4822 Chronic myeloid leukemia (CML) is characterized by the Philadelphia chromosome (Ph) observed in more than 90% of patients with CML as a result of t(9;22)(q34;q11), leading to the formation of the BCR/ABL chimeric gene. The remaining 5–10% of CML cases exhibit a variant Ph translocation generally involving a third or even a fourth chromosome in addition to chromosome 9 and 22, potentially leading to masked Ph chromosome or reveal cryptic translocations that remains undetected under conventional cytogenetic analysis. These chromosome rearrangements can be disclosed by means of fluorescence in situhybridization (FISH) or polymerase chain reaction (PCR) procedures. A very few Ph positive CML cases were reported with constitutional robertsonian translocations, i.e. translocation between two acrocentric chromomosomes (13–15, 21–22), with breakpoints in the short arms, leading to a dicentric chromosome and thus to 45 instead of 46 chromosomes Case Report. 42 year-old woman presenting with asthenia. Physical examination: Grade 1 splenomegaly. Peripheral blood count showed: hemoglobin concentration 117g/L, platelet count: 329×109/L and white blood cell count (WBC): 199×109/L. Peripheral blood smear: myelemia exhibiting 3% of myeloid blasts. Cytogenetic analysis by G-banding performed on bone marrow metaphase cells afforded the following karyotype: 45, XX, der(14;22)(q10;q10)c?, t(9;22;11)(q34;q11;q13) [20]. The analysis of the BCR-ABLfusion gene according to standard protocols detected the presence of the b3a2 isoform. FISH studies using dual color dual fusion probes in metaphases showed a 1F2G2R signal pattern. We detect a normal ABL signal on chromosome 9 and BCR signal on chromosome 22; the fusion signal was present on the der(14;22);extra-signals BCR and ABL with reduced intensities were present on der(11) and der(9) respectively: ish der(9)(ABLdim+), der(11)(BCRdim+), der(14;22)(BCR+,ABL+) [10]. FISH analysis on interphase nuclei (n=200) presented the same signal pattern. Nuc ish (ABL, BCRx3)(BCR con ABL x1) [200]. Chromosome analysis of bone marrow cells after six months of Imatinib therapy showed the following karyotype: 45, XX, der(14;22)(q10;q10)c [20] thus demonstrating complete cytogenetic remission and that der(14;22) is a robertsonian constitutional abnormality that could be inherited and thus necessitate a familial genetic councelling to inform about the familial risk of congenital malformations and miscarriage. Discussion. To explain the formation of variant chromosome Ph translocations one-step, two-step and multi-step mechanisms have been proposed. In our case complex translocations involving four chromosomes and the participation of two acrocentric chromosomes, led to the hypothesis of the presence of a constitutional or acquired Robertsonian translocation. Karyotype analysis six months after treatment confirmed the presence of a constitutional Robertsonian translocation. According to the FISH pattern, this variant Ph chromosome was formed in one step. The occurrence of Philadelphia positive CML in a patient with a constitutional Robertsonian translocation is probably coincidental. The role of constitutional chromosomes abnormalities in hematologic malignancies is well known in Down syndrome patients and in chromosome breakage syndromes such as Fanconi anemia. In the literature, only one case of CML patients with Robertsonian t(14;22) have been described. To our knowledge this is the first report showing a Robertsonian t(14;22) in a variant Ph involving four chromosomes and exhibiting the fusion FISH signal in a derivative chromosome 14, with masked Ph. Disclosures: No relevant conflicts of interest to declare.

Introduction Acute promyelocytic leukemia (APL) of the WHO classification is genetically characterized by the t(15;17)(q22;q21) chromosomal translocation involving the retinoic acid receptor alpha (RARA) located on band 17q21 and the promyelocytic leukemia gene (PML) on band 15q24 , leading to the PML/RARA fusion transcript, and by sensitivity of blast cells to all-trans retinoic acid or arsenic trioxide (ATO) targeted therapy. Although the vast majority of APL cases present with t(15;17)(q24;q21), formerly (q22;q21), a few patients have either simple or complex variants of this translocation involving chromosome 15, 17 and one or more other chromosomes. Analysis of these variant translocations is of great interest because they may mask a cryptic t(15;17) leading to misdiagnose a true APL as non APL-AML with other translocations involving RARA but not PML do exist, as mentioned in the WHO classification, and generally these cases do not respond to ATRA or ATO therapy and require more intensive chemotherapy. In these cases, the PML/RARA fusion gene can be identified by molecular analyses such as reverse transcriptase-polymerase chain reaction (RT-PCR) and fluorescence in situ hybridization (FISH). Case report 29-year-old man presented with dizziness and tiredness. Physical examination: organomegaly was not observed. Peripheral blood: hemoglobin concentration, 71 g/L; platelet count, 10x109/L white blood cell count (WBC) 0.5x109/L. Bone marrow aspirate showed 56% blast cells with Auer rods. Coagulation tests were normal. Leukemic cells were CD13+, CD33+, CD34-, CD117+ and HLA-DR-. Cytogenetic analysis by G-banding performed in bone marrow metaphase cells afforded the following karyotype: 46,XY, t(15;17)(q11;q21) with derivative (der) (15) shorter and der(17) longer than in classical t(15;17)(q24;q21). PCR analysis of the PML/RAR fusion gene according to standard protocols disclosed the presence of the L isoform. FISH studies using dual color dual fusion probes (Vysis) covering the entire PML and RARA genes , showed a classical 2F1G1R (2 fusion, 1 green, 1 red) signal pattern on nuclei. However , on metaphases, we detected a normal PML (red) and RARA (green) signals on normal chromosome 15 and 17 respectively , but the two fusion signals were located on der(17). The patient was treated with IC-APL protocols (all-trans retinoic acid plus daunorubicin) and complete remission was achieved after induction therapy. Discussion To explain the origin of the observed karyotype and molecular results, especially the double fusion signal on der(17), we propose two hypotheses: a classical t(15;17)(q24;q21) initially occurred leading to one fusion gene located on each derivative accompanied or followed either by a second translocation event implicating both derivative chromosomes with breakpoints located centromeric to the former breakpoint on der(15) and telomeric to the former breakpoint on der(17), or by an insertion of part of the der(15) containing the PML/RARA fusion gene into the der(17). Results obtained through FISH analysis support our first hypotheses. No differences in the clinical outcome between APL cases with classical t(15;17) and those with variant translocations leading to PML-RARA fusion gene have been reported. These results highlight the utility of combined cytogenetic, FISH and RT-PCR analyses to unveil the cases with variant or cryptic t(15;17). To our knowledge, this is the first report of an APL patient showing a variant t(15;17) involving only chromosomes 15 and 17 with two fusion signals on der(17). Disclosures: No relevant conflicts of interest to declare.

4839 Introduction. Chronic myeloid leukemia (CML) is characterized by the Philadelphia chromosome (Ph) observed in more than 90% of patients with CML as a result of t(9;22)(q34;q11), leading to the formation of chimeric gene BCR/ABL encoding for proteins with abnormal tyrosine kinase activity. Cytogenetic variants of Ph chromosome can be identifed in 5 to 10% of CML patients, involving additional chromosomes other than 9 and 22. To explain the formation of variant translocations one-step, two-step and multi-step mechanisms have been proposed. Rarely, the variant Ph chromosome results from a BCR insertion on the ABL region and form a BCR/ABL fusion gene, generally mapping to 9q34, instead of the usual location at 22q11. In very few variant Ph cases, the insertion of the BCR/ABL product in a third chromosome was demonstrated. Case Report 28 year-old man, with bilateral central scotoma and gingivorragia. Physical examination: Grade 4 splenomegaly. Peripheral blood count showed hemoglobin concentration 11.5 g/dl, platelet count: 300.000/mm3, and white blood cell count 590.000/mm3. Blood smear: myelemia exhibiting 30% of myeloid blasts. Bone marrow biopsy: panmyelosis showing 20% of myeloid blasts. Cytogenetic analysis by G-banding performed in peripheral blood verified the following karyotype: 46, XY, t(9;22;10)(q34;q11;q24)[20] The analysis of the BCR-ABL fusion gene according to standard protocols detected the presence of the b3a2 isoform. Fluorescence in situ hybridization (FISH) studies using dual color dual fusion probes in metaphases showed a signal pattern 1F2G1R. The fusion signal mapped to 10q24, the red signal to 9q34, and the normal green signal to chromosome 22, while a second low intensity green signal mapped to the Ph chromosome. No signal was observed in der(9). Interphase FISH analysis in nuclei (n=200) presented the same signal pattern. Instead of using whole chromosome probes for 9 and 22, we hybridised probes used to detect DiGiorge syndrome. These probes detect gene control ARSA (spectrum green) localized at 22q13 and Tuple1 at 22q11 (spectrum orange). Two signals, green and orange were identified in normal chromosome 22. Ph chromosome showed the orange signal, whereas the green signal mapped to der(10). Discussion. The localization of the hybrid BCR/ABL gene on chromosomes other than 22q is a rare event wich can only be detected by FISH techniques. When these unusual translocation occurs, the hypothesis most often put forward is that several consecutive chromosome rearrangements have taken place. In the present case the interpretation of karyotypes, FISH data and molecular evidence lead to the following hypothesis: Insertion of the BCR sequence from chromosome 22 to chromosome 9 may have ocurred, producing a BCR/ABL fusion in der(9). The Ph chromosome detected by G-banding showed a different green fluorescence intensity in the metaphase FISH signal pattern with BCR/ABL dual color dual fusion probes, as a result of an insertion on chromosome 9. This first event was followed by the translocation between the derivative 9 and chromosome 10, being the final localization of the BCR/ABL gene in 10q24. FISH analysis using a DiGeorge syndrome probe, supports the hypothesis of a multistep mechanism underlying insertion and translocations events in the present case. The relocation of BCR/ABL fusion sequence on sites other than chromosme 22q11 represent a rare type of variant Ph translocation. At least 21 cases described in the literature, showed fusion gene BCR/ABL located at 9q24. Only 12 patients with variant Ph were reported bearing BCR/ABL on a third chromosome. All of them involved a masked Ph chromosome. To our best knowledge this is the first report showing a variant Ph chromosome detected by G-banding in a CML patient due to a BCR insertion on ABL sequences and exhibiting the fusion signal in a third chromosome. Disclosures: No relevant conflicts of interest to declare.