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CMPDs are heterogeneous hematopoietic stem cell disorders caused by the constitutive activation of specific TK genes induced by mutations or, exceptionally, chromosomal translocations that often escape conventional cytogenetics (CC) identification as they often involve poorly stained regions. The demonstration of these TK gene rearrangements is twofold relevant: diagnostically, as these translocations identify specific entities within the 2016 WHO classification of CMPDs and clinically, as most TK can be targeted with specific TK inhibitors. Thus, this study employed FISH probes specific for TK genes to establish the incidence of these chromosomal translocations, to identify uncommon TK translocation partners, to establish whether eosinophils are part of the clonal cell population and to find any potential correlation with clinical parameters and outcome. From January 2005-December 2015 43 consecutive patients (pts) were analysed; 13 females and 31 males with a median age of 47 years (range 22-72). According to WHO classification, 20 pts were diagnosed as atypical chronic myeloid leukemia (aCML), 22 as chronic eosinophilic leukemia (CEL) and one as AML/T lymphoblastic lymphoma (T-LL). Median follow-up was 39 months (range 8-136). At the time of the study one pt died and one experienced disease progression. FISH probes were obtained from Kreatech (Amsterdam, NL), Abbot Molecular Inc. (Chicago, Il, USA) and from BACPAC Resources Center at C.H.O.R.I. (Oakland, USA) after determining their Mb position using UCSC genome browser on Human Feb. 2009 assembly. The commercial probes, applied according to manufacturer's guidelines were: ON FIP1L1-CHIC2-PDGFRA (4q12) Del, Break; ON PDGFRB (5q33) Break; ON FGFR1 (8p12) Break; ON JAK2 (9p24) Break; LSI BCRABL. The BAC probes RP11-484L21 and RP11-880I16 covering the PCM1 gene were labelled and applied as previously described. i-FISH, cut-off values were obtained from the analysis of 300 nuclei from ten normal samples and were fixed at 10%. An abnormal FISH pattern was revealed in 12 pts (27.9%): 5/20 (25%) with aCML, 6/22 (27.2%) with CEL and one with AML/T-LL. Two aCML pts presented a trisomy 8, one a t(9;13)(q34;q14) which involved the ABL gene and a not yet identified partner gene, one at(9;22)(p24;q11) which involved the ABL and BCR genes and one a t(8;22)(p11;q11) which involved the FGFR1 gene. Interestingly, after one month this last pt progressed to AML and on CC showed a duplication of the derivative chromosome 8. Two CEL pts showed a JAK2 rearrangement: one who carried a t(8;9)(p22;p24) on CC displayed the classical PCM1-JAK2 gene fusion, the other who carried a t(3;8)(?;p24) not revealed by CC harboured a fusion between the JAK2 gene and a not yet identified partner. Three additional CEL pts showed a PDGFRB rearrangement which escaped CC detection too. In these pts who on CC showed a t(1;5)(?;q33) with loss of the reciprocal translocation product, a t(5;8)(q33;?) and a t(5;12)(q33;?), the PDGFRB translocation partner has been not yet identified. The last chromosomally normal CEL pt showed a PDGFRA deletion. Thus, in 4 pts FISH with BAC probes is still on-going in order to search the unknown translocation partners of the JAK2 and PDGFRB genes. Noteworthy, despite the fact that all these pts presented a relevant peripheral eosinophilia (≈65%), FISH performed on peripheral blood smears always revealed a normal pattern. The sole AML/T-LL pt who carried a t(8;13)(p11;q12) which produced the classical FGFR1-ZNF198 gene fusion failed to respond to conventional chemotherapy and died of disease related complications. From a clinical point of view 3 aCML/CEL pts with TK rearrangements responded to TK inhibitors experiencing a haematological improvement including one complete remission (CR); the t(8;22) positive pt entered CR after induction chemotherapy. In conclusion, i) FISH effectively reveals cryptic TK translocations in about 36% of chromosomally normal CMPDs; ii) these rearrangements are more common in CELs than in aCML; iii) peripheral blood eosinophils may show a normal FISH pattern; iv) FISH can effectively be used to monitor the clonal cell population during disease outcome. Disclosures No relevant conflicts of interest to declare.

Recent evidence suggests that leukemia is not solely a cancer autonomous process, but rather a disease in which the bone marrow microenvironment, the niche, plays a crucial role too (Raaijmakers, 2011). MSCs are key component of the niche. Thus, several studies have tested whether these cells from haematological patients contain chromosomal defects identical or different from those present in leukemic cells. Based on these findings the principal aim of the present study was to evaluate whether leukemic and MSC from six AML patients shared the same cytogenetic defects after examination with three different technologies, conventional cytogenetics (CC), FISH and aCGH/SNPa. At the onset of the disease and after informed consent all the six patients were submitted to bone marrow (BM) aspiration. BM cells were submitted to CC and FISH analyses. In addition, MSC were isolated from BM cell suspension (10-15 ml) as previously described. Briefly, mononucleated cells were isolated from BM by density gradient centrifugation using Lympholyte®-H and seeded in 75 cm2 cell culture flasks at a cell density of 106 cells/cm2. Cells were cultured at 37°C, 5% CO2 in MEM-alpha medium containing 1% Penicillin/Streptomycin, 1% L-Glutamine and 10% fetal bovine serum. After 48-h adhesion, non-adherent cells were removed and culture medium replaced (Achille et al, 2011). Growth medium was changed every three days. MSCs were examined after the first passage and their phenotype was evaluated by flow cytometry. Cells were detached from culture using Tripsin-EDTA, washed twice with PBS and stained for ten minutes with the following fluorochrome-conjugated antibodies: anti-CD90-FITC, anti-CD105-PE, anti-CD14-FITC, anti-CD73-PE, anti-CD34-FITC, anti-CD80-PE, anti-CD133-APC, anti-CD31-PE and anti-CD45-APC-Alexa750. Stained cells were acquired with a Beckman Coulter Navios instrument and data analyzed with Kalooza software. The commercial FISH probes used were LSI D7S486/CEP7, LSI AMLETO from Abbot Molecular Inc. (Chicago, Il, USA) and ON c-Myc/SE8, SE10(D10Z1) from Kreatech (Amsterdam, NL). These probes were applied according to manufactures guidelines and cut-off values determined by applying a one-sided 95% confidence interval using a binomial distribution. aCGH/SNPa was carried out with the SureScan Microarray Scanner G4900DA (Agilent Technologies Inc. Santa Clara, CA). CC revealed a monosomy 7 in two patients, a del(7)(q31) in one, a trisomy 8 and a trisomy 10 in one patient each, a t(8;21)(q24,q22) translocation in the last patient. All these defects were confirmed by FISH. In order to establish whether leukemic cells and MSCs shared these same abnormalities, MSCs cultures were tested with FISH. MSC purity assessed by flow-cytometry was 50-87%. FISH revealed a normal pattern in all the cultures examined. In contrast, aCGH/SNPa revealed neither gains/losses nor LOH in four patients, a trisomy 5 in one and the LOH of a 3.8 Mb sized region located on 13q31.1 in one patient. This study, the first one that applied aCGH/SNPa to investigate the MSC chromosomal pattern, suggests that i) MSCs from chromosomally abnormal AML patients may show a normal FISH pattern, but may be either normal or contain chromosomal aberrations different from those present in leukemic cells on aCGH/SNPa analysis; ii) these defects are uncommonly seen in AML; iii) MSCs defects may flag that the leukemogenic event targets not only the hematopoietic tissue but also the stromal cell compartment, i.e. the niche; iii) aCGH/SNPa provides an in-depth view of MSC chromosomal pattern allowing the identification of potential clonal markers. Disclosures: No relevant conflicts of interest to declare.

Abstract 2833 The Janus kinase 2 (JAK2) gene is activated by point mutation (V617F) in patients with Philadelphia-negative myeloproliferative neoplasms (MPN), playing an important role in their pathogenesis. This has led to clinical trials on the use of JAK2 inhibitors in the treatment of MPN, and to the recent approval of ruxolitinib for treatment of primary myelofibrosis. JAK2 may also be activated by translocation and fusion with another gene. The t(8;9) (p21–23;p23–24), found in atypical myeloid and lymphoid neoplasms, fuses JAK2 with the Pericentriolar Material 1 (PCM1) gene, activating JAK2. A 31-year-old female patient was referred to our Department because of splenomegaly (12 cm below costal margin), anemia (11.5 g/dL), leukocytosis (WBC 21.6 × 109/L) with eosinophilia (eosinophils 3.5 × 109/L ), and thrombocytopenia (107 × 109/L ). The bone marrow biopsy was hypercellular (95%), and showed eosinophil proliferation and fibrosis. Studies of X-chromosome inactivation pattern demonstrated clonal hematopoiesis, while cytogenetic analysis revealed t(8;9)(p22;p24). There was no evidence of BCR-ABL1 fusion gene nor of PDGFRA or PDGFRB rearrangements, and a diagnosis of chronic eosinophilic leukemia, not otherwise specified (CEL, NOS) was made. The candidate genes for fusion were PCM1 on chromosome 8p22 and JAK2 on chromosome 9p24. Fluorescence in situ hybridization (FISH) on bone marrow cells with probes for PCM1 and JAK2 revealed the presence of two fused signals on der(8) and der(9), indicating the presence of a PCM1-JAK2 rearrangement. The presence of chimeric PCM1-JAK2 fusion transcript was confirmed by reverse transcription PCR (RT-PCR) in RNA from circulating granulocytes. Sanger sequencing was performed to define the fusion junctions, and this showed an in-frame fusion between PCM1 exon 36 and JAK2 exon 9. The fusion protein retains the coiled-coil domains of PCM1 and the tyrosine kinase domain of JAK2: this likely facilitates oligomerization of the PCM1-JAK2 chimera resulting in a constitutive activation of JAK2. The clinical course of patients with PCM1-JAK2-fusion-associated neoplasms is generally poor, and allogeneic stem cell transplantation represents the only curative treatment. Unfortunately our patient did not have a compatible stem cell donor. A SNP array evaluation did not detect any additional chromosomal aberration, and the PCM1-JAK2 fusion emerged as the unique genetic lesion. We therefore considered a treatment with a JAK2 inhibitor, and more specifically a compassionate use of ruxolitinib. Following approval by the local Ethics Committee and written informed consent, in July 2011 the patient started ruxolitinib at a dose of 15 mg BID. As of July 2012, this treatment is still ongoing without any adverse effect. The patient obtained a complete clinical remission with regression of anemia, leukocytosis, eosinophilia, splenomegaly and marrow fibrosis, and with restoration of polyclonal hematopoiesis. The cytogenetic response was assessed on bone marrow at 3, 6, and 12 months. The percentage of metaphases with t(8;9) showed a progressive decrease from baseline (80%) to the 12th month of treatment (30%). Similarly, FISH with the commercial probe ON JAK2 (9p24) Break (Kreatech Diagnostics, Amsterdam, The Netherlands), optimized to detect translocations involving JAK2 at region 9p24, revealed a progressive reduction in the proportion of rearranged nuclei. To monitor the amount of fusion transcript, we used quantitative PCR analysis for the PCM1-JAK2 rearrangement in RNA samples collected from granulocytes at 3, 6, 9 and 12 months. Quantitative PCR analysis showed an early reduction in the level of PCM1-JAK2 fusion transcript, followed by a plateau at about 20% of the baseline value. In conclusion, the identification of the PCM1-JAK2 fusion gene in our patient with CEL provided a molecular target for treatment with the oral JAK2 inhibitor ruxolitinib, which allowed a complete clinical remission and a considerable reduction in the PCM1-JAK2 clone size. As complete hematologic remissions are unlikely in MPN patients treated with ruxolitinib, our case may suggest that ruxolitinib is more effective in patients in whom JAK2 is activated by translocation than in those in whom it is activated by point mutation. Finally, since PCM1-JAK2-fusion-associated neoplasms have a poor prognosis, clinical trials on the use of ruxolitinib should be considered in patients with these disorders. Disclosures: Off Label Use: Ruxolitinib for treatment of PCM1-JAK2-fusion-associated chronic eosinophilic leukemia.

Abstract 4793 Very late recurrence (LR) of AML, defined as relapse after ≥5 years of complete remission (CR), has an incidence of 1.1–3% (Verma et al, 2010; Medeiros et al, 2010) and may be due to the re-growth of the original dormant leukemic clone which may have acquired additional genetic changes or to the emergence of a new leukemic clone favoured by previous drug exposure. In CML LRs after allogeneic haematopoietic stem cell transplantation (allo-HSCT) are also very rare (1.5% per year) and it is unknown whether rare BCR-ABL transcripts are true manifestations of the original leukemic clone or the results of a “benign” BCR-ABL rearrangement seen in healthy subjects (Sobrinho-Simòes et al, 2010). In order to highlight LR biology, most studies have focused on alterations of cellular dormancy, whereas very few on the disruption of bone marrow (BM) microenvironment. The present study, which reports three patients (two AML and one CML) who experienced LR 5–16 years after CR achievement, was aimed at establishing the incidence of LR, the origin of the leukemic clone, and a potential association between LR and infective episodes. In our AML series the incidence of LR was 0.8% (2/245). The first patient, a 23 year-old male who carried a t(1;3)(p36;q26), was diagnosed as AML M5 and achieved a I CR after four courses of Etoposide (VP16) followed by one course of Daunorubicine (DNR) and a 2-years maintenance with VP16. He remained in CR for eight years when he developed a BM relapse after a viral infection. The leukemic clone presented the same chromosomal defect revealed at the onset of the disease. A II CR was achieved after a re-induction with VP16+Mitoxantrone (M). Two courses of this same regimen were used as consolidation and were followed by an auto-HSCT preceded by a conditioning with BCNU-VP16-Ara-C. Fifteen months later a second relapse occurred. Leukemic cells presented the same original t(1;3)(p36;q26). The patient did not respond to various chemotherapy schedules and died of sepsis. The second patient, a 46 year-old male who carried a t(3;11)(q26;q23), was diagnosed as AML M5 and achieved a I CR after three courses of DNR+Ara-C. Subsequently, he received a HSCT from his daughter after a conditioning with Busulfan+Cyclophosphamide (BU+CY). Take occurred at day +14 without any sign of acute GvHD, but on day +183 the patient developed a chronic extensive GvHD. Two months post-transplant FISH with sex chromosome probes (XY-FISH) revealed 0.5% of male cells, but from day +100 onwards it revealed a complete chimera. Five years later the patient complained of fever and a chest X-ray showed a left pulmonary infection. A BM biopsy revealed the almost exclusive presence of leukemic cells which karyotype was 50,XY,t(3;11)(q26;q23),+4,+8,t(12;?)(p21;?),-14,+mar1,+mar2, +mar3[13]. XY-FISH showed 60% of male cells. The patient died of sepsis. The CML patient was the only one who relapsed 16 years after a sibling allo-HSCT in I chronic phase. HSCT was performed as the patient was intolerant to alpha-interferon which had been replaced by hydroxyurea. Pre-transplant conditioning consisted of BU+CY, take occurred at day +17 without any sign of acute GvHD, but on day +329 the patient developed a chronic limited GvHD. On day +100 XY-FISH revealed a complete chimera, a chromosomal analysis a male karyotype and a BM nested RT PCR the absence of BCR-ABL transcripts. Subsequent analyses including quantitative RT PCR confirmed these findings. However, 16 years post-transplant the patient complained of otitis and fever. Her blood count showed: Hb 11.6g/dl, WBC 8.1×109/l with 22% myelolasts, Plts 5×109/L. A BM aspiration revealed the almost exclusive presence of CD34+, TdT+, CD10+ lymphoblasts having the following karyotype: 46,XY[4]/45,X,t(9;14;22)(q34;p10;q11)[6]. A XY-FISH showed 27% of female cells and a quantitative RT PCR a BCR-ABL/ABL ratio =214,2 (International scale). Tyrosine kinase inhibitors were immediately started. In our series LR was i) a very rare event, ii) always due to the re-growth of the original leukemic clone, iii) always preceded by an infective episode which might have interrupted leukemic cells dormancy through a transient angiogenic burst within the BM microenvironment (Indraccolo et al, 2006). This suggestion is further strengthened by recent data which support a strict interaction between cancer cell metabolism and niche metabolism (Yusuf et al, 2012). Disclosures: No relevant conflicts of interest to declare.

Abstract 4867 Background: A monosomal karytype (MK) is defined by the presence of at least two or more distinct autosomal monosomies or an autosomal monosomy along with a structural defect. In AML this cytogenetic pattern has a very well-known poor prognostic significance independently of the specific chromosome involved. Currently, in MDS this negative prognostic influence is also emerging as recent data suggest that any monosomy in a complex karyotype (≥3 abnormalities) may have the same poor prognostic impact as monosomy 5 and 7 (−5,−7). Objectives: Thus, the principal goal of the present study was to test whether a MK further worsen the already poor prognostic influence of a complex karyotype and to establish whether autosomal monosomies have the same unfavourable prognostic impact on OS and progression free interval (PFI) as −5/−7. Methods: The eighty-five consecutive MDS patients with a complex karyotype analysed by the present study were included in a series of 631 patients who came at our observation in the period January 2000-December 2010. They were thirty-two females and fifty-three males with a median age of 65 years (range 25–85). Fifty-five patients were diagnosed as MDS and were subdivided in 3 RARS, 6 RA, 6 RCMD, 2 RCMDS, one MDS-u, 13 RAEB-1 and 24 RAEB-2. The IPSS score was intermediate-1 in 5, intermediate-2 in 23 and high in 27. During the follow-up 31 MDS patients died and 41 experienced disease progression (3 RARS, 5 RA, 4 RCMD, one MDS-u, 9 RAEB-1 and 19 RAEB-2). Thirty patients were diagnosed as AML evolved from MDS. Fifteen of them received supportive treatment only, the remaining single agent chemotherapy to control leukocytosis. Nineteen of these thirty patients died of disease related complications. Results: On conventional cytogenetics 37 patients (4 RA, 5 RCMD, one MDS-u, 8 RAEB-1, 12 RAEB-2 and 7 AML) presented a complex karyotype without monosomies and 48 (3 RARS, 2 RA, 2 RCMDS, one RCMD, 5 RAEB-1, 12 RAEB-2, 24 AML) a complex karyotype with monosomies. These two patients subgroups were comparable in terms of age, sex distribution, haemoglobin level, leukocyte or platelet counts, bone marrow blast cell percentage and IPSS score. However, median survival was 8 months (range 1–131) for patients with a complex karyotype without monosomies and 5 months (range 1–81) for those with a complex karyotype with monosomies (p=0.001). Twenty patients (54.0%) without monosomies died after a median time of 6 months (range 2–35), whereas 30 patients (62.5%) with monosomies died after a median time of 5 months (range 1–24). Disease progression was observed in 22 (59.4%) and 19 (39.5%) patients respectively (p=0.001). The 48 patients with a MK were further subdivided in those with −5/−7 versus those with other autosomal monosomies. The 23 patients with −5/−7 presented a median survival of 4 months (range 1–15) and the 25 with other monosomies presented a median survival of 5 months (range 1–81) (p=Not Significant). Fourteen −5/−7 patients died after a median time of 4 months (range 1–15) and 13 patients with autosomal monosomies died after a median time of 6 months (range 1–24). Disease progression occurred in 12 (52.1%) and 7 (28%) respectively. Conclusions: i) a MK further refines the prognostic stratification of MDS with a complex karyotype as it identifies a subgroup of patients with an extremely poor clinical outcome; ii) autosomal monosomies have an impact on disease outcome as detrimental as −5/−7. Disclosures: No relevant conflicts of interest to declare.

Abstract 2411 In B-CLL the precise definition of the chromosomal pattern has always been a very difficult goal as leukemic cells poorly respond to the traditionally used set of mitogens. Thus, conventional cytogenetic studies (CC) have always been replaced by interphase FISH (iFISH) analyses. However, recent studies have demonstrated that the CpG oligonucleotide plus interleukin-2 (ODN+IL-2) stimulation can effectively increase the detection rate of chromosomal abnormalities by inducing metaphase spreads from B-CLL leukemic cells. Based on these data, we decided to compare the results obtained by the ODN+IL2 combination, the pokeweed mitogen (PKWM) stimulation and iFISH in ninety-one B-CLL patients diagnosed at our Institution between January 2007 and July 2010. In addition, we evaluated any possible correlation with other prognostic markers and clinical parameters. There were thirty-eight females and fifty-three males with a median age of 64 years (range 42–83). According to Binet, sixty-three patients (69.2%) were considered as stage A, seventeen (18.7%) as stage B and eleven (12.1%) as stage C. PKWM did not provide any mitotic figure in thirteen patients (14.2%) and detected clonal defects in sixteen patients (17.5%). In contrast, the ODN+IL-2 combination was unable to provide metaphase spreads in three patients only (3.3%) and revealed clonal abnormalities in fifty-six patients (61.5%). In eight patients both cultures revealed an equal percentage of cells carrying the same abnormality. In addition, the ODN+IL-2 combination revealed a complex karyotype (≥ three defects) in twelve patients (13.2%), two chromosomal defects in fourteen patients (15.4%)(including five with band 17p13 rearrangement and three with band 11q13 rearrangement) and a single chromosomal defect in thirty patients (32.9%). Fifteen patients harboured various chromosomal rearrangements (16.4%). iFISH was carried out with the B-CLL FISH probe panel (Vysis, Downers Grove, IL, USA) and revealed clonal abnormalities in sixty-two patients (68.1%). The most common defects were: 13q-, +12, 11q- and 17p- having an incidence of 46.1%, 14.2%, 7.7% and 6.5%. Three patients with a normal FISH pattern presented a +12 when analysed with the ODN+IL-2 combination and five, who showed the loss of one p53 signal on iFISH, presented a structural 17p13 defect when investigated with the ODN+IL2 combination. In addition, considering the forty-two patients who harboured a 13q- on iFISH analyses, the ODN+IL2 combination revealed that in five patients this defect was included in a complex karyotype. From a clinical point of view, considering the fifteen patients with various chromosomal rearrangements nine were classified as stage A, four as stage B and two as stage C and considering the twelve patients with a complex karyotype three were considered as stage B and nine as stage C. In conclusion, the ODN+IL2 combination i) allows a precise definition of the chromosomal pattern in B-CLL patients and in our series identified clonal defects in 61% of patients; ii) reveals minor +12 clonal cell populations which may evade iFISH identification; iii) does not always identify a 13q deletion because of the sub-microscopic nature of this chromosomal defect; iv) reveals that the loss of one p53 signal on iFISH analyses is frequently due to an unbalanced rearrangement; iv) is the only technique which allows the identification of complex karyotypes and/or chromosomal translocations both associated with an advanced Binet stage. Thus, the ODN+IL2 combination should be used in conjunction with iFISH to achieve a more accurate karyotype definition in B-CLL patients. Disclosures: No relevant conflicts of interest to declare.

Abstract 4478 Introduction: Imatinib mesylate (IM) therapy is effective in patients with chronic myeloid leukemia (CML). However, its discontinuation or dose variation in patients who experience sustained molecular response is debated. The possibility of treating patients with an intermittent therapy could also be applied to the second-generation TK-inhibitors. We describe our single institute experience in patients with undetectable levels or in major molecular response of BCR-ABL transcript who reduced or were discontinued from IM therapy. We applied a model for the analysis of longitudinal data to study the BCR/ABL variation according to dose change or discontinuation. Methods: One hundred forty CML patients came to our observation between 1985 and 2009. Among these, 89 patients in chronic phase were eventually treated with IM. Fifty-five patients were treated with IM as naive patients. Each patient's treatment history was subdivided into time periods at constant dosage. Fifty-nine patients were followed-up for a total of 288 periods at constant dosage. At the end of each period, cytogenetic and/or molecular responses were evaluated. Thirty-eight progressions were recorded: 22 molecular, 6 cytogenetic and 10 of both types. The association between progression (molecular, cytogenetic or either) and treatment dose was assessed with the aid of generalized estimating equations (GEE) models, i.e. regression models designed to account for correlation due to repeated measurements over time on the same subject. Ten patients discontinued IM therapy for a period which ranged from three to 60 months, after the patient's individual request and informed consent. Results: We found no association between dose and progression, not even after accounting for period length. Discontinuation of treatment was not associated to an increased risk of progression. No association with a higher risk of progression was found for periods at reduced dosage ( Conclusions: It is unclear whether IM can “cure” chronic myeloid leukemia but according to our data this therapy can be safely stopped or its dose varied in patients with complete cytogenetic and major molecular response of up to 18 months. Our experience suggests that withdrawal of IM therapy in CP-CML patients after achievement of a complete molecular response may result in divergent molecular outcomes. The prompt improvement seen after the restart of therapy argues against the development of resistance. The selection of resistant clones after IM exposure, and the emergence of Ph negative clones with secondary cytogenetic abnormalities, are matters of concern, particularly in patients receiving long-term IM. The improved quality of life while off therapy, and the prompt response to restart of IM therapy, suggest that the subset of patients who have sustained complete molecular response may be candidates for a tailored approach to intermittent therapy. We suggest that the same statistical analysis can also be used for other TK-inhibitors that are under study for both retrospective or prospective trials in CML. Disclosures: No relevant conflicts of interest to declare.

Based on conventional cytogenetic studies, acquired losses of the whole or a part of chromosome 5, alone or in a complex karyotype (defined by the presence of ≥3 chromosome defects), are recurrent cyogenetic defects in MDS and AML. The size of the deleted material and the breakpoints vary among patients. Commonly deleted regions are bands 5q13, 5q31-q32, and 5q33-q34-q35. Monosomy 5 (−5), too, has always been considered a non-random chromosomal defect. However, recent evidence is changing this assumption since most patients with −5 on CC studies present a chromosome 5 fragmentation in more than two segments on FISH investigations (Herry et al, 2007). The present FISH study was aimed at establishing the incidence of true −5 and at refining chromosome 5 abnormalities in 9 patients, one MDS, classified as refractory anemia with excess of blasts type 1 and 8 AML (2 M2, 5 M4 and one M5). Monosomy 5 was the only karyotype defect in 2 patients and part of a complex karyotype in the remaining 7. CC and FISH studies were performed as already reported (Bernasconi et al, 2007). FISH analyses were carried out on mitotic figures with the following probes: the LSI CSF1R/D5S721:D5S23, mapped at 5q33 (spectrum orange) and 5p (spectrum green) from Vysis (Abbott Molecular/Vysis, North Chicago, IL, USA) and the WCP5SO probes from QBiogene (Qbiogene Inc., Carslbad, CA, USA). Additional FISH investigations were carried out with two BAC probes exploring the 5q segment comprised between the centromere and band 5q11 in order to check whether this chromosomal region was either maintained or deleted. The WCP5SO probe demonstrated that all the 9 patients presented an apparent monosomy 5 since chromosome 5 material was contained within marker chromosomes already identified by CC. In 3 patients the LSI CSF1R/D5S721:D5S23 probe provided three green signals (5p duplication) along with one red signal (5q33 monosomy). In these patients one green/red signals were mapped on the normal chromosome 5, the other two green signals were localized on marker chromosomes. In the remaining 6 patients the same probe demonstrated one green/red signal on the normal chromosome 5 and another green signal on a marker chromosome. Subsequently all the 9 patients were investigated with the 5q11 BAC probes. Five patients showed two signals revealing that band 5q11 was maintained. Considering the 4 patients with loss of band 5q11, 3 presented a fragmentation of chromosome 5 and one an internal chromosome 5 rearrangement. In conclusion our data show that true monosomy 5 is a very uncommon event in MDS/AML since in all our patients with −5 on CC 5p was either maintained or amplified. In addition, band 5q11 was maintained in 5/9 patients. In view of these results our future goal will be to check whether other 5q regions are maintained or deleted in these complex chromosome 5 rearrangements.

The t(10,11)(p12;q23) translocation is a recurrent and rare chromosomal abnormality in AML, especially associated with the M5 cytotype. An heterogeneity in 10p breakpoints has been revealed by cytogenetic studies even if molecular analysis have demonstrated that the AF10 gene is constantly involved in this translocation. Up to now, two types of chimeric transcripts the MLL-AF10 in the t(10;11)(p12;q23) and the CALM-AF10 in the t(10;11) (p13;q14) have been isolated. In 1998 the Abl-interactor 1 (ABI1) gene was identified as a new MLL partner in the t(10;11) translocation. In addition, it was demonstrated that this chromosomal rearrangement is significantly associated with complex translocations including invins(10;11) and inv(11)t(10;11). However, a duplication of the chromosome 10 derivative has never been reported. Herein we describe an additional female patient with a complex t(10;11) rearrangement and a M2 AML. She was forty-seven years old. Her blood count was the following: haemoglobin 9,4g/dl, WBC 34,2×109/l (blast cells 88%), Plts 7.0×109/l. Induction chemotherapy determined a complete remission (CR) which lasted six months. A second CR was achieved and subsequently the patient was submitted to an allogeneic bone marrow transplant. Now she is alive and well twelve months posttransplant. On clinical diagnosis cytogenetic studies on bone marrow cells revealed the following karyotype: 46,XX/46,XX,t(10;11)(p14;q23), −10, +der(10)t(10;11). FISH studies were performed with the following probes: LSI MLL and LSI ATM from Vysis, Whole Chromosome Painting (WCP) 11 and Arm Chromosome Painting (ACP) 10p from Q-Biogene. In order to better define the rearranged chromosomal region the RP11-47P2 (mapping at 10p15.1), the RP11-79F9 (corresponding to 10p14), the RP11-13C4 and the RP11-36M5 (covering the PLZF3 gene at 11q23.2), the RP11-19S45 and the RP11-48M23 (covering the ABI1 gene at 10p12.1), the RP11-46N13 and the RP-140P12 (covering the AF10 gene at 10p12.31) BAC probes were applied. Other genes investigated with the same approach were the SLC39A12 at 10p12.33, the PLXDC2 at 10p12.33-p12.32, the ARMETL1 at 10p13 and the PIP5K2A at 10p12.31. The WCP 11 and the ACP 10p probes confirmed the t(10;11) translocation, the loss of the normal chromosome 10 and the duplication of der(10)t(10;11). When analysing chromosome 11, the ATM and the PLZF3 probes were in a normal position so neither gene was rearranged. Instead, both the signals (red and green) corresponding to MLL were translocated to chromosome 10 and split by an additional rearrangement. So, we hypothesized that the t(10;11) translocation occurred first and was followed by an additional defect. This possibility was confirmed by the presence of chromosome 10 material inserted between the two MLL signals, an event which was interpreted as either an inversion or an insertion. When analysing chromosome 10, the RP11-79F9 green probe remained in the correct position, whereas the RP11-47P2 red probe was translocated to chromosome 11. Thus, in the initial t(10;11) the breakpoints on chromosome 10 fell within the region delimited by these two BAC probes. Based on the fact that either the AF10 or the ABI1 gene, located at 10p12, are MLL partners in the usually complex t(10;11) translocations we checked whether these genes were involved in the secondary rearrangement on chromosome 10. FISH studies excluded any ABI1 rearrangement. Instead, they demonstrated that the RP11-46N13 (green) and RP-140P12 (red) BAC probes, covering the AF10 gene, were still located on 10p but separated by the chromosomal material belonging to chromosome 11 carrying the two MLL signals at its extremities. Since the two signal corresponding to the AF10 gene were located in the same position of MLL split signals a AF10/MLL translocation had occurred in our patient, who is the first one up to now reported to present a duplication of the rearranged chromosome 10.

Chromosome 3q defects, namely inv(3)(q21q26)/t(3,3)(q21;q26), are revealed in about 2.5% of AML and in rare MDS patients (pts), are associated with a normal/high platelet count, tri-lineage dysplasia, resistance to intensive chemotherapy and poor survival. In these disorders ectopic EVI1 expression is the pathogenetically relevant molecular lesion. However, several pts with 3q21q26 rearrangements lack EVI1 expression and 9% of those without 3q21q26 defects present EVI1 over-expression. In addition, FISH, which can be used as an alternative tool for or an adjunct to RT-PCR to discover any EVI1 rearrangement, revealed an important breakpoint heterogeneity within this chromosomal region. Based on the above data, FISH was used to investigate 12 pts (7 MDS and 5 AML) with 3q21q26 rearrangements on conventional cytogenetics (CC). The goals of our study were to establish the incidence of EVI1 defects and to reveal any difference in morphological features and disease outcome between pts with and without EVI1 defects. The 12 pts were 4 females and 8 males with a median age of 60 years (range 35–80). Median follow-up was 22 months (range. 2–40). According to WHO classification, 2 MDS pts were diagnosed as Refractory Cytopenia with Multilineage Dysplasia (RCMD) and 5 as Refractory Anemia with Excess of Blasts type 2 (RAEB-2). Four of these 7 pts progressed into AML. Considering the 5 AML pts, 2 were diagnosed as M2 and 3 as M4. On clinical diagnosis CC showed a standard t(3,3)(q21;q26) in 5 pts, a translocation of 3p material onto band 3q26 in 2, a 3q21 deletion in 2, a complex rearrangement of band 3q26 in one, an inv(3)(q21q26) in one and a translocation involving 3p21 and 3q21 in one. FISH, carried out on cytogenetic preparation, used BAC probes obtained from Welcome Trust Sanger Institute (Cambridge, UK). The probes applied were those of the literature (Lahortiga et al, Genes Chrom & Cancer 2004; Poppe et al, Genes Chrom & Cancer 2006) and other probes covering the RPN1, EVI1, MDS1 genes. EVI1 defects had an incidence of 50%. EVI1 gene was translocated in 4 t(3;3) pts (3 RAEB-2 and one AML), amplified along with MLL in a RAEB-2 pt and deleted in the 2 AML pts with a 3q21 deletion on CC. Interestingly, no EVI1 defect was discovered in a t(3;3) RAEB-2 pt and in an inv(3)(q21q26) RCMD pt. Tri-lineage dysplasia was observed in all MDS pts independently of any EVI1 defect. Median survival of the 5 pts with either EVI1 translocation or amplification was 6 months (range 2–32), that of the 7 pts without any EVI1 defect was 26 months (range 4–40). A progression in AML occurred in all the 3 MDS pts with EVI1 defects and in one of the 4 MDS pts without any defect. A total of 8 pts, including 3 of the 4 AML progressed from MDS, were submitted to intensive chemotherapy. A complete remission (CR) was achieved in only one of the 4 pts with EVI defects and in 3 of the 4 pts without any defect. Two CR pts, one with and one without any EVI1 defect, were submitted to allogeneic bone marrow transplantation (allo-BMT). The pt with the EVI1 translocation relapsed, but succeeded in entering a second CR. In conclusion, EVI1 defects were revealed in 50% of pts, were not required for evoking the dysplastic changes associated with 3q21q26 rearrangements, were associated with a high risk of AML evolution in MDS pts, caused a short survival and resistant AML.

Defining the incidence and the prognostic relevance of cytogenetic evolution (CE) in MDS, excluding any effect of the primary defect and other well-known prognostic variables, are the main goals of the present study which included 153 patients (pts) examined between January 1990 and December 2005. They were 73 females and 80 males with a median age of 60.5 years (inter-quartile, IQ, range:49.8-68.3). FAB, IPSS score, IPSS cytogenetic categories were applied to all pts, WHO to 142. On clinical diagnosis 94 pts presented an abnormal karyotype. Median follow-up was 45.2 months (IQ range:22.8-75.6). At the time of analyses 107pts (69.9%) are alive and 46 (30.0%) have died. A clinical progression (CP) occurred in 65 (42.4%) pts and a CE in 47 (30.7%). Median progression-free survival was 65.2 months (IQ range:16.6-not reached). When a Cox model analysed CE as a time-dependent variable predicting disease outcome, a true CE occurred in a total of 40 pts who presented a survival significantly shorter than that of pts without such an evolution (Hazard ratio, HR=7.1, p i) an incidence of 30%; ii) a significant impact on survival independently of the primary defect, FAB/WHO classifications, IPSS score and IPSS cytogenetic categories; iii) a significant impact on the risk of CP independently of the primary defect, FAB/WHO classification, IPSS score and IPSS cytogenetic categories. In addition, monosomy 7, del(7q) and del(17p) were the secondary defects significantly effecting the risk of CP.

Conventional cytogenetics (CC) discovers clonal chromosomal defects in about 40–80% of de novo MDS/AML. However, due to the poor quality of metaphases, CC is often unable to reveal cryptic defects, precisely define chromosomal breakpoints and establish the nature of marker chromosomes. All these drawbacks may be overcome by FISH, a powerful technique with high sensitivity and specificity. Abnormalities of chromosome 11 long arm and of band 11p15 are seen in 5–7% and in 0.5% of de novo MDS/AML. Herein we report two patients diagnosed as AML evolved from MDS. On CC the karyotype of the first patient was 47,XX,+3,t(15;20)(q15;p11),add(11)(p15) [20] [case 1] and that of the second patient was 45,XX,t(1;?)(q12;?),−5,−7,+8,add(11)(q23),add(12)(p13),add(17)(p13),add(21)(q22) [7]/46,XX,t(1;?)(q12;?),add(5)(q11),−7,+8,add(17)(p13),add(21)(q22) [8] [case 2]. In both the patients FISH was performed with commercial probes (LSI and TCP from Vysis and QBiogene, applied following manufacturer’s guidelines) and Bacterial Artificial Chromosome (BAC) probes (kindly provided by the Wellcome Trust Sanger Institute, Cambridge UK) in order to better define chromosome 11 rearrangements. The BAC probes were cultured in LB broth for a night and the following day the DNA was extracted after lysis. Labelling with biotin and digoxigenin was carried out by nick translation reaction. Signal detection was obtained by fluorescein avidin and anti-digoxigenin. Case one was at first investigated with a BAC probe specific for the NUP98 gene, mapped at 11p15. One signal was expected on the normal chromosome 11 and a split signal on add(11)(p15). Instead, two spots on the short arm and the other on the long arm of a marker chromosome were seen. The investigation with the Cyclin D1/CEP11 probe, mapped at 11q13.3, allowed us to establish that this marker was a number 11 and, at the same time to identify the other chromosome 11, which did not show any signal corresponding to NUP98. This same chromosome showed two signals corresponding to MLL and two other signals corresponding to ATM when these probes were applied. A TCP 11 probe did not identify any other chromosome 11 fragment. Therefore, we realized that chromosomes 11 were rearranged with each others. Additional BAC probes lead us to fix the possible breakpoint area on the chromosome 11 rearranged in the long arm in between bands q21 and q13 and on the other chromosome 11 under the NUP98 gene. Case two was at first investigated with TCP 1 and 11 probes that showed chromosome 11 material on the rearranged chromosome 1 and a fragment of chromosome 1 inserted in the middle of chromosome 11 long arm, which was not totally painted by the two probes. This datum lead us to hypothesize the involvement of a third chromosome in the rearrangement. A TCP 5 probe demonstrated that the unpainted material under der(11) belong to chromosome 5. When MLL and ATM probes were applied the former was on der(1) and the second was on der(11), under the inserted chromosome 1 fragment, but over chromosome 5 material. Therefore, the breakpoint region between der(11) and chromosome 5 material lies in between band 11q22 and 11q23. In conclusion, FISH: reveals unexpected rearrangements absolutely cryptic on CC, is a very effective tool to map chromosome breakpoints and identifies new genes involved in leukemogenesis.

Different studies with conventional cytogenetics have shown that the chromosome pattern of B-CLL patients remains rather stable during disease outcome. In contrast, other reports have demonstrated that karyotypic evolution occurs in about 15% of patients, especially in those experiencing disease evolution. The aim of the present study was to evaluate the incidence of chromosome evolution in 32 B-CLL patients and to establish whether the development of additional genetic lesions correlates with clinical progression. The 32 patients entered in the study were part of a larger group of patients (253 consecutive) observed in the period January 2002–December 2005. FISH was performed on bone marrow cells in all the 32 patients on clinical diagnosis and during the follow-up, whereas CC was carried out at the onset of the disease in 7 patients and during the follow-up in only 5 patients. From a clinical point of view, these 32 patients were 11 females and 21 males with a median age of 55 years (range 36–71). On clinical diagnosis 18 were classified as stage A, 11 as stage B and 3 as stage C. Median follow-up time from clinical diagnosis was 33.5 months (range 6–88). Nineteen patients presented a stable clinical course, whereas 13 experienced disease progression after a median follow-up of 21.5 months (range 5–83). Considering these last patients, 5 progressed in stage B, 5 in stage C and 3 in Richter’s syndrome (RS). All the 32 patients were investigated with the B-CLL probe set (α12/13q14/13q34 and p53/ATM probes from Vysis) which was applied according to manufacturer’s guidelines. The cut-off value for each probe was determined by adding three times the standard deviation to the mean percentage of cells with an abnormal pattern obtained from five normal controls. A clonal p53 deletion was considered to be present if discovered in more than 20% cells. On clinical diagnosis FISH detected a normal pattern in 12 patients, a trisomy 12 in 7 and a mono-allelic loss of the D13S319 locus in 13; CC discovered a trisomy 12 in 2/7 patients. On FISH investigation only one of the 19 patients with a stable disease developed a new genetic lesion (a mono-allelic loss of the D13S319 locus). In contrast, 7/13 patients with disease progression revealed additional genetic defects. On diagnosis one of them had presented a trisomy 12 and the remaining 6 a normal FISH pattern. On clinical evolution the patient with trisomy 12 maintained three signal corresponding to α12, but developed additional defects revealed by CC; among the other 6 chromosomally normal patients one developed a bi-allelic loss of both the D13S319 loci, 3 a deletion of the ATM gene and 2 a deletion of the p53 locus in 64% and 54% cells. CC, performed in 4 of these chromosomally normal patients, revealed a normal pattern in 3 patients (one stage B disease and 2 RS) and a del(11)(q13) in the other whom showed a deletion of one ATM locus also on FISH investigation. Therefore, none of the two patients with RS developed any defects typically associated with such a disease evolution. In conclusion, our study shows that further genetic lesions may develop during disease outcome in 25% of B-CLL patients, are associated with disease evolution, frequently target the ATM and the p53 loci. In addition, in our opinion CC still has a crucial role in every B-CLL patient experiencing disease evolution since it provides information on chromosomal regions not investigated by the FISH panel probe set.

We report two HSTCL patients who were studied with conventional cytogenetics (CC) and FISH on clinical diagnosis and during the follow-up to better understand the genetic events underlying this type of lymphoma. They were a 21-year old male and a 44-year old woman who came to our clinic for evaluation because of B-symptoms. On physical examination they presented massive splenomegaly and hepatomegaly. A peripheral blood count revealed anemia and thrombocytopenia in one patient and anemia in the other. They both presented high lactic dehydrogenase levels. A bone marrow biopsy demonstrated that a malignant T-cell population constituted 30% of all marrow cells in one patient and entirely substituted normal hemopoiesis in the other. This malignant T-cell population was CD2+,CD3+,CD7+,CD56+,CD4−,CD5−,CD8−. In addition, the T-cell clone, which showed a sinusal localization, was TCRα/β + in one case and TCRγ/δ + in the other. The two patients underwent splenectomy and it was shown that the red pulp had been completely infiltrated by a malignant cell population identical to that present in the marrow of the two patients. Therefore, a diagnosis of α/β + and γ/δ+ HSTCL in stage IVB was made and the patients started treatment. The male succeeded in entering a complete remission (CR) of only three month duration and after a bone marrow relapse was unable to achieve a second CR. The female did not respond to chemotherapy and died of disease related complications. CC and FISH studies were performed on bone marrow cells. CC discovered a normal chromosome pattern in the twenty metaphases obtained from the first patient, and an abnormal pattern in the eighteen mitotic cells obtained from the second whose karyotype was: 46,XX[7]/46,XX,i(7)(q10)[5]/47,XX,i(7)(q10),+i(7)(q10)[6]. FISH on mitotic and interphase cells was performed with the 7q31/CEP11 probe (Vysis) and the Bacterial Artificial Chromosome (BAC) probes RP11-79N1, RP11-299F5, RP11-1132K14, RP11-163M21 (kindly provided by the Wellcome Trust Sanger Institute, Cambridge UK and by the BACPAC Resources Children’s Hospital, Oakland, USA), which were localized on 7p15 and covered the HOXA cluster. The commercial probes were applied according to manufacturer’s guidelines. Hybridization procedures were carried out first with the BAC probes and subsequently with the commercial probes. On clinical diagnosis a significant cell population with the aberrant pattern (1×7p/2×7cen/3×7q) corresponding to i(7)(q10) was discovered in 25% of marrow cells from the first patient and in 90% marrow cells from the second. Interestingly, in the first patient the percentage of cells displaying this pattern equalled the percentage of cells infiltrating the marrow on morphologic examination. However, when this patient relapsed we did not succeed in identifying any cell carrying the 1×7p/2×7cen/3×7q pattern (cut-off fixed at 2%), even if the percentage of malignant T-cells infiltrating the marrow was higher than on diagnosis. In addition, CC revealed an absolutely normal chromosome pattern leading us to hypothesize that a cryptic genetic event might have occurred. In conclusion, our findings further underscore the association between i(7)(q10) and HSTCL, suggest that the number of i(7)(q10) present in the malignant cell might reduce response to treatment, lead us to hypothesize that patients who relapse might develop a second more subtle genetic lesion.

An internal tandem duplication (ITD) or a point mutation of the FLT3 is detected in about one third of MDS patients at the time of clinical progression, but very few studies have determined whether these mutations are already present on clinical diagnosis. A high FLT3 expression is caused by both these as well as by other still undefined mutations. Therefore, we have decided to analyse the expression of the FLT3 gene by RT-PCR on clinical diagnosis and during disease outcome in twenty-six MDS patients. Our study was aimed at determining whether a high FLT3 expression was correlated with any peculiar clinico-haematological parameter, clinical evolution to AML and response to treatment. Fourteen patients were males and twelve females; their median age was 60 years (range 36–76). According to FAB classification seven patients were classified as refractory anemia with ringed sideroblasts (RARS), fourteen as RA and five as refractory anemia with excess of blasts (RAEB). Conventional cytogenetic studies discovered a normal karyotype in twenty patients, a del(20q) in three, a del(5q) in two and a del(12p) in one. Blast cell percentage was 0–5% in twenty patients, 6–10% in four and 11–20% in two. According to IPSS fifteen patients were considered low-risk, eight intermediate-1 risk and three as intermediate-2 risk. FLT3 expression was evaluated through a relative real-time quantification approach which used SybrGreen I as DNA binding fluorescent dye. Total RNA from mononuclear cells from a patient, who harboured an ITD of the FLT3 gene and presented a high expression of the gene, was serially diluted in order to obtain a standard curve for real-time quantification. FLT3 expression was determined by the ΔΔCt method. FLT3 levels were normalized to ABL and calibrated on a normal sample. At the onset of the disease twenty-three patients showed a FLT3 expression similar to that of the normal control, while three (one RA and two RAEB) presented a two-four fold increase. In these last patients no correlation with any particular clinico-haematological feature was noted. Nine of the twenty-six patients progressed in AML after a median time of thirty-one months (range 8–86). Three of them had already presented an increased FLT3 expression on clinical diagnosis. Considering the remaining six patients, a three-seventeen fold increase of FLT3 expression was observed in two patients and a normal FLT3 expression in the other four. Time from MDS to AML evolution was 8,22,29,33,39 months for patients with a high FLT3 expression and 31,40,42 and 86 months for those with a normal FLT3 expression. Three of the five patients with a high FLT3 expression were given different courses of intensive chemotherapy. One of them, who never responded to chemotherapy, maintained a constantly high FLT3 expression, the other two, who achieved complete remission, showed a normalization of FLT3 expression. However both of these two responsive patients again presented a six-eight fold increase of FLT3 expression on relapse. In conclusion, a high FLT3 expression i) may be observed on clinical diagnosis in about 11,5% of MDS patients, ii) does not associate with any peculiar clinico-haematological finding, iii) frequently appears at the time of AML evolution since it was detected in two of our six patients who showed a normal FLT3 expression on clinical diagnosis but a high expression on relapse.

We studied the immunophenotypic characteristics of 459 consecutive adult AML patients and their correlation with karyotype. Immunophenotype was performed using a panel of 26 directly conjugated monoclonal antibodies. Cytogenetic analysis was performed using a standard G-banding technique. Karyotype was available in 394 patients (not done in 15, failed in 50): 1) 45 (11.4%) were t(15;17) APL patients with a mature myeloid phenotype (HLA-DR-/CD13+ and/or CD33+). CD2 and CD56 were expressed in 20% and 13.3% of cases, respectively. CD11b-positivity was less frequent than in the other cytogenetic groups. The markers significantly associated with t(15;17) were: presence of CD2 (two tailed Fisher exact test: p=.003) and CD117 (p=.01), absence of CD4dim (p90% of blasts, CD34+ in 70%, MPO+ in 95.8% and HLA-DR+ in 89.3%. The association with CD14 and TdT was of borderline statistical significance. 4) 24 (6.1%) had −5/5q- with more than 80% of blasts CD117+/CD13+>CD33+. CD34 was positive in 62.9% of cases, CD7 in 33%, CD11b in 35.7%, CD11c in 67.8% and CD14 in 7.1%. CD15-positivity was less frequent than in other AML subtypes. Univariate analysis showed a trend of positive association with CD7 and CD117. 5) 40 (10.2%) showed −7/7q-, with CD13+>CD33+ detected in more than 80%, CD7 in 33.3%, CD11b in 72.7%, CD15 in 55%, CD34 in 73.3%. Abnormal CD16/CD33 and/or CD11b/CD66b myeloid cell pattern was detected in 40% of cases (p=.001). −7/7q- were significantly associated with CD34 (p=.02) and CD7 (p=.04). 6) 41 (10.4%), had complex karyotype (≥3 abn) with a similar antigenic profile than group 4) and 5) but with a more frequent expression of CD11b, CD14, CD15 and less frequent of MPO. In univariate analysis CD19 (p=.04), CD34 (p=.02) and MPO (p99%, specificity 94.7%) and CD7/CD11c/CD19/CD56/MPO/HLA-DR (sensitivity 83.3%, specificity 94.5%), respectively. We conclude that in AML patients some cytogenetic abnormalities are associated with peculiar antigenic profiles. In patients with normal karyotype the heterogeneity of antigenic pattern may reflect underlined distinct molecular abnormalities.

In B-ALL karyotype, age, leucocyte count, immunophenotype, mediastinal mass and leukemic cells in the central nervous system (CNS) are the most important prognostic parameter. The assessment of the chromosome pattern is mandatory for planning a risk-adjusted protocol-based therapy. However, even if the quality level of conventional cytogenetic (CC) analyses has progressively improved, there is a still significant failure rate in identifying karyotype defects in B-ALL. So, due to its considerable developments FISH on interphase cells has been used in addition to CC and has become the method of choice for the detection of high-risk chromosomal changes in B-ALL. In the present study iFISH was performed in 31 adult B-ALL patients (18 females and 13 males; median age 38 years, range 16–76) who presented normal G- and Q-banded karyotypes on CC. They were part of a large series of 251 consecutive adult B-ALL patients who came to our observation in a ten years period (1994–2005). In this series CC presented a failure rate of 19.9% and detected a normal karyotype in a total of 66 patients (32%), 31 of whom had cells in fixative still available for the present iFISH study. This last, which was aimed at detecting the true incidence of the BCR-ABL, ETV6-AML1, MLL rearrangements and p16/INK4A deletion, was carried out with the following commercial probes: LSI BCR/ABL1 dual color single fusion, LSI TEL/AML1 ES, LSI MLL and LSI p16 (9p21)/CEP 9 dual color (Vysis, Downers Grove, IL, USA). Hybridization procedures were carried out according to manufacturers’ guidelines. Cut-off values were determined after having analysed two-hundred cells from ten normal controls and using a one-sided binomial distribution with a 95% confidence interval. So, the cut-off values were fixed at 10% and 6% for the BCR/ABL1 and MLL probes and at 3% for both the ETV6-AML1 and the LSI p16 (9p21)/CEP 9 probes. Overall iFISH detected chromosome defects in 13/31 (41.9%) patients. The most common abnormality, which incidence was 25.8%, consisted in the loss of either one or two red signals corresponding to the LSI p16 (9p21)/CEP 9 dual color probe, caused by a cryptic del(9)(p21) deletion. Six patients presented a monosomy and 2 a nullisomy of the p16/INK4A locus. The ETV6-AML1 rearrangement was detected in no patient. In contrast, the amplification of the AML1 gene and the loss of one ETV6 gene were seen in 2 patients each. Both these defects were present in a small percentage of cells (ranging from 11% to 15%) which had escaped CC identification. A monosomy and an amplification of the MLL gene were observed in one patient each, one of whom with an already documented AML1 amplification. A cryptic BCR-ABL rearrangement was discovered in no patient. In conclusion our data suggest that i) in B-ALL iFISH plays a pivotal role in the accurate definition of karyotype since it readily discovered genetic aberrations in about 40% of our adult patients with an apparently normal karyotype, ii) the loss of either one or two p16/INK4a genes, frequently seen in T-ALL, seems to be one of the commonest defect also in adult B-ALL, iii) the ETV6-AML1 and MLL rearrangements are extremely rare in adult B-ALL, iii) since iFISH did not detect the BCR-ABL translocation in any of our chromosomally normal patients, CC seems to be effective in identifying Ph1+ B-ALLs.

In acute leukemias gene amplification occurs with incidence of 1–10%. On conventional cytogenetic (CC) studies it is usually seen either intrachromosomally as homogeneously staining regions (HSRs) or extrachromosomally as double minute chromosomes (dmins). Fluorescence in Situ Hybridization (FISH) has established that the genes most frequently amplified in ALL are AML1 and MLL. Recently, the Leukaemia Research Fund UK Cancer Cytogenetic Group has detected the amplification of the ABL gene in 5/210 childhood and in 3/70 adult T-ALL and has suggested that this genetic abnormality might identify patients with a generally poor event-free survival (EFS). The present study was aimed at determining the incidence and clinical significance of ABL amplification in a series of 31 consecutive adult T-ALL patients. All of them had been submitted to routine FISH screening for BCR/ABL and TEL/AML1 fusions and for MLL amplification. ABL amplification was detected by chance in two patients (6.4%). In one CC did not yield analysable metaphases and in the other it showed the following karyotype: 46,XX/46,XX,t(1;3)(p34;p21),del(6)(q21),del(7)(q32). FISH with a painting probe specific for chromosome 9 detected an occult trisomy in the patient without analysable mitosis and a normal pattern in the other one. In both patients the number of ABL signals varied from cell to cell and the observer was always unable to count them properly. FISH on mitotic cells showed that ABL additional copies were localized neither on chromosome 9 nor on any other chromosome and revealed that amplification was extrachromosomal in nature even if no dmins were visualized. Therefore, it was hypothesized that the amplified ABL sequences might be localized on submicroscopic extrachromosomal structures, the episomes. In order to check whether the ABL gene was really over-expressed we performed a quantitative RT PCR (Q RT PCR) assay using the β-2-microglobulin as reference gene and total RNA from a normal subject for calibration. Quantification was made using the DDCt method. By this way we found that on clinical diagnosis the two patients expressed the ABL gene nine and twelve times more than the control. From a clinical point of view both patients were males. They had a high white blood cell count (31.8 and 21.8x109/L); their blast cells exhibited a T-ALL immunophenotype and a L2 morphology; their lactic dehydrogenase level was elevated. One patient achieved a complete remission (CR) of fifteen month duration and relapsed while still on maintenance treatment, the other did not respond to chemotherapy. In the former patient ABL expression was normal in CR but increased again on disease recurrence. In conclusion our data show that i) FISH is absolutely required to identify a new subset of T-ALL patients characterized by ABL amplification, ii) the role of ABL amplification in T-ALL pathogenesis is still obscure, iii) large cooperative studies are required to better define the clinical outcome of these patients whose EFS seems to be poor, iiii) Q RT PCR might be used to quantify minimal residual disease.

The present study, carried out in two APL with multiple relapses, was aimed at determining 1) which correlation does exist between FLT3/hTERT expression levels and PML-RARA results; 2) whether high FLT3 and hTERT expression levels might be predictive of relapse; 3) whether FLT3 expression is better than FLT3 Internal Tandem Duplication (ITD) for evaluating disease outcome. Relative quantifications of FLT3/hTERT transcripts were performed by real-time PCR using SybrGreen I. For FLT3 calibration total RNA from a normal subject was used, for hTERT total RNA from K562 cells. In both cases the ΔΔCt method was used for quantification. On clinical diagnosis one patient with a WBC of 124.0x109/L and a PML-RARA fusion at PML BCR1 presented the FLT3/hTERT genes highly expressed. On qualitative PCR the patient also showed the ITD of FLT3. He was treated with the AIDA protocol and succeeded in achieving a haematological but not molecular remission. During CR FLT3/hTERT expression remained high and the ITD was never detected. Fourteen months later when on first clinical relapse FLT3 expression abruptly increased, the ITD reappeared, hTERT levels were still high. A re-induction chemotherapy induced a second haematological but not molecular remission lasting five months. FLT3 as well as hTERT expression levels became similar to those of the control, and FLT3 ITD disappeared. A progressive increase of FLT3 expression and an abrupt increase of hTERT expression preceded the second relapse which was accompanied by the reappearance of the ITD. After re-induction chemotherapy FLT3/hTERT expression dropped down to values of the control. A third CR was obtained but the patient remained PML/RARA and Flt3 ITD positive and soon after died of a CNS relapse. The other patient was treated in another Centre and came to our observation in haematological CR. At that time he was PML-RARA negative with high FLT3/hTERT expression. Eight months later he was still in clinical but not molecular CR having a PML-RARA fusion at BCR3, high FLT3/hTERT expression levels and presenting FLT3 ITD. One month later when clinical relapse occurred FLT3 expression levels were unchanged, hTERT expression dropped down to normal values and FLT3 ITD was still present. A re-induction chemotherapy induced a second CR with alternatively positive and negative PML-RARA results, high FLT3 and low hTERT expression levels. The patient underwent an allogeneic bone marrow transplant from an unrelated donor but five months later he relapsed for the second time with an abrupt rise of hTERT expression that preceded a quick increase of FLT3 expression. A third clinical but not molecular CR was achieved after chemotherapy, but the patient remained PML-RARA positive with a normal FLT3/hTERT expression. Two months later a rapid increase of hTERT expression preceded that of FLT3 and the occurrence of the third relapse. In conclusion i) increased FLT3 and hTERT levels during CR are associated with alternative positive/negative PML-RARA results on nested RT PCR and are always predictive of pending relapse; ii) on disease recurrence a marked elevation of hTERT expression often preceded that of FLT3; iii) quantitative real-time PCR of the FLT3 gene was more effective in predicting disease outcome than the ITD, this last being discovered only when FLT3 expression was already high.