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The BCR-ABL1 fusion kinase is frequently associated with chronic myeloid leukemia and B-cell acute lymphoblastic leukemia but is rare in T-cell acute lymphoblastic leukemia (T-ALL). We recently identified NUP214-ABL1 as a variant ABL1 fusion gene in 6% of T-ALL patients. Here we describe the identification of another ABL1 fusion, EML1-ABL1, in a T-ALL patient with a cryptic t(9;14)(q34;q32) associated with deletion of CDKN2A (p16) and expression of TLX1 (HOX11). Echinoderm microtubule-associated protein-like 1-Abelson 1 (EML1-ABL1) is a constitutively phosphorylated tyrosine kinase that transforms Ba/F3 cells to growth factor-independent growth through activation of survival and proliferation pathways, including extracellular signal-related kinase 1/2 (Erk1/2), signal transducers and activators of transcription 5 (Stat5), and Lyn kinase. Deletion of the coiled-coil domain of EML1 abrogated the transforming properties of the fusion kinase. EML1-ABL1 and breakpoint cluster region (BCR)-ABL1 were equally sensitive to the tyrosine kinase inhibitor imatinib. These data further demonstrate the involvement of ABL1 fusions in the pathogenesis of T-ALL and identify EML1-ABL1 as a novel therapeutic target of imatinib. (Blood. 2005;105:4849-4852)

MLL rearrangements in acute myeloid leukemia (AML) include translocations and intragenic abnormalities such as internal duplication and breakage induced by topoisomerase II inhibitors. In adult AML, FLT3 internal tandem duplications (ITDs) are more common in cases with MLL intragenic abnormalities (33%) than those with MLL translocation (8%). Mutation/deletion involving FLT3 D835 are found in more than 20% of cases with MLL intragenic abnormalities compared with 10% of AML with MLL translocation and 5% of adult AML with normal MLL status. Real-time quantification of FLT3 in 141 cases of AML showed that all cases with FLT3 D835 express high level transcripts, whereas FLT3-ITD AML can be divided into cases with high-level FLT3 expression, which belong essentially to the monocytic lineage, and those with relatively low-level expression, which predominantly demonstrate PML-RARA and DEK-CAN. FLT3 abnormalities in CBF leukemias with AML1-ETO or CBFβ-MYH11 were virtually restricted to cases with variant CBFβ-MYH11 fusion transcripts and/or atypical morphology. These data suggest that the FLT3 and MLL loci demonstrate similar susceptibility to agents that modify chromatin configuration, including topoisomerase II inhibitors and abnormalities involving PML and DEK, with consequent errors in DNA repair. Variant CBFβ-MYH11 fusions and bcr3 PML-RARA may also be initiated by similar mechanisms.

The ETV6 gene (first identified asTEL )i s a frequent target of chromosomal translocations in both myeloid and lymphoid leukemias. At present, more than 40 distinct translocations have been cytogenetically described, of which 13 have now also been characterized at the molecular level. These studies revealed the generation of in-frame fusion genes between different domains of ETV6 and partner genes encoding either kinases or transcription factors. However, in a number of cases—including a t(6;12)(q23;p13), the recurrent t(5;12)(q31;p13), and some cases of the t(4;12)(q11-q12;p13) described in this work—functionally significant fusions could not be identified, raising the question as to what leukemogenic mechanism is implicated in these cases. To investigate this, we have evaluated the genomic regions at 4q11-q12 and 5q31, telomeric to the breakpoints of the t(4; 12)(q11-q12;p13) and t(5;12)(q31;p13). The homeobox gene GSH2 at 4q11-q12 and the IL-3/CSF2 locus at 5q31 were found to be located close to the respective breakpoints. In addition, GSH2 and IL-3 were found to be ectopically expressed in the leukemic cells, suggesting that expression of GSH2 and IL-3 was deregulated by the translocation. Our results indicate that, besides the generation of fusion transcripts, deregulation of the expression of oncogenes could be a variant leukemogenic mechanism for translocations involving the 5* end of ETV6, especially for those translocations lacking functionally significant fusion transcripts. (Blood. 2002;99:1776-1784)

Single-cell polymerase chain reaction (PCR) has been used as a tool to demonstrate clonality and B-cell origin of Reed-Sternberg (RS) cells in Hodgkin disease (HD). An analogous approach was used to investigate genomic imbalances in a (cyto)genetically poorly characterized subentity: lymphocyte predominance Hodgkin disease (LPHD). Nineteen cases of LPHD were selected for a comparative genomic hybridization (CGH) study. CGH was performed with degenerate oligonucleotide primed–PCR (DOP-PCR)–amplified DNA from 4-5 microdissected CD20+ malignant cells. All analyzed cases revealed a high number of genomic imbalances (average 10.8 per case), involving all chromosomes but the excluded 19, 22, and Y, indicating a high complexity of LPHD. The majority of detected aberrations were recurrent. Gain of 1, 2q, 3, 4q, 5q, 6, 8q, 11q, 12q, and X, and loss of chromosome 17 were identified in 36.8% to 68.4% of the analyzed cases. Some of them have also been found in non-Hodgkin lymphoma (NHL), and possibly represent secondary changes associated with disease progression. Gain of 2q, 4q, 5q, 6, 11q, however, are much more rarely observed in NHL and could be more specifically associated with LPHD. Particularly interesting is a frequent overrepresentation of chromosome arm 6q, a region usually deleted in NHL. Rearrangement of theBCL6 gene (3q27) demonstrated by cytogenetics and fluorescence in situ hybridization in 2 cases in this study suggests its contribution in pathogenesis of LPHD. In conclusion, the data show a consistent occurrence of genomic alterations in LPHD and highlight genomic regions that might be relevant for development and/or progression of this lymphoma entity.

The translocation of chromosome 11, long arm, region 2, band 1, to chromosome 18, long arm, region 2, band 1 (t(11;18)(q21;q21)) represents a recurrent chromosomal abnormality in extranodal marginal zone B-cell lymphoma (MZBCL) of mucosa-associated lymphoid tissue (MALT) type and leads to a fusion of the apoptosis inhibitor-2 (API2) gene on chromosome 11 and the MALT lymphoma-associated translocation (MLT) gene on chromosome 18. A 2-color fluorescence in situ hybridization (FISH) assay, which can be used for the detection of t(11;18) in interphase nuclei and metaphase chromosomes on fresh and archival tumor tissue, was developed. The P1 artificial chromosome (PAC) clone located immediately telomeric to the MLT gene and the PAC clone spanning the API2 gene were differentially labeled and used to visualize the derivative chromosome 11 resulting from t(11;18), as evident by the overlapping or juxtaposed red and green fluorescent signals. The assay was applied to interphase nuclei of 20 cases with nonmalignant conditions and 122 B-cell non-Hodgkin's lymphomas (NHLs). The latter group comprised 20 cases of nodal follicle center cell lymphoma and diffuse large B-cell NHL, 10 cases of gastric diffuse large B-cell lymphoma, 10 cases of hairy cell leukemia, and 82 cases of MZBCL (41 extranodal from various locations, 19 nodal, and 22 splenic MZBCL) including 35 cases with an abnormal karyotype, 2 of which revealed t(11;18). By interphase FISH, t(11;18) was detected in 8 gastrointestinal low-grade MALT-type lymphomas including the 2 cytogenetically t(11;18)+ cases. In the 8 t(11;18)+ cases, the FISH results were confirmed by reverse transcriptase–polymerase chain reaction (RT-PCR) usingAPI2 and MLT specific primers. Our results indicate that t(11;18)(q21;q21) specifically characterizes a subgroup of low-grade MZBCL of the MALT-type and that the FISH assay described here is a highly specific and rapid test for the detection of this translocation.

An isochromosome of the long arm of chromosome 17, i(17q), is the most frequent genetic abnormality observed during the disease progression of Philadelphia chromosome–positive chronic myeloid leukemia (CML), and has been described as the sole anomaly in various other hematologic malignancies. The i(17q) hence plays a presumably important pathogenetic role both in leukemia development and progression. This notwithstanding, the molecular consequences of this abnormality have not been investigated in detail. We have analyzed 21 hematologic malignancies (8 CML in blast crisis, 8 myelodysplastic syndromes [MDS], 2 acute myeloid leukemias, 2 chronic lymphocytic leukemias, and 1 acute lymphoblastic leukemia) with i(17q) by fluorescence in situ hybridization (FISH). Using a yeast artificial chromosome (YAC) contig, derived from the short arm of chromosome 17, all cases were shown to have a breakpoint in 17p. In 12 cases, the breaks occurred within the Smith-Magenis Syndrome (SMS) common deletion region in 17p11, a gene-rich region which is genetically unstable. In 10 of these 12 cases, we were able to further map the breakpoints to specific markers localized within a single YAC clone. Six other cases showed breakpoints located proximally to the SMS common deletion region, but still within 17p11, and yet another case had a breakpoint distal to this region. Furthermore, using chromosome 17 centromere-specific probes, it could be shown that the majority of the i(17q) chromosomes (11 of 15 investigated cases) were dicentric, ie, they contained two centromeres, strongly suggesting that i(17q) is formed through an intrachromosomal recombination event, and also implicating that the i(17q), in a formal sense, should be designated idic(17)(p11). Because i(17q) formation results in loss of 17p material, potentially uncovering the effect of a tumor suppressor on the remaining 17p, the occurrence of TP53 mutations was studied in 17 cases by sequencing the entire coding region. In 16 cases, noTP53 mutations were found, whereas one MDS displayed a homozygous deletion of TP53. Thus, our data suggest that there is no association between i(17q) and coding TP53 mutations, and that another tumor suppressor gene(s), located in proximity of the SMS common deletion region, or in a more distal location, is of pathogenetic importance in i(17q)-associated leukemia.

Marginal zone cell lymphomas of the mucosa-associated lymphoid tissue (MALT) are the most common subtype of lymphoma arising at extranodal sites. The t(11;18)(q21;q21) appears to be the key genetic lesion and is found in approximately 50% of cytogenetically abnormal low-grade MALT lymphomas. We show that the API2 gene, encoding an inhibitor of apoptosis also known as c-IAP2, HIAP1, andMIHC, and a novel gene on 18q21 characterized by several Ig-like C2-type domains, named MLT, are recurrently rearranged in the t(11;18). In both MALT lymphomas analyzed, the breakpoint inAPI2 occurred in the intron separating the exons coding respectively for the baculovirus IAP repeat domains and the caspase recruitment domain. The breakpoints within MLT differed but the open reading frame was conserved in both cases. In one case, the translocation was accompanied by a cryptic deletion involving the 3′ part of API2. As a result, the reciprocal transcript was not present, strongly suggesting that the API2-MLT fusion is involved in the oncogenesis of MALT lymphoma.

Recently, a distinctive entity characterized by expression of the anaplastic lymphoma kinase (ALK) protein [most frequently due to the t(2;5)(p23;q35)-associated NPM-ALK fusion] has emerged within the heterogenous group of non-Hodgkin’s lymphomas (NHL) classified as anaplastic large-cell lymphoma (ALCL). Sporadic variant 2p23/ALK abnormalities identified in ALK-positive ALCL indicate that genes other than NPM may also be involved in the deregulation of ALK and lymphomagenesis. We report here three cases with an inv(2)(p23q35) detected by fluorescence in situ hybridization (FISH) in young male patients with ALK-positive ALCL. In contrast to ALCL cases with the classical t(2;5)(p23;q35) that usually show both cytoplasmic and nuclear or predominantly nuclear alone localization of the NPM-ALK chimeric product, in all three cases with an inv(2)(p23q35) the ALK protein accumulated in the cytoplasm only, supporting the previous assumption that the oncogenic potential of ALK may not be dependent on its nuclear localization. As the first step to identify theALK partner gene involved in the inv(2)(p23q35), we performed extensive FISH studies and demonstrated that the 2q35 breakpoint occurred within the 1,750-kb region contained within the 914E7 YAC. Moreover, a striking association of the inv(2)(p23q35) with a secondary chromosomal change, viz, ider(2)(q10)inv(2)(p23q35), carrying two additional copies of the putative ALK-related fusion gene, was found in all three patients, suggesting that, in contrast to the standard t(2;5)/NPM-ALK fusion, multiple copies of the putative 2q35-ALK chimeric gene may be required for efficient tumor development. In summary, we demonstrate that the inv(2)(p23q35), a variant of the t(2;5)(p23;q35), is a recurrent chromosomal abnormality in ALK-positive ALCL, the further characterization of which should provide new insight into the pathogenesis of these lymphomas. © 1998 by The American Society of Hematology.

Background. Previous studies have demonstrated that monosomal karyotype (MK) confers a particularly poor prognosis in patients with acute myeloid leukemia (AML) (Breems et al., J Clin Oncol 2008, 26:4791-7). Purpose of the study. To determine the impact of the type of remission-induction chemotherapy and the impact of having a donor on overall survival (OS) in younger ( Methods. In the EORTC/GIMEMA AML-10 trial (Mandelli et al., J Clin Oncol 2009, 27:5397-403), patients were randomized to receive either daunorubicin (DNR), mitoxantrone (MTX), or idarubicin (IDA) in addition to standard-dose cytarabine (SDAC: 10 days of 100 mg/m2 per day as continuous IV infusion) and etoposide for induction chemotherapy. In the EORTC/GIMEMA AML-12 trial, (Willemze et al., J Clin Oncol 2014, 32:219-28) patients were randomized between either SDAC or high dose cytarabine (HiDAC) (3 g/m2 every 12 hours as a 3-hour IV infusion on days 1, 3, 5, and 7) induction, both with DNR, and etoposide. In both trials, a second cycle of the same remission induction chemotherapy was administered in patients who achieved a partial remission. Patients who achieved a complete remission (CR) or a CR with incomplete counts recovery (CRi) after 1 or 2 courses of induction chemotherapy received a consolidation chemotherapy with the same anthacycline as in the induction course plus intermediate dose cytarabine. Patients in both studies were then scheduled to receive an allogeneic hematopoietic stem cell transplantation (allo-HSCT) if they had a donor or an autologous HSCT (auto-HSCT) otherwise, in first CR. For the current analyses, cytogenetics were centrally reviewed and re-classified using the European Leukemia Net (ELN) definition. MK was defined as the presence of 2 or more monosomies, or a single monosomy in the presence of structural abnormalities. Results. In the AML-10 trial, 2157 patients were randomized to receive DNR, MTX or IDA. The current analyses were performed in a subgroup of 910 patients for whom cytogenetic data were available and who did not have t(8;21), inv(16) nor t(15;17). 709 of them were classified in the ELN intermediate cytogenetic risk group and 201 in the adverse group. Out of the 910 patients, 93 had a MK. In comparison to the 817 remaining (MK-) patients, MK+ patients were less likely to achieve a CR, were more likely to have resistant disease (RD), and had worse OS and disease-free survival (DFS) (Table 1). There was no impact of the type of anthracycline on outcomes. Among the MK+ patients who achieved a CR, 5-yr OS from CR was 26% (95% CI, 10-46%) in patients with a donor (n=21, of whom, 13 received an allo-HSCT in first CR), versus 4% (95% CI, 0-18%) in those without a donor (n=24, of whom, 1 received an allo-HSCT in first CR) (P=0.07). Further, among the 10 MK+ who survived more than 1 year, 7 received an allo-HSCT in first CR (n=4) or beyond (n=3). In the AML-12 trial, 1942 patients were randomized between HiDAC or SDAC. The current analyses were performed in a subgroup of 1079 patients for whom cytogenetic data were available and who did not have t(8;21), inv(16) nor t(15;17). 893 of them were classified in the ELN intermediate cytogenetic risk group and 186 in the adverse group. Out of the 1079 patients, 98 had a MK. In comparison to the 1027 MK- patients, MK+ patients were less likely to achieve a CR, were more likely to have RD, and had worse OS and DFS (Table 1). There was a higher probability of achieving a CR and a trend for better OS in MK- patients randomized in the HiDAC arm (Table 2). In contrast, in MK+ patients, HiDAC did not increase either the CR rate as compared to SDAC, nor the 5-yr OS rate: 7% (HiDAC) vs 2% (SDAC) (Table 2). Finally, among the MK+ patients who achieved a CR, 5-yr OS from CR was 14 % (95% CI, 4-32%) in patients with a donor (n=21, of whom, 12 received an allo-HSCT in first CR), versus 4% (95% CI, 0.3-18%) in those without a donor (n=24) (P=0.60). Further, among the 4 MK+ patients who survived more than 1 year, 3 received an allo-HSCT in first CR (n=2) or beyond (n=1). Conclusions. This analysis confirms the poor prognosis of MK in younger AML patients. Shifting DNR to IDA or MTX or administration of HiDAC in the induction course failed to improve outcome of MK+ patients. Finally, there was a suggestion for a longer OS from CR in MK+ patients who had a donor, suggesting that allo-HSCT should be offered in all fit MK+ patients. FB & MSK contributed equally to this work Disclosures Thomas: Pfizer: Consultancy.

The gene on chromosome 10 at band p12 (AF10), involved in the t(10;11) translocation in acute myeloid leukemia, has been identified and shown to contain conserved zinc finger and leucine zipper domains. These regions are highly homologous to the equivalent regions on AF17, the gene involved in the t(11;17) translocations. A series of adult, childhood, and infant leukemias with either simple or complex versions of the t(10;11) has been examined by Southern analysis and shown to involve rearrangement to the HRX locus. Reverse transcriptase- polymerase chain reaction from either bone marrow or peripheral blood cells showed that HRX sequence was fused to AF10 sequence in all 8 cases and subsequent sequence analysis showed an in-frame fusion between the HRX and AF10 sequence. A consistent feature of these fusions was the juxtaposition of the leucine dimerization motif of AF10 onto the NH2-terminal region of HRX. The published data suggest that a similar conclusion can be drawn about the t(11;17) translocation, implying a critical role for this motif in the chimaeric HRX protein.

As acute nonlymphocytic leukemia (ANLL) with inv(16) (p13q22) or t(16;16)(p13;q22) has been shown to result from the fusion of transcription factor subunit core binding factor (CBFB) to a myosin heavy chain (MYH11), we sought to design methods to detect this rearrangement using reverse transcriptase-polymerase chain reaction (RT- PCR). In all of 27 inv(16)(p13q22) and four t(16;16)(p13;q22) cases tested, a chimeric CBFB-MYH11 transcript coding for an in-frame fusion protein was detected. In a more extensive RT-PCR analysis with different primer pairs, we detected a second new chimeric CBFB-MYH11 transcript in 10 of 11 patients tested. The CBFB-MYH11 reading frame of the second transcript was maintained in one patient but not in the others. We show that the different CBFB-MYH11 transcripts in one patient arise from alternative splicing. Translation of the transcript in which the CBFB-MYH11 reading frame is not maintained leads to a slightly truncated CBFB protein.

The expression of the MDR-1-encoded P-170 glycoprotein (P-170) associated with clinical multidrug resistance (MDR) was investigated in 52 consecutive patients with untreated acute myeloid leukemia (AML). P- 170 expression was analyzed in correlation with CD34 expression and clinical response. Thirty of 52 patients expressed P-170 (58%). Eight of 30 P-170+ as compared with 16 of 22 P-170- patients achieved a complete remission (CR) (27% v 73%, P = .003). In 21 of 30 P-170+ patients, expression of the CD34 antigen was observed in greater than 10% of the blast cells, as compared with 14 of 22 P-170- patients (70% v 64%, P > .05). The CR rate of CD34+ and CD34- patients was 31% and 76%, respectively (P = .006). In AMLs that simultaneously expressed both P-170 and CD34, the CR rate was worse as compared with those negative for P-170 and CD34 (5% v 63%, P = .004). In 12 patients (8 P- 170+, 4 P-170-) CD34 and P-170 expression were further characterized by double fluorescence studies. It was shown that P-170+ cells were largely, but not exclusively, restricted to the CD34+ cell population. For the 8 P-170+ AML samples, the median ratio of P-170+/P-170- in CD34+ cells was 4.845 (range, 0.60 to 25.00) as compared with 0.135 (range, 0.02 to 0.67) in CD34- cells. In these 12 AML samples, the presence of functional resistance as defined by reduced daunorubicin accumulation was evaluated in CD34+ and CD34- AML cells. In 8 of 8 P- 170+ patients, intracellular daunorubicin accumulation in CD34+ AML blast cells was lower than in CD34- cells, and it increased after cyclosporin addition. No difference of intracellular daunorubicin accumulation was observed between CD34+ and CD34- AML cells of 4 P-170- patients. These data indicate that P-170 expression in AML with a heterogeneous CD34+ phenotype seems predominantly present in CD34+ AML blast cells.

Bone marrow and blood from patients with acute myeloid leukemia and myelodysplastic syndrome were studied by simultaneous analysis of cell morphology and karyotype. A combined technique of May-Grunwald Giemsa (MGG) for cell morphology and fluorescence in situ hybridization (FISH) with chromosome-specific DNA probes for detection of cytogenetic aberrations allowed us to investigate cell-lineage-specific chromosomal abnormalities. We introduced video recordings to examine large numbers of cells. Briefly, evaluation was first performed on MGG slides, during which cell position and morphology were recorded on an S-VHS recorder. Subsequently, the same slides were used for FISH. This resulted in the identification of MGG-stained cells on the video screen and, at the same time, the interpretation of FISH signals in the fluorescence microscope. Specimens of bone marrow or blood samples from four patients with different hematologic malignancies were studied. One of these patients was studied before and after cytotoxic treatment. The gain or loss of chromosomes could be detected easily and morphologically assigned to the blasts in all patients and to a variable proportion of the myelomonocytic lineage in two patients, but not to the lymphocytes. Thus, this method provides new possibilities for investigating the clonality of hematologic malignancies.

Extensive immunologic marker analysis was performed to characterize the various leukemic cell populations in eight patients with inv(16)(p13q22) in association with acute myeloid leukemia with abnormal bone marrow eosinophilia (AML-M4Eo). The eight AML cases consisted of heterogeneous cell populations; mainly due to the presence of multiple subpopulations, which varied in size between the patients. However, the immunophenotype of these subpopulations was comparable, independent of their relative sizes. Virtually all AML-M4Eo cells were positive for the pan-myeloid marker CD13. In addition, the AML were partly positive for CD2, CD11b, CD11c, CD14, CD33, CD34, CD36, CDw65, terminal deoxynucleotidyl transferase (TdT), and HLA-DR. Double immunofluorescence stainings demonstrated coexpression of the CD2 antigen and myeloid markers and allowed the recognition of multiple AML subpopulations. The CD2 antigen was expressed by immature AML cells (CD34+, CD14-) and more mature monocytic AML cells (CD34-, CD14+), whereas TdT expression was exclusively found in the CD34+, CD14- cell population. The eight AML-M4Eo cases not only expressed the CD2 antigen, but also its ligand CD58 (leukocyte function antigen-3). Culturing of AML-M4Eo cell samples showed a high spontaneous proliferation in all three patients tested. Addition of a mixture of CD2 antibodies against the T11.1, T11.2, and T11.3 epitopes diminished cell proliferation in two patients with high CD2 expression, but no inhibitory effects were found in the third patient with low frequency and low density of CD2 expression. These results suggest that high expression of the CD2 molecule in AML-M4Eo stimulates proliferation of the leukemic cells, which might explain the high white blood cell count often found in this type of AML.

Specific rearrangements of chromosome 16 are well known in acute nonlymphocytic leukemia with abnormal eosinophils. While mapping cosmids relative to breakpoints in chromosome 16 in leukemic cells with fluorescence in situ hybridization (FISH), we have identified three areas of extensive cross-homology between 16p and 16q. Three cosmids among 99 tested showed two large signals on the short arm and one signal on the long arm of chromosome 16. A fourth cosmid showed mainly two signals on the short arm. With the 16p-specific cosmid we can demonstrate that the breakpoints of a pericentric inversion and a reciprocal (16;16) translocation, both of which are characteristic for acute leukemia, map to the most distal of two blocks on the short arm. We suggest that there may be at least two distinct repetitive elements specific for chromosome 16 interdigitated on 16p. The presence of a similar repeat in the short, as well as the long arm of the chromosome, may play a role in the origin of chromosome 16 rearrangements in acute leukemia.

The BCR-ABL1 fusion kinase is frequently associated with chronic myeloid leukemia and B-cell acute lymphoblastic leukemia but is rare in T-cell acute lymphoblastic leukemia (T-ALL). We recently identified NUP214-ABL1 as a variant ABL1 fusion gene in 6% of T-ALL patients. Here we describe the identification of another ABL1 fusion, EML1-ABL1, in a T-ALL patient with a cryptic t(9;14)(q34;q32) associated with deletion of CDKN2A (p16) and expression of TLX1 (HOX11). Echinoderm microtubule-associated protein-like 1-Abelson 1 (EML1-ABL1) is a constitutively phosphorylated tyrosine kinase that transforms Ba/F3 cells to growth factor-independent growth through activation of survival and proliferation pathways, including extracellular signal-related kinase 1/2 (Erk1/2), signal transducers and activators of transcription 5 (Stat5), and Lyn kinase. Deletion of the coiled-coil domain of EML1 abrogated the transforming properties of the fusion kinase. EML1-ABL1 and breakpoint cluster region (BCR)-ABL1 were equally sensitive to the tyrosine kinase inhibitor imatinib. These data further demonstrate the involvement of ABL1 fusions in the pathogenesis of T-ALL and identify EML1-ABL1 as a novel therapeutic target of imatinib.

The ETV6 gene (first identified as TEL) is a frequent target of chromosomal translocations in both myeloid and lymphoid leukemias. At present, more than 40 distinct translocations have been cytogenetically described, of which 13 have now also been characterized at the molecular level. These studies revealed the generation of in-frame fusion genes between different domains of ETV6 and partner genes encoding either kinases or transcription factors. However, in a number of cases-including a t(6;12)(q23;p13), the recurrent t(5;12)(q31;p13), and some cases of the t(4;12)(q11-q12;p13) described in this work-functionally significant fusions could not be identified, raising the question as to what leukemogenic mechanism is implicated in these cases. To investigate this, we have evaluated the genomic regions at 4q11-q12 and 5q31, telomeric to the breakpoints of the t(4;12)(q11-q12;p13) and t(5;12)(q31;p13). The homeobox gene GSH2 at 4q11-q12 and the IL-3/CSF2 locus at 5q31 were found to be located close to the respective breakpoints. In addition, GSH2 and IL-3 were found to be ectopically expressed in the leukemic cells, suggesting that expression of GSH2 and IL-3 was deregulated by the translocation. Our results indicate that, besides the generation of fusion transcripts, deregulation of the expression of oncogenes could be a variant leukemogenic mechanism for translocations involving the 5' end of ETV6, especially for those translocations lacking functionally significant fusion transcripts.

Background: An unmet medical need persists for elderly patients (pts) with AML who are deemed not to be fit for intensive chemotherapy. Such pts are usually treated with BSC and hydroxyurea or low-dose cytarabine, but outcomes are dismal. The immunoconjugate GO has shown single agent efficacy and tolerability in older pts with relapsed AML. The AML-19 study was designed as a sequential phase II/III trial comparing GO monotherapy to BSC (including hydroxyurea if clinically indicated) in pts age ≥ 61 yrs with previously untreated AML who were considered unfit for intensive chemotherapy (or refused it). Of the two induction schedules of GO (total dose 9 mg/m2 delivered in 2 or 3 fractions over one week) under comparison in the phase II part of the study, the 2-fraction regimen was found to have the best efficacy profile to warrant phase III comparison with BSC (BJH 2010; 149:376). We herein report the final results of the phase III part of the study. Methods: Untreated pts with de novo or secondary AML, adequate renal and hepatic function, and WBC count Results: Between 11/2004 and 03/2013, 237 pts were randomized from 35 European sites. The intention-to-treat population comprised 118 pts in the GO arm and 119 in the BSC arm. The median age was 77 years (range 62-88) with 64% of pts over 75 years. Baseline characteristics were well balanced between arms. At the final analysis (05/2014 cutoff), 228 deaths occurred: 113/118 (95.8%) in the GO arm, and 115/119 (96.6%) in the BSC arm. GO significantly improved OS (median 4.9 vs 3.6 months; 45.9% vs 29% alive at 6 months; 24.3% vs 9.7% alive at 1 year; 10.3% vs 6.9% alive at 1.5 year; HR, 0.69; 95% CI, 0.53-0.90; P=0.005) compared with BSC. Subgroup analyses based on stratification factors and other baseline characteristics showed that the OS benefit was generally consistent among subgroups, with a greater effect seen in pts with good/intermediate cytogenetics (median 7.3 vs 3.8 months; HR, 0.48; 99% CI, 0.31-0.75), in those with high CD33 expression (≥81% positive blasts; median 5.5 vs 3.8 months; HR, 0.47; 99% CI, 0.30-0.74), as well as in those with secondary AML (median 7 vs 4 months; HR, 0.55; 99% CI, 0.33-0.91). Among 111 pts who received at least the first dose of GO, the overall complete response rate was 27% (CR 15.3%, CRi 11.7%), and the overall disease control rate (including PR in 5.4% and stable disease for >30 days in 24.3%) was 56.7%. Of the 30 pts who achieved CR/CRi, 28 later relapsed or died in remission (1 infection, 2 general physical deterioration, 2 unknown cause), and the median disease-free survival (DFS) was 5.3 months with a 1-year DFS of 20%. CR/CRi pts had a median survival from remission of 8.2 months, with 40% alive at 1 year. The 30-day all-cause mortality from randomization was comparable in the GO (10.8%) and BSC (13.5%) arms. Most frequent grade 3+ non-hematologic adverse events (AEs) for GO vs BSC were infection (35.1% vs 34.3%), febrile neutropenia (18% vs 23.7%), bleeding (12.6% vs 12.3%), fatigue (11.7% vs 21%), and cardiac toxicity (6.3% vs 14%). Severe liver dysfunction occurred infrequently (2.7% vs 1.8% for GO vs BSC), and no episodes of VOD were reported. Overall, AEs led to GO discontinuation or death in 27 pts (24.3%). Conclusions: Compared with BSC including hydroxyurea as necessary, single agent GO in the dose/schedule chosen significantly improved OS in elderly AML pts not considered fit for intensive chemotherapy, with an acceptable safety profile. Of note, estimates of the benefit of GO were greater in pts presenting with better-risk cytogenetics, high CD33 expression on blast cells, or secondary disease. Disclosures Off Label Use: Gemtuzumab Ozogamicin in AML.

Introduction Decitabine has been approved for the treatment of myelodysplastic syndromes (MDS) in the United States and acute myeloid leukemia (AML) in older patients in Europe. The definitions of MDS and AML differ between the FAB and WHO classification, mainly with regards to patients with 20 to 30% blasts in blood or bone marrow having MDS according to the FAB classification (i.e. refractory anemia with excess blasts [RAEB] or RAEB in transformation), but AML according to the WHO. In the phase III trial 06011, we compared low-dose decitabine with best supportive care (BSC) in patients ≥60 years with MDS according to the FAB classification (Lübbert et al., J Clin Oncol. 2011;29:1987-96). Here, we examine trial 06011 for the efficacy and safety of decitabine in patients with AML according to WHO and low proliferation, i.e., blast counts of 20 to 30%. Patients and Methods Patients were randomly assigned to receive decitabine or BSC. Decitabine 15 mg/m2 was given intravenously over 4 hours every 8 hours for 3 consecutive days in 6-week cycles, with a maximum of 8 cycles. Results were evaluated every 2nd cycle. In case of complete remission (CR) at least 2 further courses were administered. Primary endpoint was overall survival (OS). Response rates (CR; PR, partial remission; HI, hematologic improvement; PD, progressive disease), progression-free survival (PFS; time from random assignment to PD, relapse or death), AML-free survival (AMLFS; time from random assignment to AML according to FAB [>30% bone marrow blasts] or death), and toxicity were secondary endpoints. Results Applying the WHO criteria to the 233 patients enrolled onto the trial, 164 had MDS and 50 had AML with blast counts of 20 to 30%. The remaining 19 patients were excluded from the present analyses. They comprised 14 patients with chronic myelomonocytic leukemia, 2 with AML and ≥40% blasts, and 3 with no blast counts available. Among the AML patients, 27 were in the decitabine and 23 in the BSC arm. In both arms, the median age was 70 years. Of the patients in the decitabine arm, 59% received 3 or more treatment cycles. Response rates in the decitabine and the BSC arm were as follows: CR, 11% vs 0%; PR, 11% vs 0%; HI, 11% vs 0%; and PD, 37% vs 74%. Compared with the patients receiving BSC, those receiving decitabine had longer PFS (P=0.008; Table 1). However, this did not translate into a significantly improved AMLFS or OS of the decitabine treated patients, although median OS was 9.8 months, compared to 5.9 months among patients receiving BSC only (Table 1). With regard to toxicity differences between the decitabine and BSC arms, grade 1-2 nausea was observed in 46% vs 14% and grade 3-4 febrile neutropenia in 19% vs 0%. Among the MDS patients, those receiving decitabine (n=78) had a longer PFS (P=0.07) but similar AMLFS and OS compared to the patients receiving BSC only (n=86; Table 1). The impact of decitabine on PFS, AMLFS and OS did not significantly differ between the AML and MDS patients (Table 1). Response rates among the MDS patients in the decitabine and BSC arms were as follows: CR, 14% vs 0%; PR, 4% vs 0%; HI, 18% vs 2%; and PD, 23% vs 66%. Conclusions Our data point to the clinically relevant efficacy of decitabine given in the 3-day schedule among patients with AML and low blast counts, particularly by delaying progression or relapse. No impact of decitabine, compared to BSC or low-dose cytarabine, on OS in older patients with AML and 20 to 30% marrow blasts (median, 8.0 vs 6.1 months) has been previously also reported by Kantarjian et al. (J Clin Oncol. 2012;30:2670-7). In that study, decitabine was given with 20 mg/m2/day on 5 days every 4 weeks; PFS was not presented. The prolonged PFS that we observe may be used for example as non-intensive bridge to allogeneic stem cell transplantation after reduced-toxicity conditioning. Due to the post-hoc nature of our analyses and the relatively small patient numbers, further studies appear warranted to fully establish the benefit of decitabine in AML patients with low blast counts. Disclosures: Rüter: Boehringer-Ingelheim: Employment. Platzbecker:Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Giagounidis:Celgene: Consultancy, Honoraria. Selleslag:Celgene: Consultancy; Novartis: Consultancy; Amgen: Consultancy. Baron:Genzyme: Honoraria.

Abstract 2804 Background: The “monosomal karyotype” cytogenetic subgroup (MK+), as defined by Breems et al. (1) encompasses mostly complex karyotypes (CK, >3 abnormalities) and is associated with very poor outcome in AML (1, 2) and MDS (3). Discussions are ongoing regarding treatment response prediction, and a prognosis-modifying effect within the CK+ AML subgroup (1, 4). In a study of 227 AML pts >60 years, 38 of whom were MK+, we found that particularly pts with at least 2 monosomal autosomes (MK2+, by definition also CK+) appeared to benefit from DAC (5). We now applied this question to higher-risk MDS pts randomized to either DAC or Best supportive care (BSC) in the 06011 trial (6) which explicitly enrolled pts with IPSS poor-risk cytogenetics. Methods: Of 233 randomized pts, 206 had cytogenetics informative for MK status: 63 had normal cytogenetics (CN), 143 had cytogenetic abnormalities (CA) without MK (MK-, n=73) or with MK (MK+, n=70). This last group was further subdivided into pts with 1, 2 or at least 3 monosomal autosomes (MK1, MK2, MK3+, n=17, 22, 31, resp.). Endpoints were overall survival (OS), progression-free survival (PFS) and response rate (RR, complete and partial remissions, hematologic improvement). Analysis was based on the intent-to-treat principle. Results: As recently published (6), in the overall 233 pts, PFS was significantly improved in the DAC arm as compared to BSC (median 0.55 vs 0.25 years, hazard ratio [HR] 0.68, p=0.004), but not OS (HR 0.88), probably due to post-progression treatments, suboptimal DAC schedule and treatment duration as possible confounders. In the 206 pts with informative cytogenetics, significant improvement in outcome with DAC vs. BSC was also seen for PFS (p=0.022, HR 0.72, Table 1) but not OS (HR 0.93). The improvement of PFS with DAC vs BSC was quite pronounced in the 63 pts with CN (HR 0.55, p=0.03), but less impressive and not significant in the 143 pts with CA (HR 0.76, p=0.11). When subdividing the latter by MK categories, pts with either 2 (MK2) or more than 2 (MK3+) monosomal autosomes also had significantly prolonged PFS with DAC, reflected in RR of 67% in MK2 and 33% in MK3+ DAC-treated pts (Table 1). This effect was not obvious in the MK1 subgroup, where the PFS outcome was favorable in both treatment groups, and in the MK− subgroups, either without (“MK−/CK−”) or with complex karyotype (“MK−/CK+”), the latter having a very poor outcome in both treatment groups. With caution (considering the limited size of some subgroups), these results support our observation that AML pts with a complex karyotype harboring 2+ monosomies can gain benefit from this hypomethylating agent. Conclusions: This first randomized trial addressing the predictive value of the MK genotype in the presence of 2+ monosomies embedded in a complex karyotype demonstrates a very rapid deterioration of MDS pts receiving BSC (within Disclosures: No relevant conflicts of interest to declare.

Abstract 4584 Introduction The disease course of B-cel chronic lymphocytic leukemia (CLL) is highly variable and genetic aberrations help to stratify patients with different prognosis. Therefore, detailed characterization of CLL associated chromosomal aberrations is important in order to identify patient subsets who need close monitoring and early therapy. The t(1;6)(p35.3;p25.2) involving IRF4 is extremely rare in CLL and has been associated with high-risk chromosomal aberrations [i.e. del(11q) and del(17p)], unmutated IGHV status and a rather progressive clinical course. Aim Here, we report the clinical, morphological and cytogenetic features of five new patients with a t(1;6)(p35.3;p25.2) and characterize for the first time the breakpoints at the molecular level. Materials and methods Five patients with B-NHL and a t(1;6)(p36∼p33;p25∼p23) in the karyotype were included in the study. Immunophenotype and/or morphology corresponded with CLL in 4/5 cases. The fifth case presented with follicular lymphoma (FL). The t(1;6)(p35.3;p25.2) was confirmed by FISH in all cases. Results When the 5 additional cases are considered together with 8 previously reported (Michaux et al., 2005), the female/male ratio is 3/10 with a median age of 63 (range 33–81). Four of the CLL patients presented with Binet stage A, 5 with stage B and 3 with stage C. Median leukocytosis was 11.8×109/L (range 1.8–103×109/L), LDH levels were elevated in 2 and CD38 was expressed in 7/9 patients. Nine patients showed an unmutated and 2 a mutated IGHV status (not available: n=2). Eleven patients required therapy, 0.5–62 months (median 0.5 month) after diagnosis. Only one of the 5 new cases was refractory to the therapy and died due to the disease after a follow-up of 20 months. The t(1;6) was found as the sole karyotypic change in 2 cases and was associated with one abnormality in 2 other cases (i.e. a +12 and a +15). The case with FL showed a complex composite karyotype. In addition, FISH detected a cryptic del(11q) and del(17p) in one case each. The 1p breakpoint was mapped to a 500Kb region between BACs RP11–290H1 and RP11–442N24 and the 6p breakpoint into the BAC RP11–233K4. Molecular analysis of 7 t(1;6) positive patients confirmed the presence of the translocation and demonstrated a fusion between the 5' end of RCC1 (in intron 1, 2 or 3) on 1p35, and IRF4 (in intron 1) on 6p25. The resulting fusion transcript consists of RCC1 exon 1, exon 2 or exon 3, linked to exon 2 of IRF4. In one patient, a 48 basepair long insert from intron 3 was present between RCC1 exon 3 and IRF4 exon 2. The first 4 exons of RCC1 are part of the 5'- untranslated region, whereas the open reading frame of IRF4 starts at exon 2. As such, the t(1;6) likely alters the expression of IRF4. Discussion and conclusion The RCC1 protein is widely expressed and is involved in chromatin condensation. IRF4 is a transcription factor with expression restricted to the immune system. The IRF4 protein plays a critical role in B-cell differentiation and mature T- and B-lymphocyte function. The t(6;14)(p25.2;q32) which juxtaposes IRF4 to the immunoglobulin loci (IG) resulting in IRF4 overexpression, has been reported in rare multiple myeloma (MM) cases and other mature B-NHLs. The oncogenic capacity of IRF4 has been demonstrated in MM. In CLL, the role of IRF4 remains unknown. Heterogeneous IRF4 expression has been observed between patients and examined tissues, and data on prognostic significance are conflicting. Here, we report 5 additional cases with t(1;6)(p35.3;p25.2) and demonstrate that this translocation is seen in unmutated CLL, as well as in mutated CLL and other indolent lymphoproliferative disorders. This is in contrast to the previously reported exclusive association of t(1;6) with unmutated CLL. At the molecular level, the t(1;6) results in a fusion transcript between RCC1 and IRF4 by which IRF4 expression is altered. Recognition of this and other translocations is important in order to refine the prognostic stratification of patients with CLL. Disclosures: No relevant conflicts of interest to declare.

Abstract 2609 Background: The monosomal karyotype (MK+) has recently been identified as the single most adverse cytogenetic prognosticator of AML outcome (1, 2). We made the recurrent - and seemingly paradoxical - observation that MDS/AML patients (pts) with complex karyotypes (CK+) including monosomy 7 showed encouraging responses to decitabine (DAC, ref.s 3, 4). We now evaluated a large cohort of AML pts >60 years ineligible for induction chemotherapy treated on a single phase II multicenter DAC study (trial 00331) for the effect of the MK+ genotype upon outcome. Methods: Comparisons of response rate (RR), i.e. attainment of complete and partial remissions (CR/PR) and overall survival (OS) were performed in 179 treatment-naive AML pts with available cytogenetics (median age 72 years) treated with DAC as described (ref. 3, given over 72 hours, every 6 weeks), for up to 4 courses, followed by “maintenance” with 3 daily 1-hour infusions of DAC 20 mg/m2 every 4–6-weeks. Median white blood cell counts prior to treatment was 7.500/μl (range 0.5–241,000), 79% had a performance status ECOG >1. Cytogenetic risk groups are shown in Table 1. All karyotypes were centrally reviewed and scored for MK (A.H.). Analyses were adjusted for the parameters which had a strong effect on outcome in multivariate analyses of RR and OS, i.e., performance status (on RR and OS) and platelet counts (on OS). Results: By the established definition of MK+, i.e either 1 autosomal monosomy and at least one structural change (MK1) or 2 or more autosomal monosomies (MK2), 39/179 patients were scored as MK+, with chromosomes 7 (n=17), 17 (n=17), and 5 (n=8) being most frequently lost. 17 pts had a single monosomy, as part of a median of 5 abnormalities (range, 2–21), 22 pts had multiple monosomies (median 3, range 2–11), as part of a median of 9.5 abnormalities (range, 4–17). As shown in Table 1, abnormal cytogenetics (AA) were associated with a lower RR and OS compared to pts with normal karyotype. When analyzing pts with AA (n=123) according to the presence (n=39) or absence of MK+ (n=84), MK+ patients had a higher RR than MK-, irrespective of a CK+ genotype. Since it has been shown by several groups that multiple monosomies may herald an even worse prognosis of AML than single monosomy, it was notable that pts with multiple monosomies (MK2, n=22) had a 45% RR and 25% 1-year OS (Fig. 1) compared with 24% and 12% in those with a single monosomy (MK1, n=17), both comparisons p=0.11. Conclusions: Patients with normal-karyotype AML had a better outcome with DAC treatment than those with chromosomal abnormalities. Patients with single or even multiple monosomies still appeared to respond to this treatment, supporting our previous, surprising observation that pts with highly complex karyotype including monosomies may benefit from DAC. Because of a lack of control group in our trial, it is difficult to hypothesize if this encouraging result may be related to modes of action of DAC which differ from low-dose chemotherapy, e.g., with cytarabine. The role of MK will be further studied prospectively in a randomized clinical trial in the same patient population (NCT00867672), and retrospectively in a randomized trial of higher-risk MDS patients treated with either DAC or Best Supportive Care (EORTC 06011). Disclosures: Off Label Use: Decitabine is approved for treatment of different types of MDS, in the present study its activity in AML of older patients was studied. Germing:Celgene: Consultancy, Research Funding.

Abstract 565FN2 Hyperdiploidy >50 chromosomes (HD>50) has long been recognized as favorable group in childhood B-ALL but there is still debate on the factors contributing to heterogeneity of prognosis observed within this entity. Better outcome has been reported for patients (pts) presenting DNA index (DI) >1.16 (Blood 1985;10:213), ≥56 chromosomes (Leukemia 1996;10:213), triple trisomies (TT) +4,+10,+17, double trisomies +4,+10 (Leukemia 2005;19:734) and trisomy 18 (Blood 2003;102:2756) but there is no consensus between the reports and these factors are differently applied in current protocols. We studied these factors in the pts with HD>50 enrolled in the ALL 58951 trial, BFM related. HD>50 were detected by cytogenetics (karyotype/FISH) and/or flow cytometry (DI). In order to analyze the outcome of HD>50 itself, pts with recurrent unfavorable translocations t(9;22), 11q23/MLL+, with t(1;19), t(12;21) or Down Syndrome were excluded, as well as near-triploidies/duplication of hypodiploidies 30–39 chromosomes. Pts were stratified into 4 risk groups (VLR/AR1/AR2/VHR) according to DI, Modal Number of Chromosomes (MNC), WBC, CNS/gonadal involvement, presence of VHR features (unfavorable translocations, poor response to prephase, residual disease (MRD) at the end of induction >10-2). VLR was defined as: DI>1.16 or MNC>50, WBC Overall Results: Out of 1651 B-lineage pts registered in the 58951 study over a 10-year period (1998-2008), a total of 541 pts had HD>50. Median age was 3 years and median WBC was 5.6×109/L. After prephase, 3% (N=17) were poor responders; initial risk group distribution into VLR/AR1/AR2/VHR was 45%/47%/5%/3%. After induction, 540 (99.8%) reached complete remission, 455 of whom had an MRD evaluation: MRD10-3 (N=26;6%) or MRD≥10-2 (N=13;2%). At median follow-up of 6 years, 48 pts (9%) relapsed, 22 pts (4%) died and 6 (1%) died without relapse. The 6-yr EFS was 89% (SE=1.5%). MNC was assessed in 446 pts (82%) with successful karyotypes, MNC ranged from 51 to 66 chromosomes and peaked at 55–56; 87 pts had 51 to 53 chromosomes (HD51-53), 258 pts had 54 to 57 (HD54-57) and 101 pts had ≥58 chromosomes (HD≥58). In these 3 groups, VLR regimen was given to 16%, 50% and 64% respectively. Structural abnormalities were detected in 46% of pts and associated with all MNCs. DI, assessed in 460 pts was Prognostic Factors: The only significant prognostic factors for EFS were MNC, DI, TT, DT and MRD. EFS was clearly improved (p10-2 and 2 pts: MRD≥10-3) and only 1 pt (MNC=58) relapsed. No specific profile of chromosome gains was identified in HD ≥58 since all chromosomes contributed to tri/tetrasomies, except chromosome 1. Likewise, the higher the DI the better the outcome (p=0.01): 6-yr EFS was 83% (DI TT were detected in 168 out of 468 pts, and DT in 242 out of 466 pts. There was no overlap with HD ≥58 since TT and DT were distributed from 52 to 66 chromosomes. One third of TT and of DT had MNC≥58. TT and DT were also of prognostic importance for outcome: the 6-yr EFS rate was 96% (TT) vs 86% (non-TT) (p=0.005) and 94% (DT) vs 84% (non-DT) (p=0.003). A hierarchical variable based on presence of HD≥58, TT or DT showed that HD ≥58 (N=101; 6-yr EFS: 99%) group had a better outcome than TT without HD≥58 (N=115; 6-yr EFS: 93%) and DT without HD≥58 and without +17 (N=55; 6-yr EFS: 84%) groups (p=0.04). We can infer from our results that the good outcome observed for all of the TT and DT was partially due to their association with HD '58. Consequently, the best indicator for excellent outcome was a high MNC (≥58 chromosomes). From our 58951 trial, we can assume that among children with B-ALL and HD>50, those with ≥58 chromosomes, stand every chance of being cured. Our results stress the necessity of karyotype for identifying them since this is the only way to assess MNC. They can also be detected (less accurately) by DI (DI ≥1.24). Therefore, both MNC and DI should be used for stratifying pts in the very low risk groups. Disclosures: No relevant conflicts of interest to declare.

Abstract 2922 The myelodysplastic syndromes (MDS) are a heterogeneous group of hematological malignancies characterized by ineffective hematopoiesis and a highly variable clinical course, for which novel treatments are beginning to emerge. Conventional cytogenetic studies (CCS) of bone marrow (BM) are routinely used in clinical practice to detect abnormal clones in proliferating (metaphase) cells, identifying clonal aberrations in ∼50% of de novo MDS cases. Cytogenetics is also one of the key International Prognostic Scoring System (IPSS) components used to estimate overall survival and leukemia-free survival in MDS. Chromosome abnormalities may be quantified at presentation and during treatment by the use of fluorescence in situ hybridization (FISH) with DNA probes for specific chromosome loci (e.g., chromosomes 5, 7, 8 and 20) in non-proliferating (interphase) nuclei. However, it is not clear whether or not peripheral blood CCS will yield the same diagnostic and prognostic data as bone marrow CCS at disease presentation or whether patients without apparent chromosome abnormalities by CCS have “hidden” abnormalities that can be identified by interphase FISH. To answer these questions, 15 members of the International Working Group on MDS Cytogenetics agreed to perform CCS and FISH in parallel on both peripheral blood and bone marrow samples collected from MDS patients. To be certain that all participating sites scored and interpreted their individual FISH data in a similar fashion, a quality assurance (QA) study was completed with each site studying two identical test (proficiency) samples. Concordance among sites was very good to excellent allowing for the establishment of a standardized protocol with clear scoring criteria before patient samples were processed. In the second phase of the study, a total of 77 MDS patients were accrued to the study with 61% showing an abnormal karyotype. A FISH panel consisting of eight probe sets [-5/5q-, -7/7q-/der(1;7), +8/8q-, -11/+11/11q-/add(11q), 12p-/+21/t(12;21), -13/13q-, 17p- and 20q-/i(20q)/i(20p), Abbott Molecular, Inc.] was performed on both specimen types. While CCS was frequently unsuccessful (57.5%) in the PB specimens, FISH was informative (concordant with BM/PB CCS) in 92% of cases, with 49% of PB FISH demonstrating an abnormal clone. FISH was discordant in 4 of 77 BM and PB samples (5%), while CCS and FISH on BM and PB were discordant in 6 of 77 BM specimens (8%) and 6 of 73 PB specimens (8%). The data suggest that evaluation of interphase nuclei from PB on follow-up (non-diagnostic) FISH studies on MDS patients will be equally informative (and less costly and stressful) as a BM sample. Disclosures: Slovak: PerkinElmer: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership, Research Funding. Ohyashiki:Nippon Shinyaku Co., Ltd.: Research Funding.

Abstract 1755 Poster Board I-781 Background Karyotypic abnormalities are common in myelodysplastic syndromes (MDS), and specific chromosomal abnormalities are associated with poor prognosis. The phase III AZA-001 study (Lancet Oncol, 2009) showed azacitidine (AZA) prolonged overall survival (OS) regardless of IPSS cytogenetic risk category. This analysis assessed the effects of specific cytogenetic abnormalities on OS in patient (pt) subgroups treated with AZA or a conventional care regimen (CCR). Methods Pts with higher-risk MDS (FAB RAEB, RAEB-t, or CMML and IPSS Int-2 or High) were enrolled and randomized to receive AZA or CCR. CCR comprised 3 treatments: best supportive care only, low-dose ara-C, or induction chemotherapy. Erythropoietins were prohibited. OS was determined in subgroups of pts with del 5/5q-, del 7/7q-, or trisomy 8, each as part of a non-complex karyotype ( Results A total of 358 pts were enrolled (AZA 179, CCR 179). Of them, 153 had normal karyotypes (AZA 77, CCR 76). Median OS in pts with normal karyotypes was not reached at 21.1 months with AZA vs 17.2 months (95%CI: 15.2 – 24.1 months) with CCR; HR = 0.63 (95%CI: 0.39 – 1.03). Of remaining pts, 136 had del 5/5q-, del 7/7q-, and/or trisomy 8 as part of a non-complex or complex karyotype. AZA was associated with longer OS vs CCR in all subgroups of pts with non-complex cytogenetics, with HRs ranging from 0.20 (95%CI: 0.06 – 0.65) to 0.51 (95%CI: 0.05 – 4.74) (Table). In both the AZA and CCR treatment groups, pts in all subgroups with non-complex karyotypes had substantially longer OS than pts with complex karyotypes. Pts with complex karyotypes in some subgroups had longer OS with AZA vs CCR: median OS in pts with del 5/5q-, del 5/5q- WITHOUT del 7/7q-, or trisomy 8 as part of a complex karyotype treated with AZA survived 5.1, 8.0, and 12.4 months longer, respectively, than their counterparts who received CCR. HRs with AZA vs CCR in pts with complex cytogenetics ranged from 0.42 (95%CI: 0.10 – 1.69) to 0.55 (95%CI: 0.29 – 1.05). Conclusions These findings support earlier data showing effectiveness of AZA in higher-risk MDS pts with complex or non-complex karyotypes. Major gains in OS were obtained with AZA vs CCR (12-18 months longer OS with AZA) for the following categories: del 7/7q- (non-complex), del 7/7q- WITHOUT del 5/5q- (non-complex), and trisomy 8 (non-complex and complex). Pts with trisomy 8 treated with AZA experienced a 3-fold increase in median OS compared with similar pts who received CCR. Longer OS (AZA 15.3 vs CCR 7.3 months) was also obtained for pts with del5/5q- WITHOUT del7/7q- as part of a complex karyotype. The worse cytogenetic categories, del 7/7q- and del 5/5q- AND del 7/7q-, both with complex karyotype, were associated with the poorest OS regardless of treatment. Pt subgroups in this post hoc analysis were small and heterogeneous; confirmation of these findings in larger pt samples is warranted. Disclosures Mufti: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Gore:Celgene: Consultancy, Equity Ownership, Research Funding; Johnson & Johnson: Research Funding. Santini:Celgene: Honoraria. Fenaux:Celgene: Honoraria, Research Funding; Ortho Biotech: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Cephalon: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; MSD: Honoraria, Research Funding; Epicept: Honoraria, Research Funding. Skikne:Celgene: Employment, Equity Ownership. Hellstrom-Lindberg:Celgene: Research Funding. Seymour:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Beach:Celgene: Employment, Equity Ownership. Backstrom:Celgene: Employment, Equity Ownership. Fernando:Celgene: Employment, Equity Ownership.

Chromosomal translocations have recently emerged as an important prognostic indicator in B-CLL (Mayr et al, Blood 2006). Until now however, their value had neither been determined in patients undergoing homogeneous treatment, nor compared to that of IgVH mutational status, another major prognostic factor in B-CLL. Sixty-five B-CLL pts treated with cladribine-containing regimens were included in the present analysis. All had been investigated by conventional cytogenetic analysis using TPA as a mitogen, interphase FISH (tested loci: 11q/ATM, 12cen, 17p/P53, 13q/RB1&/D13S319), IgVH mutational status and P53 mutational screening prior to inclusion. Translocations (n= 45) were detected in 42 % of the pts, and included both balanced (n=12) and unbalanced (n=33) types. Pts with translocations were more heavily pretreated (P=.05) and presented with significantly higher incidence of complex aberrations (P

The recently described episomal amplification of the NUP214-ABL1 fusion in 6% of T-ALL leads to the expression of a constitutively activated chimeric tyrosine kinase sensitive to imatinib. We collected additional cases in order to better characterize this new entity with respect to genetic presentations and clinical course. We collected 14 new NUP214-ABL1 positive cases by FISH (LSI-BCR/ABL1 ES, Vysis and ABL1 break-apart home-made probes) or RT-PCR (fusion transcript) screening. FISH analysis detected episomal amplification of NUP214-ABL1 in 11 patients with a highly variable number of nuclei with amplification ( These observations emphasize the interest of combining both (quantitative) RT-PCR and FISH for the screening and characterisation of NUP214-ABL1 fusion and amplification, demonstrate the coexistence of different NUP214-ABL1 genomic presentations in one patient (episomal amplification, 9q34 insertions, HSR) compatible with the model in which genomic amplification start with episome formation in order to create the NUP214-ABL1 fusion followed by their amplification and optional secondary reintegration, confirm occurrence of NUP214-ABL1 in T-ALL with HOX11 and HOX11L2 involvement, raise the question of the rather worse prognosis for cases with intrachromosomal amplification as previously suggested, raise the signification of minor NUP214-ABL1 clones and variable genomic presentations in the leukemogenesis of this subgroup of T-ALL that could potentially benefit from imatimib. ° on behalf of the GFCH (Groupe Francophone de Cytogénétique Hématologique) and the BCGHO (Belgian Cytogenetic Group for Hematology and Oncology).