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Abstract 1529 Poster Board I-552 Pulmonary hypertension (PAH) is an independent risk factor for death in sickle cell disease (SCD). We performed a retrospective chart review to determine the natural history of PAH in our adult SCD population. We hypothesized that increased pulmonary artery pressures seen during hospitalizations for painful crisis or acute chest syndrome would be reflected in a faster progression of PAH measured during steady state. We reviewed charts and echocardiograms of 362 patients seen at Duke University from January 1980 to March 2009. A total of 878 2D echocardiograms were reviewed, with 196 patients having 2 or more echocardiographic procedures. Fifth-three of the 81 patients who died during this period had had at least 2 or more echocardiograms. Studies were considered done at steady state if they were performed as an outpatient procedure. Out of 878 total echocardiograms, 460 had either no measurable tricuspid regurgitation jet (TR) or failed to report it and were excluded from further analysis, leaving 418 echocardiograms in 252 patients (167 at steady state and 251 as inpatient). Among patients with multiple echocardiograms and measurable TR jet performed at steady state, we used a linear mixed model to identify a mean yearly rate of progression of 0.04 m/s (p Disclosures No relevant conflicts of interest to declare.

Abstract 2567 Poster Board II-544 Surgical procedures in children with sickle cell anemia (SCA) can be complicated by vasoocclusive events (VOE) such as acute chest syndrome (ACS) and pain. Peri-operative management requires a multidisciplinary approach to provide appropriate pre-operative intravenous hydration and intra- and post-operative monitoring. Transfusion therapy has been controversial. Our institution previously described a low incidence of complications in children who received serial transfusions over 3-4 weeks prior to surgery. Subsequently, an increasing number of children have been prescribed hydroxyurea (HU) to prevent SCA complications. In general, children on HU at our institution only receive a single top-off transfusion the day prior to surgery if their hemoglobin is less than 10 g/dL. We hypothesized that children in the HU group would have a lower number of serial transfusion compared to the non-HU group and that there would be no difference in complications or days to discharge between the two groups. We conducted a single-institution retrospective cohort study of children with SCA, who were age less than 18 years and underwent at least one surgical procedure at Duke University Medical Center between January 1, 2003 and April 30, 2008. Data were abstracted from electronic and written medical records. Descriptive statistics were used to characterize the cohort. Wilcoxon test was used to compare continuous variables and Pearson test was used to compare categorical variables between the non-HU and HU groups. Fifty-three subjects were included (Table 1). The non-HU group was significantly younger than the HU group, but children in the non-HU group were significantly more likely to be transfused pre-operatively, primarily with serial transfusions or erythrocytopheresis, compared to the HU group. One subject in the non-HU group developed a pre-operative delayed hyperhemolytic transfusion reaction. Post-operative complications are detailed in Table 1; the overall rate was low. Two subjects in the HU group developed acute chest syndrome despite pre-operative transfusion; one episode was likely related to underlying asthma and poor response to hydroxyurea; the second was likely related to pain and hypoventilation after laparoscopic splenectomy and tonsillectomy/adenoidectomy. Overall, there were no significant differences in complications and no significant difference in days to discharge between the two groups. In summary, children with SCA on HU may safely undergo surgery without significantly reducing their percent HbS. Nonetheless, attention should still be made towards multidisciplinary effort to reduce intra- and post-operative complications, and clinicians should consider response to HU, pulmonary status and type of surgery when planning peri-operative management in children with SCA on HU. Disclosures: Off Label Use: hydroxyurea in young children.

Hydroxyurea is the only safe and efficacious medication to prevent complications of sickle cell disease (SCD) in children. Unfortunately, hydroxyurea requires daily dosing and frequent drug monitoring. Our objectives were to assess adherence to hydroxyurea, the impact of adherence on hematologic response, and whether we could identify those children at greatest risk for non-adherence for a future intervention. Between 2006 and 2008 children with SCD who had been taking hydroxyurea for ≥5 months were enrolled in an IRB-approved study of treatment adherence. Because adherence is difficult to measure, we used multiple estimates: attendance at clinic visits (visit within 0.5 month of recommended time frame indicating good adherence); caregiver-report with a previously validated instrument (the Modified Morisky Scale; range 0–4; ≤1 indicating good adherence) and with a visual analog scale (VAS) developed for this project (>75% of doses within the last 3–4 weeks indicating good adherence); provider estimates of adherence completed by the physician or physician extender seeing the patient in clinic (“often” or “always” adherent indicating good adherence); and pharmacy prescription refills (≥5 month-supply refills in the six months prior to enrollment indicating good adherence). Caregivers also completed a written survey regarding hydroxyurea utilization. Eighty-three children with SCD enrolled in the study (46 males and 37 females, ages 3.5–17.8 years). The average duration of hydroxyurea therapy at study entry was 4.6 years (range 0.4–11.3). The mean dose at the time of the study was 24.2 mg/kg/day (range 16.6–31.6). Between hydroxyurea initiation and enrollment in this study hemoglobin increased by 1.3 g/dL (95% CI: 1.1–1.5; p75% by 4 out of 5 measures. However, the estimate based on pharmacy refills was

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.

CNS relapse occurs in ~5% of cases of APL. Conventional treatment includes intrathecal (IT) chemotherapy with methotrexate (MTX) and/or cytarabine, systemic salvage chemotherapy, and sometimes cranial irradiation: survival is poor, ranging from weeks to a few months. Liposomal cytarabine (DepoCyte®) is a novel sustained-release formulation with a terminal half-life ~40 × longer than free cytarabine (Bleyer et al. Clin Cancer Res 99;5:3349) and is approved for the treatment of adults with lymphomatous meningitis (LM): liposomal cytarabinbe has been shown to be more effective than free cytarabine in patients with LM (Glantz et al. JCO1999;17:3110). Intrathecal (IT) injections of this agent are given once every 2 weeks during treatment, as opposed to 2–3 × per week for free cytarabine or MTX, and an Ommaya reservoir is not required. In this report we present the case of a patient with CNS relapse of APL, refractory to conventional treatment including chemotherapy with high-dose (HD) cytarabine plus idarubicin, IT MTX, and cranial irradiation, who has been treated successfully with liposomal cytarabine.In July 2003, a 33-year-old woman was admitted with low-risk APL (hypergranular leukemic promyelocytes). Blood counts showed: Hb 5.3 g/dL, leukocytes 9.3 ×109/L, platelets 53 ×109/L. Cytogenetics showed t(15;17) and RT-PCR was positive for PML/RARα (bcr1 isoform). The patient achieved a hematological remission after induction and a molecular remission after consolidation using the AIDA protocol (Mandelli et al. Blood1997;90:1014). Molecular relapse during maintenance was treated with idarubicin plus ATRA resulting in a second molecular remission. In June 05, autologous peripheral blood stem cell transplant (PML/RARα-negative) was performed during molecular remission. Following a third molecular relapse in September 2005, with headache, APL cells and PML/RARα transcript were identified in the cerebrospinal fluid (CSF). Prolonged cytopenia characterized by fever and possible fungal pneumonia followed IT MTX, HD cytarabine plus idarubicin and cranial radiation, which induced a bone marrow molecular remission, but CSF showed persistence of APL cells, confirmed by PML/RARα. In November, the patient was treated with IT liposomal cytarabine 50 mg every 2 weeks with concomitant dexamethasone. CSF was obtained before each IT treatment: 14 days after the first treatment, no APL cells or PML/RARα transcript were found in CSF. Unfortunately, despite the use of dexamethasone, after the third IT injection, arachnoiditis developed, with headache, pain, confusion, somnolence and agitation. Arachnoiditis can be caused by meningeal infiltration by tumor cells, cranial irradiation and IT administration of drugs: it is difficult to distinguish between these causes. HD steroid was started, and the patient recovered after a few days. The patient remains in CNS remission, but suffered a bone marrow relapse in May 2006, which was treated with chemotherapy. In this patient, IT liposomal cytarabine resulted in a prolonged remission of CNS disease. Unfortunately, treatment of her systemic disease proved less successful.

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.

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.

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.

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.