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Iron removal by the chelating-agent deferoxamine (DFO) arrests cell cycle progression of activated human T cells in late G1 phase, before the G1/S border. The effects of the drug on molecules that regulate progression through the cell cycle were defined. DFO (10 mumol/L) inhibited induction of transcription of the cdc2 gene, but had no effect on accumulation of cdk2, cdk4, or interleukin (IL)-2- transcripts. No detectable p34cdc2 protein accumulated, but synthesis of the p33cdk2 protein was begun. It accumulated to normal levels during the first 20 to 30 hours of incubation in the presence of DFO. Furthermore, p33cdk2 was activated as an H1 histone kinase. As active p33cdk2 primarily represents complexes of the p33 protein with cyclin E or cyclin A, the effects of DFO on these cyclins were examined. Although the induction of synthesis and early accumulation of cyclin E and cyclin E-associated kinase activity appeared normal, the appearance of cyclin A and cyclin A-associated kinase activity were inhibited by DFO. However, the production of cyclin A mRNA appeared to be normal in the presence of DFO. A major effect of DFO in blocking cell cycle progression may be mediated through inhibition of the appearance of cyclin A protein and, therefore, a major component of p33cdk2 activity. The results also indicate that the p33cdk2/cyclin E activity produced in the presence of DFO was not sufficient for completion of the G1 phase of the cell cycle.

Background: Proteasome inhibitors have a fundamental role in the treatment of multiple myeloma however there are still patients who fail to achieve a good response and have a dismal prognosis. Markers predicting the outcome of individual patients are needed to identify those who would benefit more from a different approach. Proteasome subunit beta type 1 (PSMB1) rs12717 single nucleotide polymorphism (C/G substitution resulting in a P11A change) was reported recently to be associated with greater clinical benefit in relapsed follicular lymphoma patients treated with bortezomib containing combination (Coiffier et al Clin Cancer Res 2013). To the best of our knowledge there is only one report with limited number of myeloma patients showing no association between PSMB1 P11A genotype and survival (Lichter et al Blood 2012). Aims and Methods: We analyzed the association of PSMB1 P11A polymorphism and treatment outcome of 220 consecutive myeloma patients having had first line chemotherapy in our unit. PSMB1 P11A polymorphism was tested using LightCycler melting analysis. Statistical analyses were performed using the SPSS (version 20.0) software package. Results: The distribution of presentation prognostic markers such as age, ISS and FISH were even among the three genotype groups. 149 patients had bortezomib-based chemotherapy, mainly VTD (n=76) and MPV (n=47); 63% of these patients had ASCT consolidation. 71 patients had non-bortezomib-containing induction with either thalidomide, lenalidomide or other protocols including MP and VAD; only 28% of these patients had ASCT. In a recessive model the median PFS was 727 (635-818) days in carriers of the wild type C allele either in homozygous of heterozygous form (C/C&C/G, n=188), and only 491 (366-615) days in the homozygous variant G allele carriers (G/G, n=32, p=0.01). In multivariate analysis, C allele carriers had favorable PFS compared to G/G genotype patients with a hazard ratio of 1.402 (1.093-1.799), p=0.008 besides age, ISS, FISH, bortezomib treatment and ASCT. When we analyzed separately the bortezomib-treated and non-bortezomib-treated patients the benefit in PFS was only present in the bortezomib group [Fig 1, PFS 747 (656-837) days in C/C&C/G and 448 (279-616) days in G/G patients, p=0.002], and not in patients treated without bortezomib (p=0.559). We performed statistical interaction testing which showed significant interaction between bortezomib exposure and PSMB1 P11A (p=0.035). Conclusion: PSMB1 P11A is a novel predictive marker in multiple myeloma which could help to identify patients having a suboptimal response to bortezomib who might benefit from alternative management. Disclosures No relevant conflicts of interest to declare.

Background Myeloid sarcomas are rare extramedullary manifestations of acute myeloid leukemia (AML) representing less then 1% of all cases. Since they invariably progress into systemic disease, early AML-like chemotherapy is mandated. Myeloproliferations with eosinophilia and involvement of the PDGFRA, PDGFRB or FGFR1 genes are distinct entities in the 2008 WHO classification with peculiar sensitivity to tyrosine kinase inhibitors. Extramedullary myeloid sarcomas with eosinophilia and FIP1L1-PDGFRA fusion gene expression are extremely rare with only two cases described. Here we report a case of extramedullary leukemia, eosinophilia and FIP1L1-PDGFRA rearrangement responding favourably to imatinib. Case history A 32-year old man presented with a 3-month history of subcutaneous nodules growing gradually in the occipital, chest, abdominal and perianal regions, the largest being 50 x 60 x 18 mm in size and exulcerated. There was no weight loss or fever. Complete blood count showed WBC of 6,8 x 109/L, Hb level of 104 g/dl and platelet count of 377 x 109/L. The differential revealed 42% neutrophils, 25% lymphocytes, 5% monocytes and 28% eosinophils with an absolute eosinophil count of 1,92 x 109/L. Apart from mildly elevated LDH (539 U/L, normal range: 153 - 463 U/L), other laboratory results were unremarkable. CT scan found no lymphadenopathy or hepatosplenomegaly. Histology of the nodules showed infiltration with immature monoblasts, negative for CD34, CD117 and weakly positive for MPO and CD68. Mutations of the FLT3 and nucleophosmin genes were not present and TCR gene rearrangement analysis did not reveal clonality. Bone marrow biopsy showed an increase of eosinophilic precursors without apparent blast excess. FISH analysis of the nodules performed with the LSI FIP1L1/PDGFR alpha probe detected deletion of the CHIC2 gene in most interphase nuclei. Bone marrow karyotype was normal, and FISH probes proved negative for AML/ETO and inv(16). In 15% of interphases, the FIP1L1/PDGFR alpha fusion signal could be detected. RT PCR showed FIP1L1-PDGFR alpha gene fusion in the marrow. A diagnosis of extramedullary myeloid sarcoma with concomittant eosinophilia and FIP1L1/PDGFRA fusion gene expression was established. To our knowledge, this is the third such case reported so far. Induction regimen consisted of 7 days of Ara-C (200 mg/m2) and 3 days of daunorubicin (60 mg/m2). This resulted in regression but not complete disappearance of the subcutaneous nodules. After the therapy-induced aplasia, restitution of platelets preceded neutrophils and mild thrombocytosis developed. The bone marrow showed increased eosinophils without blast excess. FISH analysis detected the presence of the FIP1L1-PDGFRA fusion signal in 11% of nuclei. Imatinib mesylate, 100 mg/day was started. In 14 days, the platelet count returned to normal and the residual nodules disappeared completely. After 5 weeks of imatinib therapy, the patient is doing well without any complaints and the bone marrow shows complete cytogenetic response. Conclusion this case of extramedullary AML, eosinophilia and FIP1L1/PDGFRA rearrangement persisting after intensive chemotherapy but responding well to low-dose imatinib emphasizes the importance to screen for mutations of the PDGFR gene in AML with major eosinophilic component due to the major therapeutic implications. Disclosures: No relevant conflicts of interest to declare.

Common HFE gene point mutations (C282Y, H63D and S65C) are responsible for hemochromatosis type 1 in homozygous or compound heterozygous states. Although HFE-related hemochromatosis is inherited in a recessive manner, heterozygous carriers were shown to have higher iron parameters (serum iron and ferritin levels, transferrin saturation) without progressive iron overload. Heterozygous HFE carriership has been implicated as a genetic modifier of several diseases, where iron homeostasis play an important role in disease susceptibility or disease progression (porphyria cutanea tarda, myelodyslasia). We investigated 336 unrelated patients with chronic myeloproliferative disease (CMPD) [176 patients with polycythemia vera (PV), 131 patients with essential thrombocythemia (ET) and 29 patients with chronic idiopathic myelofibrosis (CIMF)] and 171 first time blood donors as controls for allele frequencies (AFs) of HFE mutations by Light Cycler allele discrimination method. The presence of the acquired JAK2 V617F mutation in CMPD was investigated by allele specific PCR. Laboratory (hemoglobin, white blood cell and platelet count) and clinical features (sex, age at diagnosis, splenomegaly, presence of thrombotic, myelofibrotic or leukemic transformation) were recorded in the patient group. Compared to controls (4.7 +/− 2.3%), decreased HFE C282Y AF was found in patient group (1.8 +/− 1.0%; p=0.02; OR=0.38 [0.17–0.85]). Decreased HFE C282Y AFs were detected in all patient subgroups according to diagnosis (PV, ET, CIMF) and according to JAK2 V617F mutational status, but the difference remained significant only in the patient group with PV or with JAK2 V617F positive CMPD (PV:1.7 +/− 1.4%; p=0.04; JAK2 V617F positive patients: 1.6+/− 1.1%). H63D and S65C mutational status did not differ significantly between control (12.0+/−3.5% and 1.3+/−1.8%) and patient groups (13.1+/−2.6% and 0.5+/−0.5%). JAK2 V617F frequency was 86.9% (153/176) in PV, 60.3% (79/131) in ET, and 72.4% (21/29) in CIMF. We found significantly elevated hemoglobin levels and WBC values (measured at the time of diagnosis) in the V617F-positive PV (and ET patient groups compared to V617F-negative patients. The incidence of vascular complications (arterial and venous thrombosis or bleeding) was higher in JAK2 V617F CMPD. The age of CMPD onset and the rate of different complications were not altered by HFE mutational status. Our data confirm earlier observations that JAK2 V617F-positive ET shares clinical features (elevated hemoglobin) with PV. We found that HFE C282Y mutation may be associated with a protective role against CMPD (also CMPD associated with JAK2 positivity). Chronic iron deficiency or latent anemia may trigger disease susceptibility for CMPD, and HFE C282Y positivity can play a protective role against chronic iron deficiency.

Juvenile hemochromatosis (JH) is a rare autosomal recessive disorder of iron metabolism. The early onset of severe iron overload and the frequent occurrence of cardiomyopathy, reduced glucose tolerance, and hypogonadism distinguish JH from the more common HFE -related hereditary hemochromatosis.[1][

Treatment outcome in patients with AML is determined by a variety of prognostic factors such as WBC, LDH, age, presence of chromosomal aberration. Recently, two acquired genetic alterations, internal tandem duplications (ITD) and Asp835 codon mutations of the fms-like tyrosine kinase 3 (FLT3) gene were reported in AML as prognostic factors. 66 consecutive adult patients (39 females and 27 males) were treated with newly diagnosed AML in our institute between January 2002 and June 2004. The median age of onset was 49±15 (range 17–83) years. The gender distribution was 1.44 (female/male). We analyzed FLT3 ITD and Asp835 mutations at the time point of diagnosis by fluorescent PCR and PCR-RFLP methods. ITD was present in 22.7% (15/66) of the patients and Asp835 mutations were detected in 7.6 % (5/66). Two patients carried both mutations. 13 of 15 ITD-positive patients had myelomonocytic or monocytic leukemia (M4 and M5 in the FAB classification system). There was no difference between the numbers of ITD-positive and negative patients in complete remission (10/15 [66.7%] vs. 31/51 [60.8%]). In the ITD-positive patient group, the relapse rate was increased (5/15 [33.3%] vs. 7/51 [13.7%]) however, this difference was statistically not significant. The age of onset was greater than 40 years in all Asp835 mutation-positive patients and in this group, no relapse occurred within the follow-up time. The remission rate (4/5) of this group was high in spite of the old age of onset. The single Asp835-positive patient also carried the ITD mutation and showed primary resistance. Our results are in agreement and confirm earlier observations for the ITD mutation in a different patient population. In our relatively small patient group the Asp835 mutation seemed to have a beneficial effect.