David R. Betts

Gain of Chromosome Arm 17q and Adverse Outcome in Patients with Neuroblastoma

BACKGROUND Gain of genetic material from chromosome arm 17q (gain of segment 17q21-qter) is the most frequent cytogenetic abnormality of neuroblastoma cells. This gain has been associated with advanced disease, patients who are > or =1 year old, deletion of chromosome arm 1p, and amplification of the N-myc oncogene, all of which predict an adverse outcome. We investigated these associations and evaluated the prognostic importance of the status of chromosome 17. METHODS We compiled molecular cytogenetic analyses of chromosome 17 in primary neuroblastomas in 313 patients at six European centers. Clinical and survival information were collected, along with data on 1p, N-myc, and ploidy. RESULTS Unbalanced gain of segment 17q21-qter was found in 53.7 percent of the tumors, whereas the chromosome was normal in 46.3 percent. The gain of 17q was characteristic of advanced tumors and of tumors in children > or =1 year of age and was strongly associated with the deletion of 1p and amplification of N-myc. No tumor showed amplification of N-myc in the absence of either deletion of 1p or gain of 17q. Gain of 17q was a significant predictive factor for adverse outcome in univariate analysis. Among the patients with this abnormality, overall survival at five years was 30.6 percent (95 percent confidence interval, 21 to 40 percent), as compared with 86.0 percent (95 percent confidence interval, 78 to 91 percent) among those with normal 17q status. in multivariate analysis, gain of 17q was the most powerful prognostic factor, followed by the presence of stage 4 disease and deletion of 1p (hazard ratios, 3.4, 2.3, and 1.9, respectively). CONCLUSIONS Gain of chromosome segment 17q21-qter is an important prognostic factor in children with neuroblastoma.

Prognostic impact of specific chromosomal aberrations in a large group of pediatric patients with acute myeloid leukemia treated uniformly according to trial AML-BFM 98.

PURPOSE Because cytogenetic data are essential for risk stratification of childhood acute myeloid leukemia (AML), the impact of chromosomal aberrations is crucial. PATIENTS AND METHODS Data of a large group of patients younger than 18 years treated according to study AML-Berlin-Frankfurt-Münster (BFM) 98 (n = 454), including their cytogenetics, were analyzed. RESULTS The favorable outcome in the subgroups of patients with t(8;21), inv(16), and t(15;17), with an overall survival of 91% (SE, 4%), 92% (SE, 6%), and 87% (SE, 5%), respectively, was confirmed. Within this group, the 5-year probability of event-free survival (pEFS) of all 17 children with t(8;21) and additional aberrations apart from del(9q) or -X/-Y was 100%. As expected, the cytogenetic finding of a complex karyotype (n = 35; pEFS, 33%; SE, 8%) or a monosomy 7 (n = 12; pEFS, 17%; SE, 11%) was associated with a poor outcome. Compared with remaining patients with cytogenetic data (pEFS, 48%; SE, 2%), prognosis in patients with an MLL rearrangement (n = 91) was inferior (pEFS, 34%; SE, 5%; P = .0005). Particularly, children with t(9;11) and additional aberrations (n = 13; pEFS, 31%; SE, 14%) and MLL rearrangements other than t(9;11) and t(11;19) (n = 41; pEFS, 24%; SE, 7%) had an unfavorable outcome. Nine patients with aberrations in 12p showed an adverse prognosis (pEFS, 11%; SE, 10%). The outcome of patients with aberrations of chromosome 5 (n = 13) was better than expected (pEFS, 50%; SE, 13%). CONCLUSION Because the prognostic value of rare recurrent chromosomal aberrations still has to be elucidated, these data will contribute to future risk stratification for the treatment of pediatric AML.

Generation of an epigenetic signature by chronic hypoxia in prostate cells

Increasing levels of tissue hypoxia have been reported as a natural feature of the aging prostate gland and may be a risk factor for the development of prostate cancer. In this study, we have used PwR-1E benign prostate epithelial cells and an equivalently aged hypoxia-adapted PwR-1E sub-line to identify phenotypic and epigenetic consequences of chronic hypoxia in prostate cells. We have identified a significantly altered cellular phenotype in response to chronic hypoxia as characterized by increased receptor-mediated apoptotic resistance, the induction of cellular senescence, increased invasion and the increased secretion of IL-1 beta, IL6, IL8 and TNFalpha cytokines. In association with these phenotypic changes and the absence of HIF-1 alpha protein expression, we have demonstrated significant increases in global levels of DNA methylation and H3K9 histone acetylation in these cells, concomitant with the increased expression of DNA methyltransferase DMNT3b and gene-specific changes in DNA methylation at key imprinting loci. In conclusion, we have demonstrated a genome-wide adjustment of DNA methylation and histone acetylation under chronic hypoxic conditions in the prostate. These epigenetic signatures may represent an additional mechanism to promote and maintain a hypoxic-adapted cellular phenotype with a potential role in tumour development.

Complex karyotype newly defined: the strongest prognostic factor in advanced childhood myelodysplastic syndrome

To identify cytogenetic risk factors predicting outcome in children with advanced myelodysplastic syndrome, overall survival of 192 children prospectively enrolled in European Working Group of Myelodysplastic Syndrome in Childhood studies was evaluated with regard to karyotypic complexity. Structurally complex constitutes a new definition of complex karyotype characterized by more than or equal to 3 chromosomal aberrations, including at least one structural aberration. Five-year overall survival in patients with more than or equal to 3 clonal aberrations, which were not structurally complex, did not differ from that observed in patients with normal karyotype. Cox regression analysis revealed the presence of a monosomal and structurally complex karyotype to be strongly associated with poor prognosis (hazard ratio = 4.6, P < .01). Notably, a structurally complex karyotype without a monosomy was associated with a very short 2-year overall survival probability of only 14% (hazard ratio = 14.5; P < .01). The presence of a structurally complex karyotype was the strongest independent prognostic marker predicting poor outcome in children with advanced myelodysplastic syndrome.

The prognostic significance of cytogenetic aberrations in childhood acute myeloid leukaemia. A study of the Swiss Paediatric Oncology Group (SPOG)

In childhood‐onset acute myeloid leukaemia (AML) the clinical value of karyotypic aberrations is now acknowledged, although there is still debate concerning the prognostic significance of some events. To add to this knowledge, cytogenetic analysis was performed on a consecutive series of 84 childhood AML patients diagnosed in Switzerland. A result was obtained for all patients, with 69 (82%) showing a clonal karyotypic aberration. In the remaining 15 (18%), no karyotypic aberration was seen by either conventional or fluorescence in situ hybridisation analyses. The most frequent aberrations observed were t(11q23) (19% of all patients), t(8;21) (12%) and +8 (11%). Except for cytogenetics, no clinical parameter was shown to be significantly associated with outcome. The analysis of individual cytogenetic subgroups demonstrated that aberrations involving chromosome 16q were the strongest predictor of a good prognosis, while +8 and complex karyotypes represented the strongest predictors of a poor prognosis. It was also noteworthy that patients with the rare aberrations of del(11q) (n = 4) and t(16;21)(p11;q22) (n = 3) had a poor outcome. The results support the importance of cytogenetic analysis in childhood AML, but show that further work is required in the classification of the poor prognosis aberrations.

Clonal expansion and not cell interconversion is the basis for the neuroblast and nonneuronal types of the SK-N-SH neuroblastoma cell line

The ability of neuroblastoma (NB) cells to interconvert bidirectionally, in vitro, from a neuroblast (N) to a nonneuronal (S) form is a well-studied biologic phenomenon of great clinical importance. Differences in the morphologic/ biochemical characteristics and gene expression patterns of the two cell populations have been investigated extensively in an effort to unravel the transdifferentiation process. Subcloning of the SK-N-SH NB cell line has led to two morphologically distinct cell types: SH-SY5Y (N-type) and SH-EP (S-type). Karyotypic analysis combined with G-banding and SKY showed a difference between these two cell types in the copy number of the 2p15 approximately pter segment, including the MYC-N gene. FISH analysis showed an extra copy of MYC-N present in all three lines: in SK-N-SH and SH-SY5Y the majority of cells had three copies of MYC-N, whereas in SH-EP the majority had two copies and only a small cell population with three copies was present. We suggest that the simultaneous coexistence of both cell types and the subsequent clonal expansion of one over the other is a possible explanation for the phenomenon observed and not the accepted interconversion model. According to the clonal expansion model, both N and S cells are simultaneously present in both cell lines. Under certain conditions, the less-aggressive S cells can dominate over the highly aggressive N cells, which eventually lead to the formation of the SH-EP and vice-versa.

Prenatal origin of separate evolution of leukemia in identical twins.

Several studies involving identical twins with concordant leukemia and retrospective scrutiny of archived neonatal blood spots have shown that the TEL-AML1 fusion gene in childhood acute lymphoblastic leukemia (ALL) frequently arises before birth. A prenatal origin of childhood leukemia was further supported by the detection of clonotypic immunoglobulin gene rearrangements on neonatal blood spots of children with various other subtypes of ALL. However, no comprehensive study is available linking these clonotypic events. We describe a pair of 5-year-old monozygotic twins with concordant TEL-AML1-positive ALL. Separate leukemic clones were identified in the diagnostic samples since distinct IGH and IGK-Kde gene rearrangements could be detected. Additional differences characterizing the leukemic clones included an aberration of the second, nonrearranged TEL allele observed in one twin only. Interestingly, both the identical TEL-AML1 fusion sequence and distinct immunoglobulin gene rearrangements were identified on the neonatal blood spots indicating that separate preleukemic clones evolved already before birth. Finally, we compared the reported twins with an additional 31 children with ALL by using the microarray technology. Gene expression profiling provided evidence that leukemia in twins harbours the same subtype-typical feature as TEL-AML1-positive leukemia in singletons suggesting that the leukemogenesis model might also be applicable generally.

Karyotypic evolution pathways in medulloblastoma/primitive neuroectodermal tumor determined with a combination of spectral karyotyping, G-banding, and fluorescence in situ hybridization

Medulloblastomas (MBs) or primitive neuroectodermal tumors (PNETs) represent 15%-30% of pediatric brain tumors and are the most common brain tumors in children; they are rare in adults. Classification of these tumors is based on tissue morphology and is often controversial and problematic. Karyotypic analysis of these tumors using conventional cytogenetic methods is often a difficult process that may be hindered by a limited number of metaphase cells and poor chromosome morphology, often leading to only partial characterization of the chromosomal abnormalities. We investigated three primary human tumors and four cell lines (CHO-707, DAOY, D-341, and PFSK) utilizing a combination of conventional G-banding, spectral karyotyping (SKY), and fluorescence in situ hybridization (FISH) techniques. A high level of intratumoral heterogeneity was seen, with multiple numerical and structural chromosomal aberrations. The chromosomes most frequently involved in structural aberrations were chromosomes 1 (14 rearrangements), 7 (9 rearrangements), and 21 (9 rearrangements). The chromosomes most frequently involved in numerical aberrations were chromosomes 1, 12, and 13 (four cases) and chromosomes 14, 17, 19, 21, 22, and X (three cases). Numerous aberrant chromosomes were characterized only with the SKY analysis, and based on these findings multiple clones were identified, facilitating analysis of karyotypic evolution. The most frequent evolution mechanism was via polyploidization, followed by acquisition of additional numerical or structural aberrations (or both); however, the results showed that the karyotypic evolution process in these tumors is typically divergent and complex.

A childhood fibrolamellar hepatocellular carcinoma with increased aromatase activity and a near triploid karyotype

We report a 15-year-old boy with hepatocellular carcinoma (HCC) of the fibrolamellar type. He presented with advanced disease and a non-resectable tumor. Clinical features included marked gynecomastia which had been present for 3 years, failure to enter puberty, and failure to thrive. These features might have been due to a high aromatase activity of the tumor. The course of the illness suggested that the tumor had been present for at least 3 years prior to diagnosis. At diagnosis the patient had multiple metastases which included infiltrated ascites. Cytogenetic analysis of the ascites revealed a near triploid karyotype with cell-to-cell variation and an abnormality of chromosome 1 q. This to our knowledge is the first karyotype report of fibrolamellar HCC in a child.

Loss of single HLA Class I allospecificities in melanoma cells due to selective genomic abbreviations

Expression of human leucocyte antigen (HLA) Class I molecules is essential for the recognition of malignant melanoma (MM) cells by CD8+ T lymphocytes. A complete or partial loss of HLA Class I molecules is a potent strategy for MM cells to escape from immunosurveillance. In 2 out of 55 melanoma cell cultures we identified a complete phenotypic loss of HLA allospecificities. Both patients have been treated unsuccessfully with HLA‐A2 peptides. To identify the reasons underlying the loss of single HLA‐A allospecificities, we searched for genomic alterations at the locus for HLA Class I α‐chain on chromosome 6 in melanoma cell cultures established from 2 selected patients with MM in advanced stage. This deficiency was associated with alterations of HLA‐A2 gene sequences as determined by polymerase chain reaction‐sequence specific primers (PCR‐SSP). Karyotyping revealed a chromosomal loss in Patient 1, whereas melanoma cell cultures established from Patient 2 displayed 2 copies of chromosome 6. Loss of heterozygosity (LOH) using markers located around position 6p21 was detected in both cases. By applying group‐specific primer‐mixes spanning the 5′‐flanking region of the HLA‐A2 gene locus the relevant region was amplified by PCR and subsequent sequencing allowed alignment with the known HLA Class I reference sequences. Functional assays using HLA‐A2‐restricted cytotoxic T‐cell clones were performed in HLA‐A2 deficient MM cultures and revealed a drastically reduced susceptibility to CTL lysis in HLA‐A2 negative cells. We could document the occurrence of selective HLA‐A2 deficiencies in cultured advanced‐stage melanoma metastases and identify their molecular causes as genomic alterations within the HLA‐A gene locus.