Hidden chromosome abnormalities in haematological malignancies detected by multicolour spectral karyotyping.

Cytogenetic analysis provides critical information of diagnostic and prognostic importance for haematological malignancies. In fact, the identification of recurring chromosomal breakpoints in leukaemias and lymphomas has expedited the cloning of genes whose translocation-induced deregulation causes malignant transformation. The pillar of karyotype analysis rests on chromosome banding techniques that have the distinct advantage that the entire genome can be analysed in a single experiment. However, poorly spread or contracted metaphase chromosomes and highly rearranged karyotypes with numerous marker chromosomes, common in tumour cell preparations, are often difficult to interpret unambiguously and subtle chromosomal aberrations, in particular the exchange of telomeric chromatin or small insertions remain elusive. Fluorescence in situ hybridization (FISH) overcomes some of these limitations, but is mainly utilized to confirm the presence of previously characterized or suspected aberrations. We have developed a novel approach, termed spectral karyotyping or SKY based on the hybridization of 24 fluorescently labelled chromosome painting probes that allows the simultaneous and differential colour display of all human chromosomes. We have used SKY to complement conventional banding techniques in haematological malignancies by analysing 15 cases with unidentified chromosome aberrations. In all instances SKY provided additional cytogenetic information, including the identification of marker chromosomes, the detection of subtle chromosomal translocations and the clarification of complex chromosomal rearrangements. Thus, SKY in combination with standard chromosome banding allows the characterization of chromosomal aberrations in leukaemia with unprecedented accuracy.

The detection of subtelomeric chromosomal rearrangements in idiopathic mental retardation.

A major challenge for human genetics is to identify new causes of mental retardation, which, although present in about 3% of individuals, is unexplained in more than half of all cases. We have developed a strategy to screen for the abnormal inheritance of subtelomeric DNA polymorphisms in individuals with mental retardation and have detected three abnormalities in 99 patients with normal routine karyotypes. Pulsed-field gel electrophoresis and reverse chromosome painting showed that one case arose from an interstitial or terminal deletion and two from the de novo inheritance of derivative translocation chromosomes. At least 6% of unexplained mental retardation is accounted for by these relatively small chromosomal abnormalities, which will be an important resource in the characterization of the genetic basis of neurodevelopment.

Computer-assisted analysis of chromosomal abnormalities: detection of a deletion in aniridia-Wilms' tumor syndrome.

A chromosome translocation, t(8p + ; 11q -), in a patient with aniridia and Wilms' tumor, appeared balanced by standard techniques, including trypsin banding. Computer analysis of optical microscope scanning profiles of chromosome pairs 8 and 11 revealed an interstitial deletion of the short arm of 8. Computer analysis coupled to the new banding techniques provides greater resolution for the detection of subtle chromosomal variations not recognized by banding methods alone.

MN blood-group locus: data concerning the possible chromosomal location.

Combined data derived from clinical, cytogenetic, and blood-grouping studies of one family suggest that the MN locus is on the long arm of either the No. 2 or the No. 4 chromosome.

Prenatal diagnosis of genetic disorders.

Sampling of amniotic fluid, visualization of the fetus, fetal blood sampling, and screening of maternal blood represent successive approaches to the diagnosis of specific genetic disorders in the second trimester of pregnancy. Clinical and ethical concerns about the appropriateness, safety, and efficacy of the techniques have led to multidisciplinary assessments at an early stage. A major growth in demand for medical and educational genetic services can be anticipated.

Gains, losses, and amplification of genomic material in rhabdomyosarcoma analyzed by comparative genomic hybridization.

In this study, 10 embryonal and 14 alveolar rhabdomyosarcoma (RMS) tumor samples, including 4 cell lines derived from tumors of the alveolar subtype, were analyzed by comparative genomic hybridization. In the embryonal tumors, the gain of whole or most of various chromosomes, notably chromosomes 2 (60% of cases), 13 (60%), 12 (60%), 8 (60%), 7 (50%), 17 (40%), 18 (40%), and 19 (40%), and the loss of chromosomes 16 (40%), 10 (30%), 15 (20%), and 14 (20%) were found. One case showed evidence of genomic amplification at 12q13-15. In contrast, the alveolar tumors and cell lines showed consistent evidence of genomic amplification, with multiple amplicons in some cases. The amplicons were localized to l2q13-15 (50%), 2p24 (36%), 13q14 (14%), l3q32 (14%), 1q36 (14%), 1q21 (7%), and 8q13-21 (7%). Four cases had additional copies of chromosome 17 or l7q. These changes were in addition to the presence of fusion gene transcripts that are associated with translocations specific to alveolar RMS. The results show that distinct patterns of primarily gains of specific chromosomal material are associated with the embryonal subtype of RMS, and that genomic amplification seems to play an important role in the alveolar subtype. Notably, these distinct changes predominantly involved chromosomes 2, 12, and 13 in both subtypes.

Cytogenetic analysis of 124 prospectively ascertained male germ cell tumors.

We report the cytogenetic analysis of 124 adult male germ cell tumors ascertained consecutively at the Memorial Sloan-Kettering Cancer Center between 1988 and 1990. Biopsies from testicular and extragonadal primary and metastatic lesions studied included all histological subtypes of germ cell tumors and cases of malignant transformation. Nonrandom numerical and structural chromosomal abnormalities including i(12p), the previously described characteristic marker of these tumors, were determined, and their frequency was compared between histological subtypes, between gonadal and extragonadal lesions, and between primary and transformed lesions. The frequency and copy number of i(12p) were found to be higher in nonseminomas compared with seminomas. Nonrandom sites of chromosome rearrangements associated with specific histologies comprised 1p32-36 and 7q11.2 in teratomas and 1p22 in yolk sac tumors. Some tumors that underwent malignant differentiation exhibited chromosome changes previously described to be nonrandomly associated with de novo tumors with the same histological characteristics. Cytological evidence of gene amplification in the form of homogeneously staining regions and/or double minutes was detected in 24% of extragonadal lesions, mainly metastatic tumors, suggesting amplification of a gene(s) associated with metastatic progression of these tumors. While a number of previous small cytogenetic series or individual case reports of germ cell tumors identified several of the features of these tumors reported here, this series comprises analysis of the largest group of tumors ascertained consecutively at a single institution, defines the incidence of nonrandom abnormalities in tumor subsets, and addresses their biological significance.

An integrated microsatellite length analysis using an automated fluorescent DNA sequencer.

Analyzing microsatellite instability (MI) in malignant tumors is thought to be useful for screening cancer patients to identify those patients with a higher risk of developing second malignant tumors. In this paper, we report a new, accurate, and efficient method of detecting MI using an automated fluorescent DNA sequencer and a computer that automatically calculates the size, height, and area of each fluorescent product, making it possible to assess MI more accurately and more rapidly. The primers for amplification of each microsatellite locus are labeled by two different fluorescent dyes, rox (red) and fam (blue). The rox-labeled primer was used for the tumor, whereas the fam-labeled primer was used for the corresponding normal tissue. Two amplified products from both the tumor and the normal tissue were co-loaded into a single lane of the sequencing gel and were analyzed. MI could be detected based on the presence of different waving patterns. Furthermore, several loci could also be analyzed simultaneously for MI in a single lane. Using this method, we examined the frequency of MI in gastric cancer. The results showed that 5 of 22 (22.7 %) gastric cancers were MI-positive, which corresponds to the findings of previous reports that used the radioisotopic method. The improved method may open up the possibility of performing routine examination of MI in many cancer patients and offers hope for the potential clinical application of Ml analysis as a follow-up evaluation of cancer patients.

Current urinary mass screening for catecholamine metabolites at 6 months of age may be detecting only a small portion of high-risk neuroblastomas: a chromosome and N-myc amplification study.

We studied 96 infants and children with untreated neuroblastomas. Chromosomes of tumor cells were analyzed in 68, and N-myc copy numbers were determined in 67 patients. Patients found by a mass screening program for 6-month-old infants (group A1, 39 patients) or those less than 12 months of age found clinically (group A2, 13 patients) were rarely in the disseminated stage (A1, three of 39; A2 one of 13); their tumors usually had near-triploid (3n) or hypertetraploid (greater than 4n) karyotypes (A1, 28 of 37; A2, nine of 11), and never had N-myc amplification (A1, zero of 34; A2, zero of 11). In contrast, children 12 months or over (group B, 27 patients) were usually in the disseminated stage (19 of 27) (P less than .0001); their tumors usually had near-diploid (2n) or near-tetraploid (4n) karyotypes (16 of 20) (P = .0027), and often had N-myc amplification (nine of 22) (P less than .0001). Of the 40 clinically found patients (A2 and B), six had undergone the screening with a negative result at the age of 6 months. Two of the six patients had N-myc amplification in the tumors. Most tumors found by the screening showed known characteristics predicting a good prognosis, and the majority of tumors showing characteristics predicting a poor prognosis were found in patients aged between 12 and 36 months. Our chromosome and N-myc amplification studies suggest that a low-risk tumor does not usually evolve to a high-risk tumor. Thus, the current mass screening program may be detecting only a small portion of highly malignant neuroblastomas at the earliest stage. Infants should be screened twice, at 6 months as well as at 12 months of age, for the early detection of high-risk neuroblastomas.

Arrhythmic disorder mapped to chromosome 1q42-q43 causes malignant polymorphic ventricular tachycardia in structurally normal hearts.

OBJECTIVES: The purpose of this study was to provide clinical and anatomical characteristics as well as genetic background of a malignant arrhythmogenic disorder. BACKGROUND: An inherited autosomally dominant cardiac syndrome causing stress-induced polymorphic ventricular tachycardia and syncope in the absence of structural myocardial changes was detected in two families. METHODS: Two unrelated families with six victims of sudden death and 51 living members were evaluated. Resting and exercise electrocardiograms (ECG), echocardiography, magnetic resonance imaging (MRI), cineangiography, microscopic examination of endomyocardial biopsies and a drug testing with a class IC antiarrhythmic agent flecainide were performed. A genetic linkage analysis was carried out to map the gene locus. RESULTS: Of the 24 affected individuals, 10 had succumbed with six cases of sudden death, and 14 survivors showed evidence of disease. Exercise stress test induced ventricular bigeminy or polymorphic ventricular tachycardia in affected individuals. Three children initially examined before 10 years of age developed arrhythmias during a four-year follow-up. Resting ECGs were normal in affected subjects except a slight prolongation of the QT intervals adjusted for heart rate (QTc) (430 +/- 18 vs. 409 +/- 19 ms, affected vs. nonaffected, p < 0.01). Administration of flecainide did not induce ECG abnormalities encountered in familial idiopathic ventricular fibrillation. Ventricular volumes, contractility and wall measurements were normal by echocardiography, right ventricular cineangiography and MRI. Histopathological examination showed no fibrosis or fatty infiltration. The cumulative cardiac mortality by the age of 30 years was 31%. The disease locus was assigned to chromosome 1q42-q43, with a maximal pairwise lod score of 4.74 in the two families combined. Only one heterozygous carrier was clinically unaffected suggesting high disease penetrance in adulthood. CONCLUSIONS: A distinct cardiac disorder linked to chromosome 1q42-q43 causes exercise-induced polymorphic ventricular tachycardia in structurally normal hearts and is highly malignant. Delayed clinical manifestation necessitates repeated exercise electrocardiography to assure diagnosis in young individuals of the families.

Clinical significance of chromosome abnormalities in childhood acute lymphoblastic leukemia in Japan.

Of 240 Japanese children with acute lymphoblastic leukemia (ALL) treated between 1983 and 1990, 75 (31%) had normal diploidy, 47 (20%) hyperdiploidy with more than 50 chromosomes, 18 (8%) hyperdiploidy with 47-50 chromosomes, 77 (32%) pseudodiploidy, 22 (9%) hypodiploidy and one hypotetraploidy in the leukemic cells. Event-free survival (EFS) +/- standard error (SE) at 4 years was 74 +/- 7% in patients with hyperdiploidy > 50, 68 +/- 6% in those with normal diploidy, 55 +/- 13% in those with hyperdiploidy 47-50, 54 +/- 11% in those with hypodiploidy, and 22 +/- 5% in those with pseudodiploidy (log-rank, p < 0.0001). Seventy-four patients with translocation and 166 patients without translocation had EFS +/- SE of 26 +/- 6% and 64 +/- 4%, respectively, at 4 years (p < 0.0001). The overall prognosis of our patients was comparable to that of the patients mainly treated in the late 1970s and reported by the Sixth International Workshop on Chromosomes in Leukemia, but was much poorer than that of the patients more recently treated and reported from some American institutions. These findings may possibly reflect less appropriate chemotherapy having been applied to Japanese children with ALL.

High rate of chromosome abnormalities detected by fluorescence in situ hybridization using BCR and ABL probes in adult acute lymphoblastic leukemia.

The value of dual-color fluorescence in situ hybridization (FISH) with BCR and ABL probes for the detection of the Philadelphia (Ph) translocation and of other alterations involving ABL and/or BCR was evaluated in adult acute lymphoblastic leukemia (ALL). One hundred and four patients were studied prospectively using interphase nuclei FISH, chromosome analysis (CA), and PCR assays for the chimeric BRC/ABL transcript. FISH detected a Ph translocation in 24 cases (23.1%), as was confirmed by CA and/or PCR. FISH revealed a false positive diagnosis of a Ph translocation in four cases (5% false positive rate). Among 54 cases with combined FISH, CA and PCR assays, FISH failed to establish a correct diagnosis in 3.7%, PCR in 5.6%, and CA in 7.4%. The combination of two screening methods led to discrepant results in 9.3% (FISH + PCR), 11.1% (FISH + CA), or 13% (CA + PCR) of the cases. In seven of 80 (8.8%) Ph-negative patients, gain of BCR and/or ABL was identified. Overall, FISH detected alterations of the BCR and/or ABL genes with an incidence of 29.8% of the current study. Due to the possibility of false positive diagnosis of a Ph translocation using dual-color FISH the combination with chromosome and/or RT-PCR analyses is recommended in adult ALL patients.

Detection of minimal residual disease using fluorescence DNA in situ hybridization: a follow-up study in leukemia and lymphoma patients.

We used fluorescence DNA in situ hybridization (FISH) to detect chromosomal abnormalities as an indicator of minimal residual disease in follow-up samples from the bone marrow (BM), or peripheral blood, of 25 patients with leukemia, lymphoma and myelodysplastic syndromes. Trisomies were detected by interphase FISH with repeat-sequence probes (RSP) or by using metaphase FISH with whole-chromosome paint probes (WCP). Specific translocations were detected using WCP probes. Translocations were observed using metaphase FISH in two patients in uncertain or complete remission (CR), who both later suffered relapse. Five patients with no abnormal cells remained in CR. Four patients with trisomies detected during CR suffered relapse; metaphase FISH detected the trisomy in 0.17-16% of metaphase cells. Five patients for whom the trisomy occurred in 0.034% of cells remained in CR. Trisomic nuclei were observed in 0.27-2.3% of interphase cells, by means of RSPs, in four patients who later suffered relapse. Five patients with trisomic nuclei in 0.061% remained in CR. When two probes were used simultaneously in a sample from one patient, 1% of the residual cells were abnormal. The patient later suffered relapse. In one patient with anaplastic large cell lymphoma, CD30-positive interphase cells were shown to have trisomic chromosome 7 by immunophenotyping and FISH. Our results suggest that metaphase FISH using WCP probes is a sensitive and specific method for detecting minimal residual disease especially in patients with translocations.

Rapid synthesis of hybrid RNA molecules associated with leukemia-specific chromosomal translocations.

A number of gene arrangements have been described as characteristic abnormalities associated with different types of leukemia, and this list is still growing. In view of the biological, clinical and prognostic relevance of the pathological fusion products, techniques permitting their detection are of paramount importance in the clinical setting. In some instances, permanent leukemic cell lines carrying the abnormality of interest are available for the establishment and standardization of molecular assays. For a number of newly discovered gene rearrangements, however, this may not be the case. It is therefore of great interest for clinical laboratories to have alternative technical possibilities for the set-up of standardized molecular tests. This problem provided the stimulus to design a simple and rapid method for in vitro generation of chimeric RNA molecules corresponding to pathological fusion transcripts typical for chromosomal translocations in leukemias. Two separate fragments are generated in a four-primer multiplex PCR. Due to a PCR-generated overlap, a chimeric fragment can be synthesized in a second round of PCR. This PCR product is then purified with the help of magnetic beads. Due to the SP6 promotor sequence incorporated during the second round of PCR, transcription into RNA is easily facilitated while the template DNA is still bound to the solid phase. Following this strategy we were able to synthesize the fusion transcripts m-BCR/ABL, CBF beta/MYH11, and MLL/AFp1 which are the molecular equivalents of t(9;22)(q34,q11), inv16(p13;q22) and t(1;11)(p32;q23), respectively. The chimeric RNA will be useful as a control template in diagnostic RT-PCR strategies. It can also be further processed in translation systems leading to the corresponding chimeric oncoprotein. This approach can be easily used to create any hybrid RNA of interest.

Heterogeneity of VH-JH gene rearrangement patterns: an insight into the biology of B cell precursor ALL.

Oligoclonal B cell proliferation, as defined by the presence of more than one leukemic clone, has been detected in approximately 20% to 30% of patients with acute lymphoblastic leukemia (ALL) using PCR or Southern blotting. An accurate assessment of these populations is required to avoid false negative measurements of minimal residual disease (MRD) in follow-up bone marrow (BM) samples of ALL patients. In this study, we analysed 29 ALL patients with two or more immunoglobulin heavy (IGH) chain gene rearrangements in the presentation samples using IGH fingerprinting PCR and sequence analysis. Thirty-nine (51%) of 76 sequences (from 15 patients), shared no VNDNJ homology (ie different CDR3 regions). In the remaining 14 patients, at least two related VH sequences were identified in each patient (identical DNJ sequences). Numerical abnormalities of chromosome 14 was detected in 10 patients. Eight patients were analysed at presentation and relapse. In four of them, expansion of a minor presentation-clone was detected at relapse while the major presentation clone disappeared, confirming 'subclonal evolution'. Finally, in our cohort of patients, the presence of related or unrelated IGH clones did not influence overall survival.

Effect of the Philadelphia chromosome on minimal residual disease in acute lymphoblastic leukemia.

The Philadelphia translocation is associated with a poor prognosis in adults and children with acute lymphoblastic leukemia, even though the majority of patients achieve remission. To test the hypothesis that the translocation leads to drug resistance in vivo, we studied 61 children and 20 adults with acute lymphoblastic leukemia and used the level of minimal residual disease at the end of induction as the measure of drug resistance in vivo. In children the presence of the translocation was associated with a significant increase in residual disease, indicating higher drug resistance in vivo; five of seven Philadelphia-positive children but only five of 54 Philadelphia-negative children had a minimal residual disease level >10(-3), a level which is associated with a high risk of relapse in childhood acute lymphoblastic leukemia of standard risk. By contrast, in adults, residual disease and hence drug resistance was already higher than in children, and the presence of the Philadelphia translocation in seven patients had no obvious additional effect. We conclude that the Philadelphia chromosome may increase resistance to drugs in vivo in children, but not detectably in adults.

Minimal residual disease in leukemia: state of the art 1991.

Recent achievements in decreasing the detection level of "minimal residual disease" in leukemia are summarized both in a relevant rat model for acute myelocytic leukemia (BNML) and in the human leukemias. Detection levels as low as 1 leukemic cell per 10(8)-10(9) and 10(5)-10(6) normal bone marrow cells have become realistic in the animal model and in human leukemia, respectively. The implications of these new tools for detecting hidden leukemic cells are discussed.

Karyotype abnormalities and their clinical significance in blast crisis of chronic myeloid leukemia.

We examined karyotypes and their prognostic significance in a series of 122 patients with chronic myeloid leukemia in blast crisis. Of 73 patients cytogenetically examined at the onset of blast crisis 63% had developed secondary cytogenetic abnormalities in addition to the Philadelphia chromosome. These newly emerging abnormalities included a double Philadelphia chromosome in 20 patients, a trisomy 8 in 17, and an isochromosome 17q in 11 patients. Development of such additional karyotypic abnormalities was significantly associated with a shorter median survival and less response to cytoreductive treatment and was significantly more common in nonlymphoid blast crisis than in the lymphoid-type blast crisis. Thus, assessment of karyotypes at the onset of chronic myeloid leukemia blast crisis appears to be of prognostic significance for both remission duration and survival.

Perfect endings: a review of subtelomeric probes and their use in clinical diagnosis.

Chromosomal rearrangements involving the ends of chromosomes (telomeres) are emerging as an important cause of human genetic diseases. This review describes the development of first and second generation sets of telomere specific clones, together with advances in fluorescence in situ hybridisation (FISH) technology, which have made the prospect of screening for telomeric rearrangements a realistic goal. Initial FISH studies using the telomere specific clones indicate that they will be a valuable diagnostic tool for the investigation of mental retardation, the characterisation of known abnormalities detected by conventional cytogenetic analysis, spontaneous recurrent miscarriages, infertility, haematological malignancies, and preimplantation diagnosis, as well as other fields of clinical interest. In addition, they may help investigate telomere structure and function and can be used in the identification of dosage sensitive genes involved in human genetic disease.