Proto-oncogene amplification and human breast tumor phenotype

Amplification of c-myc, c-erbB-2, hst and int-2 proto-oncogenes was investigated in two independently collected breast tumor series comprising 292 carcinomas. Differences in the frequencies of amplification could be observed between these two series for c-myc (9.3% vs. 20.8%) and hst/int-2 (21.5% vs. 15.6%) whereas similar values were found for c-erbB-2 (22.5% vs. 20.3%). Statistical correlations between amplification and disease parameters were also dependent on population sampling. Therefore we performed our statistical analysis on the pooled populations and focused on the 219 primary breast carcinomas from patients without therapy prior to surgery. Amplification of c-erbB-2 was strongly correlated to the absence of either estrogen (ER-, P = 0.003) or progesterone (PR-, P = 0.004) receptors. An amplified c-myc was significantly associated with PR- (P = 0.005) and was prevalent in high grade tumors. On the contrary, hst/int-2 amplification was correlated to PR+ tumors (P = 0.01) and was more frequent in ER+ and low grade tumors, and was also correlated with lymph node involvement (P = 0.04). Our data suggest that amplification of each of these proto-oncogenes could be representative of a particular subset of breast tumors. Therefore, proto-oncogene amplification may be helpful in characterizing new biological subclasses in human breast cancer.     

Genetic imbalances in preinvasive tissue of hypopharynx provide evidence for cytogenetic heterogeneity

Multiple chromosomal aberrations have been reported in head and neck squamous cell carcinoma (HNSCC). But less information is available on specific patterns of chromosomal amplifications which distinguish different areas of head and neck tumors. To elucidate genetic mechanisms causing the aggressive growth and high proliferation of hypopharyngeal squamous cell carcinoma (SCC), we performed reverse chromosome painting (RCP) on a total of eight hypopharyngeal SCC including invasive carcinoma and preinvasive tissue. Five hypopharyngeal invasive carcinomas showed amplifications on chromosome 3q. Furthermore, we detected gains on chromosomes 11q and 6p. Compared to the histologically classified preinvasive tissues, we found amplified alterations on chromosome 6p, 11q and 12q, but none of them showed gains on chromosome 3q. This observed heterogeneity in hypopharyngeal SCC might reflect a specific role of chromosome 3q as a late event in the highly invasive capacity of these SCC.     

Correlation between long-term survival in breast cancer patients and amplification of two putative oncogene-coamplification units: hst-1/int-2 and c-erbB-2/ear-1

The incidence and association with 10-year survival of amplification in five protooncogenes or transforming genes were retrospectively examined using DNAs extracted from formalin-fixed, paraffin-embedded blocks of tissues obtained from 176 consecutive patients surgically treated for primary breast carcinoma. The incidences of greater than threefold amplification of hst-1, int-2, c-erbB-2, ear-1 (one of c-erbA), and c-myc were 12, 13, 16, 10, and 4.0%, respectively. hst-1 and int-2 were almost always coamplified (21/22), while c-erbB-2 and ear-1 were frequently coamplified (18/28) with almost the same copy number. The hst-1 and int-2 pair and the c-erbB-2 and ear-1 pair, localized on chromosomes 11q13 and 17q21-22, respectively, in normal cells, were inferred to be constituents of different amplification units. Amplification of hst-1 and/or int-2 was detected preferentially in the younger age group, and was correlated with poorer prognosis in cases carrying four or more copies of the genes. Amplification of c-erbB-2 and/or ear-1 was strongly correlated with poor prognosis in all 176 patients, especially those with lymph node metastasis. Amplification of c-myc was also correlated with poor prognosis. Cox's life-table regression analysis showed that amplification of c-erbB-2 had a prognostic value, which was independent of other known prognostic factors such as lymph node status and tumor size.     

WNT and FGF gene clusters (review)

Mouse mammary tumor virus (MMTV) is a retrovirus, activating Wnt genes (Wnt1/int-1, Wnt3/int-4, Wnt10b), Fgf genes (Fgf3/int-2, Fgf4, Fgf8) and other genes (Notch4/int-3, Eif3s6/int-6) due to proviral integration. Among 19 WNT genes, WNT3 and WNT14B genes are clustered in human chromosome 17q21, WNT3A and WNT14 in human chromosome 1q42, WNT10A and WNT6 in human chromosome 2q35, and WNT10B and WNT1 in human chromosome 12q13. Among 22 FGF genes, FGF19, FGF4 and FGF3 genes are clustered in human chromosome 11q13, while FGF23 and FGF6 in human chromosome 12p13. WNT and FGF gene clusters are conserved between the human genome and the mouse genome. Activation of mouse Wnt or Fgf genes due to proviral integration of MMTV occurs in 5 out of 13 clustered genes, and in 1 out of 28 solitary genes (p=0.0033), which clearly indicates that mouse Wnt or Fgf gene clusters are recombination hot spots associated with carcinogenesis. Recombination results in retroviral integration as well as in chromosomal translocation, gene amplification and deletion during carcinogenesis. The CCND1-FGF19-FGF4-FGF3 gene cluster in human chromosome 11q13 is amplified in breast cancer, squamous cell carcinoma of head and neck, and bladder tumors, and is also translocated in parathyroid tumors and B-cell lymphoma. WNT gene clusters on human chromosome 1q42, 2q35, 12q13, and 17q21 as well as FGF gene cluster on human chromosome 12p13 might be amplified or translocated in human cancer just like FGF gene cluster on human chromosome 11q13.     

Amplification of c-erbB-2, int-2 and c-myc Genes in Node-Negative Breast Carcinomas: Relationship to Prognosis

Primary tumours from 100 Norwegian node-negative breast carcinoma patients were examined for c-erbB-2, int-2, and c-myc proto-oncogene amplification. c-erbB-2, int-2, and c-myc amplification was found in 12.1% (12 of 99), 8.6% (8 of 93), and 1.1% (1 of 89) of the samples respectively. All the c-erbB-2 amplified tumours were of the ductal type, and all the int-2 amplified tumours were oestrogen receptor positive. No other significant or borderline significant associations between gene amplification and clinical or histopathological parameters were found. Relapse occurred more frequently in patients with c-erbB-2 gene amplification (relapse in 33.3% of the patients with c-erbB-2 amplification compared to 20.7% in the non-amplified group), but the difference was not statistically significant. int-2 amplification was not associated with increased risk of relapse, whereas the prognostic value of the c-myc amplification could not be evaluated.     

High incidence of coamplification of hst-1 and int-2 genes in human esophageal carcinomas

We analyzed the alteration of the hst-1 and int-2 genes in 36 cases of esophageal squamous cell carcinoma, 42 cases of gastric adenocarcinoma, and 52 cases of colorectal adenocarcinoma. Coamplification of the hst-1 and int-2 genes was observed in 19 of 36 esophageal carcinomas (52%), 16 of 34 primary tumor tissues (47%), and 10 of 10 metastatic tumors (100%). The degree of amplification ranged from 4- to 8-fold. The incidence of hst-1 and int-2 gene coamplification was significantly higher in male patients than that in female patients (P less than 0.05). The coamplification of the hst-1 and int-2 genes had a tendency to correlate with clinical stage. The progesterone receptor gene, which is mapped to chromosome 11 at band q21-23, was not amplified in these esophageal carcinomas. Coamplification of the hst-1 and int-2 gene does not seem to imply increased numbers of chromosome 11, and the hst-1 and int-2 genes appear to be in same amplification unit on chromosome 11 at band q13. No coamplification of the hst-1 and int-2 genes was detected in gastric carcinomas and colorectal carcinomas. These results suggest that amplification of chromosomal locus of the hst-1 and int-2 genes might participate in carcinogenesis, in progression, and particularly in metastasis of esophageal carcinomas.     

Gene Amplification of int-2 and erbβ in Human Esophageal Cancer: Relationship to Clinicopathological Variables

Gene amplification is a relatively frequent event in human malignant tumors and is believed to play an important role in tumor progression. The int-2 and erbB genes are amplified more frequently than any other genes in human esophageal cancer. In order to investigate the correlation between these two proto-oncogenes and the clinical behavior of esophageal cancer, we examined DNA amplification of int-2 and erbB and analyzed their relationship to clinicopathological variables. Genomic DNA was extracted from 21 esophageal squamous carcinomas and normal esopfiageal mucosa, as well as from 4 metastatic tumors. We used Southern blot analysis for detection of gene amplification. Amplification of int-2 was observed in 13 of 21 cases (62%) and in all the metastatic tumors (4/4; 100%). We found a significant correlation between amplification of int-2 and the length of the primary lesion. Amplification of erbB was detected in 3 of 18 patients (17%). All patients who showed amplification of erbB also demonstrated amplification of int-2. These results suggest that amplification of int-2 or neighboring genes on 11q may participate in tumor progression and metastasis in patients with esophageal squamous cancer.     

Expression of int-2 mRNA in human tumors amplified at the int-2 locus

Gene amplification is a relatively frequent event in human malignant tumors and is believed to have an important function in neoplastic transformation and tumor progression. Our attention has been focused on the amplification and the expression of the int-2 gene for several reasons: (1) In the mouse mammary tumorigenesis int-2 is frequently activated by MMTV proviral integration. (2) The human homolog of int-2, located on chromosome 11q13, is frequently amplified in human primary tumors and is comprised in an amplification unit encompassing the hst gene, which is often coamplified; the amplification at the 11q13 locus in breast carcinomas correlates with a poor outcome of the disease. (3) int-2 and hst belong to the basic FGF gene family. All these observations raise the possibility that the human int-2 gene plays an active role in the neoplastic process, but this will prove to be true only if int-2 is expressed in human tumors. In the present study we used RNA:RNA in situ hybridization and Northern blot analysis to show that int-2 gene is expressed in a number of human carcinomas amplified at the same locus.     

Analysis of protooncogenes in squamous-cell carcinomas of the head and neck (scchn)

We have analysed DNA and RNA from 40 untreated head and neck cancers for amplified and overexpressed proto-oncogenes by Southern and Northern blot hybridisation. Coamplification but no expression of the bcl-1 and the hst genes was found in 12.5% of the patients. Furthermore, amplifications of c-myc were found in 10%, of Ha-ras and c-erbB-2 in 5%. c-erbB-2 amplification was accompanied by gene expression but no overexpression. Correlating our results with clinicopathological data of the patients amplifications were only found in stage III and IV disease (p=0.0164). No correlation was found between amplification and primary tumour site, histopathological differentiation of the tumours, response to induction chemotherapy, or survival. Our results indicate that oncogene amplification in advanced SCCHN reflects more the general genomic instability of advanced tumours than be a reason for tumour growth.     

Amplification and expression of the bcl-1 gene in human solid tumor cell lines.

The bcl-1 gene maps to chromosome 11q13 and has recently been shown to be a member of the cyclin gene family. Amplification of the chromosome region containing bcl-1 occurs frequently in breast cancer, squamous cell cancer, and other tumor types. We have hypothesized that amplification results in altered expression of the bcl-1 gene, contributing to carcinogenesis. In this work, we studied bcl-1 gene amplification and expression in a panel of human cell line. bcl-1 is expressed in all cell lines studied. The level of expression tends to be higher in amplified cell lines. We also screened these cell lines for int-2 and hst-1 expression, genes which are frequently coamplified with bcl-1. No int-2 expression was detected, and the two cell lines expressing hst-1 were unamplified. Our data provide support for the importance of bcl-1 in carcinogenesis.     

Identification and cloning of two overexpressed genes, U21B31/PRAD1 and EMS1, within the amplified chromosome 11q13 region in human carcinomas.

Amplification of the chromosome 11q13 region is frequently found in human breast cancer and in squamous cell carcinomas of the head and neck, and has been associated with an unfavourable clinical course of disease. The known oncogenes within the amplified 11q13 region, INT2 and HSTF1, are rarely expressed in these tumours, indicating that another, hitherto unidentified, gene or genes confer(s) the biological (prognostic) significance to the amplification of the 11q13 region. To identify the gene or genes, we have constructed a cDNA library from a cell line with an 11q13 amplification and have performed differential cDNA cloning using [32P]dCTP-labelled cDNAs from human squamous cell carcinoma cell lines with and without an 11q13 amplification. We isolated two cDNA clones, U21B31 and U21C8, which recognize two genes amplified and overexpressed in cell lines harbouring an 11q13 amplification. In breast carcinomas and in squamous cell carcinomas amplification of both the U21B31 and the U21C8 gene was found in most tumours with an amplification of the 11q13 region, despite the large distance between both genes. Sequence analysis of the U21C8 cDNA clone revealed no homology to known genes; we call this gene EMS1. The U21B31 cDNA clone corresponded to the 3' end of the PRAD1 proto-oncogene, recently cloned from a parathyroid adenoma. Both gene products are of interest as potential markers to identify tumours with an 11q13 amplification.     

Analysis of gene amplification in head-and-neck squamous-cell carcinoma

The presence of gene amplification was determined in 66 fresh head-and-neck SCC specimens using a battery of 9 different probes. Amplification of at least one gene was found in 12 samples (18%), of which 7 were amplified at multiple loci (58%). We observed amplifications for EGFR (10% of samples) and c-myc (9%), as well as co-amplification of bcl-1/int-2 (7%). No amplifications were demonstrated for c-Ha-ras-1, TGF alpha, c-mos, c-erbB-2, or c-erbA-2. The incidence of proto-oncogene amplification in head-and-neck SCC patients is comparable to that reported for other solid tumours. There was no statistically significant difference in survival between patients with or without gene amplification. However, the presence of multiple amplifications in several patients with advanced primary tumours suggests that the accumulation of genetic changes may correlate more closely with tumour size than with inherent biologic aggression.     

Visualization of the timing of gene amplification during multistep head and neck tumorigenesis

Head and neck tumorigenesis is thought to represent a multistep process whereby carcinogen exposure leads to genetic instability in the tissue and the accumulation of specific genetic events, which result in dysregulation of proliferation, differentiation, and cell loss and the acquisition of invasive capacity. Chromosome 11q13 amplification is frequently observed in head and neck squamous cell carcinoma (HNSCC), and the amplified gene products are assumed to play important functional roles in the tumor phenotype. However, it is not well understood whether gene amplification precedes carcinoma development or results from the unstable nature of intact tumors. To determine the timing of gene amplification during tumorigenesis, tissue sections from amplified HNSCC specimens (containing a contiguous transition from normal epithelium to hyperplasia to dysplasia to carcinoma) were probed for INT2 gene copy number by chromosome in situ hybridization. In addition, representative epithelia were microdissected from the tissue sections, and the DNA was isolated and assessed for INT2 gene copy number by semiquantitative PCR. In those cases containing amplified INT2 in the carcinoma, gene amplification appeared to precede HNSCC development. In one case, INT2 gene amplification appeared in the hyperplasia to dysplasia transition, whereas in two other cases, gene amplification was apparent at dysplasia. These results suggest that gene amplification can occur early during head and neck tumorigenesis and that genetic instability is an important driving force in the tumorigenesis process.     

Amplification and protein over-expression of the neu/HER-2/c-erbB-2 protooncogene in human breast carcinomas: relationship to loss of gene sequences on chromosome 17, family history and prognosis

c-erbB-2 gene amplification and protein over-expression were investigated in 89 primary tumours and 24 metastases from Norwegian breast cancer patients. Amplification occurred in 22.5% of the primary tumours and 50% of the metastases. The amplification was negatively correlated to the oestrogen receptor (ER) content in both the primary tumours and the metastases. No significant differences between amplified and non-amplified tumours were observed with regard to node status, clinical stage, tumour size or menopausal status, although correlations of borderline significance were found between node status, clinical stage and high degree of gene amplification. All the amplified tumours were of the invasive ductal type. Follow-up data of patients observed for more than 1 year showed a significantly higher recurrence rate in the c-erbB-2 amplified group. Allele loss of chromosome 17p and of 7q was seen in 55% and 48% of the tumours respectively. No significant correlation was found between these losses and clinico-histological parameters. More than 50% of the tumours with a loss of 17q sequences had an amplification of c-erbB-2 which is located on 17q12-21, indicating that only one of the chromosomes may be involved in the amplification of the c-erbB-2. A trend towards a correlation between loss of 17q and high degree of amplification were found. No correlation was found between positive family history of breast cancer and c-erbB-2 gene amplification, nor loss of 17p or 17q sequences. Our data support the hypothesis that amplification correlates with aggressive tumour behaviour, and thus may be used as a prognostic factor in breast carcinomas. The allele losses on 17p and 17q points to tumour suppressor gene or genes on this chromosome, although not as predisposing genes in families.     

Amplification on chromosomes 1p31, 1q21-24, 5p13-14, and 11p12-14 in ovarian-carcinoma detected by reverse chromosome painting

Recently, amplifications of several genes including c-myc, HER-2/neu, and FGF-3 (int-2) have been identified in ovarian carcinomas. We analyzed Il tumor samples from ovarian carcinoma for gene amplification using reverse chromosome painting and standard Southern blot analysis. Reverse chromosome painting detected four amplified domains on chromosome bands 1p31, 1q21-24, 5p13-14, and 11p12-14. None of the amplified domains contained genes previously reported to be amplified in ovarian carcinomas. Southern blot analysis revealed amplifications of genes HER2/neu, N-ras and H-ras. Tumor T3711 showed three independent amplifications including chromosome bands 1p31, 11p12-14 and the HER-2/neu gene (17q11.2-12).     

Prognostic significance of accumulation of gene and chromosome alterations and histological grade in node-negative breast carcinoma

The histological grade of atypia is a known prognostic indicator for breast cancer patients and correlates with many gene and chromosome alterations. To investigate the independent prognostic significance of gene and chromosome alterations in axillary node-negative (n0) breast cancers of the invasive ductal and invasive lobular types, the prevalence of eight gene and chromosome alterations and their association with histological grade and recurrence was studied in 129 consecutive patients who had undergone resection over an average follow-up period of 43.4 months. Loss of heterozygosity on 16q, 17p, 16p, 17q and 18q, p53 gene mutation and c-erbB-2 and int-2 gene amplifications were detected in 55%, 37%, 25%, 24%, 22%, 23%, 15% and 11% respectively. Individual alterations in 17p, 17q, 18q, c-erbB-2 and p53 were detected most frequently and gene and chromosome alterations tended to be accumulated in Grade 3 n0 invasive ductal/lobular carcinomas. Histological grade, 18q, int-2 and the number of these gene and chromosome alterations were significant prognostic indicators on Cox's univariate proportional hazard model, and 18q and int-2 were still significant after adjustment for histological grade. Results suggested that examination of the presence of certain gene and chromosome alterations, as well as histological grade, were effective in identifying n0 breast cancer patients at high risk of early recurrence.      

BCL-1 participates in the 11q13 amplification found in breast cancer

In an attempt to probe the significance of HST and INT-2 gene amplification in human breast carcinomas, we have surveyed the amplification status of five molecular markers located on the long arm of chromosome 11 (BCL-1, HST, INT-2 & SEA on 11q13, and ETS-1 on 11q23) in a population of 297 mammary tumors. ETS-1 was rarely amplified and always independently from the other proto-oncogenes. Concerning band q13: (i) 50 tumors (approximately 17%) were co-amplified for BCL-1, HST & INT-2; (ii) in 3 cases, amplification extended to the SEA gene; (iii) in 6 carcinomas, BCL-1 was the only amplified marker. The fact that we never observed amplification of HST & INT-2 independently of BCL-1, which in turn can be amplified solely, suggests the presence, between HST/INT-2 and BCL-1, of a genetic element which could be important in the development of a subset of mammary tumors.     

Frequent amplification of the bcl-1 locus in poorly differentiated squamous cell carcinoma of the lung. The Lung Cancer Study Group

The bcl-1 locus on chromosome 11 at band q13 has been shown to be rearranged in some chronic B lymphoid malignancies with the t(11;14)(q13;q32). Chromosome 11 abnormalities occur in solid tumors including squamous cell cancers and adenocarcinomas. We have recently reported the frequency amplification of the bcl-1 locus in squamous cell carcinomas of head and neck origin, and increased copy number also has been found in some breast adenocarcinomas. In this investigation, we analyzed the arrangement, copy number and expression of this locus in 111 primary non-small cell lung cancers. Bcl-1 was found to be amplified three- to 10-fold in seven fresh tumors. Whereas none of 51 adenocarcinomas showed bcl-1 amplification, six of 46 squamous cell carcinomas revealed an increase in bcl-1 copy number. This amplification was more frequently associated with larger and more poorly differentiated tumors. None of the cancers showed rearrangement of this locus.     

Mapping of chromosome 17 breakpoints in acute myeloid leukemias.

The 17q11-21 chromosomal region is frequently involved in non-random structural rearrangements associated with the M1 and M2 subtypes of acute myeloid leukemias (AML), as well as with the 15;17 translocation typical of the promyelocytic subtype. A number of genes have been localized in this region including the c-erbA-1 and c-erbB-2 proto-oncogenes, the genes coding for the granulocyte-colony stimulating factor (G-CSF), the retinoic acid receptor alpha (RAR alpha) and the myeloperoxidase enzyme (MPO). However, the precise location of these genes in relationship to the 17q11-21 breakpoint(s) has not been determined. Using in situ hybridization on metaphase chromosomes, we established the position of the breakpoints in relationship to the c-erbA-1, c-erbB-2, G-CSF, RAR alpha and MPO loci in a series of AML cases bearing 17q11-21 rearrangements. We report: (i) that the respective position of the five genes is centromere - c-erbA-1 - G-CSF - c-erbB-2 - RAR alpha - MPO - telomere; (ii) that the breakpoints of the various AML subtypes are variably located between the centromere and c-erbB-2 in M1 and M2; (iii) that the breakpoints are consistently located between c-erbB-2 and RAR alpha/MPO in M3; and (iv) that the breakpoint on chromosome 17 in the 15;17 translocation is located on 17q21 and not on 17q11-12 as previously reported.