Cryptic Translocation Research Articles

Prenatal Diagnosis of a Satellited Chromosome 8p Results from a de novo Cryptic Translocation between Chromosomes 8 and 22

The authors of the present study report the prenatal detection of a chromosomal abnormality with additional satellites on the distal short arm of chromosome 8. A 35-year-old woman was referred for amniocentesis because of her advanced maternal age and positive result for maternal serum screening test. Cytogenetic analysis of cultured amniocytes showed a satellite 8p chromosome. The satellite 8p chromosome was positive for nucleolus organizer region (NOR) staining. The parents karyotypes were normal. Fluorescence ’ in situ hybridization (FISH) study for metaphases of fetal amniocytes revealed a cryptic translocation of chromosomes 8p and 22p. The fetal karyotype was described as 46,XY,8ps.ish t(8;22)(p23.3;p11.2) (D8S504-;D8S504+)dn. The parents decided to continue the pregnancy and a phenotypically normal boy was born at 38 weeks of gestation. In case of de novo terminal NORs detected prenatally, more accurate cytogenetic and molecular analysis should be performed in order to rule out cryptic chromosomal rearrangement among other chromosomes.

NUP98-NSD1 Fusion Gene Is Strongly Associated with a Poor Prognosis in Pediatric Acute Myeloid Leukemia: A Study of the Japanese Childhood AML99 Cooperative Study Group

Abstract 1475 Background:Acute myeloid leukemia (AML) is a complex disease caused by mutations, deregulated gene expression, and epigenetic modifications of genes leading to increased proliferation and decreased differentiation of hematopoietic progenitor cells. In hematological malignancies with 11p15 translocations, the nucleoporin 98-kDa (NUP98) protein regulates nucleocytoplasmic transport of protein and RNA. More than 20 different chromosomal rearrangements involving NUP98 have been identified. The cryptic translocation t(5;11)(q35;p15.5), which creates a fusion gene between the NSD1 and the NUP98. The NUP98-NSD1 fusion gene has been associated with a deletion of the long arm of chromosome 5, del(5q), in pediatric AML patients. Recently, NUP98-NSD1 was identified in childhood AML by fluorescence in situ hybridization (FISH) with subtelomeric probes. However, the frequency and clinical impact of NUP98-NSD1 in pediatric AML remain uncertain. Methods:To estimate the frequency and clinical impact of the NUP98-NSD1 in pediatric AML, we examined the NUP98-NSD1 fusion transcript in a consecutive series of 157 newly diagnosed AML patients enrolled in the AML99 protocol in Japan clinical trial. Total RNA extracted from the leukemia cells of the patient at diagnosis was reverse transcribed to cDNA. The PCR was performed using the primer sets for detection of NUP98-NSD1. The PCR amplification was performed with a DNA thermal cycler followed by direct sequencing with an ABI PRISM 310 Genetic Analyzer. We also examined somatic mutations of the FLT3, KIT, WT1, NPM1, NRAS, KRAS and MLL genes, which are prevalent in AML, in these AML patients with NUP98-NSD1. Results:In the current study, we identified the NUP98-NSD1 by RT-PCR followed by direct sequencing in 6 (3.8%) out of 157 AML patients, and clustered significantly in the cytogenetically normal AML subgroup (6/33, 18.2%). Significant differences between patients with and without NUP98-NSD1 were observed in 4-year OS (33.3% versus 80.0%), and EFS (33.3% versus 69.2%). NUP98-NSD1 was highly seen in males (5 males and 1 female). The statistical differences between patients with and without NUP98-NSD1 was not significant in age (median: 7 years [range: 2–15 years] versus 6 years [range: 0–15 years]), and the initial WBC count (median: 115.9 × 109/L [range: 9.0–329 × 109/L] versus 51.8 × 109/L [range: 1.0–621 × 109/L]). Interestingly, NUP98-NSD1 was observed in 4 patients with normal karyotype, and was observed in 2 patients with del(9q). NUP98-NSD1 could not be identified by conventional G-banding technique in any cases. Patients with NUP98-NSD1 had any mutation of FLT3 -ITD (66.7%), WT1 (50%), KIT (16.7%) or RAS (33.3%). Furthermore, NUP98-NSD1 was frequently found in M4 or M5 patients according to FAB classification. Conclusions:We identified the NUP98-NSD1 in 6 out of 157 AML patients, and clustered significantly in the cytogenetically normal AML subgroup. Patients with NUP98-NSD1 had any FLT3 -ITD, WT1, KIT or RAS mutations. These results suggest that simultaneous occurrence of FLT3 -ITD, WT1, KIT mutations and RAS aberrations in NUP98 -related leukemia may be associated with poor prognosis. The NUP98-NSD1 was not identified by conventional G-banding technique, confirming that this translocation can exist as the only cytogenetic alteration. Therefore, FISH and RT-PCR analyses for NUP98-NSD1 fusion gene is recommended at the onset of disease in larger series of AML patients presenting an apparently normal karyotype, del(9q) or del(5q) to gain insight into the actual incidence and clinical features of patients with t(5;11)(q35;p15.5). Disclosures:No relevant conflicts of interest to declare.

Distinctive Spectra of Cytogenetic Alterations in Over Five Hundred East Asian Patients with Acute Leukemia

Abstract 4408 Background:Racial difference of spectra of chromosomal aberrations in various hematological malignancies was previously reported between Asian and western countries. Therefore, characterization of the cytogenetic profile is still a matter of interests for identifying cytogenetic risk factors in the current risk-adapted chemotherapy. The aim of this study was to investigate the spectrum of chromosomal aberrations in East Asian (Korean) acute leukemia patients, and to propose a panel of leukemic fusion genes suitable for the development of new molecular detection systems. Patients and Methods:We prospectively analyzed bone marrow samples from 502 patients with acute leukemia referred for screening leukemic fusion genes by simultaneous analysis of conventional cytogenetics, FISH and multiplex RT-PCR system in Chonnam National University Hwasun Hospital (Hwasun, Korea) for last 5 years. Results:A total of 334 patients were diagnosed with AML, 155 with ALL and 13 with mixed phenotype acute leukemia. Twenty types in 28 fusion genes were detected in 42.4% of the total patients by multiplex RT-PCR system and in 33.9% by conventional cytogenetics including FISH. In the group of AML, the common fusion genes were 57 PML/RARa, 38 AML1/MGT8, 12 CBFb/MYH11, 11 MLL1, 5 BCR/ABL1, SET/CAN, PLZF/RARA and TLS/ERG in 2 cases each, and one of each AML1/MDS1, TEL/ABL and TEL/MN1. In the group of ALL, the following fusion genes were detected: 33 BCR/ABL1, 22 TEL/AML1, 10 MLL1, 7 E2A/PBX, 2 SET/CAN and SIL/TAL, one TEL/ABL. In addition, cytogenetically cryptic translocations were detected in 14.8% patients with normal karyotype or numerical aberrations by conventional cytogenetics. Among 288 patients with negative results by multiplex RT-PCR system, one child with early pre-B-ALL harbored MLL1/AF4, and 8 patients had 3 types of clinically significant chromosomal aberrations such as t(3;3)(q21;q26.2), t(8;14)(p24.1;q32) and i(17)(q10), which were all confirmed by conventional cytogenetics. Conclusions:The current study demonstrated the spectrum and frequency of chromosomal aberrations in Korean patients with acute leukemia, which are different from Caucasian data. Also these results may offer important implications for the development of new multiplex molecular detection system. Disclosures:No relevant conflicts of interest to declare.

BCR-ABL Translocation in Pediatric Acute Lymphoblastic Leukemia in Southern India

Cytogenetics and polymerase chain reaction (PCR) based assays provide important information regarding biologically defined and prognostically relevant subgroups in acute leukemias. We utilized karyotyping and molecular analysis by reverse transcriptase PCR for the BCR-ABL translocation, in addition to morphological study, cytochemistry and immunophenotyping, to study 24 cases of pediatric acute lymphoblastic leukemia (ALL). Our objective was to determine the frequency of the BCRABL translocation in childhood ALL from southern India. Karyotyping showed one case of hyperdiploidy, one case of t (12; 21) translocation and one case of 46, XY-21+mar. The BCR-ABL translocation was found in 8.3% of these cases. One of these was a cryptic translocation, the karyotype being normal. BCR-ABL positivity in ALL is associated with aggressive disease and has been shown to be a poor prognostic factor, especially in children.

A Comparative Analysis of Molecular Genetic and Conventional Cytogenetic Detection of Diagnostically Important Translocations in More Than 400 Cases of Acute Leukemia, Highlighting the Frequency of False-Negative Conventional Cytogenetics

In this study, we correlated the results of concurrent molecular and cytogenetic detection of entity-defining translocations in adults with acute leukemia to determine the frequency of cryptic translocations missed by conventional cytogenetics (CC) and of recurrent, prognostically relevant translocations not detectable by multiplex reverse transcriptase-polymerase chain reaction (MRP). During a 5.5-year period, 442 diagnostic acute leukemia specimens were submitted for MRP-based detection of 7 common recurrent translocations: t(8;21), t(15;17), inv(16), t(9;22), t(12;21), t(4;11), and t(1;19), with a detection rate of 15.2% (67/442). CC was performed in 330 (74.7%) of 442 cases. In 7 of these 330 cases, CC missed the translocation detected by MRP. In 50 additional cases, CC revealed 1 of the MRP-detectable translocations (all were also MRP positive), yielding a false-negative rate of 12% (7/57) for the CC assay. The remaining 140 of 190 cases with clonal cytogenetic changes harbored abnormalities that were not targeted by the MRP assay, including 8 that define specific acute myeloid leukemia entities. This study revealed the frequent occurrence of false-negative, entity-defining CC analysis and highlighted the complementary nature of MRP and CC approaches in detecting genetic abnormalities in acute leukemia.

Cryptic X; Autosome Translocation in a Boy—Delineation of the Phenotype

Chromosome X-to-autosome translocations [t(X;A)] are rare rearrangements with an estimated occurrence of 1 to 3 per 10,000 live births. Occurrences of Xq duplications have been observed in male and female subjects in whom the X chromosome segment escapes inactivation and results in functional disomy. We report a case of X;6 translocation in a 7-year-old boy with severe mental retardation, hypotonia, and recurrent respiratory tract infections. High-resolution chromosome analyses (fluorescence in situ hybridization, multiplex ligation probe-dependent amplification, and whole-genome array) revealed a terminal duplication of chromosome X at q28-qter (approximately 3.246 Mb in size) involving gene MECP2 and a terminal deletion (approximately 1.89 Mb) with the breakpoint at 6q27. This is the second report of a boy with a cryptic unbalanced Xq-autosome translocation. This case increases our understanding of mental disability caused by terminal Xq duplication.

Combined 22q11.1-q11.21 deletion with 15q11.2-q13.3 duplication identified by array-CGH in a 6 years old boy

BackgroundDeletions of chromosome 22q11 are present in over 90% of cases of DiGeorge or Velo-Cardio-Facial syndrome (DGS/VCFS). 15q11-q13 duplication is another recognized syndrome due to rearrangements of several genes, belonging to the category of imprinted genes. The phenotype of this syndrome varies but has been clearly associated with developmental delay and autistic spectrum disorders. Co-existence of the two syndromes has not been reported so far.ResultsHere we report a 6-year-old boy presenting growth retardation, dysmorphic features and who exhibited learning difficulties. Fluorescence in situ hybridization (FISH) analysis of the proband revealed a deletion of DiGeorge Syndrome critical region (TUPLE). Array-CGH analysis revealed an interstitial duplication of 12 Mb in size in the area 15q11.2-q13.3, combined with a 3.2 Mb deletion at region 22q11.1-q11.21. FISH analysis in the mother showed a cryptic balanced translocation between chromosome 15 and chromosome 22 (not evident by classic karyotyping).DiscusionThe clinical manifestations could be related to both syndromes and the importance of array-CGH analysis in cases of unexplained developmental delay is emphasized. The present case further demonstrates how molecular cytogenetic techniques applied in the parents were necessary for the genetic counseling of the family.

CALLA negative precursor B lymphoblastic leukemia with MLL gene translocation and an unusual FISH signal pattern

Rearrangements of the mixed lineage leukemia (MLL) gene at 11q23 commonly occur in infants with CALLA negative B lymphoblastic leukemia (B-ALL). Most often, these are detected by conventional karyotyping; however, fluorescent in-situ hybridization (FISH) with the help of a dual-color break-apart probe is used to identify cryptic translocations. When there is an MLL gene translocation, the usual FISH signal pattern is 1 red-1 green-1 yellow fusion signal pattern. We present a case of an infant with CALLA negative precursor B-ALL with a characteristic translocation t(4;11) (q21;q23), however, with an unusual MLL FISH signal pattern.

Unbalanced translocation 9;16 in two children with dysmorphic features, and severe developmental delay: Evidence of cross-over within derivative chromosome 9 in patient #1

We describe 2 children with dysmorphic features, and severe developmental delay presenting with overlapping unbalanced translocations of 9q34.3 and 16p13. Patient #1: A 4 year old African-American female with normal karyotype with a pericentric inversion on one chromosome 9 known to be a benign variant. Low resolution array CGH revealed a single BAC clone loss at 9q34.3 and a single BAC clone gain at 16p13.3, confirmed by FISH. Whole genome SNP array analysis refined these findings, identifying a terminal 1.28 Mb deletion (138,879,862–140,164,310) of 9q34.3 and a terminal 1.62 Mb duplication (45,320–1,621,753) of 16p13.3. Sub-telomeric FISH showed an unbalanced cryptic translocation involving the inverted chromosome 9 and chromosome 16. FISH of the father showed a balanced t(9;16)(q34.3;p13.3) involving the non-inverted chromosome 9, and a pericentric inversion on the normal 9 homologous chromosome. The presence of two rearrangements on chromosome 9, both an unbalanced translocation and a pericentric inversion, indicates recombination between the inverted and derivative 9 homologues from her father. Patient #2: A 1 year old Iraqi-Moroccan female with normal karyotype. Array-CGH identified a 0.56 Mb deletion of 9q34.3 (139,586,637–140,147,760) and an 11.31 Mb duplication of 16p13.3p13.13 (31,010–11,313,519). Maternal FISH showed a balanced t(9;16)(q34.3;p13.13). Both patients present with similar clinical phenotype.

Incidence of ETV6 Cryptic Translocations In Myeloid Disorders

AbstractAbstract 2733Rearrangements affecting band 12p13 are found as recurring chromosomal changes in myeloid malignancies. The main target of these rearrangements is the ETV6 gene, a member of the ETS family of transcription factors that is required for hematopoiesis within the bone marrow. To date, more than 41 translocations affecting ETV6 have been described, most of them encoding chimeric proteins with oncogenic properties. However, some of the translocations do not produce a functional fusion protein, but may induce ectopic expression of oncogenes located in the neighbourhood of the breakpoint.In order to investigate the incidence of ETV6 aberrations in myeloid disorders and to search for novel ETV6 translocations, we performed fluorescence in situ hybridization (FISH) screening with a specific breakapart probe for the ETV6 gene (Abbott Vysis) on 134 acute myeloid leukemia (AML) cases, of which 20 cases were secondary AML evolved from myelodysplatic (MDS) or myeloproliferative syndromes (MPS), 10 MDS, and 3 MPS.Conventional cytogenetic analysis of the cases identifed abnormal karyotypes in 72 cases, of which 20 cases presented del(12p), 5 cases add(12p), and 8 cases a translocation involving 12p. The remaining 39 abnormal karyotypes did not present alterations in the short arm of chromosome 12. Normal karyotypes were detected in 53 cases and cytogenetic analyses failed in 22 cases.FISH detected 22 cases with heterozygous deletions of ETV6, of which 15 correlated with previous cytogenetic findings, 4 were detected in patients without evaluable mitoses, 2 were associated with add(12p), and one resulted from an unbalanced translocation involving chromosomes × and 12. In the remaining 7 translocations, FISH confirmed the t(3;12)/EVI1-ETV6, t(4;12)/CHIC2-ETV6, and t(5;12)/PDGFRβ-ETV6 in 4 cases, excluded an ETV6 involvement in 2 translocations, and revealed in one patient a novel t(3;12;17)(q21;p13;q25) with breakpoint in ETV6, previously described as t(3;12)(q21;p13).Interestingly, cryptic translocations involving ETV6 were revealed in 3 patients with cytogenetically described 12p deletions: the t(12;17)(p13;p12–13) in a secondary AML1; inv(12)(p13q24) in a secondary AML; and t(2;12)(p16.1;p13) in an AML FAB subtype M1.In one additional patient with del(12p), FISH revealed a cryptic interstitial deletion with breakpoint in ETV6, del(12)(pterp13). In the latter patient, hybridization signals centromeric to ETV6 were deleted, whereas the probe hybridizing telomeric to ETV6 was retained on the derivative chromosome 12. Finally, one further ETV6 cryptic translocation, t(1;12)(p36;p13), was found in patient with MDS and an apparently normal karyotype.The ETV6 translocation partner could be identified in the t(2;12)(p16.1;p13) and showed a novel fusion transcript derived from the ETV6 gene and the BAC clone RP11-440P5, on chromosome 2p16.1. DNA sequencing revealed an in-frame fusion of exon 7 of ETV6 to sequences derived from BAC 44OP5. We further investigated whether the ETV6 translocation partner in the t(1;12) was the recently described MDS2 gene of the t(1;12)(p36;p13).2 However, RT-PCR with specific primers for ETV6 and MDS2 failed to amplified an ETV6-MDS2 fusion transcript. The molecular cloning of the inv(12) and the t(3;12;17) is in progress.In conclusion, we have identified 6 new ETV6 rearrangements in patients with myeloid neoplasias, of which 5 were not seen by standard cytogenetic analyses. The prevalence of ETV6 rearrangements assessed by FISH (10/147, 6,8%) was 50% higher than by conventional cytogenetic (5/147, 3,4%). This study shows the relatively high frequency of cryptic translocations involving ETV6 on 12p13 and underlines the importance of FISH to detect and characterize these translocations.This work was supported by the Deutsche José Carreras Leukämie-Stiftung eV.

A Cell Line SYRMO Derived From AML with EVI 1 Rearrangements Following Imatinib Mesylate Therapy for Chronic Myeloid Leukaemia

AbstractAbstract 4470Chronic Myeloid Leukaemia (CML) is a malignant disorder of the haematopoeitic stem cell, usually characterised by the t(9;22) giving rise to the Philadelphia chromosome (Ph), and by the BCR-ABL gene rearrangement at the molecular level. Imatinib mesylate (IM), which targets the tyrosine kinase (TK) activity of BCR-ABL, has become the first line therapy for CML patients. Dasatinib or Nilotinib is now indicated as a second line therapy for patients who develop resistance or intolerance to IM.Here we report the case of a 51 year old woman who was diagnosed in Cyprus (2007) with BCR/ABL1 positive CML. After failing to respond to IM therapy, she was treated with Dasatinib achieving complete cytogenetic remission (CCR), and subsequent complete molecular remission. In July 2009 the patient presented with 88% blasts in the bone marrow. Immunophenotyping showed the presence of CD34+, CD117+ myeloblasts, and a diagnosis of AML was confirmed. Analysis of G banded metaphases from cultured peripheral blood revealed a complex karyotype: 46,XX, t(2;3)(p21;q26),t(4;8)(q12;q22), del(6)(q13;q23)[1]/45,idem,der(6)ins(6;6)(q23;),-7[9]. FISH revealed EVI 1 rearrangement resulting from the t(2;3) in the absence of BCR/ABL1 fusion and confirmed the secondary monosomy 7. The patients failed to respond to treatment and passed away shortly afterwards. Whole genome screening by aCGH using a 244K platform (Agilent) confirmed the loss of chromosome 7 and the 6q22/23 region.Clonal cytogenetic abnormalities (CCA) in Ph-negative metaphases are known to develop in some patients during IM therapy. The most frequent of these abnormalities are trisomy 8, -7, del(7q), and del(20q), which are also associated with MDS and AML. These abnormalities are usually transient and disappear after a short time, or have no clinical consequence (Deininger et al., Cancer 2007). However, reports of development of high risk MDS or AML in association with CCA in Ph-negative cells are rare, but patients with chromosome 7 abnormalities appear to be at greater risk. The t(2;3) is a non random abnormality in MDS and AML, including therapy-related leukaemias, frequently associated with -7 and a complex karyotype with a poor prognosis (Stevens-Kroef et al Leukemia 2004).This is the first report of a CML patient on TKI treatment with t(2;3) and EVI1 gene rearrangements in Ph(-) cells. This cryptic translocation could easily be overlooked in the presence of a monosomy 7 with a dramatic effect on patient management. Disclosures:No relevant conflicts of interest to declare.

The Spectra of Chromosomal Aberrations In Acute Leukemia and Implications for the Development of Optimal Profiles of Leukemic Fusion Genes In Multiplex RT-PCR System.

AbstractAbstract 3362 Background:Cytogenetic aberrations have been identified as important prognostic parameters for acute leukemia, in addition to diagnosis and treatment. The aim of the current study was to investigate the spectrum of chromosomal abnormalities in East Asian (Korean) patients with acute leukemia. Based on the spectrum of genetic aberrations found in our study, we proposed a revised list of chromosomal aberrations for the development of optimal profiles of leukemic fusion genes in multiplex RT-PCR system. Patients and Method:We prospectively analyzed blood or bone marrow samples from 348 patients (260 adults, 88 children) with leukemia from 2006 to 2009 at the Chonnam National University Hwasun Hospital (Hwasun, Korea). Simultaneous analyses of conventional cytogenetics, FISH and a commercially available multiplex RT-PCR system (HemaVision) were performed. Result:Thirty-six types of chromosomal abnormalities were found in the total patient population. Twenty types of chromosomal abnormalities were detected in 42% of all patients by multiplex RT-PCR system and in 33% of all patients by conventional cytogenetics including FISH. Within the group of 213 AML patients, the common cytogenetic abnormalities were t(15;17)(q21;q22) (n=35), t(8;21)(q22;q22) (n=31), 11q23 abnormalities (n=7), inv(16)(p13;q22) (n=3), t(9;9)(q34:q34) (n=2), and the abnormalities t(9;22)(q34;q11), t(9;12)(q34;p13), t(3;21)(q26;q22), t(11;17)(q23;q21), t(12;22)(p13;q11) and t(16;21)(p11.2;q22.3) were each involved in one case. In the group of 104 ALL patients, the following fusion transcripts were detected: 21 t(9;22)(q34;q11), 15 t(12;21)(p13;q22), nine 11q23 abnormalities, four t(1;19)(q23;p13), with t(9;9)(q34:q34), t(9;12)(q34;p13) and del(1p34) each involved in one case. In addition, cryptic translocations were detected in 9% of patients with normal karyotypes or numerical aberrations by conventional cytogenetics. Among 53 patients showing negative results for multiplex RT-PCR, one child patient with early pre-B-ALL harbored MLL1/AF4 fusion genes and 5 patients had 3 types of clinically significant chromosomal aberrations such as t(3;3)(q21;q26.2), t(8;14)(p24.1;q32) and i(17)(q10). Conclusion:The current study demonstrated the spectrum and frequency of chromosomal abnormalities in Korean leukemia patients, which were slightly differed from previous studies including western and some Asian countries. These results may offer important implications in understanding characteristics of molecular pathophysiology, the development of a new RT-PCR system as well as revision of the current commercial available RT-PCR system. Disclosures:No relevant conflicts of interest to declare.

Cytoplasmic Immunoglobulin Fluorescence In Situ Hybridization (cIg FISH) Enhances the Quantitative Detection of Chromosome Abnormalities In Plasma Cell Neoplasms Compared to Conventional FISH

AbstractAbstract 1198Risk stratification of plasma cell neoplasms (PCNs) has become an integral part of clinical management along with the rapid advances in treatment. Cytogenetics is widely performed at diagnosis but has a low abnormality detection rate due to poor proliferation of plasma cells (PCs) in culture and inability to identify cryptic translocations. Conventional interphase FISH improves detection sensitivity, but lacks specificity since the percentage of PCs is diluted by the large number of normal bone marrow cells due to the indiscriminative analysis of all cells by FISH. The International Myeloma Working Group recommends all FISH tests for myeloma be PC specific by either PC enrichment or identifying PCs by cytoplasmic immunoglobulin staining (cIg FISH). However, the real benefits of cIg FISH compared to conventional FISH have never been demonstrated in a controlled study. In this objective comparative study, 75 paired samples from patients with PCNs were analyzed concurrently by conventional FISH and cIg FISH with probes for t(4;14), t(11;14), t(14;16), -13/13q (RB1/LAMP1), 17p- (TP53) and CEP3. For conventional FISH, a minimum of 200 nuclei were scored per probe set. For cIg FISH, 100 cIg positive cells were scored per probe with a minimum of 25 cIg+ cells required for conclusive reporting. In this study, 51/75 cases met the above criteria for direct comparison (see Table). cIg FISH results demonstrated an improved overall detection rate [-13/13q: 53% vs. 45%; 17p-: 14% vs. 12%; t(11;14): 25% vs. 24% and t(4;14): 14% vs.12%]. Impressively, cIg FISH quantitatively identified a much higher proportion of abnormal PCs in all cases. The median % of abnormal PCs with all probes was >50% by cIg FISH but no more than 10% by conventional FISH. The abnormal PCs detected by cIg FISH were >90% of cIg+ cells in 18 cases with at least one probe. In contrast, the abnormal PCs detected by conventional FISH were <5% of scored cells in 12 cases with all probes. Of 11 cases with discordant results between cIg FISH and conventional FISH, cIg FISH detected more abnormalities in 8/11 cases. In 2 cases from the same myeloma patient during follow up, t(11;14) was detected by both methods but del(13q) was detected only by conventional FISH, which was related to a concomitant myelodysplastic syndrome rather than myeloma, confirmed by cytogenetic study. In the third case, multiple abnormalities were detected by both methods but +3 was detected only by conventional FISH; its nature remains undetermined. Out of the remaining 24 cases, cIg+ cells were absent or sparse in 18 due to low PC counts. The results of conventional FISH were normal in all 18 cases. Six cases had only <25 cIg+ cells per probe, but most of the cIg+ cells were abnormal in 4/6 cases with probes consistent with the concurrent results of conventional FISH. The other 2 cases had a single abnormality only by conventional FISH at the level close to its detection threshold. Collectively, these results suggest the percentage of PCs in cIg FISH samples need to reach a threshold in order to ensure unequivocal results and performance consistency, which appears to be similar to the detection limit of conventional FISH. In summary, cIg FISH tends to have a greater detection rate and consistently identifies a higher proportion of abnormal PCs in all samples compared to conventional FISH. It can be used not only at diagnosis for accurate prognostic assessment, but also at relapse and during active disease for better monitoring expansion of abnormal clones and clonal evolution.Table 1Comparison of results of cIg FISH and conventional FISH for same PCN samplesSide-by-side Conventional FISH & cIg FISH N=51-13/13q RB1/LAMP117p- TP53+3 CEP3t(11;14) BCL1/IGHt(4;14) FGFR3IGHt(14;16) IGH/MAFNumber (%) of positive cases27 (53)7 (14)17 (33)13 (25)7 (14)1 (2)cIg FISH Conventional FISH23 (45)6 (12)17 (33)12 (24)6 (12)1 (2)Median % pos cells/case765456918378cIg FISH Conventional FISH79109610Range of % pos cells/case14-10019-9626-8663-10021-9678cIg FISH Conventional FISH4-806-114-461-461-2210 Disclosures:Jagannath:Celgene: Honoraria; Millenium/Takeda Pharma: Honoraria; J&J Family: Honoraria; Onyx: Honoraria; Merck: Honoraria.

Activation of a Novel Palmitoyltransferase through a t(6;14) Translocation In T/Myeloid Biphenotypic Leukemia and Subsets of Acute Myeloid Leukemia.

AbstractAbstract 1663The current World Health Organization (WHO) classification of hematologic malignancies includes a number of entities defined by specific genetic abnormalities. Most known abnormalities result from chromosomal translocations and mutations involving genes encoding transcription factors or tyrosine kinases. We identified the activation of a novel palmitoyltransferase, ZDHHC14, through a chromosomal translocation, t(6;14)(q25;q32). This translocation was initially documented by three independent groups in two children and two adult patients with T/myeloid biphenotypic leukemia, a poorly differentiated form of acute leukemia with a single population of leukemic blasts co-expressing antigens of both T-lymphoid and myeloid lineages. Recently, we detected the same translocation in two additional patients with T/myeloid biphenotypic leukemia. Molecular characterization of the translocation breakpoints revealed the juxtaposition of a regulatory region downstream of BCL11B (at chromosome region 14q32) upstream of an uncharacterized gene, ZDHHC14 (at chromosome region 6q25), leading to transcriptional activation of the ZDHHC14. Notably, this regulatory region from 14q32 has been found to be responsible for transcriptional activation of a homeobox gene, TLX3, as a result of a cryptic translocation t(5;14)(q35;q32) in T-cell acute lymphoblastic leukemia. Our subsequent survey of ZDHHC14 protein levels in blasts from patients with various subtypes of acute myeloid leukemia (AML) showed that the protein was not detectable in AML patients with t(8;21), t(15;17), or inv(16), but was detected in AML patients with FLT3 mutations or with complex karyotypes, indicating a possible association of increased expression of this protein with unfavorable prognostic features of the disease. We also demonstrated that overexpression of ZDHHC14 could inhibit TPA-induced cellular differentiation of K562 cells. Interestingly, the Drosophila homolog of ZDHHC14 has been found to be a palmitoyltransferase regulating Fat signaling. Fat is a Drosophila tumor suppressor gene, encoding a large cadherin that regulates growth and a form of tissue organization. While the function of ZDHHC14 and the role of protein palmitoylation in mammalian hematopoiesis remain unknown, recent studies in neuronal development have demonstrated that palmitoylation regulates diverse aspects of protein trafficking. Our findings suggest that palmitoyltransferase plays a previously unappreciated role in hematopoiesis and that increased expression of ZDHHC14 may be associated with a primitive status of leukocytes. Further characterization of ZDHHC14 may broaden our knowledge of leukemogenesis, contribute to risk stratification of acute leukemia, and provide a novel therapeutic target. Disclosures:No relevant conflicts of interest to declare.

Three Novel AML Cases Harboring the Semi-Cryptic t(7;21)(p22;q22) Translocation Expressing RUNX1-USP42 Fusion Transcripts Associated with Diploidy/Tetraploidy and/or 5q Alterations: a Probably Underestimated Combination

AbstractAbstract 2708RUNX1 is implicated in numerous chromosomal abnormalities acquired in acute myeloid leukemia (AML). The most frequent one, the t(8;21) is associated with a particular morphology together with a favorable prognosis. This is not the case for other 21q abnormalities, that are much less frequent and for which the prognosis is quite different. Moreover, beside point mutations, conventional cytogenetics failed to detect some of chromosomal alterations involving RUNX1.Recently 3 cases of the rare and semi-cryptic t(7;21)(p22;q22) translocation expressing the RUNX1-USP42 fusion transcripts have been reported, demonstrating the recurrence of this abnormality in AML. We describe here 3 additional cases with the same translocation and fusion transcripts, associated to 5q alterations leading to EGR1 and CSF1R heterozygous losses.In all our patients, the t(7;21)(p22.1;q22.3) was initially detected by the systematic FISH evaluation of the blastic populations using ETO-AML1 Dual Fusion probe. Patient#1 bone marrow karyotype was characterized by a tetraploid clone (89,XXYY) with loss of chromosomes 15, 17 and 18 in addition to the t(7;21), and a unbalanced translocation der(5)t(5;13)(q23;q?) between long arms of chromosomes 5 and 13, resulting in a heterozygous loss of EGR1 and CSF1R. Patient #2 blood and bone marrow karyotypes revealed a diploid clone with a del(5)(q31q33) associated with the t(7;21). The FISH analysis confirmed EGR1 and CSF1R deletions. In patient #3, the bone marrow karyotype showed diploid/tetraploïd clones, both harboring the t(7;21)(p22;q22), confirmed by FISH experiments (WCP7, AML1 probes). In addition, a der(5)t(1;5)(q3?2;q21-23) was identified within the tetraploïd clone, resulting in the loss of EGR1 and CSF1R, confirmed by FISH.In all three cases a RUNX1-USP42 fusion transcript was detected using RT-PCR, as well as the reciprocal transcript. Sequence analysis of RT-PCR products showed that the breakpoints occurred exactly in the same introns of USP42 and RUNX1 as in the previously described cases. For patient #1 and #3 a chimeric transcript was found formed of the RUNX1 exon 7 fused to the USP42 exon 3. In patient #2, a shorter chimeric transcript arised from the fusion of the RUNX1 exon 5 to the exon 3 of USP42. As already noticed in the previous reports, an alternative splicing of the RUNX1 exon 6 has been detected in these three cases.The description of these 3 novel t(7;21) confirm the recurrence of this balanced translocation in AML, and shows that this chromosomal abnormality is often associated with diploid/tetraploid clones and/or 5q alterations. Special attention should be paid in karyotype analysis of AML with diploid or tetraploid clones harboring 5q alterations. In such cases RUNX1 rearrangements should be explored using FISH analysis, and RUNX1-USP42 fusion transcript should be searched by RT-PCR in positive cases. Prospective and retrospective studies of AML have now to be settled in order to assess the incidence and clinical relevance of this cryptic translocation. Disclosures:No relevant conflicts of interest to declare.

Interstitial insertion of RARα gene into PML gene in a patient with acute promyelocytic leukemia (APL) lacking the classic t(15;17)

The diagnosis of APL is based on clinical and morphological tests though the final diagnosis is at the molecular level. An accurate diagnosis is important as it mandates targeted therapy to improve survival. We report a case of APL without t(15;17) in conventional cytogenetic study and with initially negative fluorescence in situ hybridization (FISH) study on cells in interphase. Reverse transcription polymerase chain reaction (RT-PCR) for the promyelocytic/retinoic acid receptor alpha gene (PML/RARα) fusion oncogene proved the clinical diagnosis as well as FISH study on cells in metaphase. The cause was a cryptic translocation of the RARα gene into PML. We reviewed 36 additional cases of APL diagnosed in our hospital since 1992. This was the only case that failed to show t(15;17) in cytogenetics. However, three cases with t(15;17) in cytogenetics had negative RT-PCR for PML/RARα. Our case emphasizes that cytogenetics, FISH and RT-PCR studies are complementary studies for the molecular diagnosis of APL.

Familial Subtelomeric Rearrangement of Chromosomes 19 and 20: A New Contribution to Partial Distal 19q Trisomy

The role of cryptic translocations in human syndromes is a matter of fact, though this phenomenon is apparently rare. Apart from episodic case reports due to the increasing application of new molecular cytogenetic techniques, no data on its frequency in the general population are currently available. Rearrangements due to the unbalanced segregation of cryptic translocations are found in many anomalies responsible for different clinical pictures. In nearly 50% of cases, subtelomeric abnormalities are inherited from a parent carrying a balanced cryptic chromosome rearrangement. To date, very few cases of partial trisomies of 19q have been reported, with different breakpoints. Involvement of the distal region 19q is even more rare, and the delineation of its main clinical characteristics is still vague and awaiting better definition. We report two new cases of partial 19q13.42-qter trisomy associated with a partial 20p13-pter monosomy in a family found to have the cryptic translocation t(19;20)(q13.42;p13). We investigated a 5-year-old boy and his 49-year-old paternal uncle, and both had a similar, previously unrecognized mental retardation pattern, associated with the same subtelomeric rearrangement.

Myelodysplastic syndromes with deletions of chromosome 11q lack cryptic MLL rearrangement and exhibit characteristic clinicopathologic features

Deletions of chromosome 11q[del(11q)] as part of a non-complex karyotype are infrequent in myelodysplastic syndromes (MDS), leaving the clinicopathologic and genetic features largely undefined. From three large medical centers over a 10-year period, we identified 32 MDS cases where del(11q) was present either as a sole ( n = 23) or associated with another abnormality ( n = 9), showing an overall 0.6% frequency in MDS. These patients included 15 men and 17 women, with a median age of 68 years. Three were therapy-related, and 29 were primary MDS. These cases were characterized by transfusion-dependent anemia (65%); frequent ring sideroblasts (RS) (59%); bone marrow hypocellularity (22%), and less severe thrombocytopenia. With a median follow-up of 32 months, 9/24 (38%) cases progressed to acute myeloid leukemia (AML), and the overall survival (OS) was 35 months (3–105). Fluorescence in situ hybridization (FISH) showed MLL deletion in 6/10 cases, but no cryptic MLL translocations in all 15 MDS cases tested. In contrast, FISH performed in AML with del(11q) showed MLL rearrangement in 3/17 (18%) cases. In summary, del(11)q occurring in a non-complex karyotype is predominantly associated with primary MDS, lack of cryptic MLL rearrangements, and shows characteristic clinicopathological features. These clinicopathological features are likely attributed to commonly deleted regions of 11q and their involved genes.

A new stripe rust resistance gene transferred from Thinopyrum intermedium to hexaploid wheat (Triticum aestivum)

Wheat stripe rust (Puccinia striiforis f. sp. tritici) races CYR31 and CYR32, prevalent in China, are virulent to many wheat stripe rust resistance genes (Yr genes). To expand the availability of effective resistance to CYR31 and CYR32, stripe rust resistance was transferred from intermediate wheatgrass (Thinopyrum intermedium) to common wheat (Triticum aestivum). The susceptible wheat cultivar CM107 was crossed with amphiploid TAI7047, derived from the wide cross Taiyuan768/Thinopyrum intermedium//76(64). Two wheat lines originating from the cross, YU24 and YU25, were resistant to CYR31 and CYR32. Pedigree analysis showed that the resistance to stripe rust in YU24 and YU25 originated from intermediate wheatgrass. Genetic analyses indicated that the resistance to stripe rust is controlled by a single dominant gene. Allelic tests determined that the resistance gene(s) in YU24 and YU25 are identical. The new gene has temporarily been designated as YrYU25. SSR and RAPD analyses showed that YrYU25 was introduced by cryptic translocation into common wheat.

Abstract 324: A NUT-variant midline carcinoma showing a complex karyotype

Abstract NUT-midline carcinomas (NMC) are rare, almost uniformly lethal malignant tumours arising in the midline organs. Originally diagnosed predominantly in the young, they have since been identified in people ranging in age from 3 to 78 years. The tumours are typically very aggressive in behaviour, with an average survival of only nine months. NMCs are typified by a single, conserved underlying genetic translocation t(15;19)(q14;p13.1), that results in a fusion of the BRD4 and NUT genes. There are a few cases described in which NUT is fused to a different gene (primarily BRD3) and these are known as NUT-variants. It is very rare however, for translocations other than the primary one, to be present. We present an NMC case with a far more complex karyotype, with the translocations t(6;19)(q14;p13.1) and t(1;18;7)(q42;q11.2;q21) present in all cells, as well as approximately 10% of cells demonstrating t(12;15)(q13;q22). Initial FISH experiments, using break-apart probes, established a translocation of the 19p13.1 (BRD4) region, and demonstrated no translocation of the 15q14 (NUT) region. Subsequent experiments, using BRD4 specific probes in a cell line derived from the patient specimen, have shown that BRD4 was inserted into chromosome 15, in a region proximal to the NUT gene. We are currently determining whether BRD4 was fused directly to NUT through a cryptic translocation in this case. Illumina Human-Cyto-SNP-12 BeadChip array analysis has also shown that a 3.3MB region of 6q14 (adjacent to the region translocated onto chromosome 19) was deleted in the tumour cells. The array indicated no deletions or duplications at the sites of any of the other translocations. Given the aggressive nature of these tumours, a better understanding of the underlying genetic causes and precipitating factors of the disease processes remains imperative to improving the outcome for such patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 324.