High-throughput tissue microarray analysis of cyclin E gene amplification and overexpression in urinary bladder cancer

Studies by comparative genomic hybridization revealed that the 19q13 chromosomal region is frequently amplified in bladder cancer. The cyclin E gene (CCNE), coding for a regulatory subunit of cyclin-dependent kinase 2, has been mapped to 19q13. To investigate the role of cyclin E alterations in bladder cancer, a tissue microarray of 2,317 specimens from 1,842 bladder cancer patients was constructed and analyzed for CCNE amplification by fluorescence in situ hybridization and for cyclin-E protein overexpression by immunohistochemistry. Fluorescence in situ hybridization analysis showed amplification in only 30 of the 1,561 evaluable tumors (1.9%). Amplification was significantly associated with stage and grade (P: < 0.0005 each). Immunohistochemically detectable cyclin E expression was strong in 233 (12.4%), weak in 354 (18.9%), and negative in 1, 286 of the 1,873 interpretable tumors. The majority (62.1%) of CCNE-amplified tumors were strongly immunohistochemistry-positive (P: < 0.0001). The frequency of protein expression increased from stage pTa (22.2%) to pT1 (45.5%; P: < 0.0001) but then decreased for stage pT2-4 (29.4%; P: < 0.0001 for pT1 versus pT2-4). Low cyclin E expression was associated with poor overall survival in all patients (P: < 0.0001), but had no prognostic impact independent of stage. It is concluded that cyclin E overexpression is characteristic to a subset of bladder carcinomas, especially at the stage of early invasion. This analysis of the prognostic impact of CCNE gene amplification and protein expression in >1,500 arrayed bladder cancers was accomplished in a period of 2 weeks, illustrating how the tissue microarray technology remarkably facilitates the evaluation of the clinical relevance of molecular alterations in cancer.     

Comparing fluorescence in situ hybridization and chromogenic in situ hybridization methods to determine the HER2/neu status in primary breast carcinoma using tissue microarray.

Identification of HER2/neu status is important for predicting response to specific chemotherapy in breast carcinoma. Chromogenic in situ hybridization was performed using tissue microarray technology on 188 primary breast carcinomas. To validate the reliability of novel chromogenic in situ hybridization technology, the results of chromogenic in situ hybridization were correlated with the results of two-color fluorescence in situ hybridization done with the same tumors. On tissue microarray panels containing 188 breast carcinoma tissues, fluorescence in situ hybridization and chromogenic in situ hybridization were conducted simultaneously. HER2/neu amplification was detected in 46 tumors (24.5%) by fluorescence in situ hybridization and in 43 tumors (22.9%) by chromogenic in situ hybridization. Results of each method agreed with each other in 177 tumors (concordance: 94.1%). HER2/neu amplification by fluorescence in situ hybridization was associated with nuclear pleomorphism (P =.021), and HER2/neu amplification by chromogenic in situ hybridization was associated with poor nuclear grade (P =.037). High concordance between fluorescence in situ hybridization and chromogenic in situ hybridization indicated that chromogenic in situ hybridization can be a tempting alternative to fluorescence in situ hybridization for the detection of HER2/neu amplification in breast carcinoma because of its accuracy and relative low cost. HER2/neu appeared to have a prognostic implication because its amplification was associated with aggressive biologic features of the breast carcinoma. Integration of tissue microarray technology enabled high-throughput determination of HER2/neu amplification profile with rapidity and accuracy in large cohorts of the breast carcinoma.     

Chromosome 8p deletions are associated with invasive tumor growth in urinary bladder cancer.

Alterations of chromosome 8, including deletions of 8p, occur frequently in many tumors. In this study, fluorescence in situ hybridization was used to study the relationship between 8p deletions, 8q gains, and phenotype in bladder cancer. Cells from 87 tumors were examined by dual-labeling fluorescence in situ hybridization with a centromere 8 probe (pJM12) and P1 probes for 8p22, 8p12, 8q12, and 8q24. Both 8p22 deletions and 8q24 gains were strongly associated with tumor phenotype. There was a marked difference in 8p22 deletions between noninvasive (pTa) tumors (3/33) and minimally invasive (pT1) tumors (8/19; P = 0.005) whereas there was no significant difference between pT1 and muscle-invasive (pT2-4) tumors (19/35; P = 0.3926). Six tumors with 8p22 deletion were examined at 8p12. Three of these tumors showed no 8p12 deletion, narrowing down the site of a putative tumor suppressor gene distal to 8p12. In one other case, there was a marked increase in 8p12 copy number (> 40 per cell; amplification), suggesting the presence of an oncogene involved in bladder cancer at 8p12. The marked difference in 8p22 deletions between noninvasive (pTa) and minimally invasive (pT1) tumors is consistent with a role of a putative tumor suppressor gene on 8p for development of invasive tumor phenotype.     

c-myc copy number gains in bladder cancer detected by fluorescence in situ hybridization.

Amplification and overexpression of c-myc have been suggested as prognostic markers in human cancer. To assess the role of c-myc gene copy number alterations in bladder cancer, 87 bladder tumors were examined for c-myc aberrations by fluorescence in situ hybridization. Dual labeling hybridization with a repetitive pericentromeric probe specific for chromosome 8 and a probe for the c-myc locus (at 8q24) was performed to analyze c-myc copy number in relation to chromosome 8 copy number on a cell by cell basis. A clear-cut c-myc amplification (up to 40 to 150 copies per cell) was found in 3 tumors. There was a low level c-myc copy number increase in 32 of the remaining 84 tumors. There was no association of low level c-myc copy number increase with c-myc protein overexpression. This suggests that a c-myc gene copy number gain as detected by fluorescence in situ hybridization does not necessarily reflect a disturbed c-myc gene function but may indicate a structural chromosome 8 abnormality including gain of distal 8q. The strong association of low level c-myc (8q) gains with tumor grade (P < 0.0001), stage (P < 0.0001), chromosome polysomy (P < 0.0001), p53 protein expression (P = 0.0019), p53 deletion (P = 0.0403), and tumor cell proliferation (Ki67 labeling index; P = 0.0021) is consistent with a role of chromosome 8 alterations in bladder cancer progression.     

High-throughput tissue microarray analysis of 11q13 gene amplification (CCND1, FGF3, FGF4, EMS1) in urinary bladder cancer

Gene amplification is a common mechanism for oncogene overexpression. High-level amplifications at 11q13 have been repeatedly found in bladder cancer by comparative genomic hybridization (CGH) and other techniques. Putative candidate oncogenes located in this region are CCND1 (PRAD1, bcl-1), EMS1, FGF3 (Int-2), and FGF4 (hst1, hstf1). To evaluate the involvement of these genes in bladder cancer, a tissue microarray (TMA) containing 2317 samples was screened by fluorescence in situ hybridization (FISH). The frequency of gains and amplifications of all genes increased significantly from stage pTa to pT1-4 and from low to high grade. In addition, amplification was associated with patient survival and progression of pT1 tumours. Among 123 tumours with amplifications, 68.3% showed amplification of all four genes; 19.5% amplification of CCND1, FGF4, and FGF3; and 0.8% co-amplification of FGF4, FGF3, and EMS1. Amplification of CCND1 alone was found in 9% of the tumours, while EMS1 alone was amplified in 1.6% and FGF4 in 0.8%. Overall, the amplification frequency decreased with increasing genomic distance from CCND1, suggesting that, among the genes examined, CCND1 is the major target gene in the 11q13 amplicon in bladder cancer.     

BIRC2 amplification in squamous cell carcinomas of the uterine cervix

Oncogene amplification is a key step in cell transformation towards malignancy. Chromosomal aberrations involving the long arm of chromosome 11, including amplifications at 11q13 and 11q22, have been previously reported in cervical cancer. While the role of the CCND1 gene as the driver gene for 11q13 amplification is well established in different tumor types, the significance of the 11q22 amplicon is less clear. The 11q22 amplicon corresponds to several putative target genes including the apoptose inhibitor BIRC2, recently detected as amplified in cervical cancer cell lines. To better understand the distribution and frequency of 11q amplification sites in uterine cervical carcinomas, we analyzed BIRC2 and CCND1 copy number changes using fluorescence in situ hybridization in a tissue microarray containing 238 cervical cancers. High-level amplification of BIRC2 was found in 12.9?% of tumors. Amplification of BIRC2 in cervical carcinomas was homogeneous as shown in corresponding whole tissue sections of amplified tumors at the tissue microarray. BIRC2 amplification was significantly more frequent than CCND1 amplification (2.1?%) in our cohort (p?相似文献   

EGFR and gastrointestinal stromal tumor: an immunohistochemical and FISH study of 82 cases.

Gastrointestinal stromal tumor is the most common mesenchymal neoplasm of the gastrointestinal tract. Mutually exclusive KIT or platelet-derived growth factor receptor-alpha mutations are key events in gastrointestinal stromal tumor pathogenesis, and specific treatment targeting KIT/platelet-derived growth factor receptor-alpha activation is available. Epidermal growth factor receptor plays an important role in cancer biology and also constitutes a promising molecular target of therapy. Very few reports have been published in the literature about the relationship between gastrointestinal stromal tumor and epidermal growth factor receptor. The aim of this study was to investigate epidermal growth factor receptor immunohistochemical expression and epidermal growth factor receptor gene amplification in 82 consecutive gastrointestinal stromal tumor cases using tissue microarray technique. Hematoxylin- and eosin-stained sections and clinical information were reviewed, and expression of CD117 (KIT), CD34 and epidermal growth factor receptor was investigated by immunohistochemistry. Epidermal growth factor receptor gene copy number was determined using fluorescence in situ hybridization. Immunohistochemistry revealed that CD117 and CD34 were expressed in 96 and 57% of tumors, respectively. Variable epidermal growth factor receptor protein immunohistochemical overexpression was detected in 96% of gastrointestinal stromal tumor cases, but none of the 75 cases with represented tumor tissue cores and countable fluorescence signals exhibited epidermal growth factor receptor gene amplification by fluorescence in situ hybridization. These results show that there is no correlation between epidermal growth factor receptor protein overexpression by immunohistochemistry and epidermal growth factor receptor gene amplification by fluorescence in situ hybridization. Considering that the mechanisms of epidermal growth factor receptor protein overexpression are not well understood and the possibility that anti-epidermal growth factor receptor therapy may be beneficial for patients with gastrointestinal stromal tumor that overexpresses epidermal growth factor receptor, additional studies are encouraged.     

Frequent amplifications and abundant expression of TRIO, NKD2, and IRX2 in soft tissue sarcomas

Copy number gains and high-level amplifications of the short arm of chromosome 5 are frequently observed in soft tissue sarcomas. To identify genes from this region possibly involved in tumor progression, we analyzed 34 soft tissue sarcomas (10 pleomorphic and 8 dedifferentiated liposarcomas, 6 malignant fibrous histiocytomas, and 10 malignant peripheral nerve sheath tumors (MPNST)) using a DNA microarray including 418 BAC clones representing 99% of chromosome arm 5p. In seven tumors, distinct high-level amplifications were identified affecting four different subregions. From these regions, genes TERT, TRIO, SKP2, FBXO32, NKD2, SLC6A3, IRX2, POLS, FYB, PTGER4, and FGF10 were selected for detailed quantitative expression analysis (RQ-PCR) based on their potential tumorigenic function. Of these, TRIO, coding for a guanidine nucleotide exchange factor, was consistently overexpressed in all cases, while IRX2 and NKD2, both involved in the regulation of developmental processes via the WNT pathway, showed a characteristic expression only in MPNSTs. Detailed nonparametric multidimensional scaling analysis further showed that the expression of TRIO, IRX2, and NKD2 strongly correlated with the gene copy number. In conclusion, we found TRIO, IRX2, and NKD2 frequently affected by high-level amplifications as well as up-regulated in a gene-dosage dependent manner. Thus, these genes represent candidate targets of 5p amplifications in soft tissue sarcomas and might play a crucial role during the progression of this disease.     

CDK4 is a probable target gene in a novel amplicon at 12q13.3-q14.1 in lung cancer

Several chromosomal regions are recurrently amplified or deleted in lung tumors, but little is known about the underlying genes, which could be important mediators in tumor formation or progression. In lung cancer, the RB1-CCND1-CDKN2A pathway, involved in the G1-S transition, is damaged in nearly all tumors. In the present study, we localized a novel amplicon in lung tumors to a fragment of less than 0.5 Mb at 12q13.3-q14.1 by using comparative genomic hybridization (CGH) on cDNA microarrays. This approach enabled us to identify 10-15 genes with the most consistent amplifications. Semiquantitative RT-PCR analyses of 13 genes in this region showed that four of them (CDK4, CYP27B1, METTL1, and TSFM) were also highly up-regulated. Immunohistochemical (IHC) analysis of 141 tumor samples on a tissue microarray showed that CDK4 was expressed at a high level in 23% of lung tumors. Six (21.4%) of the tumors with high CDK4 expression (n = 28) were shown by fluorescence in situ hybridization (FISH) to contain the 12q13.3-q14.1 amplification. For CDK4, a positive correlation was found between gene copy number (FISH and CGH array), mRNA expression (RT-PCR), and level of protein expression (IHC). CDK4 expression did not correlate with CDKN2A methylation status. Amplification of CDK4 has been described in other tumor types, but its role in lung cancer remains to be elucidated. Although CDK4 amplification seems to be a relatively rare event (4.3%) in lung tumors, it indicates the significance of the RB1-CCND1 pathway in lung tumorigenesis.     

Association of TSC1/hamartin, 14-3-3sigma, and p27 expression with tumor outcomes in patients with pTa/pT1 urothelial bladder carcinoma

TSC1/hamartin is a tumor suppressor gene involved in the development of various malignancies, including bladder cancer. In vitro studies showed that hamartin controls cell proliferation partly by up-regulating p27 and 14-3-3sigma. This study was designed to explore the value of these biomarkers in predicting outcome in pTa/pT1 tumors and validate the regulation of p27 and 14-3-3sigma by hamartin in vivo using human bladder cancer tissue. A tissue microarray of 134 pTa and pT1 tumors was constructed, and sections were stained with hamartin, 14-3-3sigma, and p27 antibodies. In multiple Cox regression analysis, pTa/pT1 tumors with reduced or low expression of hamartin tended to have higher risk of progression (P = .030). High-grade tumors tended to be at higher risk of progression in comparison with low-grade tumors (P < .001). The combination of expression of these 3 biomarkers did not add predictive value regarding disease outcomes. Low hamartin expression and high tumor grade are independent factors in predicting faster pTa/pT1 tumor progression. In a subset of pTa/pT1 tumors, hamartin has a role in bladder carcinogenesis by positively regulating 14-3-3sigma and p27.     

Amplification of EIF3S3 Gene Is Associated with Advanced Stage in Prostate Cancer

Gain of the long arm of chromosome 8 (8q) is one of the most common gains found in the advanced prostate cancer by comparative genomic hybridization. We have previously identified a putative target gene for the 8q gain, EIF3S3, that encodes a p40 subunit of eukaryotic translation initiation factor 3 (eIF3). Here, we studied the frequency of the EIF3S3 amplification in different stages of prostate cancer and co-amplification of EIF3S3 and oncogene MYC. In addition, prognostic utility of the EIF3S3 copy number alteration was evaluated. The analyses were done with fluorescence in situ hybridization and tissue microarray technology. High-level amplification of EIF3S3 was found in 11 of 125 (9%) of pT1/pT2 tumors, 12 of 44 (27%) of pT3/pT4 tumors, and 8 of 37 (22%) of lymph node metastases as well as in 26 of 78 (33%) and 15 of 30 (50%) of hormone refractory locally recurrent tumors and metastases, respectively. The amplification was associated with high Gleason score (P < 0.001). One of the 79 tumors with EIF3S3 amplification had only two copies of MYC, whereas all tumors with amplification of MYC had also amplification of EIF3S3 indicating common co-amplification of the genes. Gain of EIF3S3 was associated with poor cancer-specific survival in incidentally found prostate carcinomas (P = 0.023). In the analyses of prostatectomy-treated patients, the amplification was not statistically significantly associated with progression-free time. In conclusion, amplification of EIF3S3 gene is common in late-stage prostate cancer suggesting that it may be functionally involved in the progression of the disease.     

Deletions of chromosome 8p and loss of sFRP1 expression are progression markers of papillary bladder cancer

Many molecular alterations are known to occur in urothelial carcinoma of the bladder, but their significance for tumor progression is poorly understood. Deletions of chromosome 8p are frequently found in several tumor types and are often associated with progressive disease. In all, 99 bladder tumors were screened for deletions at 8p using loss of heterozygosity (LOH) and multicolor fluorescence in situ hybridization FISH analyses. Allelic loss on chromosome 8p in at least one marker was found in 25/99 (25%) tumors. There was a significant correlation of 8p deletions with invasive tumor growth and a highly significant association with papillary growth pattern in patients with invasive disease. cDNA array analyses revealed that secreted Frizzled-related protein 1 (sFRP1), an antagonist of Frizzled receptors and Wnt pathway activation on chromosome 8p12-11.1, is frequently downregulated in bladder cancer. To investigate sFRP1 as a candidate for a putative progression-related gene on 8p, urothelial cell lines and primary urothelial carcinomas were screened for sFRP1 expression using quantitative real-time PCR, Northern blot, immunofluorescence and immunohistochemistry (IHC). Of the investigated bladder cancers, 38% showed loss of sFRP1 expression by quantitative RT-PCR. Evaluation of the protein expression by IHC using tissue microarrays containing 776 bladder cancers revealed loss or strong reduction of sFRP1 expression in 66% of cases. SFRP1 loss was associated with higher tumor stage and grade and shorter overall survival. In addition, loss of sFRP1 was an independent indicator of poor survival in patients with papillary but not with muscle invasive bladder cancer. There were neither mutations in the coding region of sFRP1 nor homozygous deletions at 8p12-11.21. However, promoter methylation was detected using methylation-specific PCR in 29% of cases. In conclusion, we could show a close correlation of chromosome 8p deletions and progression of papillary bladder tumors. The sFRP1 gene on chromosome 8p12-11.1 could be a candidate gene for the predicted, progression-related tumor suppressor gene in bladder cancer and could contribute to urothelial carcinogenesis.     

miR-143, miR-222, and miR-452 are useful as tumor stratification and noninvasive diagnostic biomarkers for bladder cancer

Altered microRNA (miRNA) expression may occur early in bladder cancer and may play a role in carcinogenesis and tumor behavior. We evaluated whether alterations in miRNA expression could improve disease stratification and outcome prognosis in bladder tumors and noninvasive diagnosis in urinary samples. miR-143, miR-222, and miR-452 expression levels were analyzed by quantitative RT-PCR (RT-qPCR) in paired urinary and matching tumors and in two independent prospective series of tumors and urinary specimens. Differential expression of miR-143, miR-222, and miR-452 in urine were verified by in situ hybridization in matching tumors. Tumor miRNA expression by RT-qPCR correlated with tumor grade, size, and presence of carcinoma in situ for miR-222, recurrence (miR-222 and miR-143), progression (miR-222 and miR-143), disease-specific survival (miR-222), and overall survival (miR-222). Protein expression patterns of potential miRNA targets, including vascular endothelial growth factor, BCL2, v-erb-b2 erythroblastic leukemia viral oncogene (ERBB) homolog 3, and ERBB4, were evaluated by IHC in tissue arrays containing tumors for which miRNAs were assessed by RT-qPCR. Target expression correlated with expression of their predicted regulatory miRNAs, recurrence (ERBB3), progression (ERBB4), disease-specific survival (ERBB3 and ERBB4), and overall survival (ERBB3 and ERBB4). Furthermore, RT-qPCR of miR-452 (area under the curve, 0.848) and miR-222 (area under the curve, 0.718) in urine provided high accuracies for bladder cancer diagnosis. Thus, bladder tumors were characterized by changes in miRNA expression that could aid in tumor stratification and clinical outcome prognosis, and miRNAs were detected in urinary specimens for noninvasive diagnosis.     

Genetic abnormalities and HPV status in cervical and vulvar squamous cell carcinomas

Cervical and vulvar cancers are diseases of the female lower genital tract, and high-risk human papillomavirus (HPV) infection is the most important risk factor for the development of both cancers. However, it is clear that additional genetic events are necessary for tumor progression, particularly in HPV-negative cases. We detected the presence of high-risk HPV16 and HPV18 genomes by gene-specific polymerase chain reaction and searched for common genetic imbalances by comparative genomic hybridization (CGH) in 28 cervical and 8 vulvar tumor samples and 7 cancer cell lines. The presence of the HPV genome was detected in 25/28 (89%) cervical tumors and 6/8 (75%) vulvar tumors. CGH of cervical and vulvar tumor samples revealed a consistent pattern of genetic changes in both cancers. Frequent gains were found in 1q, 3q, 5p, and 8q, and less consistent losses were detected in 2q, 3p, 4p, and 11p. Notably, a high-level amplification of 3q was found in 9/28 (32%) cervical tumors and 1/8 (12.5%) vulvar tumors, indicating a pivotal role of gain of 3q in cervical and vulvar carcinogenesis. Furthermore, gains of 5p identified in 9/28 (32%) cervical tumors and 3/8 (37.5%) vulvar tumors were seldom described, particularly in vulvar tumors. Our findings suggest that cervical and vulvar carcinomas bear similar chromosomal alteration hot spots that largely coincide with common genomic lesions during tumor progression, besides the initiation by infection and integration of oncogenic HPV.     

MYC amplification and TERT expression in breast tumor progression

The complex roles of genomic instability, MYC oncogene amplification, activation of telomerase, and p53 function still remain to be fully described in breast tumors. MYC stimulates the telomerase catalytic subunit, TERT, which interacts with p53. Oncogene MYC amplification analysis was performed on 27 paraffin-embedded breast tumor samples by fluorescence in situ hybridization, selected on the basis of chromosomal instability. TERT immunostaining was performed on a larger group of breast tumor sections. All tumor samples were analyzed for TP53 mutation, genomic index, S-phase fraction, and pathological stages. Amplification of MYC was detected in 16 of 27 tumors (59%) and found to be associated with TNM stages I and II (P = 0.018), genomic index > 1.5 (P = 0.033), and S-phase fraction > 5% (P = 0.020). No association was found between MYC amplification and TERT immunostaining or TP53 mutations. Analysis of TERT in 103 primary breast tumors showed > 50% nuclei immunostaining in 58% of cases. High TERT immunostaining associated with genomic index > 1.5 (P = 0.017), high S-phase fraction (P = 0.056), and TP53 mutations (P = 0.030). No association was found between TERT staining and TNM stages. This study supports early involvement of MYC amplification in breast tumor progression. Both MYC amplification and TERT expression appear to be associated with high genomic instability and proliferation. TERT association with TP53 mutations indicates that TERT activity is downregulated by functional p53 protein in breast tumors.     

RAD21 and KIAA0196 at 8q24 are amplified and overexpressed in prostate cancer

To detect genes that are overexpressed in prostate cancer, a subtracted cDNA library was first constructed from the PC-3 cell line and subsequently screened by using cDNA microarray hybridization. Sixty-eight genes were found to be overexpressed (ratio>3) in PC-3. Half of these genes were in chromosomal regions, which, using comparative genomic hybridization, we previously showed to be gained in PC-3. Subsequently, the expression and copy number of selected genes were studied by quantitative RT-PCR and fluorescence in situ hybridization in prostate cancer cell lines, xenografts, and clinical tumor specimens of benign prostate hyperplasia and untreated as well as hormone-refractory prostate carcinomas. Two genes from chromosomal region 8q24-RAD21 and KIAA0196-showed increased expression in clinical prostate carcinomas and were also amplified in 30-40% of xenografts and hormone-refractory tumors. In addition, the expression of KIAA0196 was significantly (P=0.0051) higher in tumors with the gene amplification than in those without it. The data suggest that KIAA0196 and possibly RAD21 are putative target genes for the common amplification of 8q23-24 in prostate cancer.     

EGFR gene amplification in breast cancer: correlation with epidermal growth factor receptor mRNA and protein expression and HER-2 status and absence of EGFR-activating mutations.

The human epidermal growth factor receptor (HER) family of receptor tyrosine kinase has been extensively studied in breast cancer; however, systematic studies of EGFR gene amplification and protein overexpression in breast carcinoma are lacking. We studied EGFR gene amplification by chromogenic in situ hybridization (CISH) and protein expression by immunohistochemistry in 175 breast carcinomas, using tissue microarrays. Tumors with >5 EGFR gene copies per nucleus were interpreted as positive for gene amplification. Protein overexpression was scored according to standardized criteria originally developed for HER-2. EGFR mRNA levels, as measured by Affymetrix U133 Gene Chip microarray hybridization, were available in 63 of these tumors. HER-2 gene amplification by fluorescence in situ hybridization (FISH) and protein overexpression by immunohistochemistry were also studied. EGFR gene amplification (copy number range: 7-18; median: 12) was detected in 11/175 (6%) tumors, and protein overexpression was found in 13/175 (7%) tumors. Of the 11 tumors, 10 (91%) with gene amplification also showed EGFR protein overexpression (2+ or 3+ by immunohistochemistry). The EGFR mRNA level, based on Affymetrix U133 chip hybridization data, was increased relative to other breast cancer samples in three of the five tumors showing gene amplification. Exons 19 and 21 of EGFR, the sites of hotspot mutations in lung adenocarcinomas, were screened in the 11 EGFR-amplified tumors but no mutations were found. Three of these 11 tumors also showed HER-2 overexpression and gene amplification. Approximately 6% of breast carcinomas show EGFR amplification with EGFR protein overexpression and may be candidates for trials of EGFR-targeted antibodies or small inhibitory molecules.     

Deletions of 17p are associated with transition from early to advanced colorectal cancer

Several chromosome defects parallel morphologic evolution in colorectal tumor progression. Allelic losses in the short arm of chromosome 17, the majority encompassing the 17p13.3 band, have been found in advanced cancer in the absence of TP53 mutations, suggesting that loss of genes in this chromosome region is relevant for tumorigenesis. The aim of this study was to investigate 17p13.3 deletions throughout the colorectal tumor progression using two-color fluorescence in situ hybridization. Histologic sections from 20 colorectal adenomas containing early invasive carcinoma were analyzed by interphase fluorescence in situ hybridization using a centromeric probe for chromosome 17 simultaneously with a subtelomeric probe mapping to the 17p13.3 band. Separate evaluation was made for sectors corresponding to adenoma tissue with low-grade dysplasia, high-grade dysplasia, and early cancer. The same technique was also used in 20 cases of advanced adenocarcinoma of the large bowel. Loss of one centromeric signal was observed in 20, 40, 50, and 10% of low-grade dysplasia, high-grade dysplasia, early cancer, and advanced cancer, respectively (P<0.02 early vs. advanced cancer). Subtelomeric 17p deletions were seen in 60% of advanced cancer and in 15% of early cancer (P<0.01). These findings indicate that loss of genes from the 17p13.3 chromosome region may play an important role in sustaining the transition from early to advanced cancer in colorectal tumor progression.