To characterize cytogenetic alterations found in Barrett's adenocarcinoma (BA) and, more importantly, its premalignant stages, we studied chromosomal imbalances in various lesions in the histologically proposed metaplasia-dysplasia-carcinoma sequence using comparative genomic hybridization (CGH). Using 30 esophageal adenocarcinoma resection specimens, we were able to study 30 areas of Barrett's adenocarcinoma and 8 lymph node metastases (LN). In addition, we investigated 25 premalignant lesions adjacent to BA derived from a subset of 14 resection specimens including 11 areas of high grade dysplasia (HGD), 8 areas of low grade dysplasia (LGD), and 6 areas of intestinal metaplasia (IM), which were laser-microdissected and studied with CGH. To validate the CGH findings, fluorescence in situ hybridization analysis on 13 BA with probes specific for HER-2/neu and 20q13.2 were performed. The chromosomal alterations most often identified in BA were: gains on 8q (80%), 20q (60%), 2p, 7p and 10q (47% each), 6p (37%), 15q (33%) and 17q (30%). Losses were observed predominantly on the Y-chromosome (76%), 4q (50%), 5q and 9p (43% each), 18q (40%), 7q (33%) and 14q (30%). High-level amplifications were observed on 8q23-qter, 8p12-pter, 7p11-p14, 7q21-31, 17q11-q23. Recurrent chromosomal changes were also identified in metaplastic (gains on 8q, 6p, 10q, losses on 13q, Y, 9p) and dysplastic epithelium (gains on 8q, 20q, 2p, 10q, 15q, losses on Y, 5q, 9p, 13q, 18q). Novel amplified chromosomal regions on chromosomes 2p and 10q were detected in both Barrett's adenocarcinoma and premalignant lesions. An increase of the average number of detected chromosomal imbalances from IM (7.0 +/- 1.7), to LGD (10.8 +/- 2.2), HGD (13.4 +/- 1.1), BA (13.3 +/- 1.4), and LN (22 +/- 1.2) was seen. Although the detection of common chromosomal alterations in premalignant lesions and adjacent carcinomas suggest a process of clonal expansion, the occurrence of several chromosomal changes in an apparently random order relative to one another is striking evidence that clonal evolution is more complex than would be predicted by linear models. This is probably a reflection of the existence of many divergent neoplastic subpopulations and highlights one of the main problems associated with surveillance of Barrett's patients, namely sampling error.

DNA in situ hybridization with human chromosome specific DNA libraries was applied to compare the karyotypes of humans (Homo sapiens, 2n = 46) and cats (Felis catus, 2n = 38). For the autosomes alone, 30 segments of conserved synteny were revealed. The arrangement of these segments in the feline karyotype differs by only seven single chromosome breaks and one intrachromosomal inversion from their arrangement in humans. Comparison of these data with those recently obtained for pig and those available from conventional gene mapping studies in mice and cattle has allowed us to develop a model of karyotype evolution in mammals. The cat and human karyotypes, with 36 and 44 autosomes respectively, were found to be very similar to a putative ancient mammalian founder karyotype. It would appear that during evolution to the human karyotype the status quo has been conserved for at least some 100-120 million years. There has been no need to alter the well-balanced gene arrangement of the mammalian founder karyotype.

The fusion transcript AML1/ETO corresponding to translocation t(8;21)(q22;q22) can be found in approximately 7-12% of childhood de novo AML. Despite the favorable prognosis, some of these patients relapse. Most of MRD studies so far were performed on adults treated not uniformly. Therefore, we analyzed the follow-up of 15 AML1/ETO-positive children using real-time quantitative reverse transcription PCR (RQ-RT-PCR), all enrolled in the multicenter therapy trial AML-BFM 98. AML1/ ETO copy numbers were normalized to the control gene ABL and the results were expressed in copy numbers AML1/ETO per 10 000 copies ABL. At diagnosis, a median of 10 789 copies AML1/ETO was found. A linear decrease to about 10 copies (2-4 log) could be seen in most of the children by the start of consolidation. In the majority of cases they remained positive at this low level during the ongoing therapy. Four children relapsed and two of them had a decrease of less than 2 log before starting consolidation. Three of the relapsed children showed, prior to relapse, an increase of the AML1/ETO fusion transcript at 6, 9, and 11 weeks, respectively. These results suggest that monitoring of minimal residual disease using RQ-RT-PCR could be helpful in detecting patients with a higher risk of relapse.

Deletions of chromosome 6q have been reported in several hematological malignancies, but data are not conclusive regarding their biological and prognostic impact. Therefore, we focused on pediatric patients diagnosed with T-cell lymphoblastic lymphoma (T-LBL) treated uniformly according to the NHL-BFM95 protocol. We used loss-of-heterozygosity (LOH) analysis of 25 microsatellite markers located on chromosome 6q14-q24. Fragment-length analysis was performed on ABI-PRISM3100 Genetic-Analyzer. Eligibility criterion was X3 informative markers. Between April 1995 and March 2003, 185 T-LBL patients were treated according to the NHL-BFM95 protocol. Five-year event-free (EFS) and disease-free survival (DFS) were 7973 and 8773% (median follow-up 4.7 [1.2-10.1] years). Sixty-one patients were evaluable for LOH analysis, including 18 out of 23 patients with relapse. EFS and DFS were 6776 and 6976% for these 61 patients. Testing of 853 markers in the 61 patients identified the presence of LOH in 19 patients (31%): 13 of the 18 relapse patients and five of the 41 in complete remission (odds ratio 18.7, 95% confidence interval 4.7-75.3). One LOH-positive patient died from treatment-related toxicity. We conclude that LOH on chromosome 6q14-q24 may have conferred a high risk of relapse on our group of children with T-LBL treated according to the NHL-BFM95 protocol.

Burkitt's lymphomas (BLs) are characterized by an activated MYC gene that provides a constitutive proliferative signal. However, activated myc can initiate ARF-dependent activation of p53 and apoptosis as well. Data derived from cell culture and animal models suggest that the inactivation of the ARF-MDM-2-p53 apoptotic signaling pathway may be a necessary secondary event for the development of BL. This has not been tested in freshly excised BL tissue. We investigated the ARF-MDM-2-p53 pathway in tumor specimen from 24 children with sporadic BL/B-ALL. Direct sequencing revealed a point mutation in the p53 gene in four BL. Overexpression of MDM-2 was evident in 10 of the BL samples analyzed by real-time quantitative PCR. Deletion of the CDKN2A locus that encodes ARF or reduced expression of ARF could not be detected in any BL by fluorescence in situ hybridization analysis or real-time quantitative PCR, respectively. Our results indicate that the ARF-MDM-2-p53 apoptotic pathway is disrupted in about 55% of the cases of childhood sporadic BL. We suggest that in addition to the inactivation of the ARF-MDM-2-p53 protective checkpoint function other antiapoptotic mutations may occur in a substantial part of children with sporadic BL.