 2 complex t(8;21), which have not been previously described:t(8;21), der(8) t(8;21) (8pter→8q22::21q22→21qter), der(21) t(8;21;8) (8qter→ 8q22::21p13→ 21q22::8q22→ 8qter) and t(21;8;18;1), der(8) t(8;21) (8pter→ 8q22::21q22→ 21qter), der(21) t(21;8;18;1) (21p13→ 21q22::8q22→ 8q24::18?::1q?q?). In 10 cases of MDS, 37 kinds of structural rearrangements were detected by M-FISH including insertion, deletion, translocation and derivative chromosomes, and among them 34 kinds were unbalanced rearrangements, only 3 were balanced rearrangements including t(6;22)(q21;q12), t(9;19)(q13;p13) and t(3;5)( ?;?), 7 abnormalities were never reported before. The CCAs invloved nearly all chromosomes, of which the chromosome 17, 5 and 7 were invloved more frequent than the rest. Chromosomes 5, 17, 7 were involved in 15 cases (62.5%), 12 cases (50%) and 6 cases (25%) respecrively. We conclude that M-FISH could refine CCAs of AML and MDS patients, find or correct the missed or misidentified aberrations by CC analysis. Our findings confirm that M-FISH is a powerful tool to characterize complex karyotypes in AML and MDS.
Peroxidase Activity and Nuclear Density Analysis in the Diagnosis of Acute Myeloid Leukemia.
Authors: Gibbs Graham
Published: October 2019

Awareness of the range of additional parameters offered by many modern hematology analysers is increasing and some have found applications in today’s clinical diagnostic laboratories. In order to determine the white blood cell (WBC) count and differential, the Advia 120 (Bayer Diagnostics, Tarrytown, NY) uses low angle (0–5°) and high angle (5–15°) light scatter measurements to determine size and density of the cellular nuclei following removal of the cytoplasm by phthalic acid, as well as a cytochemical reaction involving hydrogen peroxide and a substrate (4-chloro-1-naphthol) to measure peroxidase activity in white blood cells. The latter are then displayed visually as 2-dimensional cytograms. The utility of these parameters to provide an early indication of the presence of leukemic cells was assessed retrospectively in 25 newly presenting cases of acute myeloid leukemia (AML) without peripheral blood pancytopenia. Using the French-American-British (FAB) classification, the cases comprised M0 (n=1), M1 (n=2), M2 (n=2), M3 (n=2), M4 (n=1), M5 (a+b) (n=4), and M6 (n=1). The remaining 12 patients were unclassified AML (although not M3). Using the classification system described by d’Onofrio (Bloodline Reviews2001; 2-R: 3–6), peroxidase activity was scored from P0 (peroxidase negative) to P6 (intense peroxidase positivity), and nuclear density was scored as D0 (normal profile) or D1 (abnormal mononuclear cell population shifted down and to the left - a region corresponding to the presence of blast cells). 23 out of 25 cases were correctly classified as probable AML (92%), comparing well with the findings of d’Onofrio (95.2%). 21 out of these 23 cases were also categorised correctly according to probable FAB type (91%). Of the 2 cases incorrectly classified, 1 case was classified as probable acute lymphoblastic leukemia (ALL), and 1 case was classified as probable myelodysplastic syndrome (MDS). Of note, only cases of M3 AML are allocated to the P6/D1 classification category. Both cases of M3 AML in this study were P6/D1and none of the remaining cases were misclassified into this category. The risk of associated coagulopathy and the unique response to treatment with all-trans retinoic acid (ATRA) makes prompt detection particularly important within this AML patient subgroup. Although not diagnostic in itself, the use of peroxidase activity and nuclear density analysis cytograms in routine complete blood counts (CBC) provides a valuable early indication of a possible case of AML, allowing the prompt initiation of confirmatory tests.