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.

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.

Tumor cytogenetics is an essential tool for diagnosis and prognosis differentiation, especially in malignancies of the peripheral blood. As metaphase spreads are sometimes difficult to obtain in leukemia and lymphoma preparations, interphase cytogenetics is currently the method of choice in this regard. To save time and/or to obtain additional information on the cytogenetic status of a malignant disease, interphase nuclei from peripheral blood or bone marrow can be prepared directly, or a blood or bone marrow smear can be made on a slide. Several protocols that allow such direct preparation are presented here.

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.

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.

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.

During the last decade not only multicolor fluorescence in situ hybridization (FISH) using whole chromosome paints as probes, but also numerous chromosome banding techniques based on FISH have been developed for the human and for the murine genome. This review focuses on such FISH-banding techniques, which were recently defined as ‘any kind of FISH technique, which provide the possibility to characterize simultaneously several chromosomal subregions smaller than a chromosome arm. FISH-banding methods fitting that definition may have quite different characteristics, but share the ability to produce a DNA-specific chromosomal banding’. While the standard chromosome banding techniques like GTG lead to a protein-related black and white banding pattern, FISH-banding techniques are DNA-specific, more colorful and, thus, more informative. For some, even high-resolution FISH-banding techniques the development is complete and they can be used for whole genome hybridizations in one step. Other FISH-banding methods are only available for selected chromosomes and/or are still under development. FISH-banding methods have successfully been applied in research in evolution- and radiation-biology, as well as in 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 the future.

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.

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.

We report on three cases with a cytogenetically identical ring chromosome containing euchromatin from the long arm of chromosome 1 (r[1][::p11.1 -> q21.1::]). Two cases were newborn males (Cases 1 and 2) and the third one was prenatally identified as female (Case 3). Mosaicism was present in all three cases in different degrees, i.e. 48%, 25% and 14% of the cells, respectively. Clinical signs and symptoms vary between the three cases.The results of our three cases are compared with those from the literature.

We report on the case of a pregnant woman with hyposomia who was previously suspected of having Turner syndrome. Prenatal cytogenetic diagnostics showed a fetal karyotype of 46,XX,dup(13)(q14.2q21.1) ish.13q14(RB1 x 3). Parental and grandparental chromosome analyses were performed and the dup(13) was found to be of maternal origin (de novo). The pregnancy was continued and a healthy female child was born with normal development apart from growth retardation. The reported chromosomal aberration is, together with two other cases reported in the literature, the first hint of a short stature-like phenotype due to dup(13)(q14.2q14.3).

To clarify the nature of chromosome sub-bands in more detail, the multicolor banding (MCB) probe-set for chromosome 5 was hybridized to normal metaphase spreads of GTG band levels at ∼850, ∼550, ∼400 and ∼300. It could be observed that as the chromosomes became shorter, more of the initial 39 MCB pseudo-colors disappeared, ending with 18 MCB pseudo-colored bands at the ∼300-band level. The hierarchically organized splitting of bands into sub-bands was analyzed by comparing the disappearance or appearance of pseudo-color bands of the four different band levels. The regions to split first are telomere-near, centromere-near and in 5q23→q31, followed by 5p15, 5p14, and all GTG dark bands in 5q apart from 5q12 and 5q32 and finalized by sub-band building in 5p15.2, 5q21.2→q21.3, 5q23.1 and 5q34. The direction of band splitting towards the centromere or the telomere could be assigned to each band separately. Pseudo-colors assigned to GTG-light bands were resistant to band splitting. These observations are in concordance with the recently proposed concept of chromosome region-specific protein swelling.

Prenatal DiagnosisVolume 24, Issue 12 p. 1022-1024 Letter to the Editor Inherited cryptic chromosomal aberrations may be more easily detected in their balanced forms: a case report with hidden der(1)t(1;17)(q44;p13.2) Thomas Liehr, Corresponding Author Thomas Liehr Institute of Human Genetics and Anthropology, Jena, GermanyInstitute of Human Genetics and Anthropology, Jena, GermanySearch for more papers by this authorAnita Heller, Anita Heller Institute of Human Genetics and Anthropology, Jena, GermanySearch for more papers by this authorKarl-Heinz Eichhorn, Karl-Heinz Eichhorn Practice of Gynecology, Weimar, GermanySearch for more papers by this authorVolkmar Beensen, Volkmar Beensen Institute of Human Genetics and Anthropology, Jena, GermanySearch for more papers by this authorEberhart Schulze, Eberhart Schulze Institute of Pathology, Jena, GermanySearch for more papers by this authorHeike Starke, Heike Starke Institute of Human Genetics and Anthropology, Jena, GermanySearch for more papers by this authorUwe Claussen, Uwe Claussen Institute of Human Genetics and Anthropology, Jena, GermanySearch for more papers by this authorIsolde Schreyer, Isolde Schreyer Institute of Human Genetics and Anthropology, Jena, GermanySearch for more papers by this author Thomas Liehr, Corresponding Author Thomas Liehr Institute of Human Genetics and Anthropology, Jena, GermanyInstitute of Human Genetics and Anthropology, Jena, GermanySearch for more papers by this authorAnita Heller, Anita Heller Institute of Human Genetics and Anthropology, Jena, GermanySearch for more papers by this authorKarl-Heinz Eichhorn, Karl-Heinz Eichhorn Practice of Gynecology, Weimar, GermanySearch for more papers by this authorVolkmar Beensen, Volkmar Beensen Institute of Human Genetics and Anthropology, Jena, GermanySearch for more papers by this authorEberhart Schulze, Eberhart Schulze Institute of Pathology, Jena, GermanySearch for more papers by this authorHeike Starke, Heike Starke Institute of Human Genetics and Anthropology, Jena, GermanySearch for more papers by this authorUwe Claussen, Uwe Claussen Institute of Human Genetics and Anthropology, Jena, GermanySearch for more papers by this authorIsolde Schreyer, Isolde Schreyer Institute of Human Genetics and Anthropology, Jena, GermanySearch for more papers by this author First published: 21 December 2004 https://doi.org/10.1002/pd.971Citations: 6AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article. REFERENCES Boehm D, Herold S, Kuechler A, Liehr T, Laccone F. 2004. Rapid detection of subtelomeric deletion/duplication by novel real-time quantitative PCR using SYBR-green dye. Hum Mutat 23: 368–378. Cavani S, Perfumo C, Faravelli F, et al. 2003. Cryptic 1p36.3/6q25.2 translocation in three generations ascertained through a foetus with IUGR and cerebral malformations. Prenat Diagn 23: 819–823. Gentile M, Di Carlo A, Volpe P, et al. 2003. FISH and cytogenetic characterization of a terminal chromosome 1q deletion: clinical case report and phenotypic implications. Am J Med Genet 117A: 251–254. Harada N, Hatchwell E, Okamoto N, et al. 2004. Subtelomere specific microarray based comparative genomic hybridization: a rapid detection system for cryptic rearrangements in idiopathic mental retardation. J Med Genet 41: 130–136. Jalal SM, Harwood AR, Sekhon GS, et al. 2003. Utility of subtelomeric fluorescent DNA probes for detection of chromosome anomalies in 425 patients. Genet Med 5: 28–34. Liehr T, Heller A, Starke H, et al. 2002. Microdissection based high resolution multicolor banding for all 24 human chromosomes. Int J Mol Med 9: 335–339. Mantripragada KK, Buckley PG, de Stahl TD, Dumanski JP. 2004. Genomic microarrays in the spotlight. Trends Genet 20: 87–94. Villa N, Sala E, Colombo D, Dell'Orto M, Dalpra L. 2000. Monosomy and trisomy 1q44-qter in two sisters originating from a half cryptic 1q;15p translocation. J Med Genet 37: 612–615. Citing Literature Volume24, Issue1215 December 2004Pages 1022-1024 ReferencesRelatedInformation

Reports on patients with pure and complete trisomy 12p are rare. Up to now, 12 cases have been described in the literature. Here, we report on the genotype/phenotype-correlation of a female patient with a pure trisomy 12p. Conventional cytogenetic studies on peripheral blood chromosomes as well as molecular cytogenetic (fluorescence in situ hybridization, FISH) techniques including whole chromosome painting (WCP), comparative genomic hybridization (CGH), multicolor-banding (MCB) detected a female karyotype with an abberant chromosome 12:46,XX,der(12).ish dup(12)(pter --> q24.3::p11.2 --> pter). In addition to the trisomy 12p specific clinical hallmarks, the patient showed some features of Pallister-Killian syndrome (PKS) such as sparse hair, macroglossia, and epilepsy. These findings contribute to the genotype/phenotype correlation in trisomy 12p patients.

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.

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->5q13-q14:: 11q24->11q25::1q12->1qter),+7 or +der(7)t(7;1)(7qter->7p15:: 1p31.1->1pter),+8,+der(9)t(1;9)(1qter->1q12::9q12->9pter), der(11)t(1;11;1)(1pter->1p31.1::11p15.5->11q25::1q12->1qter), -13,der(14)t(X;14)(Xqter->Xq21.3::14pter->14qter), +15,+18,der(19)t(9;19)(9qter->9q12::19q11->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.

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.