ALBERT

Description: Abstract 3375 Introduction. Telomere are effective sensors of cell integrity and their accelerated shortening is a marker of genetic and/or proliferative stress in several tissues including the hematopoietic compartment. Severe telomere attrition has been indeed observed in aplastic anemia and post-transplant setting. Little is currently known on the genetic integrity of Ph-negative hematopoietic cells (HCs) repopulating the bone marrow (BM) after successful chronic myeloid leukemia (CML) treatment. We thus decided to verify whether severe telomere shortening might occur in this setting and to assess whether its presence might correlate to genetic and functional impairment of Ph-negative hematopoiesis. Patients and methods. We investigated 81 CML patients with persistent (≥12 months) complete cytogenetic remission (CCyR). Median age was 62 years (23-88), M/F ratio was 1.5. Median time from diagnosis and CCyR were 4 years (1-18) and 3 years (1-12) respectively. 15 patients had acquired cytogenetic abnormalities (CA) (del7: 4 patients, +8: 5 patients, del5q: 2 patients, del or +Y: 2 patients, other CA: 2 patients). Telomere length (TL) analysis was performed by Southern Blotting on polymorphonucleates (PMN) and on monocyte-depleted PBMC (MD-PBMC) to monitor both the myeloid and lymphoid compartments. As control group we analyzed 76 age-matched healthy donors. Prospective follow-up monitoring of TL was performed on 56 CML patients with a median time of 22 months from the first determination (range 12–20). Results. PMN (but not MD-PBMC) from CML patients showed a major erosion of their telomeric DNA (median loss 1294 bp p

Description: Abstract 2196 Poster Board II-173 BACKGROUND: Imatinib (IM) 400 mg daily is the standard treatment for Chronic Myeloid Leukemia (CML) in early chronic phase (ECP). The European LeukemiaNet (ELN) recommendations were designed to help identify ECP CML patients responding poorly to front-line IM, suggesting, at given time points, when the treatment strategy should be changed (”failure”), or when “the long-term outcome of the treatment would not likely be as favourable” (“suboptimal response”). Suboptimal response is a “grey zone”: the patient may still have substantial benefit from continuing IM, but other therapies should be considered. AIM: To assess the outcome of “failure” and “suboptimal responders” Philadelphia-positive (Ph+) CML patients in a large multicentric, nationwide experience. METHODS: Between January 2004 and April 2007, 559 patients were enrolled in an observational study and in 2 independent intervention studies of the GIMEMA CML WP (Clin Trials Gov. NCT00514488 and NCT00510926). Response monitoring was based on conventional cytogenetic examination of bone marrow cell metaphases every 6 months and RT Q-PCR evaluations of blood cells after 3, 6, 12 months, and every 6 months thereafter. Definitions: major molecular response (MMR): BCR-ABL/ABL ratio 〈 0,1%IS; failure (according to ELN criteria): no hematologic response (HR) at 3 months, no complete HR (CHR) at 6 months, no cytogenetic response (CgR) at 6 months, no partial CgR (PCgR) at 1 year, no complete CgR (CCgR) at 18 months, loss CHR or CCgR, progression or death; suboptimal response (according to ELN criteria): no CHR at 3 months, no PCgR at 6 months, no CCgR at 12 months, no MMR at 18 months ; optimal response: non-suboptimal and non-failure at each time-point; event: failure or treatment discontinuation for any reason. All the calculations have been made according to the intention-to-treat principle. RESULTS: The patients who fitted the ELN criteria for failure had a significantly lower probability of subsequently achieving a CCgR and a MMR, and had a significantly lower overall survival (OS), failure-free survival (FFS) and event-free survival (EFS). The patients who fitted the ELN definitions of suboptimal response at 6 months (data not shown) and at 12 months (figure 1) had a significantly lower probability than “optimal” responders of subsequently achieving a CCgR and a MMR, and a significantly poorer FFS and EFS (figure 1), while the OS was not different in the two groups (90% and 95%, p= 0.35). CONCLUSIONS Our data confirms that suboptimal responders at 6 and at 12 months have a poorer outcome with respect to “optimal” responders, comparable to the outcome of failure patients. Acknowledgments: European LeukemiaNet, COFIN, University of Bologna and BolognAIL. Disclosures: No relevant conflicts of interest to declare.

Description: Background The outcome of 129 pregnancies, in 97 women with ET diagnosed in seventeen Italian hematological centres from June 1998 to July 2007, were retrospectively analyzed by the RIT. Diagnosis was made according to criteria (PVSG or WHO) in use at the time of first observation. The median age was 27 years (range 17–45) at ET diagnosis and 32.5 years (range 21–45) at the beginning of the pregnancy. The diagnosis of ET was established in 87 patients before the first pregnancy (median interval 27 months, range 1–96), in 9 patients during a pregnancy and in one patient during an in vitro fertilization treatment. JAK2 (V617F) mutation was documented in 16 out of the 33 women studied till now. Platelet count ranged from 489 to 2,140 × 109/L (median 867) at diagnosis, and from 232 to 2,000 × 109/L (median 564) at delivery. Results: Only 120 out of the 129 pregnancies are valuable (six voluntary abortions and three ongoing pregnancies are not considered).The pregnancies outcome was: live birth in 90 cases (75%), spontaneous abortion in 26 cases (22%) (19 in the first trimester and 7 in the second trimester), still-birth in 4 cases (3%). Twelve (10%) premature births were reported, and two pregnancies ending in live birth were complicated by eclampsia or pre-eclampsia. The foetal growth was within normal limits in 76 out of the 78 full term births. The delivery was by caesarean section in 40% of cases. Thirty-six pregnancies (30%) occurred during cytoreductive treatment, but the outcome was not different than in the other ET patients (p 0.49). Aspirin treatment (mainly 100 mg/ day) was reported in 92 pregnancies (76.6%), associated in 18 cases to prophylactic LMWH during the last week before delivery and six weeks post-partum. The rate of successful pregnancy in the group receiving ASA was not different respect to the group not receiving ASA (p 0.62,). JAK2 mutation was not significantly (p 0.08) associated with a higher risk of foetal loss, but the analysis has to be completed since the study is still ongoing. The IFN treatment (as single agent or combined with ASA) was performed in 21 high-risk pregnancies. Nineteen of these pregnancies were valuable (1 ongoing and 1 elective abortion): 18 pregnancies (95%) ended in live births, and this outcome was significantly better than in patients not receiving IFN (p 0.04). Conclusion This retrospective study shows that in ET women: the IFN therapy seems able to protect against fetal loss; low-dose aspirin has not a statistically significant favourable impact on the pregnancy outcome; the unfavourable effect of JAK2 mutation could be confirmed after completion of the study. A prospective study has been recently activated by the RIT (GIMEMA project), with aim to improve the diagnostic and therapeutic approach of pregnant ET.

Description: Resistance to imatinib in Philadelphia-positive (Ph+) leukemia patients is often associated with selection of point mutations in the Bcr-Abl kinase domain (KD). Dasatinib and nilotinib are second-generation tyrosine kinase inhibitors (TKIs) with different binding modes with respect to imatinib, that have been shown to confer in vitro and in vivo activity against many Bcr-Abl mutated forms. However, both dasatinib and nilotinib have been shown to retain some ‘Achilles heels’, and they include both imatinib-resistant mutations (e.g., T315I) and some novel, inhibitor-specific ones. Selection of either type of KD mutations has frequently been observed in patients (pts) who relapse after an initial response to dasatinib or nilotinib and represents one of the major hurdles on the road to successful treatment of imatinib-resistant pts. We have monitored Abl KD mutation status in a total of 121 pts who received dasatinib (n= 78) or nilotinib (n=43) as 2nd TKI after imatinib failure since February 2005. Fifty-eight (48%) pts had chronic phase (CP) chronic myelogenous leukemia (CML), 63 pts (52%) had accelerated phase (AP) or blast crisis (BC) CML or Ph+ acute lymphoblastic leukemia (ALL). Median age was 55 years (range, 18–76); median time from diagnosis was 49 months (range, 4–181); median time on imatinib was 32 months (range, 4–66). Median follow-up of all pts who received a 2nd TKI is 7 months (range, 1–38). Median follow-up of pts who are still on 2nd TKI treatment is 32 months (range, 28–38). Relapses after an initial response have so far been observed in 46/121 pts. Thirty-eight out of these 46 pts had AP/BC CML or Ph+ ALL at the time 2nd TKI was started. Forty-one out of 121 (34%) pts have experienced relapse after an initial response during the first 12 months of 2nd TKI treatment (median time to relapse, 6,5 months; range 4–12 months), while only five of the 45 (11%) pts who were still on 2nd TKI treatment after 〉12 months have relapsed (at 13, 15, 18, 20 and 33 months, respectively). Interestingly, none of these 5 pts had never achieved more than a minor cytogenetic response (CgR), and 4/5 pts were receiving a reduced TKI dose because of toxicity. In 36/46 (78%) cases, relapse was associated with newly acquired Abl KD mutations. In particular 26/30 (87%) pts who relapsed on dasatinib and 10/16 (63%) pts who relapsed on nilotinib had evidence of a newly acquired KD mutation presumably responsible for treatment failure. Newly acquired mutations in pts who relapsed on dasatinib as 2nd TKI were T315I (n= 12 pts) F317L (n= 8 pts) T315A (n=3 pts); V299L (n=3 pts); F317I (n=2 pts); 2 pts had multiple mutations. Newly acquired mutations in pts who relapsed on nilotinib as 2nd TKI were E255K (n=3); E255V (n=2); Y253H (n=2); T315I (n=1); F359V (n=1); F359C (n=1). Sixteen pts (but none of those harboring the T315I) switched to dasatinib or nilotinib or high-dose imatinib as 3rd TKI and this rescued hematologic or even cytogenetic responses in a proportion of cases. Our observations suggest that: newly acquired mutations leading to relapse in Ph+ leukemia pts receiving dasatinib or nilotinib as 2nd TKI usually arise rapidly; the likelihood of mutation selection consistently decreases over time, and seems mainly confined to advanced phase pts and to pts with no or minor CgR; almost all (87%) cases who developed resistance to dasatinib had newly acquired KD mutations - suggesting that the higher potency with respect to imatinib can overcome Bcr-Abl gene amplification and that Src kinase inhibition may turn off Bcr- Abl-independent resistance mechanisms; a lower incidence (63%) of newly acquired KD mutations was observed in pts who developed resistance to nilotinib; with the exception of T315I, there is little if no overlap between dasatinib and nilotinib-resistant mutants, which may allow to regain responses by switching TKIs.

Description: Abstract 2164 Poster Board II-141 Background and Aims. Little is known on the functional and genetic integrity of Ph-negative hematopoietic cells (HC) repopulating the bone marrow after successful chronic myeloid leukemia (CML) treatment, although the frequent detection of cytogenetic abnormalities (CA), reminiscent of those seen in myelodysplastic syndromes (MDS), suggests the presence of functional and genetic defects. Telomere attrition represents a useful marker of proliferative and oxidative stress and might provide useful insights to monitor the genetic integrity of the hematopoietic compartment. This approach has been used in combination with a functional study of short and long term progenitors. Patients and methods. We investigated 78 CML patients with persistent (〉12 months) complete cytogenetic remission (CCR). Median age was 64 (23-88), M/F ratio was 1.5. Median time from diagnosis and CCR were 64 months (25-915) and 39 months (12-150) respectively. Sokal score was low in 36 patients, intermediate in 28, and high in 14. Six patients were received IFN only, 45 Imatinib only, while 27 were currently on Imatinib, but received previous treatment with INF and/or chemotherapy. Complete and partial molecular responders were 35 and 28 respectively. Fifteen patients had acquired CA (del7: 4 patients, + 8: 5 patients, del5q: 2 patients, del or +Y: 2 patients, and 2 patients had other CA). Short term progenitors (CFU-GM, BFU-E CFU-Mix) and long-term culture-initiating cells (LTC-ICs)(Sutherland HJ et al Blood 1994) have been performed on 30 patients (requiring bone marrow examination for clinical purposes). Telomere length (TRF-L) analysis was performed by Southern Blotting as previously described (Ladetto M et Al, Blood 2004), both on polymorphonucleates (PMN) and on monocyte-depleted PBMC (MD-PBMC) (Rocci et al Exp Hematol 2007) to monitor both the myeloid and lymphoid compartment. Sixty four patients were assessed on repeated samples to monitor the kinetics of telomeric loss (median time 8 months, range 6-20). A control database of 109 healthy subjects has been used for comparison. Results. Ph-negative HC of CML patients were functionally impaired compared to controls, with reduced number of CFU-Mix (median 2,62 vs 4, p=0,010), CFU-GM (median 99,5 vs 181, p

Description: Imatinib (Glivec, Novartis) is a tyrosine kinase specific inhibitor used for the treatment of CML. The occurrence of cytogenetic abnormalities in Ph-negative cells emerging after suppression of the Ph-positive clone has been described; however the origin as well as the biological and clinical significance are unknown. We collected data on 32 patients through the GWP in CML registry. Bone marrow cell segregation, cell culture and morphologic features in a subgroup of these patients were studied to acquire insights into the origin of the Ph-negative clone. Patient characteristics and clinical follow up (up to 49 months) are presented together with hypothesis regarding their biological significance. The emergence of a cytogenetic abnormal clone in Ph-negative cells was evidenced in 32 patients after a median of 16.2 months after starting Imatinib. Median age was 51 years, F:M=18:14, median time from CML diagnosis 35 months. All patients but one have started Imatinib while in chronic phase and were in chronic phase at detection of the abnormal Ph-negative clone. Eight patients were treated with Imatinb at onset. At diagnosis no additional abnormalities were evidenced except for one patient which presented with the Ph and a dup(1q)(q11q21). All patients achieved a good response to Glivec with 21 complete, 5 major and 6 minor cytogenetic remissions when additional abnormalities were noticed in Ph-negative cells. The clonal cytogenetic abnormalities included +8 in 14 patients, −Y in 5 patients,, three del(20q), two del(5q) and del(7q), one −7, del(13q), t(6;7)(p24;q21), t(2;6)(p25;q23), with one patient presenting with both +8 and +21, and one three clones with +8, double +8, and double +8 and −X. Retrospective analyses of stored pellet using FISH did not evidence abnormalities in previous samples. Patients that lost cytogenetic response showed that the percentage of the Ph+ cells inversely correlated to the abnormal clone. In 7 patients the abnormal clone was not evidenced in subsequent controls, suggesting the possibility that the abnormalities could be temporary. Two patients that lost response to Glivec were treated with a second generation tyrosine kinase inhibitor Dasatinib (Sprycel, Bristol-Myers Squibb) and, at reduction of Ph+ clone, the del (7q) reappeared in one patient, while the +8 clone of the second patient was not further evidenced. FISH analyses on separated CD34+ and CD34-negative cells evidenced that the abnormal clone was present into the CD34+ compartment suggesting the stem cells involvement. Bone marrow biopsies presented with reduced cellularity, normal differential and mild dysplastic signs as documented in patients responding to Imatinib. No increased angiogenesis was evidenced. We performed cell culture on a subgroup of 6 patients demonstrating normal growth in five patients and an abnormal growth pattern in one patient with reduced CFU formation affecting BFU-Es, CFU-GM, and colony size microclusters. While a longer follow up observation and laboratory analyses are required, we remark that after 〉4 years follow up the Ph-negative abnormal clone did not tend in our patients to evolve in MDS/AML, nor it seems to be associated with CML clonal evolution and disease progression.

Description: Epidemiological, diagnostic, prognostic and therapeutical data were retrospectively obtained by the Italian Registry in over 2000 Essential Thrombocythemia (ET) patients who mainly were diagnosed according to the PVSG criteria and were treated with potentially leukemogenic drugs. The Registro Italiano Trombocitemie (RIT), that is a GIMEMA project, has been activated in order to registry italian ET patients, to improve the diagnosis appropriateness (WHO criteria), to promote the acquisition of biological data, to evaluate the compliance to the therapeutical guidelines of the Italian Society of Hematology (SIE), to monitor in particular the ET patients receiving Interferons alpha and Anagrelide, to evaluate cases of pregnancy, pediatric age and familiarity, to define the prognostic value of the biological factors as JAK2 mutation, clonality, etc, to create a network for activation of new clinical and biological studies. The RIT, co-ordinated by the Hematology Unit of Reggio Emilia, is a web based registry that beside a public area comprehends a database of italian ET patients. The data, with respect of the privacy rules, are object of validation and analysis by various RIT expert subcommittees. Eighty hematological Institutions adhered to the RIT and 801 patients have been registered since June 2005. The ET diagnosis was done according to the PVSG (90%) and WHO (10%) criteria. The patients, 492 females and 309 males, had age 70 yr (30%), and the median age was 59 years. At diagnosis the platelet count was 〉1000 ×109/L in 29% of cases (mean 932). Few patients had prior thrombosis (4%; major 2%) and prior hemorrhage (2.6%). Patients at high risk of thrombosis were 55% on considering age 〉60 yr and/or previous thrombosis and/or PLT count 〉1500 ×109/L, and 65% on considering the PLT count cut-off of 1000 ×109/L. The patients shown general thrombotic risk factors (70%), disease related symptoms (40%) and splenomegaly (25%). By applying the WHO diagnostic criteria, the picture of true ET was found in 27% of cases. Sixty pregnancies have been reported. Aspirin was administered in 72% of patients and cytoreduction was performed in 62% of them, with use of Hydroxyurea (65%), Anagrelide (10%), Interferons alpha (10%), Pipobroman (4%), Busulfan (2%). A separate analysis for patients treated with Anagrelide and Iterferons alpha is in progress. To improve the diagnostic approach, the RIT has promoted the bone marrow biopsy revision (WHO criteria) and the acquisition of the new biological parameters.

Description: Sphingolipids (SPLs), classically known as structural components of cellular membranes, are bioactive mediators of many processes. New data are available on SPLs as valid targets to induce death in leukemia cells and to overcome drug resistance. In this direction, an important role has been attributed to deregulation of sphingosine kinase 1 (SK1) that phosphorylates sphingosine (Sph) to Sphingosine-1-Phosphate (S1P). S1P in turn acts as a second messenger promoting cell survival and proliferation or as a ligand for the G-protein-coupled receptors S1P1–5, controlling physiological functions such as immunity, vasculogenesis and inflammation. SK1 is released into the cytoplasm from where, upon phosphorylation, it translocates to the plasma membrane where Sph is located. SK1 was shown to be oncogenic and growing evidences assigned it a role in solid as well as in hematological malignancies. In this study we aimed to define the role of SK1 in the growth and survival of myeloid leukemia cells and to identify target genes involved in the kinase signaling pathway. As in vitro models, cell lines representing different subtypes of myeloid leukemia were used: AR230, K562, RWLeu4, HL-60 and Eol-1. We observed a statistical correlation between the levels of SK1 expression and activity. Exposure of cells to “SK Inhibitor” (SKI, Calbiochem) caused an evident decrease of cell proliferation and viability in a time- and dose-dependent fashion, which was associated to a significant inhibition of kinase activity in all cell lines. When the in vitro effect of SKI was tested on the clonogenic potential of CD34+ cells from 3 healthy donors and 2 CML patients in chronic phase, a more effective inhibition was observed on leukemic than on normal progenitors (IC50: 3,9 and 7,5 μM respectively). Next, we focused on K562 as an in vitro model of CML. We demonstrated that SKI affects the activity but not the expression of SK1 in a time- and dose-dependent manner, and that inhibition regarded about 50% of kinase activity already after 6 hrs of treatment at the dose corresponding to the IC50, increasing up to 80% after 48 hrs. Concomitantly we observed an increase of the phosphorylated form of ERK1/2, known to phosphorylate SK1 at Ser225. Additionally, gene expression profiling of K562 exposed to SKI was investigated after 12 hrs of treatment: supervised analysis identified 11 genes down- and 99 genes up-regulated and functional analysis indicated involvement in protein biosynthesis, transcription regulation and cell cycle progression control. Finally, we tested the effect of Imatinib Mesylate (IM) on SK1. Treatment of K562 with IM at the IC50 for 48 hrs reduced SK1 activity compared to untreated cells, with no changes in kinase expression. Moreover, when K562 cells were exposed to the combination of IM and SKI, a strong synergistic effect was observed after 24 hrs, when cell viability was about 48% of control. We conclude that SK1 does have a role in the survival and proliferation of myeloid leukemia cells and that pharmacological inhibition of SK1 represents a possible novel strategy for the treatment of leukemias. Our results suggest that there might be a functional link between the Bcr/Abl and SPLs pathways, and support further investigations on possible treatment based on the combination of IM and SKI.

Description: Point mutations in the kinase domain (KD) of the Bcr-Abl gene are generally regarded as the most frequent mechanism of resistance to the tyrosine kinase inhibitor (TKI) imatinib mesylate (IM) in patients (pts) with chronic myeloid leukemia (CML). Nearly all studies, however, have focused mainly on pts with advanced disease, where resistance is most often observed. Nowadays, the great majority of pts on IM are early chronic phase (ECP) pts receiving IM as front-line treatment. If, on one hand, the IRIS study demonstrated that response rates are high and relapse is infrequent in ECP, on the other hand we still know very little on the contribution of KD mutations to resistance in this subset of pts. Between January 2005 and July 2007 we analyzed for the presence of Abl KD mutations one hundred and two ECP pts on IM who were referred to our laboratory because their response was defined either as ‘failure’ (n=70 pts) or as ‘suboptimal’ (n=32 pts) according to recently published recommendations (Baccarani et al, Blood 2006). Twenty mutations were detected in 17/70 (24%) pts who failed IM. In particular, mutations were observed in 1/2 pts who showed no hematologic response (HR) at 3 months, 1/10 (10%) pts who showed less than partial cytogenetic response (PCgR) at 12 months, 4/25 (16%) pts who showed less than complete cytogenetic response (CCgR) at 18 months, 6/23 (26%) pts who lost CCgR, 5/10 (50%) pts who lost HR. Mutations were M244V (n=2), G250E (n=1), Y253H (n=4), E255K (n=1), T277A (n=1), E279K (n=1), F311I (n=1), T315I (n=1), M351T (n=3), E355D (n=1), F359V (n=1), H396R (n=3). In 7 pts who progressed to accelerated or blastic phase shortly after, four had mutations: Y253H (n=2 pts), E255K (n=1 pt) and T315I (n=1 pt). Four mutations were detected in 4/32 (13%) pts who had a suboptimal response to IM. In particular, a mutation was observed in 1/11 (9%) pts who showed less than PCgR at 6 months and in 3/21 (14%) pts who showed less than CCgR at 12 months. Mutations were E255K, F317L, M351T, F359V. In both groups no correlation was observed between likelihood of mutation selection and Sokal risk score. We conclude that in ECP pts who receive IM as front-line treatment Abl KD mutations are not the major mechanism of drug-resistance, probably because mutations tend to accumulate during the natural course of the disease as a result of a progressively increasing genetic instability and are therefore a feature of CML clinical deterioration rather than a phenomenon observed only against a background of IM exposure. Our data highlight the need to find out which is the actual predominant mechanism(s) of resistance acting in the setting of ECP - which now gathers the overwhelming majority of CML pts on IM therapy - as a mandatory step towards the development of effective second-line treatment strategies.

Description: Angiogenesis is a dynamic process regulated by a number of pro- and anti-angiogenic molecules. The most important pro-angiogenic factor is vascular endothelial growth factor (VEGF), which is capable of stimulating mitogenic activity and the proliferation of endothelial cells, increasing vascular permeability, and inducing vasodilation. VEGF carries out many of its functions by means of the VEGF receptor 1 (VEGFR-1) and receptor 2 (VEGFR-2). Increased microvessel density (MVD) has been demonstrated in many haematological malignancies, including Philadelphia chromosome-negative chronic myeloproliferative disorders (Ph−CMPDs). Recently, we firstly reported a direct correlation between VEGF immunohistochemical expression and MVD in the bone marrow biopsies (BMBs) of Ph−CMPDs patients. Increased protein and RNA expression of VEGFR-1 and VEGFR-2 have been documented in acute myeloid leukemia and myelodysplastic syndrome but, at present, no data have been reported on their expression in Ph−CMPDs. In this study we examined MVD and the immunohistochemical expression of VEGF, VEGFR-1 and VEGFR-2 in BMBs of a large series of Ph−CMPDs. The study population included 83 consecutive patients (52 males, 31 females, M/F=1.6/1; median age 61 years, range: 18–85), 27 affected by essential thrombocythemia (ET), 21 by polycythemia vera (PV) and 35 by chronic idiopathic myelofibrosis (CIMF), according to the WHO classification. The normal controls (NCs) consisted of 10 BMBs obtained for staging purposes which were free of neoplasias and other abnormalities. MVD was analysed using the “hot-spot” method with the anti-CD34 (QB-END/10) antibody. Immunohistochemical VEGF and VEGFR-1 immunoreactivity was expressed as index, based on the formula [n (i) = (% of BMB cellularity x % n-positive cells)/104]. VEGFR-1(i) resulted significantly higher in Ph−CMPDs than in NCs (0.25 ± 0.17 vs 0.07± 0.03; p=0.001) and, in particular, in PV (0.30±0.19) and CIMF (0.31±0.17) (Bonferroni’s LSD test; p