Identification of a novel inherited ALK variant M1199L in the WNT type of medulloblastoma.

Rearrangements involving the ALK gene were identified in a variety of cancers, including paediatric tumour neuroblastoma where presence of ALK expression is also associated with adverse prognosis. Microarrays data indicate that ALK is expressed in another paediatric tumour - medulloblastoma. Therefore, we investigated if the ALK gene is mutated in medulloblastoma and performed simultaneously the molecular profiling of tumours. Tumours from sixty-four medulloblastoma patients were studied for detection of ALK alterations in exons 23 and 25 using Sanger method. The molecular subtypes of tumours were identified by detection of mutations in the CTNNB1 gene, monosomy 6 and by immunohistochemistry using a panel of representative antibodies. Among three ALK variants detected two resulted in intron variants (rs3738867, rs113866835) and the third one was a novel heterozygous variant c.3595A>T in exon 23 identified in the WNT type of tumour. It resulted in methionine to leucine substitution at codon position 1199 (M1199L) of the kinase domain of ALK protein. Results of analysis using three in silico algorithms confirmed the pathogenicity of this single nucleotide variation. The same gene alteration was detected in both patient and maternal peripheral blood leukocytes indicating an inherited type of the detected variant. Presence of ALK expression in tumour tissue was confirmed by immunohistochemistry. The tumour was diagnosed as classic medulloblastoma, however with visible areas of focal anaplastic features. The patient has been disease free for 6 years since diagnosis. This is the first evidence of an inherited ALK variant in the WNT type of medulloblastoma, what altogether with presence of ALK expression may point towards involvement of the ALK gene in this type of tumours.

Minimal disease monitoring by QRT-PCR: guidelines for identification and systematic validation of molecular markers prior to evaluation in prospective clinical trials.

Real-time RT-PCR (QRT-PCR) is a sensitive method for the detection of minimal disease (MD) and may improve monitoring of disease status and stratification of patients for therapy. Where tumour-specific mRNAs have not been identified, the selection of which target(s) is(are) optimal for the detection of MD remains a challenge. This reflects the heterogeneity of tumour cells, the stability of mRNAs and low-level of transcription in cells of the normal haemopoietic compartments. The aim of this study was to establish for the first time guidelines for the systematic prioritization of potential markers of MD detected by QRT-PCR prior to evaluation in multicentre prospective clinical outcome studies. We combined microarray analysis, ESTs gene expression profiles, improved probe-sets sequence annotation, and previously described standard operating procedures for QRT-PCR analysis to identify and prioritize potential markers of MD. Using this methodology, we identified 49 potential markers of MD in neuroblastoma (NB), of which 11 were associated with neuronal function. We found that, in addition to TH, Phox2B and DCX mRNA may be useful targets for the detection of MD in children with NB. This same strategy could be exploited to select MD markers of other solid tumours from the large number of potential targets identified by microarray gene expression profiles.

Histological profile of tumours from MYCN transgenic mice.

BACKGROUND: MYCN is the most commonly amplified gene in human neuroblastomas. This proto-oncogene has been overexpressed in a mouse model of the disease in order to explore the role of MYCN in this tumour. AIMS: To report the histopathological features of neuroblastomas from MYCN transgenic mice. METHODS: 27 neuroblastomas from hemizygous transgenic mice and four tumours from homozygous mice were examined histologically; Ki67 and MYCN immunocytochemistry was performed in 24 tumours. RESULTS: Tumours obtained from MYCN transgenic mice resembled human neuroblastomas, displaying many of the features associated with stroma-poor neuroblastoma, including heterogeneity of differentiation (but no overt ganglionic differentiation was seen), low levels of Schwannian stroma and a high mitosis karyorrhexis index. The tumours had a median Ki67 labelling index of 70%; all tumours expressed MYCN with a median labelling index of 68%. The most striking difference between the murine and human neuroblastomas was the presence of tingible body macrophages in the transgenic mouse tumours reflecting high levels of apoptosis. This has not previously been described in human or other murine neuroblastoma models. CONCLUSIONS: These studies highlight the histological similarities between tumours from MYCN transgenic mice and human neuroblastomas, and reaffirm their role as a valuable model to study the biology of aggressive human neuroblastoma.

Identification of candidate genes involved in neuroblastoma progression by combining genomic and expression microarrays with survival data.

Identifying genes, whose expression is consistently altered by chromosomal gains or losses, is an important step in defining genes of biological relevance in a wide variety of tumour types. However, additional criteria are needed to discriminate further among the large number of candidate genes identified. This is particularly true for neuroblastoma, where multiple genomic copy number changes of proven prognostic value exist. We have used Affymetrix microarrays and a combination of fluorescent in situ hybridization and single nucleotide polymorphism (SNP) microarrays to establish expression profiles and delineate copy number alterations in 30 primary neuroblastomas. Correlation of microarray data with patient survival and analysis of expression within rodent neuroblastoma cell lines were then used to define further genes likely to be involved in the disease process. Using this approach, we identify >1000 genes within eight recurrent genomic alterations (loss of 1p, 3p, 4p, 10q and 11q, 2p gain, 17q gain, and the MYCN amplicon) whose expression is consistently altered by copy number change. Of these, 84 correlate with patient survival, with the minimal regions of 17q gain and 4p loss being enriched significantly for such genes. These include genes involved in RNA and DNA metabolism, and apoptosis. Orthologues of all but one of these genes on 17q are overexpressed in rodent neuroblastoma cell lines. A significant excess of SNPs whose copy number correlates with survival is also observed on proximal 4p in stage 4 tumours, and we find that deletion of 4p is associated with improved outcome in an extended cohort of tumours. These results define the major impact of genomic copy number alterations upon transcription within neuroblastoma, and highlight genes on distal 17q and proximal 4p for downstream analyses. They also suggest that integration of discriminators, such as survival and comparative gene expression, with microarray data may be useful in the identification of critical genes within regions of loss or gain in many human cancers.

17q gain in neuroblastoma predicts adverse clinical outcome. U.K. Cancer Cytogenetics Group and the U.K. Children's Cancer Study Group.

BACKGROUND: It is now recognized that gain of chromosome 17 material is the most frequent genetic abnormality of neuroblastoma cells. Several studies have linked 17q gain with known adverse prognostic factors: patient age >1 year, advanced stage disease, deletion of chromosome arm 1 p, and amplification of the MYCN oncogene. We sought to further investigate the clinical and prognostic associations of chromosome 17 status in relation to other well-established predictive factors. PROCEDURE: In a collaborative study by UK cytogenetics centres, we compiled a series of 104 neuroblastoma tumours for which the status of chromosome 17 was confidently defined by cytogenetics, metaphase or interphase FISH, or CGH analysis. The results were correlated with data on 1p and MYCN, and with centrally collated clinical and survival information. RESULTS: Gain of 17q (i.e., unbalanced gain of segment 17q21-qter) was found in 66.3% of tumours, while 33.7% showed a '17q normal' status (i.e., no gain at all, or gain of whole chromosome 17 relative to ploidy). Gain of 17q was strongly associated with advanced stage disease, patient age >1 year, 1p deletion, and MYCN amplification (all P< 0.01). In univariate analysis, 17q gain was a significant predictor of adverse outcome (projected 5 year relapse-free survival 15.6% compared to 75.2% in cases lacking this feature in tumour cells; (P < 0.0001). In multivariate analysis, 17q gain was more strongly associated with adverse outcome than was either stage (Stage 4 vs other combined) or 1p status. CONCLUSION: We conclude that gain of chromosome segment 17q21-qter is of great biological and clinical importance in neuroblastoma, and that its detection at diagnosis should be a priority.

Molecular cytogenetic definition of 17q translocation breakpoints in neuroblastoma.

BACKGROUND: Unbalanced translocations resulting in the gain of material from 17q are the most common chromosomal changes in neuroblastoma and are associated with poor patient survival, and are established indicators of bad prognosis. PROCEDURE: We have used 13 fluorescent in situ hybridisation probes to map 17q translocation breakpoints in ten neuroblastoma cell lines and 21 primary tumours. RESULTS: At least seven different breakpoints have been identified, all localised within the proximal half of 17q (53-68 cM, 17cen-17q22). CONCLUSION: These results suggest that the dosage of a gene, or genes, in 17q22-qter is responsible for the clinical effects of 17q gain, rather than the disruption of a specific gene.

Multicentre analysis of patterns of DNA gains and losses in 204 neuroblastoma tumors: how many genetic subgroups are there?

PROCEDURE: Analysis of comparative genomic hybridization (CGH) data of 120 tumors from four different studies, and data of 84 previously unpublishied tumors, allowed delineation of at least six different genetic subsets of neuroblastomas. RESULTS AND CONCLUSIONS: A small number of tumors show no detectable imballances. A second group of tumors presents with gains and losses of whole chromosomes and is found predominantly in prognostically favorable stage 1 and 2 tumors. The remaining groups are characterized by the presence of partial chromosome imbalances, and are found mostly in stage 3, 4, and 4S tumors. The third group shows 17q gain without 11q loss, 1p loss, or MYCN amplification (MNA). The fourth group has 1p deletion or MNA, and finally, a fifth group shows 11q loss without 1p deletion or MNA, and is found mainly in stage 4 tumors. The latter group is significantly associated with losses of 3p, 4p, and 14q.

Comprehensive genetic and histopathologic study reveals three types of neuroblastoma tumors.

PURPOSE: To determine the relationship between multiple genetic features, tumor morphology, and prognosis in neuroblastoma. PATIENTS AND METHODS: The genetic alterations and morphologic features that underpin three histopathologic risk classifications were analyzed in 108 neuroblastoma patients. Tumors were subdivided into four groups based on the three most frequent and prognostically significant genetic alterations (17q gain, 1p deletion, and MYCN amplification), and all other genetic, morphologic, and clinical data were analyzed with respect to these groups. RESULTS: Our analyses identify three nonoverlapping tumor types with distinct genetic and morphologic features, defined here as types 1, 2, and 3. Type 1 tumors show none of the three significant genetic alterations and have good prognosis. Both type 2 (17q gain only or 17q gain and 1p del) and type 3 (17q gain, 1p del, and MYCN amplification) tumors progress. However, these tumor types are distinguished clinically by having significantly different median age at diagnosis and median progression-free survival (PFS). Multivariate analysis indicates that 17q gain is the only independent prognostic factor among all genetic, histopathologic, and clinical factors analyzed. Among histopathologic risk systems, the International Neuroblastoma Pathology Classification was the best predictor of PFS. CONCLUSION: Our results indicate that specific combinations of genetic changes in neuroblastoma tumors contribute to distinct morphologic and clinical features. Furthermore, the identification of two genetically and morphologically distinct types of progressing tumors suggests that possibilities for different therapeutic regimens should be investigated.

Comparative genomic hybridization (CGH) analysis of stage 4 neuroblastoma reveals high frequency of 11q deletion in tumors lacking MYCN amplification.

We have studied the occurrence and association of 11q deletions with other chromosomal imbalances in Stage 4 neuroblastomas. To this purpose we have performed comparative genomic hybridization (CGH) analysis on 50 Stage 4 neuroblastomas and these data were analyzed together with those from 33 previously published cases. We observed a high incidence of 11q deletion in Stage 4 neuroblastoma without MYCN amplification (59%) whereas 11q loss was only observed in 15% of neuroblastomas with MYCN-amplification (p = 0.0002) or 11% of cases with 1p deletion detected by CGH (p = 0.0001). In addition, 11q loss showed significant positive correlation with 3p loss (p = 0.0002). Event-free survival was poor and not significantly different for patients with or without 11q deletion. Our study provides further evidence that Stage 4 neuroblastomas with 11q deletions represent a distinct genetic subgroup that typically shows no MYCN-amplification nor 1p deletion. Moreover, it shows that neuroblastomas with 11q deletion also often present 3p deletion. This genetic subgroup shows a similar poor prognosis as MYCN amplified 4 neuroblastomas.

MYCN amplification and 17q in neuroblastoma: evidence for structural association.

MYCN oncogene amplification in neuroblastoma is statistically associated with gain of chromosome segment 17q21-qter. In neuroblastoma cell lines and primary tumors with MYCN amplification in the form of homogeneously staining regions (hsrs), juxtaposition of chromosome 17 material with MYCN sequences has occasionally been reported, raising the possibility of a physical affinity between MYCN and chromosome arm 17q. We used FISH to test for association between chromosome 17 segments and MYCN in eight neuroblastoma cell lines and two neuroblastoma primary tumors known to include hsrs. Evidence of an association was found in the chromosomes of both primary tumors; in one, a MYCN hsr was inserted into a structurally abnormal chromosome 17, in the other, an hsr in 16p was shown to be flanked by 17 material. In cell line NCG, hsrs in 4q and 16p were flanked by 17q material. These observations confirm the juxtaposition of 17q material with MYCN sequences in some neuroblastomas, and imply that there may be a physical or functional relationship between these two features in MYCN amplified neuroblastoma.

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.

Molecular cytogenetic delineation of 17q translocation breakpoints in neuroblastoma cell lines.

It has recently been recognized that unbalanced translocations resulting in the gain of material from 17q are the most common chromosomal changes in neuroblastoma. These rearrangements are associated with established indicators of bad prognosis and poor patient survival. We have used 13 fluorescence in situ hybridization (FISH) probes to map 12 translocation breakpoints on 17q in 10 neuroblastoma cell lines, identifying at least seven different breakpoints, all localized within the proximal half of 17q (268-369 cR, 53-68 cM). These results suggest that the dosage of a gene, or genes, in 17q22-qter is responsible for the clinical effects of 17q gain, rather than the disruption of a specific gene. This region contains two genes, nm23-H1 and NGFR, already implicated in neuroblastoma biology.

Gain of chromosome arm 17q predicts unfavourable outcome in neuroblastoma patients. U.K. Children's Cancer Study Group and the U.K. Cancer Cytogenetics Group.

Gain of chromosome arm 17q has recently been reported in neuroblastoma tumours. We analysed 17q status in relation to other known prognostic features and clinical outcome in a series of 45 tumours. Chromosome 17 status was detected by cytogenetic analysis, fluorescence in situ hybridisation (FISH) anc comparative genomic hybridisation (CGH) and correlated with other clinical and genetic factors. Survival analysis was calculated by the Kaplan-Meier estimation. Twenty-eight out of 45 tumours showed 17q gain, and this was associated with established indicators of poor prognosis; stage 4 disease (P < 0.001), age above 1 year at diagnosis (P < 0.001), 1p deletion (P < 0.01), MYCN amplification (P = 0.03) and diploidy/tetraploidy (P = 0.04). 17q gain was associated with poor outcome: 3-year survival was 13.5% compared with 100% for tumours without 17q gain (P = 0.0001); and progression-free survival (PFS) was 8.1% after 3 years compared with 83% for 17q normal tumours (P = 0.0001). PFS in 28 MYCN non-amplified patients indicated that 17q status has discriminatory power within this group: PFS 0% for 17q gain (n = 14) versus 100% for normal 17q (n = 14) (P = 0.0001). This study indicates that 17q changes have prognostic significance in neuroblastoma and should be a target for molecular cytogenetic detection at diagnosis.

Promiscuous translocations of chromosome arm 17q in human neuroblastomas.

Deletions of chromosome arm 1p and amplification of the MYCN oncogene are well-recognized genetic changes in neuroblastoma cells. Technical difficulties in cytogenetic analysis of this tumour have hampered the recognition of other recurring abnormalities, but recent use of molecular cytogenetic techniques has indicated significant involvement of chromosome arm 17q. In primary tumours and in cell lines, a recurrent unbalanced translocation t(1p;17q) has been identified by fluorescence in situ hybridization. We confirm the occurrence of this translocation in primary tumours and, in addition, we describe seven new structural rearrangements all of which result in gain of 17q in tumour cells. These rearrangements involved chromosome arms 9p, 10q, 11p, 14q, and 16q. Triplication of the 17q arm was seen in one case. The 17q breakpoint was most commonly q21. All these 17q changes were found in near-diploid tumours. We have also reviewed the literature for neuroblastoma karyotypes involving 17q abnormalities; taken in conjunction with our findings this indicates a remarkable promiscuity of translocation partners, with more than 20 different chromosome regions involved in 17q translocations.

Comparative genomic hybridization study of primary neuroblastoma tumors. United Kingdom Children's Cancer Study Group.

Neuroblastoma tumors show a complex interaction of genetic abnormalities, among which some are of significant prognostic importance; however, analysis of chromosome changes in this tumor is often unsuccessful. Twenty primary tumors were studied by comparative genomic hybridization (CGH), and abnormalities were found in 19. While these changes included deletions of chromosome arm Ip (45%) and MYCN oncogene amplification (30%), gains of chromosome 17 material were much more frequent (75%). We also found evidence in two cases of a new amplification site at band 2p23.

Fluorescence in situ hybridization analysis using cosmid probes to define chromosome 6q abnormalities in ovarian carcinoma cell lines.

Deletion of 6q is a frequent finding in ovarian carcinoma, which would suggest that this region contains one or more putative tumor suppressor genes. Chromosome 6q abnormalities in six ovarian carcinoma cell lines were analyzed by G-banding and fluorescence in situ hybridization (FISH). Using a variety of probes, including a chromosome 6 paint, a probe specific for the chromosome 6 centromere, and cosmids that map to q24 (cCI6-115), q25 (cCI6-4), q26 (cCI6-91, cCI6-119), and q27 (cCI6-13, cCI6-24, and cCI6-111), abnormalities of 6q were found in three cell lines. In cell line OAW42 (hypotetraploid), the sequences complementary to cCI6-119, cCI6-91, and cCI6-13 probes were lost in two homologues of chromosome 6, which indicates the deletion of genetic material from bands q26-27. The same bands were translocated in cell line PEO1 (hypertriploid). The probes from this region were absent on two copies of chromosome 6, but hybridized to two or three markers. In cell line 59M (hyperdiploid) an interstitial deletion proximal to q24 was detected in one chromosome 6. We conclude that it is very likely that a gene or genes localized in bands 6q26-27, and perhaps in the region proximal to 6q24, play a critical role in the development or progression of ovarian carcinoma.

Non-Hodgkin's lymphoma and Klinefelter syndrome.

Patients with a 47, XXY karyotype (Klinefelter syndrome) appear to have an increased risk of developing a malignancy in adulthood, usually cancer of the breast, extragonadal germ cell tumor, and acute nonlymphoblastic leukemia. There is growing evidence to show that these patients also have an increased risk of developing a malignancy in childhood. There are reports describing the development of acute lymphoblastic leukemia, retinoblastoma, and rhabdomyosarcoma in children with a 47, XXY or mosaic 47, XXY/46, XY karyotype. We report a child with a bone metastasizing, B-cell lineage, non-Hodgkin's lymphoma (NHL) who was found to have a 47, XXY karyotype in both the tumor and constitutional cells.

[Myasthenic syndrome associated with relapse of Hodgkin's disease]. / Zespól miasteniczny w przebiegu nawrotu choroby Hodgkina.

Hodgkin's Disease in 12-years old boy was described. After 1-year remission, the relapse of disease localizes in the mediastinum occurred. At the same time myasthenic syndrome was diagnosed. During chemotherapy of H.D. relapse, symptoms of myasthenic syndrome regressed.

[Neuroblastoma in children. Results of the treatment and analysis of selected prognostic factors]. / Zwojak zarodkowy u dzieci. Wyniki leczenia i analiza wybranych czynników prognostycznych.

Results of treatment of 46 children within 6 months-16 years limits of age, with neuroblastoma hospitalized at the I-st Clinic of Children Diseases in Poznan, are presented. In 44 of patients the III-rd or IV-th stage of clinical advancement has been diagnosed, hence the analysis is concerned mainly with advanced forms of disease. 3 year survival of patients treated since 1985 with application of an intensive GPO-NBL programme appeared to be higher (p = 0.37 +/- 0.15) as compared with a group of children treated earlier (p = 0.17 +/- 0.07). The difference appeared however is not significant. Among analysed prognostic factors only age has been of importance. The probability of survival of younger children-below 2 years of a age (p = 64 +/- 0.23) is significantly higher as compared with older age group (p = 0.03 +/- 0.04).