Vincenzo Federico

Inhibition of Glycogen Synthase Kinase-3 Increases NKG2D Ligand MICA Expression and Sensitivity to NK Cell–Mediated Cytotoxicity in Multiple Myeloma Cells: Role of STAT3

Engagement of NKG2D and DNAX accessory molecule-1 (DNAM-1) receptors on lymphocytes plays an important role for anticancer response and represents an interesting therapeutic target for pharmacological modulation. In this study, we investigated the effect of inhibitors targeting the glycogen synthase kinase-3 (GSK3) on the expression of NKG2D and DNAM-1 ligands in multiple myeloma (MM) cells. GSK3 is a pleiotropic serine–threonine kinase point of convergence of numerous cell-signaling pathways, able to regulate the proliferation and survival of cancer cells, including MM. We found that inhibition of GSK3 upregulates both MICA protein surface and mRNA expression in MM cells, with little or no effects on the basal expression of the MICB and DNAM-1 ligand poliovirus receptor/CD155. Moreover, exposure to GSK3 inhibitors renders myeloma cells more efficient to activate NK cell degranulation and to enhance the ability of myeloma cells to trigger NK cell–mediated cytotoxicity. We could exclude that increased expression of β-catenin or activation of the heat shock factor-1 (transcription factors inhibited by active GSK3) is involved in the upregulation of MICA expression, by using RNA interference or viral transduction of constitutive active forms. On the contrary, inhibition of GSK3 correlated with a downregulation of STAT3 activation, a negative regulator of MICA transcription. Both Tyr705 phosphorylation and binding of STAT3 on MICA promoter are reduced by GSK3 inhibitors; in addition, overexpression of a constitutively active form of STAT3 significantly inhibits MICA upregulation. Thus, we provide evidence that regulation of the NKG2D-ligand MICA expression may represent an additional immune-mediated mechanism supporting the antimyeloma activity of GSK3 inhibitors.

Evaluation of comorbidities at diagnosis predicts outcome in myelodysplastic syndrome patients

Recent data suggest that proper assessment of comorbidities is useful to predict the outcome of MDS patients receiving allogeneic stem cell transplantation. However, the results obtained in this highly selected subset of patients cannot be applied to the whole MDS population. We evaluated the impact of comorbidities in 418 consecutive MDS patients diagnosed at our institute from 1992 to 2005. All patients were classified according to WHO criteria and all received only conservative and supportive treatment. One or more comorbidities were detected in 390 patients (93%) at the time of diagnosis, with a higher incidence in older patients. Cardiac diseases were the most frequent comorbidities (30%) while diabetes and correlated adverse events were the second cause of comorbidity (20%). We applied 3 comorbidity prognostic scores (CCI, HCT-CI and a MDS-CI score proposed by Della Porta et al.). According to CCI score, 253 patients had a score 0, 111 patients had a score 1 and 54 patients had a score >2. According to HCT-CI, 209 patients had a score 0, 105 patients had a score 1 and 106 patients had a score >2. With MDS-CI score, 288 patients had a score 0 and 129 patients had a score >1. We found a significant correlation between survival and stratification according to CCI and MDS-CI scores (p=0.01 and 0.02, respectively), but not according to HCT-CI score. The number of comorbidities as evaluated according to CCI was directly correlated to the development of RBC transfusion-dependency and was associated to a significantly higher risk of death not related to leukemic evolution (HR = 2.12, p ≤ 0.001). Conversely, higher risk of non-leukemic death did not correlate with higher transfusional requirement according to HCT-CI and MDS-CI scores (p = 0.3 and 0.43, respectively). As suggested by Della Porta et al., also in our experience the presence of cardiac, liver, renal, pulmonary diseases and solid tumours was found to independently affect the risk of death in a multivariable Cox regression analysis (p values from <0.01 to 0.004). In conclusion, assessment of comorbidities at diagnosis in MDS patients may improve the ability of therapeutic decisions.

Che-1-induced inhibition of mTOR pathway enables stress-induced autophagy

Mammalian target of rapamycin (mTOR) is a key protein kinase that regulates cell growth, metabolism, and autophagy to maintain cellular homeostasis. Its activity is inhibited by adverse conditions, including nutrient limitation, hypoxia, and DNA damage. In this study, we demonstrate that Che‐1, a RNA polymerase II‐binding protein activated by the DNA damage response, inhibits mTOR activity in response to stress conditions. We found that, under stress, Che‐1 induces the expression of two important mTOR inhibitors, Redd1 and Deptor, and that this activity is required for sustaining stress‐induced autophagy. Strikingly, Che‐1 expression correlates with the progression of multiple myeloma and is required for cell growth and survival, a malignancy characterized by high autophagy response.

Refractory cytopenia with unilineage dysplasia: analysis of prognostic factors and survival in 126 patients

According to the revised WHO classification of 2008, dysplasia in ≥10% of one bone marrow lineage and one cytopenia constitutes the low-risk category of unilineage cytopenia and unilineage dysplasia (UCUD). We retrospectively reclassified, according to WHO, low-risk MDS from our database and found 126 subjects with these features at diagnosis: 79 patients were categorized as refractory anemia (RA), 23 patients as refractory neutropenia (RN), and 24 as refractory thrombocytopenia (RT). We did not find differences between the three subgroups as regards sex, median age, and cytogenetic aberrations. Lower PMN count (0.8 × 109/L) was observed in the RN category, as well as lower platelet count in the RT category (51 × 109/L). Moreover, we found a lower rate of patients requiring RBC transfusions, during the disease course, in the RT category (45.8%) as compared to RA (62%) and RN (69%) groups (p = 0.05); a lower incidence of infections at diagnosis in the RT category (20.8%) compared to RA (32%) and RN (43%) categories (p = 0.03); and a higher incidence of hemorrhagic symptoms at diagnosis in the RT category (41.6%) and RN category (26%) as compared to the RA group (5%) (p = 0.001). Application of different scoring systems (Bournemouth and Spanish scores, WPSS) revealed a low OS in high-risk patients within the RT category, compared to RA and RN categories, although unlikely to reflect the consequences of low OS found in the former category. Statistically significant differences were also evidenced in the incidence of acute myeloid leukemia (AML) evolution and overall survival: 7/79 (8%) patients with the RA category evolved to AML in a median time of 89 months, whereas 4/23 (17%) of the RN category and 1/24 (4%) of the RT category experienced disease progression, in a median time of 33.8 and 12.8 months, respectively (p = 0.03). The RT category had a lower overall survival (15.9 months) as compared to RA (48.2 months) and RN (35.9 months) categories (p < 0.001). In conclusion, in our study, application of the revised 2008 WHO classification confirmed the importance of separating patients with unilineage dysplasia for prognostic disease assessment; from our results it seems that the RT category has a worse outcome.

Evaluation of overall survival according to myelodysplastic syndrome-specific comorbidity index in a large series of myelodysplastic syndromes

We read with interest the article by Della Porta and colleagues[1][1] and the editorial of Prof. Cazzola[2][2] relating to the role of comorbidities in myelodysplastic syndromes (MDS). We have previously published a paper on the role of comorbidities in MDS patients[3][3] and we found it intriguing

Suboptimal response to imatinib according to 2006–2009 European LeukaemiaNet criteria: a ‘grey zone’ at 3, 6 and 12 months identifies chronic myeloid leukaemia patients who need early intervention

Barker, J.N., Weisdorf, D.J., DeFor, T.E., Blazar, B.R., McGlave, P.B., Miller, J.S., Verfaillie, C.M. & Wagner, J.E. (2005) Transplantation of 2 partially HLA-matched umbilical cord blood units to enhance engraftment in adults with hematologic malignancy. Blood, 105, 1343–1347. Barker, J.N., Scaradavou, A. & Stevens, C.E. (2010) Combined effect of total nucleated cell dose and HLA match on transplantation outcome in 1061 cord blood recipients with hematologic malignancies. Blood, 115, 1843–1849. Blume, K.G., Forman, S.J., O’Donnell, M.R., Doroshow, J.H., Krance, R.A., Nademanee, A.P., Snyder, D.S., Schmidt, G.M., Fahey, J.L., Metter, G.E., Hill, L.R., Findley, D.O. & Sniecinski, I.J. (1987) Total body irradiation and high-dose etoposide: a new preparatory regimen for bone marrow transplantation in patients with advanced hematologic malignancies. Blood, 69, 1015–1020. Brunstein, C.G., Barker, J.N., Weisdorf, D.J., DeFor, T.E., Miller, J.S., Blazar, B.R., McGlave, P.B. & Wagner, J.E. (2007) Umbilical cord blood transplantation after nonmyeloablative conditioning: impact on transplantation outcomes in 110 adults with hematologic disease. Blood, 110, 3064–3070. Delaney, C., Gutman, J.A. & Appelbaum, F.R. (2009) Cord blood transplantation for haematological malignancies: conditioning regimens, double cord transplant and infectious complications. British Journal of Haematology, 147, 207–216. Kurtzberg, J., Laughlin, M., Graham, M.L., Smith, C., Olson, J.F., Halperin, E.C., Ciocci, G., Carrier, C., Stevens, C.E. & Rubinstein, P. (1996) Placental blood as a source of hematopoietic stem cells for transplantation into unrelated recipients. New England Journal of Medicine, 335, 157–166. Laughlin, M.J., Barker, J., Bambach, B., Koc, O.N., Rizzieri, D.A., Wagner, J.E., Gerson, S.L., Lazarus, H.M., Cairo, M., Stevens, C.E., Rubinstein, P. & Kurtzberg, J. (2001) Hematopoietic engraftment and survival in adult recipients of umbilical-cord blood from unrelated donors. New England Journal of Medicine, 344, 1815–1822. MacMillan, M.L., Weisdorf, D.J., Brunstein, C.G., Cao, Q., DeFor, T.E., Verneris, M.R., Blazar, B.R. & Wagner, J.E. (2009) Acute graftversus-host disease after unrelated donor umbilical cord blood transplantation: analysis of risk factors. Blood, 113, 2410–2415. Rocha, V. & Broxmeyer, H.E. (2010) New approaches for improving engraftment after cord blood transplantation. Biology of Blood and Marrow Transplantation: Journal of the American Society for Blood and Marrow Transplantation, 16, S126–132. Wagner, J.E., Barker, J.N., DeFor, T.E., Baker, K.S., Blazar, B.R., Eide, C., Goldman, A., Kersey, J., Krivit, W., MacMillan, M.L., Orchard, P.J., Peters, C., Weisdorf, D.J., Ramsay, N.K. & Davies, S.M. (2002) Transplantation of unrelated donor umbilical cord blood in 102 patients with malignant and nonmalignant diseases: influence of CD34 cell dose and HLA disparity on treatment-related mortality and survival. Blood, 100, 1611–1618.

Cytogenetic and molecular responses in chronic phase chronic myeloid leukaemia patients receiving low dose of imatinib for intolerance to standard dose.

Imatinib mesylate is the gold standard treatment of chronic myeloid leukaemia (CML) and 400 mg/day is considered the standard dose. Although it is generally well tolerated, some patients require temporary drug discontinuation and permanent dose reduction because of haematological or non‐haematological toxicities. Whether or not reduced doses are effective as the standard dose in inducing and/or maintaining complete cytogenetic and molecular response is not clear. We report the outcome of 45 CML patients in early (17) and late (28) chronic phase (CP) in whom, within a series of 250 patients treated with imatinib, reduced the dose of the drug after experiencing adverse events. Median time interval between the start of therapy and dose reduction was 58 days, whereas median administered dose was 300 mg/day. At 6 months from reduction, major cytogenetic responses (MCRs) were observed in 67% of patients, with 58% being complete cytogenetic remission (CCR), and complete molecular response (CMR) were obtained in 18% of patients. At 12 months, all patients who had obtained MCR reached CCR: this was significantly higher in low risk patients (87%) versus non‐low risk patients (66 and 46%), and in early phase (82%) versus late phase (53.5%). CMR and major molecular response (MMR) were detected in 20 and 22% of patients, respectively. Low dose imatinib appears effective in patients with intolerance to standard dose, even though long‐term effects remain to be established. Copyright

Cost of illness in patients with multiple myeloma in Italy: The CoMiM study

AIMS AND BACKGROUND Multiple myeloma is the second most common hematological cancer. Although it accounts for only a relatively small percentage of all cancer types, the costs associated with managing multiple myeloma are considerable. Available studies are mainly focused on health care costs. The Costo del Mieloma Multiplo (Cost of MM, CoMiM) study investigated the cost of illness of multiple myeloma in Italy during one year of disease management. METHODS CoMiM is a retrospective, prevalence-based, multi-center, cross-sectional study based on a stratified sample of patients seen during normal clinical practice (asymptomatic; symptomatic on drugs; symptomatic receiving autologous stem cell transplantation; plateau/remission). Demographics, clinical history, health care and non-health care resource consumption data were collected. Costs were evaluated from the societal viewpoint and expressed in Euro 2008. RESULTS Data on 236 patients were analyzed (39 asymptomatic, 17%; 29 symptomatic receiving autologous stem-cell transplantation, 12%; 105 symptomatic receiving drugs, 44%; 63 plateau/remission, 27%). The total cost of illness reached € 19,267.1 ± 25,078.6 (asymptomatic, € 959.3 ± 1091.6; symptomatic receiving drugs, € 21,707.8 ± 21,785.3; symptomatic receiving autologous stem-cell transplantation, € 59,243.7 ± 24,214.0; plateau/remission, € 8130.7 ± 15,092.5). The main cost drivers of total cost of illness were drugs and hospital admissions (46.1% and 29.4%, respectively). Antineoplastics and immunomodulators drove the cost of drugs (21.6% and 21.1% of the total cost of illness). Cost of antineoplastics was led by bortezomib (97.4%), whereas the cost driver for immunomodulators was lenalidomide (99.4%). Cost of hospitalization funded by the Italian National Health Service was strongly influenced by transplantation (94.6%), whereas chemotherapy and skeletal fractures did not exceed 1% and 2%, respectively. CONCLUSIONS Despite some limitations, the CoMiM study provides Italian health care decision-makers with an insight into the stratified cost of illness of multiple myeloma patients.

Isolated central nervous system relapse after nine years of complete molecular remission in a lymphoid blast crisis of chronic myeloid leukemia treated with imatinib

Extramedullary blast crisis (BC) of chronic myeloid leukemia CML) has been observed in 7–10% of patients, with or withut other manifestation of disease progression [1]. Cerebrospinal nvolvement as a site of extramedullary BC is rare, but since the dvent of imatinib as first-line treatment for CML, there have een several reports describing patients with isolated central nerous system (CNS) involvement [1–5], because the drug poorly enetrates the blood-brain barrier. Here we report a case of a ymphoid BC, developed under IFN therapy, which reached comlete molecular remission with imatinib and presented an isolated elapse of the same disease in CNS after nine years of imatinib herapy. A 71-year-old female was diagnosed with Philadelphia+ CML in anuary 2001 and was initially treated with interferon (IFN ) at he maximum tolerated dose of 5 MU/day. After 6 months of treatent, she developed a sudden leucocytosis (WBC 52 × 109 /l). At hat time bone marrow examination showed a total infiltration by 0% blast cells: immunophenotypic analysis revealed a blastic poplation, which was positive for the following superficial antigens: D34, CD79-a, CD19 and CD38, CD13, CD33, CD66-C, TdT+, and egative for CD10, CD20, leading to a diagnosis of lymphoid BC. ytogenetic analysis at the time of acute transformation showed uplication of Ph+ chromosome in 20 analysed metaphases. Cererospinal fluid (CSF) analysis at that time did not reveal evidence of isease. The patient was started on imatinib at the dose of 600 mg aily and achieved a complete haematological response after 1onth and a complete cytogenetic response (CCR) after 3 months. complete molecular response (CMR) was reached after 3 years f treatment (ratio BCR-ABL1/ABL1 < 0.001IS), with imatinib being ontinued at 400 mg/day for the following nine years, without any ign of toxicity. In September 2010, the patient suddenly develped left hypoacusia, left lower limb weakness and right visual loss. MRI showed a solid tissue around the left facial nerve. Optical xamination revealed bilateral papilloedema. Bone marrow examnation confirmed CCR and RQ-PCR showed a residual disease with BCR-ABL1/ABL1 ratio of 0.2%IS. CSF analysis demonstrated high cell ount (1,300 cells/mm3): cytological examination revealed lymhoblast infiltration. Immunophenotypic analysis performed on SF showed the same phenotype detected at diagnosis (positivty for CD34, CD79-a, CD19 and CD38, CD13, CD33, CD66-C, TdT, egativity for CD10, CD20) and FISH analysis detected duplicaion of Ph+ chromosome. RQ-PCR performed on CSF showed a CR-ABL1/ABL1 ratio of 120% IS. A diagnosis of extramedullary BC

Isodicentric duplication of Philadelphia chromosome as a mechanism of resistance to dasatinib in a patient with chronic myeloid leukemia after resistance to imatinib

Imatinib mesylate, the first selective inhibitor of BCR–ABL1 tyrosine kinase protein, has shown clinical efficacy in the treatment of chronic myeloid leukemia (CML) by inducing a high rate of complete remission and decreased mortality of patients with CML. Despite the beneficial effects of imatinib, some patients may develop primary or acquired resistance [1]. The IRIS study (International Randomized Study of Interferon vs. STI571) at 8-year follow-up provided an indication of imatinib resistance: 31% of patients discontinued imatinib, with an estimated resistance of 16% [1]. Several mechanisms may contribute to this phenomenon, including increased expression of BCR–ABL1 kinase through gene amplification or duplication, decreased intracellular drug concentration caused by altered drug efflux/influx proteins (OCT1 or P-glycoprotein; Pgp), clonal evolution, or overexpression of Src kinases (Lyn, Hck) involved in BCR–ABL1 independent activation of alternative pathways. However, 40% of resistance in chronic phase (CP) is attributed to the emergence of clones expressing mutant forms of BCR–ABL1 [2]. Alternative treatments for resistant patients have been indicated by the recently updated European LeukemiaNet recommendations [3]: in a case of failure or suboptimal response to imatinib, dasatinib or nilotinib has been proposed as a valid therapeutic strategy. Development of mutations in the ABL1 kinase domain has been reported as a cause of resistance to second-generation tyrosine kinase inhibitors (TKIs), but to date no case of Philadelphia chromosome positive (Phþ) duplication has been reported as a possible mechanism of resistance to dasatinib. In the present study, we determined through G-banding and fluorescence in situ hybridization (FISH) analysis the presence of an acquired isodicentric Ph chromosome occurring in a dasatinib-resistant patient. The impact of this chromosome anomaly on genomic instability and TKI resistance is discussed. A 69-year-old female was referred to our center in June 1998 for the finding of leukocytosis during routine analysis. A peripheral blood complete cell count revealed white blood cells (WBC) 158 6 10/ L with the presence of intermediate forms, hemoglobin 8.3 g/dL, and platelets 450 6 10/L. Physical examination revealed mild hepatosplenomegaly. Bone marrow analysis showed hypercellular marrow with a granuloblastic hyperplastic population in the absence of undifferentiated cells, which was compatible with a chronic myeloproliferative disorder. Conventional cytogenetic analysis revealed a standard t(9;22)(q34;q11.2) translocation in 100% of analyzed metaphases. Conventional cytogenetic analysis was performed on bone marrow cells according to standard methods. Chromosomes were G-banded with trypsin-Giemsa stain (GTG-band), and karyotype was described according to International System for Human Cytogenetic Nomenclature (ISCN 2009) recommendations [4]. The patient