Preferred co-localization of chromosome 8 and 21 in myeloid bone marrow cells detected by three dimensional molecular cytogenetics.

The impact of chromosome architecture in the formation of chromosome aberrations is a recent finding of interphase directed molecular cytogenetic studies. Also positive correlation of translocation frequencies and spatial proximity of chromosomes was described. Thus, disease specific chromosomal translocations could be due to tissue specific genomic organization. However, no three-dimensional interphase fluorescence in situ hybridization (FISH) studies for the nuclear architecture of bone marrow (BM) cells have previously been done. In this study, BM of three secondary acute myelogenous leukemia (AML) cases with trisomy 8 and otherwise normal karyotype were evaluated. Bone marrow cells of one AML and one ALL (acute lymphoblastic leukemia) case, peripheral blood lymphocytes and human sperm, all of them with normal karyotype, served as controls. Multicolor banding (MCB) probes for chromosomes 8 and 21 were applied in suspension-FISH (S-FISH). Interestingly, in myeloid bone marrow cells chromosomes 8 (di- and trisomic) and 21 tended to co-localize with their homologue chromosome(s), rather than to be separated. Thus, the co-localization of chromosomes 8 and 21 might promote a translocation providing a selective advantage of t(8;21) cells in AML-M2. In summary, the concept that tissue specific spatial proximity of chromosomes leads to enhanced translocation frequencies was further supported.

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.

Delineation of yet unknown cryptic subtelomere aberrations in 50% of acute myeloid leukemia with normal GTG-banding karyotype.

Acute myeloid leukemia (AML) is a heterogeneous disease with respect to clinical prognosis and acquired chromosomal aberrations. After routine banding cytogenetic analysis 45% of AML patients show a normal karyotype (NK-AML). For a better understanding of development and progression in AML, it is important to find markers which could be primary genetic aberrations. Therefore, in this study 31 patients with NK-AML were analyzed by new high resolution molecular cytogenetic approaches. A combination of multitude multicolor banding and metaphase microdissection-based comparative genomic hybridization revealed deletions of the subtelomeric regions in 6% of the studied cases. According to these results, locus-specific probes for the subtelomeric regions of chromosomes 5, 9, 11, 12 and 13 were applied on 22 of the studied 31 NK-AML cases. Surprisingly, 50% of them showed deletions or duplications. These aberrations occurred in the in vitro proliferating as well as in the non-proliferating cells. Meta-analysis of the aberrant regions revealed that they often include genes known to be associated with tumors, e.g. RASA3 on chromosome 13. These results implicate that aberrations in the subtelomeric regions of NK-AML occur quite often and may be considered as primary genetic changes, and should not be neglected in future diagnostic approaches.

Clinical impact of nucleophosmin mutations and Flt3 internal tandem duplications in patients older than 60 yr with acute myeloid leukaemia.

BACKGROUND: Nucleophosmin (NPM1) and Flt3 internal tandem duplications (Flt3-ITD mutations) represent the most frequent molecular aberrations in patients with acute myeloid leukemia (AML). While NPM1 mutations are associated with favourable prognosis in younger AML patients, Flt3-ITD mutations reflect an unfavourable prognostic factor in these patients. So far, especially NPM1 mutations have not yet been evaluated exclusively in older patients. PATIENTS AND METHODS: We retrospectively analysed the prevalence of NPM1 and Flt3-ITD mutations and its association with complete remission (CR), and survival in 99 elderly patients (median age 71 yr, range 60-85 yr) newly diagnosed for AML. Primary treatment approach was curative in 54, and palliative in 38 patients, while seven patients received best supportive care only. The mean follow-up of surviving patients was 600 d. RESULTS: Sixty-seven patients were tested negative for NPM1 and Flt3-ITD mutations (group 1), 16 patients carried only a NPM1 mutation (group 2) and nine patients had only a Flt3-ITD mutation (group 3) while additional seven patients were positive for both aberrations (group 4). We can demonstrate a significant higher rate of CR comparing wildtype vs. NPM1 positive patients (40.5% for group 1 vs. 80.0% for group 2, P = 0.03) for patients receiving curative therapy. Interestingly, there is no significant difference in overall survival between group 1 and group 2 (Log-rank test P = 0.22, median 440 d vs. 1125 d). In contrast, patients carrying a Flt3-ITD mutation had a significant worse overall survival compared to wildtype patients (P = 0.03, median 210 d for group 3 + 4 vs. 634 d for group 1 + 2) while no difference of CR rate could be observed (42.8% vs. 48.9%, P = 0.91). CONCLUSION: As elderly but medically fit patients with AML carrying a NPM1 mutation have a high CR rate, age itself should not be a barrier for induction treatment. However, new therapeutic concepts of postremission therapy (e.g. allogeneic stem cell transplantation after dose-reduced conditioning) should be considered for these patients in first CR.

Tapping an unexploited repository: Carnoy's fixed cell pellets for proteomic biomarker research in leukemia.

For chromosomal analysis in tumor genetics, cells from blood and bone marrow are prepared and preserved virtually indefinitely in Carnoy's fixative (methanol/acetic acid). Numerous samples are stored unvalued in hospitals and institutes worldwide. We developed a method to analyze proteins from even a small amount of these cells by mass spectrometry using affinity chromatographic surfaces (SELDI), and demonstrated the application of proteomic biomarker research in cases of acute myeloid leukemia.

New rearrangement t(3;17)(q26.3;q12) in an AML patient with a poor outcome.

In more than 90% of acute promyelocytic leukemia (APL) cases a reciprocal translocation t(15;17)(q22;q12) can be observed. The RARalpha gene on 17q12 is known to have other translocation partners than PML (in 15q22) in a minority of APL cases. Here, we describe a previously unrecorded chromosomal translocation involving the RARalpha gene and an unknown partner on chromosome 3. The chromosomal rearrangement was studied in detail by 24-color-FISH using whole chromosome painting probes plus multicolor banding. Thus, the breakpoint could be characterized as t(3;17) (q26;q12). In this case 10% of blasts showed AML-M3 characteristics although typical rearrangements with RARalpha were not detected by molecular methods. The characterization of the present and other comparable APL-cases with exceptional translocation partners of PML or RARalpha will help to enlighten the understanding of the pathogenesis of APL.

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.

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.

Small supernumerary marker chromosomes (SMCs): genotype-phenotype correlation and classification.

Small supernumerary marker chromosomes (SMCs) are present in about 0.05% of the human population. In approximately 30% of SMC carriers (excluding the approximately 60% SMC derived from one of the acrocentric chromosomes), an abnormal phenotype is observed. The clinical outcome of an SMC is difficult to predict as they can have different phenotypic consequences because of (1). differences in euchromatic DNA-content, (2). different degrees of mosaicism, and/or (3). uniparental disomy (UPD) of the chromosomes homologous to the SMC. Here, we present 35 SMCs, which are derived from all human chromosomes, apart from chromosome 6, as demonstrated by the appropriate molecular cytogenetic approaches, such as centromere-specific multicolor fluoresence in situ hybridization (cenM-FISH), multicolor banding (MCB), and subcentromere-specific multicolor FISH (subcenM-FISH). In nine cases without an aberrant phenotype, neither partial proximal trisomies nor UPD could be detected. Abnormal clinical findings, such as psychomotoric retardation and/or craniofacial dysmorphisms, were associated with seven of the cases in which subcentromeric single-copy probes were proven to be present in three copies. Conversely, in eight cases with a normal phenotype, proximal euchromatic material was detected as partial trisomy. UPD was studied in 12 cases and subsequently detected in two of the cases with SMC (partial UPD 4p and maternal UPD 22 in a der(22)-syndrome patient), indicating that SMC carriers have an enhanced risk for UPD. At present, small proximal trisomies of 1p, 1q, 2p, 6p, 6q, 7q, 9p, and 12q seem to lead to clinical manifestations, whereas partial proximal trisomies of 2q, 3p, 3q, 5q, 7p, 8p, 17p, and 18p may not be associated with significant clinical symptoms. With respect to clinical outcome, a classification of SMCs is proposed that considers molecular genetic and molecular cytogenetic characteristics as demonstrated by presently available methods.

Highly complex karyotypic changes in acute myelogenous leukemia: a case report.

We report on a patient with a clinically diagnosed acute myelogenous leukemia (AML) with so far unrecorded complex translocations. As GTG-banding was not able to resolve all karyotypic changes, spectral karyotyping (SKY) and multiplex-fluorescence in situ hybridization (M-FISH) were performed for comparison. Both methods gave nearly identical results, however, they were unable to characterize all involved chromosomal breakpoints in detail. Thus, multicolor banding (MCB) technique was applied and its results were confirmed for two large derivative chromosomes by microdissection and reverse painting. Using this battery of molecular cytogenetic approaches the karyotype of this AML case could be described as 40 approximately 44,XY,der(1)t(1;5;8;20) (1qter-->1p12::5q14.3-->5q15 or 5q15-->5q14.3::8p11.2-->8p23.? 3::20p11.1-->20p13),del(2)(q12),der(3)t(3;6),der(5)t(5;18) (5p15.33-->5q11::18q21.3-->18q23),del(6),-8,der(9)t(9;17;15),der(10)t(3;10),del(11)(q24),-15,-16,del(17),der(18) t(8;18;5;2;20)(8q24.3-->8q24.2 or 8q24.2-->8q24.3::18p11.22-->18q21.3::5q14.3-->5q11::2q32-->2q12::20q13.2-->20q13.33), der(20)t(1;20;18)(1p36.33-->1p31.3-22.3::20p11.1-->20q11.2 or 20q11.2-->20p11.1::18p11.22-->18p11.32).

A multiple translocation event in a patient with hexadactyly, facial dysmorphism, mental retardation and behaviour disorder characterised comprehensively by molecular cytogenetics. Case report and review of the literature.

UNLABELLED: We report a 13-year-old female patient with multiple congenital abnormalities (microcephaly, facial dysmorphism, anteverted dysplastic ears and postaxial hexadactyly), mental retardation, and adipose-gigantism. Ultrasonography revealed no signs of a heart defect or renal abnormalities. She showed no speech development and suffered from a behavioural disorder. CNS abnormalities were excluded by cerebral MRI. Initial cytogenetic studies by Giemsa banding revealed an aberrant karyotype involving three chromosomes, t(2;4;11). By high resolution banding and multicolour fluoresence in-situ hybridisation (M-FISH, MCB), chromosome 1 was also found to be involved in the complex chromosomal aberrations, confirming the karyotype 46,XX,t(2;11;4).ish t(1;4;2;11)(q43;q21.1;p12-p13.1;p14.1). To the best of our knowledge no patient has been previously described with such a complex translocation involving 4 chromosomes. This case demonstrates that conventional chromosome banding techniques such as Giemsa banding are not always sufficient to characterise complex chromosomal abnormalities. Only by the additional utilisation of molecular cytogenetic techniques could the complexity of the present chromosomal rearrangements and the origin of the involved chromosomal material be detected. Further molecular genetic studies will be performed to clarify the chromosomal breakpoints potentially responsible for the observed clinical symptoms. CONCLUSION: This report demonstrates that multicolour-fluorescence in-situ hybridisation studies should be performed in patients with congenital abnormalities and suspected aberrant karyotypes in addition to conventional Giemsa banding.

FISH banding methods: applications in research and diagnostics.

Recently, several chromosome banding techniques based on fluorescence in situ hybridization (FISH) have been developed for the human and the mouse genome. In contrast to the standard chromosome banding techniques presently used, giving a protein-related banding pattern, those FISH techniques are DNA-specific. Currently the FISH banding methods are still under development and no high resolution banding technique is available that can be used for a whole genome in one hybridization. Nevertheless, FISH banding methods were used successfully for research in evolution- and radiation-biology, as well as for studies on the nuclear architecture. Moreover, their suitability for diagnostic purposes has been proven in prenatal, postnatal and tumor cytogenetics, indicating that they are an important tool with the potential to partly replace the conventional banding techniques in future.

A complex translocation event between the two homologues of chromosomes 5 leading to a del(5)(q21q33) as a sole aberration in a case clinically diagnosed as CML: characterization of the aberration by multicolor banding.

We report on a patient with a clinically diagnosed Philadelphia negative chronic myelogenous leukemia (CML) with a so far unrecorded complex translocation event between the two homologue chromosomes 5. At the GTG-band level the karyotype was normal, apart from an enlarged chromosome 5 and an extremely shortened second chromosome 5. Both derivative chromosomes 5 consisted exclusively of #5 derived material as proven by 24-color FISH. To characterize the complex aberration in more detail the multicolor banding (MCB) technique using a chromosome 5 specific probe set was applied. Using this DNA-based high resolution banding procedure, the karyotype could be described as 46,XX,del(5)(pterright curved arrow q12::q33right curved arrow qter),ins(5)(pterright curved arrow q15::q12right curved arrow q21::q21right curved arrow qter). In consequence, the aberration leads to a partial deletion of the long arm of chromosome 5: del(5)(q21q33), which would not have been identified using conventional banding techniques or 24-color FISH.

Heterogenic molecular basis for loss of ABL1-BCR transcription: deletions in der(9)t(9;22) and variants of standard t(9;22) in BCR-ABL1-positive chronic myeloid leukemia.

The objective of this study was to characterize the ABL1-BCR fusion gene in 76 BCR-ABL1-positive chronic myeloid leukemia (CML) patients regarding expression as well as genomic status, to assess the frequency of ABL1-BCR gene deletion in these patients, which has been reported to be an adverse prognostic factor in Philadelphia chromosome-positive CML. Patients were analyzed for ABL1-BCR 1b-b3 and/or 1b-b4 transcription by RT-PCR analysis. ABL1-BCR gene status was analyzed by FISH in 16 CML patients with no ABL1-BCR transcript. FISH revealed a partial or total deletion of the ABL1-BCR gene in 9/16 and localized the 5' portion of ABL1 and the 3' portion of BCR at separated loci in 5/16 patients. The latter FISH pattern resulted from a nonreciprocal translocation in two and a complex translocation in three individuals. In 2/16 patients, FISH could not exclude an intact ABL1-BCR fusion gene. Thus, most CML patients without ABL1-BCR transcript could be characterized cytogenetically to belong to two major subgroups: a silent ABL1-BCR gene was attributed to a deletion in der(9)t(9;22) in 56% of the investigated patients or to variants of a standard t(9;22) (approximately 31%). Conversely, none of the 50 patients with an ABL1-BCR transcript exhibited a variant t(9;22) in GTG-banding analysis. Thus, genomic aberrations such as deletions or complex genomic rearrangements are the basic and most frequent cause for ABL1-BCR RNA negativity in CML. The heterogeneity of the underlying molecular mechanisms may explain divergent clinical implications described for patients with an ABL1-BCR deletion and those with no ABL1-BCR transcript.