Homogeneously Staining Regions Research Articles

Analysis of Telomeric Repeats and Telomerase Activity in Human Colon Carcinoma Cells with Gene Amplification

COLO320DM and COLO320HSR are cell lines derived from a human malignant neuroendocrine colon carcinoma. Both lines have a 30–40-fold amplification of a large DNA domain containing the MYC oncogene. By using fluorescence in situ hybridization techniques with a MYC probe, we could demonstrate that MYC amplicons are contained in a large marker chromosome in COLO320HSR cells, in double minutes (dmin) of COLO320DM cells, and in the interstitial regions of 3–4 additional chromosomes in both cell lines. Amplicons in homogeneous staining regions (HSRs) comprise normal MYC genes, while dmin chromosomes contain PVT/MYC chimeras. Although both cell lines showed similar levels of telomerase activity, the telomere length and telomere distribution in chromosomal termini were considerably lower in COLO320DM than in COLO320HSR cells. This indicates that the average telomere length in cancer cells is regulated no only by the rates of telomerase activity but also by some other non-enzymatic mechanisms.

Unusual chromosome architecture and behaviour at an HSR.

Amplification of sequences within mammalian chromosomes is often accompanied by the formation of homogeneously staining regions (HSRs). The arrangement of DNA sequences within such amplicons has been investigated, but little is known about the chromosome structure or behaviour of these unusual regions. We have analysed the metaphase chromosome structure of the dihydrofolate reductase (DHFR) amplicon of CHOC400 cells. The chromatin in this region contains hyperacetylated nucleosomes yet, at the same time, appears to be densely packed like heterochromatin. The region does not bind heterochromatin proteins. We show that the dense packing of the region is restricted to DNA located close to the chromosome core/scaffold. In contrast, levels of the chromosome scaffold protein topoisomerase II at HSRs are the same as those found at other euchromatic locations. Metaphase chromosome condensation of the HSR is shown to be sensitive to topoisomerase II inhibitors, and sister chromatids often appear to remain attached within the HSRs at metaphase. We suggest that these features underlie anaphase bridging and the aberrant interphase structure of the HSR. The DHFR amplicon is widely used as a model system to study mammalian DNA replication. We conclude that the higher-order chromosome structure of this amplicon is unusual and suggest that caution needs to be exercised in extrapolating data from HSRs to normal chromosomal loci.

Amplification of C- MYC as the Origin of the Homogeneous Staining Region in Ovarian Carcinoma Detected by Micro-FISH

Homogeneous staining region (hsr), a cytogenetic indicator of gene amplification, has been frequently found in ovarian carcinoma (ovc). To identify the origin of the hsr, chromosome microdissection combined with polymerase chain reaction and fluorescence in situ hybridization (FISH) was applied to two human ovarian cancer cell lines, GR and MLS/P. The hsr probes were labeled with biotin or digoxigenin and hybridized to normal metaphase spreads to elucidate the chromosomal origin and regional localization of the amplified genes. FISH to normal metaphase spreads with the probe generated from the whole hsr-bearing chromosome from GR hybridized to 8q24, 2p13 →2q11.2, 10pter →10p15, 10p12 →10q11.2, 5q23 →5q31, and 5q33 →5qter. For MLS/P, the hsr-bearing marker chromosome hybridized to 8q and 15q. In both cases, detailed FISH analysis revealed enhanced signal intensity at the 8q24 locus, which coincides with the chromosomal location of the C- MYC oncogene. To verify the involvement of C- MYC in hsr formation, in situ hybridization with a probe specific for the C- MYC oncogene was conducted and confirmed the amplification of C- MYC as the origin of the hsr. The whole hsr-bearing chromosome for GR is designated as rev ish der(10) (10pter →10p15::8q24hsr:: 10p12 →10q11.2::8q24::2q11.2 →2p13::2p13 →2q11.2::8q24::10q11 →10p11.2::5q23 →5q31::5q33 →5qter (wcp10 +,D10Z1 ++,wcp2 +,D2Z ++,wcp5 +,wcp8 +,C-MYC ++/hsr). The hsr-bearing marker for MLS/P is designated as rev ish der(8)(qter →8q24::8q24::8q24 →8q10::8q10 →8q24::8q24::8q24::8q24 →8qter:: 15q11 →15qter)(wcp8 +,D8Z1 +,wcp15 +,C- MYC+++++++). FISH with the probe generated from the hsr of GR also painted the hsr in MLS/P, indicating that the two hsrs have shared homology, which indicates that the amplification of 8q24/C- MYC as the origin of hsr may be a nonrandom genomic alteration in ovc.

Diagnosis and Classification of the Small Round-Cell Tumors of Childhood

The small-round-cell tumors of childhood include neuroblastoma, the Ewing family of tumors, rhabdomyosarcoma, lymphoma, and desmoplastic small-round-cell tumor. Although classical histological features are generally highly suggestive of tumor type, on occasion these tumors may be indistinguishable by light microscopy, making a definitive diagnosis difficult. Accurate diagnosis of pediatric small-round-cell tumors has become increasingly crucial, as disparate approaches to therapy are used for distinct tumor types. In addition, because for many pediatric cancers, therapy is also tailored according to patient risk, it has become important to further classify tumors biologically, using cytogenetic or molecular studies to identify chromosome translocations, gene amplification, gene expression patterns, and/or mutations. In this issue of The American Journal of Pathology, Gilbert and colleagues used a reverse transcriptase polymerase chain reaction (RT-PCR) assay to analyze the expression of two genes involved in the catecholamine biosynthetic pathway, tyrosine hydoxylase and dopa decarboxylase, in 84 pediatric malignancies. 1 Their studies demonstrate that the expression of these two genes is highly specific for neuroblastoma. Of the 29 non-neuroblastoma tumor samples examined, only pheochromocytomas expressed clearly detectable levels of the genes. These results suggest that analysis of tyrosine hydoxylase and dopa decarboxylase expression may help distinguish neuroblastoma from other small-round-cell childhood tumors. Despite recent advances in immunohistochemistry and molecular pathology, some cases of small-round-cell tumors of childhood remain diagnostically problematic. Thus, additional diagnostic tools, such as the ones described by Gilbert and co-workers, are needed to ensure that every child with a small-round-cell tumor is diagnosed correctly. The value and limitations of current immunohistochemical, cytogenetic, and molecular studies as diagnostic aids for the small-round-cell tumors of childhood are highlighted below.

Origin of the chromosome 1 HSR of the house mouse detected by CGH.

The polymorphic Sp100-rs repeat cluster in chromosome band 1D of the house mouse, Mus musculus, makes up as much as 0.1-5% of the haploid genome. 'High-copy' versions of this long-range repeat cluster are cytogenetically apparent as DAPI-negative chromomycin-A3-positive homogeneously staining regions (HSRs). The cluster is a relatively recent acquisition in the genus Mus; the related species M. caroli possesses neither the Sp100-rs cluster nor even the Sp100-rs gene. Except for chromosomes with high-copy clusters, no major rearrangements are visible in chromosomes 1 from M. musculus and M. caroli: they have the same order of G-bands, DAPI-bands and chromomycin A3-bands. Comparative genomic hybridization (CGH) visualizes the cluster in M. musculus and detects a single region of sequence homology to the cluster in M. caroli chromosome band 1D. This indicates that the M. musculus cluster has evolved in situ from sequences originally present in the same chromosome band.

Homogeneously staining regions in 223 breast carcinomas: cytogenetic and clinicopathological correlations.

A correlation analysis was performed on 223 breast carcinomas to assess the relationships between gene amplification, karyotypic and clinicopathological features. Homogeneously staining region (HSR) is the most frequent form of amplification found in breast cancer. HSR-containing tumours accounted for 60% of the cases. Although up to 40% of tumours with slightly altered karyotype contained HSRs, an excess of HSRs was found within the tumours whose karyotype showed the highest rates of rearranged chromosomes. HSRs were also found to be particularly frequent in small tumours of high histological grade and with a low expression of progesterone receptors. An excess of HSRs seems to be observed in younger patients, however, significant correlation could be demonstrated only for patients below 55 years and below 60 years, compared with older ones. With a 120-month follow-up for 152 patients, a significant association between the presence of HSRs and a shortened overall survival was observed. Altogether, the presence of HSRs appears to be a good indicator of poor prognosis. Further studies are needed to determine whether amplification of specific genes or cell ability to amplify is the most important parameter for tumour progression.

A New Role for Hypoxia in Tumor Progression: Induction of Fragile Site Triggering Genomic Rearrangements and Formation of Complex DMs and HSRs

Genome rearrangements including gene amplification are frequent properties of tumor cells, but how they are related to the tumor microenvironment is unknown. Here, we report direct evidence for a causal relationship between hypoxia, induction of fragile sites, and gene amplification. Recently, we showed that breaks at fragile sites initiate intrachromosomal amplification. We demonstrate here that hypoxia is a potent fragile site inducer and that, like fragile sites inducing drugs, it drives fusion of double minutes (DMs) and their targeted reintegration into chromosomal fragile sites, generating homogeneously staining regions (HSRs). This pathway operates efficiently for DMs bearing different sequences, suggesting a model of hypoxia-driven formation of the HSRs containing nonsyntenic sequences frequently observed in solid tumors.

FISH characterization of head and neck carcinomas reveals that amplification of band 11q13 is associated with deletion of distal 11q

In order to characterize homogeneously staining regions (HSR) and other 11q13 rearrangements identified cytogenetically, we performed fluorescence in situ hybridization (FISH) using a CCND1 cosmid and five YAC clones spanning chromosomal bands 11q13-14 on metaphase cells from 14 primary and one metastatic head and neck carcinomas. At the cytogenetic level, a total of 17 HSR were detected in ten cases: five were in derivative chromosomes 11 in band 11q13, and 12 were located in other derivative chromosomes. Other forms of 11q13 rearrangements were observed in five cases, whereas two cases had normal chromosomes 11. FISH analysis demonstrated that all HSR but two were derived from the 11q13 band. The size of the amplicon varied from case to case, but the amplification always included the region covered by YAC 55G7, which contains the CCND1 locus. The amplification of CCND1 was confirmed by use of a CCND1 cosmid. We also showed that most of the cases (9 of 11) with 11q13 amplification had lost material from distal 11q. The breakpoints were mapped by FISH and were shown to cluster to the region between YACs 55G7 and 749G2. We conclude that loss of gene(s) in distal 11q may be as important as amplification of genes in 11q13 for the biological aggressiveness of head and neck carcinomas.

Restoration of the Mendelian transmission ratio by a deletion in the mouse chromosome 1 HSR.

The house mouse, Mus musculus, harbours a variable cluster of long-range repeats in chromosome 1. As shown in previous studies, some high-copy clusters such as the MUT cluster are cytogenetically apparent as a homogeneously staining region (HSR) and are associated with a distortion of the Mendelian recovery ratio when transmitted by heterozygous females. The effect is caused by a decreased viability of +/+ embryos. It is compensated by maternal or paternal MUT. In this study, a deletion derivative of MUT, MUTdel, shows normal transmission ratios and no compensating capability. In this respect, MUTdel behaves like a wild-type cluster. Hence, both properties--transmission ratio distortion and compensating capability--map to the deleted region. The deletion comprises three-quarters of the MUT HSR and does not extend to the nearest markers adjacent to the HSR.

Extensive genetic heterogeneity in the neuroblastoma cell line NB(TU)1

A neuroblastoma cell line displaying genetically unique features was established from a stage III case of a 20-month-old girl. Southern blotting by the probe pTNB6, which contains exon 1 of the N-myc gene, showed that the primary tumor had in total 4 aberrant bands beside the normal amplified band. The established cell line NB(TU)1 had an aberrant N-myc band (9.0 kb) in addition to the normal band (2.9 kb). Cytogenetic analysis revealed that NB(TU)1 has a composite karyotype composed of at least 7 related karyotypes, which are pseudo-diploid and contain complex chromosomal abnormalities, including translocations, deletions and homogeneously staining regions (HSRs). Such extensive abnormalities were considered to be prominent among known neuroblastoma cell lines, and it was suggested that NB(TU)1 had acquired a certain type of genetic instability. Analysis of N-myc bands in 11 clones of NB(TU)1 showed that the intensity ratio of the normal-sized band (2.9 kb) and the aberrant one (9.0 kb) markedly varied among clones. Moreover, 3 clones showed an additional band with the size of 3.7 kb, which was detectable neither in the parent NB(TU)1 nor in the primary tumor. Thus, NB(TU)1 was shown to be composed of heterogeneous cell components. To further detect such ongoing chromosomal instability, we examined micronuclei formation. NB(TU)1 yielded a larger number of micronuclei than 5 other neuroblastoma cell lines. We conclude that NB(TU)1 has acquired genetic instability detectable by both Southern blotting and cytogenetic analysis.

Cytogenetic and molecular genetic analysis of a pediatric pleomorphic sarcoma reveals similarities to adult malignant fibrous histiocytoma

Cytogenetic and molecular genetic studies were performed on a pleomorphic sarcoma removed from the left atrium of a 15-year-old girl. Histologic analysis was consistent with a storiformpleomorphic malignant fibrous histiocytoma (MFH). Although MFH is the most common soft-tissue sarcoma of late adulthood, it is extremely rare in childhood and its existence in the pediatric population remains controversial. Cytogenetic analysis revealed several alterations previously associated with adult MFH, including abnormalities of chromosomal bands 11p11 and 19p13. Moreover, the tumor demonstrated homogeneously staining regions (HSR) and double minute chromosomes (dmin) suggestive of gene amplification. We therefore screened the case for amplification of genes localized to chromosomal bands 12q13-14, including the putative protooncogenes MDM2, CDK4, SAS, CHOP, and GLI, which are frequently amplified and overexpressed in adult MFH. Southern and Northern blot analysis confirmed the coamplification of MDM2, CDK4, SAS, and CHOP. To our knowledge, such coamplification studies of the 12q13-14 amplicon have not been previously detected in pediatric MFH. Our results provide cytogenetic and molecular genetic evidence that pediatric and adult MFH are histogenetically related entities.

Amplification of the c-erbB-2 gene detected by FISH in gastric cancers.

The amplification and overexpression of the c-erbB-2 gene are considered to be implicated in the process of carcinogenesis of a variety of human tumors. The amplification and overexpression of c-erbB-2 were investigated in 48 surgically resected human gastric cancers by means of fluorescence in situ hybridization and immunohistochemistry. DNA ploidy was determined by flow cytometry. The c-erbB-2 amplification was demonstrated as a cluster of signals, suggesting homogeneously staining region (HSR), in three tumors (6.3%) accompanied by the overexpression of its protein. Such overexpression was detected in another tumor without amplification of the c-erbB-2 gene. All tumors with amplification and overexpression of c-erbB-2 were differentiated adenocarcinoma histologically, but only 10.3 and 13.8% of differentiated carcinomas showed amplification and over-expression of the c-erbB-2 gene, respectively. There was no relationship between the amplification and overexpression of c-erbB-2 and the depth of tumor invasion and lymph node involvement. Three of four cases with overexpression of c-erbB-2 were classified into DNA aneuploid tumor.

Genomic instability and poor prognosis associated with abnormal TP53 in breast carcinomas. Molecular and immunohistochemical analysis.

Alterations of the TP53 gene were analyzed in samples from 87 primary breast cancer patients, using molecular and immunohistochemical approaches. Mutations were detected in 17% of the samples, using polymerase chain reaction (PCR) and constant denaturant gel electrophoresis (CDGE) on exons 5-8 of the TP53 gene, and were confirmed by sequencing. Abnormal TP53 protein staining was found in 55% of the primary samples, using the monoclonal TP53 antibody DO7. A statistically significant association was found between TP53 mutations and abnormal protein staining (p = 0.002). Our results suggest that dysfunction of the TP53 protein is associated with tumor progression, as we found an association between TP53 abnormalities and accumulation of genetic lesions, measured as overall allelic imbalance (AI), homogeneously staining regions (HSR) and strong ERBB2 overexpression. Furthermore, patients with TP53 mutation had a highly elevated risk of dying from breast cancer during the study period (p < 0.001, RR = 10.68) at a median follow-up time of 42 months. Abnormal TP53 staining was much more frequent than the mutations, but it was not of prognostic significance, whereas strong staining was an independent prognostic factor. We therefore conclude that loss of functional TP53 leads to genetic instability, resulting in poorer short-term prognosis, and that only strong staining of TP53, and not abnormal protein staining in general, is of prognostic significance.

In vivo localization of DNA sequences and visualization of large-scale chromatin organization using lac operator/repressor recognition.

We report a new method for in situ localization of DNA sequences that allows excellent preservation of nuclear and chromosomal ultrastructure and direct, in vivo observations. 256 direct repeats of the lac operator were added to vector constructs used for transfection and served as a tag for labeling by lac repressor. This system was first characterized by visualization of chromosome homogeneously staining regions (HSRs) produced by gene amplification using a dihydrofolate reductase (DHFR) expression vector with methotrexate selection. Using electron microscopy, most HSRs showed approximately 100-nm fibers, as described previously for the bulk, large-scale chromatin organization in these cells, and by light microscopy, distinct, large-scale chromatin fibers could be traced in vivo up to 5 microns in length. Subsequent experiments demonstrated the potential for more general applications of this labeling technology. Single and multiple copies of the integrated vector could be detected in living CHO cells before gene amplification, and detection of a single 256 lac operator repeat and its stability during mitosis was demonstrated by its targeted insertion into budding yeast cells by homologous recombination. In both CHO cells and yeast, use of the green fluorescent protein-lac repressor protein allowed extended, in vivo observations of the operator-tagged chromosomal DNA. Future applications of this technology should facilitate structural, functional, and genetic analysis of chromatin organization, chromosome dynamics, and nuclear architecture.

Detection of 11q13 amplification as the origin of a homogeneously staining region in small cell lung cancer by chromosome microdissection

Chromosomal homogeneous staining region (hsr), which is a cytogenetic indication of gene amplification, was found in the pleural effusion (BHII) of a patient with small cell lung cancer (SCLC) after failure of multiple drug treatments. Amplification of band 11q13 was identified as the origin of the hsr by means of chromosomal microdissection combined with G-banding, DNA amplification by polymerase chain reaction, and fluorescence in situ hybridization (micro-FISH). In situ hybridization with the biotin-labeled DNA probe generated from the hsrs of BHII to the cell line BHI, which was established from the lymph node metastasis prior to chemotherapy, revealed the preexistence of 11q13 amplification. This ruled out the possibility of therapeutic induction of the 11q13 amplification. However, the hsr-bearing marker chromosomes were identified by the micro-FISH approach as der(21)(Xqter-->Xq24::hsr(11)(q13)::21p11-->21qter+ ++) in BHII but as der(11)t(3;11)(q21;q13)hsr(11)(q13) in BHI. This suggests that 11q13 DNA sequence amplification may occur first and is then followed by various types of structural rearrangements. FISH analysis with INT2/FGF3 and HST1/FGF4 probes revealed that these protooncogenes were coamplified in the hsrs of BHI and BHII. The results obtained suggest that 11q13 amplification and the successive structural rearrangement may play an important role in the progression of the disease and its therapeutic response.

Chromatin organization in the homogeneously staining regions of a methotrexate-resistant mouse cell line: interspersion of inactive and active chromatin domains distinguished by acetylation of histone H4.

We have analyzed the organization of the homogeneously staining regions (HSRs) in chromosomes from a methotrexate-resistant mouse melanoma cell line. Fluorescence in situ hybridization techniques were used to localize satellite DNA sequences and the amplified copies of the dihydrofolate reductase (DHFR) gene that confer drug-resistance, in combination with immunofluorescence using antibody probes to differentiate chromatin structure. We show that the major DNA species contained in the HSRs is mouse major satellite, confirming previous reports, and that this is interspersed with DHFR DNA in an alternating tandem array that can be resolved at the cytological level. Mouse minor satellite DNA, which is normally located at centromeres, is also distributed along the HSRs, but does not appear to interfere with centromere function. The blocks of major satellite DNA are coincident with chromatin domains that are labelled by an autoantibody that recognizes a mammalian homologue of Drosophila heterochromatin-associated protein 1, shown previously to be confined to centric heterochromatin in mouse. An antiserum that specifically recognizes acetylated histone H4, a marker for active chromatin, fails to bind to the satellite DNA domains, but labels the intervening segments containing DHFR DNA. We can find no evidence for the spreading of the inactive chromatin domains into adjacent active chromatin, even after extended passaging of cells in the absence of methotrexate selection.

Incidence of homogeneously staining regions in non-Hodgkin lymphomas

We report a series of 86 non-Hodgkin's lymphomas (NHL) studied cytogenetically in which two cases with a homogeneously staining region (HSR) located on chromosome 19 and on chromosome 1, respectively, were observed. The low incidence detected (2.3%) suggests that HSR are rare events in NHL. An oncogenetic amplification study was performed with probes for genes that are currently known to undergo amplification and with probes for two genes located on chromosome 19q (AKT2 and BCL-3). We detected no amplification except for AKT2 putative oncogene in the lymphoma in which the HSR was located on chromosome 19. Because amplification of AKT2 putative oncogene has been detected in ovarian carcinomas bearing HSR and in this case NHL, we believe that this gene may be implicated in the pathogenesis of other neoplasias in cooperation with other genes. Further investigation is needed to confirm the low incidence of HSR in NHL and the origin of the amplified material.

A highly amplified mouse gene is homologous to the human interferon-responsive Sp100 gene encoding an autoantigen associated with nuclear dots.

In human cells, three proteins are currently known to colocalize in di screte nuclear domains (designated nuclear dots): Sp100, a transcription-activating protein autoantigenic primarily in patients with primary biliary cirrhosis; PML, a tumor suppressor protein involved in development of acute promyelocytic leukemia; and NDP52, a protein of unknown function. Here we report sequence similarities between the Sp100 protein and a putative protein encoded by a highly amplified mouse gene which is visible as an inherited homogeneously staining region (HSR) on chromosome 1 of some mouse populations. By in situ hybridization, the Sp100 gene was mapped to locus 2q37, the syntenic region of the HSR on mouse chromosome 1. Unlike the highly amplified mouse gene, Sp100 was found to be a single-copy gene and showed no restriction fragment length polymorphisms. Sequence similarities in the promoter regions and similar exon-intron organizations of the two genes were revealed. As for Sp100, steady-state levels of the mRNAs of the HSR-encoded genes could be greatly increased by interferon (IFN) treatment. As in human cells, IFN treatment led to an enlargement in both size and number of nuclear dots in mouse cells as visualized by immunofluorescence staining with autoimmune sera from patients with primary biliary cirrhosis. These data indicate that a gene located in the inherited HSR of mice, designated mSp100, is homologous to the human Sp100 gene, has a similar gene organization, and responds similarly to IFN treatment.

Sequential emergence of MRP- and MDR1-gene over-expression as well as MDR1-gene translocation in homoharringtonine-selected K562 human leukemia cell lines.

To investigate the mechanism of resistance to an antineoplastic natural product homoharringtonine (HHT) in leukemic cells, we have established 5 sub-lines of human myeloid leukemia K562 cells, designated as K-H30, K-H100, K-H200, K-H300 and K-H400, which showed progressive resistance to different concentrations of HHT. These sub-lines were cross-resistant to daunorubicin, vincristine, etoposide and mitoxantrone, but not to melphalan. Immunofluorescence with monoclonal anti-Pgp antibody MRK16 and Northern-blot analysis demonstrated that resistance to HHT is related to the sequential emergence of MRP- and MDR1-gene over-expression. In the low-level-resistant K-H30 sub-line, the MDR1 gene was not over-expressed, but the MRP gene was over-expressed 2.1-fold. In the intermediate-level-resistant K-H100 and K-H200 sublines, both the MRP and the MDR1 genes were over-expressed. However, in the high-level-resistant K-H300 and K-H400 sublines, MDR1-gene over-expression predominated (20- and 21-fold respectively). On the other hand, GST pi-gene expression was decreased in all 5 sub-lines. Southern-blot analysis revealed no MRP-gene amplification in any of the 5 sub-lines, whereas the MDR1 gene was amplified in the high-level-resistant K-H300 and K-H400 sub-lines. The most interesting observation is a homogeneously staining region (HSR) found in chromosome 2 of the K-H300 and K-H400 sub-lines. Chromosome painting and in situ hybridization demonstrated that this HSR was translocated from chromosome 7 and consisted of the amplified MDR1 gene, suggesting that there is a relationship between MDR1-gene, translocation and MDR1-gene amplification.

Genetic clinical markers of human neuroblastoma with special reference to N-myc oncogene: amplified or not amplified?--An overview.

Neuroblastoma is the most common extracranial tumor in children, and cytogenetically, chromosome 1p deletions, extrachromosomal double minutes, and homogeneously staining regions (HSRs) are commonly observed in cell lines and in tumors in advanced stages. It is found that an HSR represents genomic amplification of N-myc, which plays a key role in determining the aggressiveness of neuroblastoma. However, stage IV neuroblastomas or cell lines which lack N-myc amplification are also progressive, and some of them show evidence of N-myc expression in terms of mRNA and/or N-Myc oncoprotein. It was recently shown that a small proximal locus mapped between 1p35-36.1 and 1p36.23 may function as a suppressor gene of N-myc amplification. In neuroblastoma, a pattern of diploidy is associated with rapid tumor growth and poor survival. Expression of bcl-2 proto-oncogene is strongly associated with unfavorable histology, while expressions of Ha-ras and trk-A proto-oncogenes indicate a favorable prognosis. trk-A proto-oncogene encodes a receptor for nerve growth factor. Genetic characteristics of neuroblastomas found by urinary catecholamine mass screening are also discussed.