Global screening and extended nomenclature for 230 aphidicolin-inducible fragile sites, including 61 yet unreported ones.

Since the first description of human fragile sites (FS) more than 40 years ago, a variety of substances were reported to induce chromosomal breaks at non-random, breakage-prone regions. According to information available from human genome browsers aphidicolin, an inhibitor of DNA replication induces 77 of 88 known common FS. However, in the literature additional FS are reported, which are also, at least in part, inducible by aphidicolin. To the best of our knowledge, here we present the first and largest ever done systematic, whole genome-directed and comprehensive screening for aphidicolin-inducible breakage-prone regions. The study was performed on stimulated peripheral blood lymphocytes of 3 unrelated healthy individuals. Twenty-five thousand metaphase spreads were analyzed and overall 22,537 FS located in 230 different loci were recorded. Sixty-one of those FS were never observed before and 52 were already previously reported but not included in genome browsers and yet verified. Interestingly, aphidicolin was able to induce all types of rare and common FS, suggesting that these breakage-prone regions are less dependent on the inducing chemicals than originally supposed. Overall, we provide the first comprehensive genome wide map for FS and studied possible correlations of chromosome length and GTG-banding level with FS-frequency. To handle FS better in future, an extension of the already existing alphabetical nomenclature for FS on single chromosomes is suggested.

Automated detection of residual cells after sex-mismatched stem-cell transplantation - evidence for presence of disease-marker negative residual cells.

BACKGROUND: A new chimerism analysis based on automated interphase fluorescence in situ hybridization (FISH) evaluation was established to detect residual cells after allogene sex-mismatched bone marrow or blood stem-cell transplantation.Cells of 58 patients were characterized as disease-associated due to presence of a bcr/abl-gene-fusion or a trisomy 8 and/or a simultaneous hybridization of gonosome-specific centromeric probes. The automatic slide scanning platform Metafer with its module MetaCyte was used to analyse 3,000 cells per sample. RESULTS: Overall 454 assays of 58 patients were analyzed. 13 of 58 patients showed residual recipient cells at one stage of more than 4% and 12 of 58 showed residual recipient cells less than 4%, respectively. As to be expected, patients of the latter group were associated with a higher survival rate (48 vs. 34 month). In only two of seven patients with disease-marker positive residual cells between 0.1-1.3% a relapse was observed. Besides, disease-marker negative residual cells were found in two patients without relapse at a rate of 2.8% and 3.3%, respectively. CONCLUSION: The definite origin and meaning of disease-marker negative residual cells is still unclear. Overall, with the presented automatic chimerism analysis of interphase FISH slides, a sensitive method for detection of disease-marker positive residual cells is on hand.

Protein profiling of microdissected pancreas carcinoma and identification of HSP27 as a potential serum marker.

BACKGROUND: Patients with pancreatic adenocarcinomas have a poor prognosis because of late clinical manifestation and the tumor's aggressive nature. We used proteomic techniques to search for markers of pancreatic carcinoma. METHODS: We performed protein profiling of microdissected cryostat sections of 9 pancreatic adenocarcinomas and 10 healthy pancreatic tissue samples using ProteinChip technology (surface-enhanced laser desorption/ionization). We identified proteins by use of 2-dimensional gel electrophoresis, peptide fingerprint mapping, and immunodepletion and used immunohistochemistry for in situ localization of the proteins found. We used ELISA to quantify these proteins in preoperative serum samples from 35 patients with pancreatic cancer and 37 healthy individuals. RESULTS: From among the differentially expressed signals that were detected by ProteinChip technology, we identified 2 proteins, DJ-1 and heat shock protein 27 (HSP27). We then detected HSP27 in sera of patients by use of ELISA, indicating a sensitivity of 100% and a specificity of 84% for the recognition of pancreatic cancer. CONCLUSIONS: The detection of DJ-1 and HSP27 in pure defined tissue and the retrieval of HSP27 in serum by antibody-based methods identifies a potential marker for pancreatic cancer.

Identification of specific protein markers in microdissected hepatocellular carcinoma.

At present, the molecular mechanisms of hepatocellular carcinogenesis are not well-understood, and hepatocellular carcinoma (HCC) stays one of the most frequent and high-risk metastatic visceral neoplasms worldwide. For the identification of tumor-relevant proteins, we analyzed microdissected cells from nontumorous liver tissue (n = 28) and tissue derived from hepatic tumor center (n = 25), as well as tumor margin (n = 23). We unequivocally identified 53 proteins from hepatic tumor tissues by peptide fingerprint mapping and SELDI mass spectrometry that were separated using two-dimensional gel electrophoresis. Among a number of signals that were detected as significantly different in the protein profiling analysis, we identified for the first time ferritin light subunit (FLS) and adenylate kinase 3 alpha-like 1 (AK3), showing decreased expressions in hepatic tumor, as well as biliverdin reductase B (BVRB) that was upregulated in HCC. The use of ProteinChip technology in combination with tissue microdissection gives insight of the complex changes occurring at the protein level in hepatocellular cancer associated with tumor development and progression and resulted in three new potential diagnostically useful markers.

Comet fluorescence in situ hybridization analysis for oxidative stress-induced DNA damage in colon cancer relevant genes.

Our objective was to study whether products of oxidative stress, such as hydrogen peroxide (H(2)O(2)), trans-2-hexenal, and 4-hydroxy-2-nonenal (HNE), cause DNA damage in genes, relevant for human colon cancer. For this, total DNA damage was measured in primary human colon cells and colon adenoma cells (LT97) using the single-cell gel electrophoresis assay, known as "Comet Assay." APC, KRAS, and TP53 were marked in the comet images using fluorescence in situ hybridization (Comet FISH). The migration of APC, KRAS, or TP53 signals into the comet tails was quantified and compared to total DNA damage. All three substances were clearly genotoxic for APC, KRAS, and TP53 genes and total DNA in both types of cells. In primary colon cells, TP53 gene was more sensitive toward H(2)O(2), trans-2-hexenal, and HNE than total DNA was. In LT97 cells, the TP53 gene was more sensitive only toward trans-2-hexenal and HNE. APC and KRAS genes were more susceptible than total DNA to both lipid peroxidation products but only in primary colon cells. This suggests genotoxic effects of lipid peroxidation products in APC, KRAS, and TP53 genes. In LT97 cells, TP53 was more susceptible than APC and KRAS toward HNE. Based on the reported gatekeeper properties of TP53, which in colon adenoma is frequently altered to yield carcinoma, this implies that HNE is likely to contribute to cancer progression. This new experimental approach facilitates studies on effects of nutrition-related carcinogens in relevant target genes.

Uranyl nitrilotriacetate, a stabilized salt of uranium, is genotoxic in nontransformed human colon cells and in the human colon adenoma cell line LT97.

Previous uranium mining in the "Wismut" region in Germany enhanced environmental distribution of heavy metals and radionuclides. Carryover effects may now lead to contamination of locally produced foods. Compounds of "Wismut" origin are probably genotoxic via their irradiating components (radon) or by interacting directly with cellular macromolecules. To assess possible hazards, we investigated the genotoxic effects of uranyl nitrilotriacetate (U-NTA) in human colon tumor cells (HT29 clone 19A), adenoma cells (LT97), and nontransformed primary colon cells. These are target cells of oral exposure to environmentally contaminated foods and represent different cellular stages during colorectal carcinogenesis. Colon cells were incubated with U-NTA. Cell survival, cytotoxicity, cellular glutathione (GSH) levels, genotoxicity, and DNA repair capacity (comet assay), as well as gene- and chromosome-specific damage combination of comet assay and fluorescence in situ hybridization [FISH], 24-color FISH) were determined. U-NTA inhibited growth of HT29 clone 19A cells (75-2000 microM, 72 h) and increased GSH (125-2000 microM, 24 h). U-NTA was genotoxic (1000 microM, 30 min) but did not inhibit the repair of DNA damage caused by hydrogen peroxide (H(2)O(2)), 4-hydroxynonenal, and 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]-pyridine. U-NTA was also genotoxic in LT97 cells and primary colon cells, where it additionally increased migration of TP53 into the comet tail. In LT97 cells, 0.5-2mM U-NTA increased chromosomal aberrations in chromosomes 5, 12, and 17, which harbor the tumor-related genes APC, KRAS, and TP53. It may be concluded that uranium compounds could increase alimentary genotoxic exposure in humans if they reach the food chain in sufficient amounts.

Characteristic genomic imbalances in pediatric pheochromocytoma.

Pheochromocytoma (PCC) in children is rare, genetically not well described, and often related to a poor prognosis. We detected genomic imbalances in all 14 tumors from children analyzed by comparative genomic hybridization. A combinatorial loss of chromatin from 3p and 11p was a common feature in 10 of 14 (72%) patients, which was a result of either a loss of a total chromosome 3 and a total chromosome 11 in 6 of 10 patients, or confined deletions of their p arms in 4 of 10 patients. All patients exhibiting a loss of 3p and 11p carried VHL mutations. The VHL mutations were constitutive in 9 cases and somatic and restricted to tumor DNA in the remaining tumor. On the other hand, VHL mutations were absent in 4 patients, 2 who had other familial syndromes (NF1, SDHD) and 2 with unknown etiology. Our data show that the pattern of imbalances in the tumor DNA of PCC patients strongly correlated with an underlying familial VHL mutation. Furthermore, we show that true sporadic PCC is rare in childhood. Thus, children with PCC should be checked for a related predisposing gene. This would also identify familial syndrome patients requiring long-term monitoring for other syndrome-related malignancies.

Minimal phenotype in a girl with trisomy 15q due to t(X;15)(q22.3;q11.2) translocation.

Few cases of de novo unbalanced X;autosome translocations associated with a normal or mild dysmorphic phenotype have been described. We report a 3-year-old dizygotic female twin with prenatally ascertained increased nuchal translucency. Prenatal chromosome studies revealed nearly complete trisomy 15 due to a de novo unbalanced translocation t(X;15)(q22;q11.2) confirmed postnatally. A mild phenotype was observed with normal birth measurements, minor facial dysmorphic features (hypertelorism, short broad nose, and a relatively long philtrum), and moderate developmental delay at the age of 3 years in comparison to her male fraternal twin. Replication timing utilizing BrdU and acridine-orange staining showed that the der(X) chromosome was late-replicating with variable spreading of inactivation into the translocated 15q segment. The der(X) was determined to be of paternal origin by analyses of polymorphic markers and CGG-repeat at FMR1. Methylation analysis at the SNRPN locus and analysis of microsatellites on 15q revealed paternal isodisomy with double dosage for all markers and the unmethylated SNRPN gene. The Xq breakpoint was mapped within two overlapping BAC clones RP11-575K24 and RP13-483F6 at Xq22.3 and the 15q breakpoint to 15q11.2, within overlapping clones RP11-509A17 and RP11-382A4 that are all significantly enriched for LINE-1 elements (36.6%, 43.0%, 26.6%, 22.0%, respectively). We speculate that the attenuated phenotype may be due to inactivation spreading into 15q, potentially facilitated by the enrichment of LINE-1 elements at the breakpoints. In silico analysis of breakpoint regions revealed the presence of highly identical low-copy repeats (LCRs) at both breakpoints, potentially involved in generating the translocation.

Molecular cytogenetic characterization of the mouse cell line WMP2 by spectral karyotyping and multicolor banding applying murine probes.

The Moloney murine leukemia virus-transformed suspension cell line WMP2 is derived from wild mice (Mus musculus) of the WMP/WMP strain. These mice carry nine pairs of metacentric Robertsonian translocation chromosomes. As the chromosomes of the wild-type mouse are all acrocentric, metaphase spreads of the WMP2 cells seam to be highly suited for physical gene mapping. Here we studied the WMP2 line using spectral karyotyping (SKY) combined with new established mouse specific multicolor banding (mcb) probes for the chromosomes X, 3, 4, 6 and 18. SKY revealed that the WMP2 cell line developed further four derivative chromosomes. After application of mcb five previously unrecognizable intrachromosomal rearrangements with 9 breakpoints were detected for the studied chromosomes.

2-Dodecylcyclobutanone, a radiolytic product of palmitic acid, is genotoxic in primary human colon cells and in cells from preneoplastic lesions.

The irradiation of fat results in the formation of 2-alkylcyclobutanones, a new class of food contaminants. Results of previous in vitro studies with primary human colon cells and in vivo experiments with rats fed with 2-alkylcyclobutanones indicated that these radiolytic derivatives may be genotoxic and enhance the progression of colon tumors. The underlying mechanisms of these effects, however, are not clearly understood. Therefore we performed additional investigations to elucidate the genotoxic potential of 2-dodecylcyclobutanone (2dDCB) that is generated from palmitic acid. In particular, we explored the relative sensitivities of human colon cells, representing different stages of tumor development and healthy colon tissues, respectively. HT29clone19A cells, LT97 adenoma cells and primary human epithelial cells were exposed to 2dDCB (150-2097 microM). We determined cytotoxic effects using trypan blue exclusion. Genotoxicity, reflected as strand breaks, was assessed using the alkaline version of the comet assay and chromosomal abnormalities were investigated by 24-color fluorescence-in-situ-hybridization. 2dDCB was cytotoxic in a time- and dose-dependent manner in LT97 adenoma cells and in freshly isolated primary cells but not in the human colon tumor cell line. Associated with this was a marked induction of DNA damage by 2dDCB in LT97 adenoma cells and in freshly isolated colonocytes, whereas in the HT29clone19A cells no strand breaks were detectable. A long-term incubation of LT97 adenoma cells with lower concentrations of 2dDCB revealed cytogenetic effects. In summary, 2dDCB was clearly genotoxic in healthy human colon epithelial cells and in cells representing preneoplastic colon adenoma. These findings provide additional evidence that this compound may be regarded as a possible risk factor for processes in colon carcinogenesis related to initiation and progression.

Characterization of pepsinogen C as a potential biomarker for gastric cancer using a histo-proteomic approach.

We analyzed 74 cryostat sections of central gastric tumor, tumor margin, and normal gastric epithelium using ProteinChip Arrays and SELDI-TOF MS. One peak was significantly down-regulated in tumor tissue (P = 1.43 x 10(-6)) and identified as pepsinogen C using MS/MS analysis and immunodepletion. This signal was further characterized by immunohistochemistry. This work demonstrates that differentially expressed signals can be identified and assessed using a proteomic approach comprising tissue-microdissection, protein profiling, and immunohistochemistry.

The gut fermentation product butyrate, a chemopreventive agent, suppresses glutathione S-transferase theta (hGSTT1) and cell growth more in human colon adenoma (LT97) than tumor (HT29) cells.

PURPOSE: The gut fermentation product of dietary fiber, butyrate, inhibits growth of HT29, an established tumor cell line. It also induces detoxifying enzymes belonging to the glutathione S-transferase family (GSTs), namely hGSTM2, hGSTP1, hGSTA4, but not of hGSTT1 . Here we investigated kinetics of effects in HT29 and compared sensitivities with preneoplastic LT97 colon adenoma cells, to assess mechanisms of colon cancer chemoprevention in two stages of cell transformation. METHODS: We determined cell growth after butyrate treatment by quantifying DNA, GST expression by Northern/Western Blotting or biochemical analysis and butyrate consumption by measuring the residual concentrations in the cell culture supernatants. Stability of GST-theta (hGSTT1) mRNA was assessed in HT29 cells after inhibition of transcription with actinomycin D. RESULTS: LT97 adenoma cells consumed twofold more butyrate and were more sensitive to growth inhibition than HT29 (EC(50)1.9 mM and 4.0 mM, respectively). Butyrate did not induce GSTs, but instead reduced hGSTT1 in LT97 and HT29. CONCLUSIONS: Butyrate has suppressing-agent activities in human colon cells by inhibiting two survival factors, namely hGSTT1 and cell growth, with LT97 more sensitive than HT29. These findings indicate that butyrate formation in the gut lumen of humans could be protective by reducing survival of transformed colon cells.

Discovery and identification of alpha-defensins as low abundant, tumor-derived serum markers in colorectal cancer.

BACKGROUND & AIMS: Although colorectal cancer is one of the best characterized tumors with regard to the multistep genetic progression, it remains one of the most frequent and deadly neoplasms in Western countries. This is mainly due to the fact that, up to now, no clinically relevant serum markers could be established in an early routine diagnostic procedure. METHODS: We comparatively analyzed microdissected normal and tumorous colonic epithelium by ProteinChip technology to detect proteins specific for the tumor directly in the tissue. Immunohistochemistry (IHC) was used for the in situ localization of the discovered proteins, and an ELISA was performed to quantify these proteins in serum. RESULTS: By this approach, we found and identified alpha-defensins 1-3 (HNP1-3) to be more highly expressed in the tumor than in normal epithelium. These findings could be confirmed by IHC. Detection of these peptides in the corresponding serum samples was subsequently performed with ELISA, resulting in an average sensitivity of 69% and specificity of 100% for the recognition of colorectal cancer when using the HNP1-3 level in the serum of the patients. CONCLUSIONS: The direct analysis of microdissected tissue for the discovery of tumor-specific markers followed by the specific detection of these markers in serum by antibody-based methods proved to be a successful strategy in this study. Therefore, we can conclude that these promising markers would not have been found in serum without the information gained through the analysis of microdissected tissue by ProteinChip technology.

Detection of cryptic chromosomal aberrations in the in vitro non-proliferating cells of acute myeloid leukemia.

A total of 22 acute myeloid leukemia (AML) cases were analyzed by cell-specific comparative genomic hybridization (micro-CGH). Conventional banding analysis identified a monosomy 7 in six (group I), a trisomy 8 in eight (group II) and a normal karyotype in eight cases (group III). A total of 32 additional chromosomal imbalances was detected and confirmed in two independent micro-CGH experiments. However, only in 9 of the 22 cases (group I: 4 cases; group II: 1 case; group III: 4 cases) the existence of 11 of the 32 (34.5%) detected copy number alterations could be confirmed by other fluorescence in situ hybridization (FISH) approaches. These results lead to two conclusions: i) in the in vitro non-proliferating population of AML tumor cells one can detect cryptic chromosomal aberrations, which might constitute tumor markers of diagnostic and prognostic value; ii) The results of CGH need to be checked by other approaches.

Identification of protein pattern in kidney cancer using ProteinChip arrays and bioinformatics.

Tumor biology of renal cell carcinoma (RCC) is not very well understood, although many studies on molecular and cellular biology have been performed. It is accepted now that cancer research has to be performed also with proteomic tools, because proteins are the real actors in the genesis and progression of cancer. Therefore, we used a ProteinChip System(R) (SELDI) which is able to detect minute amounts of protein and moreover to analyze a complex protein pattern. We analyzed 37 cases of clear cell RCC as a training set including corresponding normal tissue. From all samples protein lysates were made and spotted directly on different chip surfaces (SAX2, WCX). After a washing procedure the arrays were analyzed in the ProteinChip Reader. All profiles were subjected to a bioinformatical analysis including normalization, clustering, rule extraction and rating. Defined rules (markers) were evaluated using a test set of 24 samples (13 tumor tissues and 11 normal kidney tissues). The generated rule base for the SAX2 surface showed a sensitivity of 100% and a specificity of 97.3%. For the WCX arrays the optimal rule base showed worse results. A combined rule base for SAX2 and WCX did not result in a higher sensitivity or specificity. Using the optimal rule base for the SAX2 chip in the test set, sensitivity and specificity reached 76.9% and 100%, respectively. The ProteinChip System represents a key technology for the rapid detection of cancer specific proteomic patterns. It is possible to identify clear cell renal cancer with high sensitivity and specificity from minimal amounts of cells.

Two further AHO-like syndrome patients with deletion of glypican 1 gene region in 2q37.2-q37.3.

In this report, we describe two unrelated patients with mental retardation and brachydactyly E classified as patients suffering from Albright hereditary osteodystrophy-like (AHO-like) syndrome. Fluorescence in situ hybridization (FISH) analysis using 8 different subtelomeric probes in 2q36-37 proved that the patients had subtelomeric 2qter deletions of similar size. The recently proposed candidate gene glypican 1 (GPC1) is deleted in both reported patients.

Characterization of a highly aberrant plasma cell leukemia karyotype: a case report.

We report on a 72-year-old patient with a clinically diagnosed plasmocytoma which developed to a plasma cell leukemia (PCL) with so far unrecorded complex translocations. As GTG-banding was not able to resolve all karyotypic changes, multiplex-fluorescence in situ hybridization (M-FISH) in combination with microdissection based comparative genomic hybridization (micro-CGH) and multicolor banding (MCB) have been done. Using these molecular cytogenetic approaches the karyotype of the PCL case can be described as: 51,XY,-1,-1,+3,+der(5)t(5;11;1)(5pter right curved arrow 5q13-q14::11q24 right curved arrow 11q25::1q12 right curved arrow 1qter),+7 or +der(7)t(7;1)(7qter right curved arrow 7p15::1p31.1 right curved arrow 1pter),+8,+der(9)t(1;9)(1qter right curved arrow 1q12::9q12 right curved arrow 9pter),der(11)t(1;11;1)(1pter right curved arrow 1p31.1::11p15.5 right curved arrow 11q25::1q12 right curved arrow 1qter),-13,der(14)t(X;14)(Xqter right curved arrow Xq21.3::14pter right curved arrow 14qter),+15,+18,der(19)t(9;19)(9qter right curved arrow 9q12::19q11 right curved arrow 19pter),+i(19)(q10). The case shows one of the most complex karyotypic rearrangements ever described in PCL and indicates two additional chromosomal regions which may contain genes of interest for the development of this hematological disorder: loss of 1p10-p31.1 material and gain of Xq21.3-qter.

Breakpoint differentiation in chromosomal aberrations of hematological malignancies: Identification of 33 previously unrecorded breakpoints.

Routine cytogenetic analysis provides important information of diagnostic and prognostic relevance for hematological malignancies. In spite of this, poorly spread metaphase chromosomes and highly rearranged karyotypes with numerous marker chromosomes, are often difficult to interpret. In order to improve the definition of chromosomal breakpoints multicolor banding (MCB) was applied on 45 bone marrow samples from patients suffering from hematological malignancies like myelodysplastic syndrome (MDS), acute myelocytic leukemia (AML), chronic myelocytic leukemia (CML) or acute lymphoblastic leukemia (ALL). The breakpoints defined by GTG banding were confirmed by MCB in 8 cases, while in the remaining 37 cases the breakpoints had to be redefined. In 20/45 cases the breakpoints could only be characterized after application of MCB. In summary, 73 different breakpoints were characterized, thereof 33 were previously undescribed. Eleven cases showed known acquired aberrations and 21 cases had previously described aberration types such as del(5q-), del(7q-), del(13q-) or t(1;5) as sole rearrangement or in connection with other complex ones. In a total of 11 cases 19 breakpoints as described before were involved in hematological malignancies, while in 14 cases 33 breakpoints were identified which have not been described previously. Thus, MCB has proven to be a powerful and reliable method for screening of chromosomal aberrations, which considerably increased the accuracy of cytogenetic diagnosis.

Karyotyping of human synaptonemal complexes by cenM-FISH.

The purpose of this work was to adapt the recently described centromere-specific multicolour (cenM-) FISH technique to human meiotic cells, and evaluate the usefulness of this multiplex fluorescence method for karyotyping human synaptonemal complex (SC), previously analysed by immunocytogenetic approaches. The results obtained demonstrate that cenM-FISH is a reliable one-single-step method, which allows for the identification of all SC present in pachytene spreads. Moreover, when cenM-FISH is applied after immunocytogenetic analysis, the number and distribution of MLH1 foci per chromosome can be established and recombination analysis for each chromosome can be performed easily.

Complex chromosomal rearrangements in a secondary acute myeloblastic leukemia after chemotherapy in TRAPS.

We report on the cytogenetic findings from a patient with a de novo TNF-receptor-associated periodic syndrome (TRAPS), who showed first symptoms at the age of four months. Thus, he obtained a long-term therapy with cortisone, chlorambucile, methotrexate and cyclophosphamide. At the age of 14 he developed a secondary acute myeloblastic leukemia. Highly complex chromosomal rearrangements were detected after banding analysis. The exact definition of karyotype and the involved breakpoints could only be resolved after application of sophisticated multicolor-FISH techniques: 44,XY,-5,der(6)t(6;7)(6pter right curved arrow 6q12::7p22.2 right curved arrow 7pter or 7pter right curved arrow 7p22.2), dic(7;19)t(6;19;6;7;19;7;19)(19qter right curved arrow 19q12::7p13 right curved arrow 7p11.1::19q12 right curved arrow 19p12 or 19p12 right curved arrow 19q12::7p11.1 right curved arrow 7q21.3::6q12 right curved arrow 6q26::19p13.3 right curved arrow 19p12::6q26-6qter),dic(12;13)(13qter right curved arrow 13p11.2::12p13.1 right curved arrow 12qter),ace(12;13)(13pter right curved arrow 13p11.2::12p13.1 right curved arrow 12pter), -19. The simultaneous presence of two dicentric chromosomes has not been reported previously and is striking, as such chromosomes are suggested to be instable. However, such chromosomes are observed frequently after chemo- or radiotherapy and in secondary, i.e. therapy related AML (tAML). Thus, AML in this case may result from a long-term therapy of TRAPS with methotrexate, cyclophosphamide, chlorambucile and cortisone.