Genotype-outcome correlations in pediatric AML: the impact of a monosomal karyotype in trial AML-BFM 2004.

We conducted a cytogenetic analysis of 642 children with de novo acute myeloid leukemia (AML) treated on the AML-Berlin-Frankfurt-Münster (BFM) 04 protocol to determine the prognostic value of specific chromosomal aberrations including monosomal (MK+), complex (CK+) and hypodiploid (HK+) karyotypes, individually and in combination. Multivariate regression analysis identified in particular MK+ (n=22) as a new independent risk factor for poor event-free survival (EFS 23±9% vs 53±2% for all other patients, P=0.0003), even after exclusion of four patients with monosomy 7 (EFS 28±11%, P=0.0081). CK+ patients without MK had a better prognosis (n=47, EFS 47±8%, P=0.46) than those with MK+ (n=12, EFS 25±13%, P=0.024). HK+ (n=37, EFS 44±8% for total cohort, P=0.3) influenced outcome only when t(8;21) patients were excluded (remaining n=16, EFS 9±8%, P<0.0001). An extremely poor outcome was observed for MK+/HK+ patients (n=10, EFS 10±10%, P<0.0001). Finally, isolated trisomy 8 was also associated with low EFS (n=16, EFS 25±11%, P=0.0091). In conclusion, monosomal karyotype is a strong and independent predictor for high-risk pediatric AML. In addition, isolated trisomy 8 and hypodiploidy without t(8;21) coincide with dismal outcome. These results have important implications for risk stratification and should be further validated in independent pediatric cohorts.

An international study of intrachromosomal amplification of chromosome 21 (iAMP21): cytogenetic characterization and outcome.

Intrachromosomal amplification of chromosome 21 (iAMP21) defines a distinct cytogenetic subgroup of childhood B-cell precursor acute lymphoblastic leukaemia (BCP-ALL). To date, fluorescence in situ hybridisation (FISH), with probes specific for the RUNX1 gene, provides the only reliable detection method (five or more RUNX1 signals per cell). Patients with iAMP21 are older (median age 9 years) with a low white cell count. Previously, we demonstrated a high relapse risk when these patients were treated as standard risk. Recent studies have shown improved outcome on intensive therapy. In view of these treatment implications, accurate identification is essential. Here we have studied the cytogenetics and outcome of 530 iAMP21 patients that highlighted the association of specific secondary chromosomal and genetic changes with iAMP21 to assist in diagnosis, including the gain of chromosome X, loss or deletion of chromosome 7, ETV6 and RB1 deletions. These iAMP21 patients when treated as high risk showed the same improved outcome as those in trial-based studies regardless of the backbone chemotherapy regimen given. This study reinforces the importance of intensified treatment to reduce the risk of relapse in iAMP21 patients. This now well-defined patient subgroup should be recognised by World Health Organisation (WHO) as a distinct entity of BCP-ALL.

Array-based comparative genomic hybridization and copy number variation in cancer research.

Array-based comparative genomic hybridization (aCGH) is a molecular cytogenetic technique used in detecting and mapping DNA copy number alterations. aCGH is able to interrogate the entire genome at a previously unattainable, high resolution and has directly led to the recent appreciation of a novel class of genomic variation: copy number variation (CNV) in mammalian genomes. All forms of DNA variation/polymorphism are important for studying the basis of phenotypic diversity among individuals. CNV research is still at its infancy, requiring careful collation and annotation of accumulating CNV data that will undoubtedly be useful for accurate interpretation of genomic imbalances identified during cancer research.

[Cytogenetic alterations in renal tumors. Applications for comparative genomic hybridization and fluorescence in situ hybridization]. / Zytogenetische Veränderungen bei Nierentumoren. Einsatzmöglichkeiten von Chromosomen-CGH und Fluoreszenz-in-situ-Hybridisierung.

The WHO classification of renal cell carcinomas (RCC) takes into account chromosomal alterations. New cytogenetic techniques such as comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH) offer alternative methods to the classic cytogenetic banding technique. Clear cell (classic) RCC frequently show the loss of 3p. Papillary RCC are characterized by trisomies and tetrasomies as well as loss of the Y chromosome. CGH analysis demonstrates that DNA copy increase is more common in type I papillary RCC compared to type II. Chromophobe RCC are characterized by losses in chromosomes 1, 2, 6, 10, 13, 17, and 21. Oncocytomas can be divided into cases with rearrangements in the 11q13 region and those with loss of chromosome 1 and the sex chromosomes. Translocations involving chromosome 3, such as t(3;8)(p14;q24.13) and t(2;3)(q35;q21) have been described in familial clear cell RCC. The most recent class of RCC, seen only in men, is referred to as translocation tumors. These tumors demonstrate a tubulopapillary growth pattern and have a t(X;1)(p11.2;q21.2) translocation. Although not required for most clinical diagnoses, CGH and FISH complement the standard histologic diagnosis of RCC and may provide a definitive diagnosis in a small number of challenging cases.

Spectral karyotyping and fluorescence in situ hybridization detect novel chromosomal aberrations, a recurring involvement of chromosome 21 and amplification of the MYC oncogene in acute myeloid leukaemia M2.

Recurring chromosomal aberrations are of aetiological, diagnostic, prognostic and therapeutic importance in acute myeloid leukaemia (AML). However, aberrations are detected in only two thirds of AML cases at diagnosis and recurrent balanced translocations in only 50%. Spectral karyotyping (SKY) enables simultaneous visualization of all human chromosomes in different colours, facilitating the comprehensive evaluation of chromosomal abnormalities. Therefore, SKY was used to characterize 37 cases of newly diagnosed AML-M2, previously analysed using G-banding. In 15/23 patients it was possible to obtain metaphases from viably frozen cells; in 22 additional cases, fixed-cell suspensions were used. Of the 70 chromosomal aberrations identified by SKY, 30 aberrations were detected for the first time, 18 aberrations were redefined and 22 were confirmed. SKY detected two reciprocal translocations, t(X;3) and t(11;19). In five cases, eight structural aberrations resulted in partial gains of chromosome 21, six of which were undetected by G-banding. In 4/5 cases, these resulted in copy number increases for AML1. Amplification of MYC was detected in three cases. Using SKY and FISH, clonal aberrations were identified in 5/18 cases with a presumed normal karyotype; 3/5 aberrations were of known unfavourable prognostic significance. Karyotypes were entered into a custom-designed SKY database, which will be integrated with other cytogenetic and genomic databases.

Aberrations of chromosomes 5 and 8 as recurrent cytogenetic events in anaplastic carcinoma of the thyroid as detected by fluorescence in situ hybridisation and comparative genomic hybridisation.

Comparative genomic hybridisation (CGH) is a technique which identifies gains and losses of DNA sequence copy number in tumours. We used CGH to search for genetic changes in one of the most aggressive malignancies--anaplastic thyroid carcinoma (ATC). For this purpose, we analysed tumour specimens of nine ATCs and DNA of two ATC cell lines. CGH detected aberrations in 10 of 11 samples, with a mean number of gains or losses per carcinoma of 4.8 (range 0-13). Total or partial changes of chromosome 8 (n=6), including gains or losses of 8p (n=6) or 8q (n=5) were those detected most frequently. Chromosome 5p was amplified in five cases. Gains in two of three samples were found for 3q, 7p, 11q and 20q. Gains in a fewer number were seen for 1p (1 case), 1q (1), 7q (2), 9q (2), 11p (2), 12q (1), 14 (1), 15 (1), 17q (2), 18p (2), 18q (1), 20p (1), 21 (2), Xp (2) and Xq (2). Losses were less frequent than gains and observed for 1p (2 cases), 1q (1), 2p (1), 2q (2), 3p (2), 3q (1), 4q (2), 6q (1), 9p (2), 9q (1), 18p (1), 18q (1) and Y (2). Examples of analysis of tumour sections and cell lines performed by fluorescence in situ hybridisation (FISH) confirmed the gains and losses found by CGH and detected additional signals for 8q21 in tumour cells in a sample with no gains or losses normally in CGH. The results suggest that aberrations of 5p, 8p and 8q, which are rarely found in differentiated thyroid carcinoma, may play an important role in the development of ATC. Therefore, these chromosomes could harbour gene loci potentially involved in the aggressiveness of neoplastic tumours, as shown in tumours such as in this study for ATC.

Cytogenetic aberrations in myelodysplastic syndrome detected by comparative genomic hybridization and fluorescence in situ hybridization.

Conventional cytogenetics (CC) is proven as a diagnostic and prognostic factor in myelodysplastic syndrome (MDS). However, CC may be hampered by insufficient metaphase preparation and cannot analyze interphase nuclei. These problems are solved by using comparative genomic hybridization (CGH). The CGH was applied to samples from 45 patients with MDS, and the results were compared with CC and fluorescence in situ hybridization (FISH). The CC detected aberrations in 12 of 45 samples, including chromosomes 3 (n = 1), 5 (n = 9), 7 (n = 2),8(n = 1), 18(n = 1),21 (n = 1), X (n = 1), and Y(n = 2). In one patient, loss of B and C group chromosomes and a marker chromosome were seen. The CGH revealed chromosomal imbalances in 18 of 45 samples, including chromosomes 5 (n = 11), 7 (n = 2), 8 (n = 1), 18(n = 1), 20(n = 1), 21 (n = 1), X (n = 1), and Y (n = 2). All unbalanced aberrations found by CC were detected by CGH, too. In two patients, the CGH found additional aberrations and redefined the aberrations of the chromosomes of the B and C group in one sample. The FISH confirmed these aberrations. Additionally performed FISH for chromosomes 5, 7, and 8 gave normal findings in all patients found to be normal in CC and CGH. The CGH and FISH confirmed the results obtained by CC. All three techniques showed changes of chromosomes 5 and 7 as the most frequent aberrations, emphasizing the importance of these chromosomes in the development of MDS. Furthermore, the CC is proven as the basic technique for cytogenetic evaluation of MDS.

Analysis of hematologic diseases using conventional karyotyping, fluorescence in situ hybridization (FISH), and comparative genomic hybridization (CGH).

Comparative genomic hybridization (CGH) has been proven to be an important tool in interphase cytogenetics of solid tumors. Although, because of methodological implications, balanced aberrations are not detected by CGH, the technique has uncovered a variety of new and interesting imbalanced karyotype changes. However, only a few studies deal with its application to hematologic disorders, although this is a main topic of cytogenetics. The aim of our study was, therefore, to evaluate the usefulness of CGH in the examination of hematologic neoplasms. For this purpose, bone marrow aspirates of 33 patients with different hematologic disorders were examined with CGH and the results compared with conventional cytogenetics (CC) and fluorescence in situ hybridization (FISH). CGH showed chromosome changes in 8 of 33 cases. CC found balanced aberrations in 4 of 33 and unbalanced changes in 9 of 33 samples. Differences between CGH and CC in unbalanced aberrations were seen in four cases. In these samples, either the number of aberrant cells found by CC was low and, therefore, difficult to detect by CGH, or complex aberrations in different cell clones as seen in CC were lumped together as one karyotype by CGH. In one sample, CC was not capable of giving any results at all, whereas CGH showed trisomy 8. CGH was also helpful in defining the bands involved in the structural aberrations, which was difficult by CC in some cases because of the low quality of metaphase spreads. All results obtained by CGH were confirmed by FISH, whereas CC and FISH were discordant in one case. Although CGH was not able to detect all aberrations, it gave important additional information for the correct localization of the aberrations found in CC, and it was most helpful in samples not processed successfully in CC. These advantages would open up a new field of application for CGH not only for research, but also for diagnostic purposes.

Applications of single-color and double-color oligonucleotide primed in situ labeling in cytology.

Interphase cytogenetics is mostly performed with use of fluorescence in situ hybridization (FISH), using long DNA probes of several hundred or thousand base pairs in length. Recently, oligonucleotide primed in situ labeling (PRINS) was established for staining centromeres and telomeres of chromosomes in metaphase spreads by Taq-polymerase-mediated incorporation of labeled nucleotides. We investigated the use of PRINS in intact interphase cells of various cytologic preparations, targeting chromosomes 1, 8, and 9. Examining cell smears (n = 3), touch preparations (n = 20), and cytospins (n = 11) of non-neoplastic and neoplastic tissues, PRINS was as sensitive and reliable as the FISH method in assessing the exact chromosome number. Aneuploidy in tumor cells was confirmed by double-color PRINS in a part of the specimens. The PRINS reaction, which requires heating of the cell preparations to as high as 96 degrees C, did not affect the cytomorphologic details. Because PRINS is much faster and approximately 10 times less expensive than FISH, this method allows an increased application of interphase cytogenetics in diagnostic cytopathology.

Single- and double-color oligonucleotide primed in situ labeling (PRINS): applications in pathology.

Molecular cytogenetics is mostly performed by fluorescence in situ hybridization using long DNA probes that are generated by vector cloning. Oligonucleotide primed in situ labeling (PRINS) is a recent method that has been established for the detection of the centromeric or telomeric region in metaphase chromosomes. In this overview, we demonstrate the possible applications of PRINS and provide elaborated protocols for its use in intact interphase cells of routine cytological preparations, e.g., cell smears, touch preparations, and cytospins of non-neoplastic and neoplastic tissues. Moreover, the various modifications of the PRINS method, such as multi-color PRINS for targeting different chromosomes within one cell or the enzymatic detection of the PRINS product instead of the more commonly used fluorochromes, are discussed.

Detection of karyotype changes in interphase cells: oligonucleotide-primed in situ labelling versus fluorescence in situ hybridization.

Interphase cytogenetics is a rapidly developing technique which is usually performed by fluorescence in situ hybridization (FISH). Recently, oligonucleotide-primed in situ synthesis (PRINS) has become established as a method of labelling centromeric regions of chromosomes in metaphase spreads. We tested the suitability of PRINS in detecting the exact copy number of chromosomes 1, 3, 7 and 8 in intact interphase cells of 17 cytological preparations derived from normal and neoplastic tissues. Control procedures consisted in preparation of metaphase spreads of lymphocytes of healthy donors, conventional cytogenetics in some of the specimens, and omission of the primers or Taq polymerase from the reaction mixture. All specimens were additionally examined by FISH and analysed blind by two experienced observers. Both PRINS and FISH revealed a corresponding distribution of hybridization signals for all chromosomes examined in specimens of normal bone marrow (n = 5), normal liver cells (n = 5), three samples of acute nonlymphocytic leukaemia in which conventional chromosome analyses had shown monosomy 7 or trisomy 8, and in four hepatocellular carcinomas that displayed trisomy 1. Overall, statistical analysis revealed no significant difference in the signal distribution between the two techniques. Our results demonstrate that PRINS is as reliable as FISH for detecting chromosome copy numbers in interphase nuclei of intact cells. The PRINS method, however, is easier to perform, faster and less expensive, holding great potential for future applications in diagnostic pathology.

Value of fluorescence in situ hybridization for detecting the bcr/abl gene fusion in interphase cells of routine bone marrow specimens.

Fluorescence in situ hybridization (FISH) is a new technique that allows demonstrating of the bcr/abl gene fusion in bone marrow cells of patients with Philadelphia translocation (Ph)-positive chronic myeloid leukemia (CML). In this study, bone marrow samples of 150 patients were investigated routinely by interphase FISH, cytogenetics, and bone marrow histopathology. In 20 patients with reactive hyperplasia of the granulopoiesis and normal karyotypes, FISH revealed nonspecific bcr/abl fusion signals at a mean frequency of 2.7% of the cells examined. The cutoff level for specific fusion signals was set at three times the standard deviation (9.0%). None of the 29 cytogenetically Ph-negative patients with myeloproliferative disease other than CML had fusion signals exceeding 9%. The mean frequency of specific fusion signals in nontreated patients with CML (n = 59) was 92.7%, and 49.3% in patients with CML who received therapy (n = 42). For diagnosing Ph-positive CML, interphase FISH has been faster, more reliable, and more sensitive than cytogenetics, which was successful in 54 of 59 patients investigated at first diagnosis but only in 27 of 42 patients receiving therapy, and it failed to detect Ph-positive cells in three patients with CML. However, small percentages of less than 9.0% of cells with bcr/abl fusion signals were below the threshold of interphase FISH, thereby limiting its use for detecting minimal residual disease.