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Minichromosome Maintenance Protein 2 (MCM2) is critical in initiating DNA replication during the cell division process. As expressed intensively in all phases of the active cell cycle, MCM2 has been proposed as a novel biomarker to determine cellular proliferation. We aimed at clarifying the prevalence and clinical significance of MCM2 in HER2-amplified breast cancer subtype. MCM2 expression was studied in 142 primary HER2-amplified breast carcinomas by applying a novel fluoro-chromogenic immunohistochemistry and tailored digital image analysis to determine labelling index (MCM2-LI). The presence of MCM2 was detected with HRP-conjugated polymer and visualized with 3, 3'-diaminobenzidine tetrahydrochloride, in cytokeratin (CK)-positive and Cy2-IgG-labelled breast cancer cells of epithelial origin. Stained slides were digitized by scanning sequentially under bright field (for MCM2) and fluorescence (for CK) illumination. Multilayer JPEG2000 images were analyzed with ImmunoRatio 2.5 (accessory in SlideVantage 1.2 software) utilizing its bright field and fluorescence image-blending mode to display MCM2-CK dual-positive cells. MCM2-LI was retrospectively compared with histopathologic characteristics and patients' clinical outcome. MCM2 protein-expressing cells (median MCM2-LI, 63.5%) were more frequent than those of Ki67 (median Ki67 labelling index, 33%). Significant correlations were found between high MCM2-LI, high Ki67 labelling index, negative hormone receptor (ER, PR) statuses, high grade of malignancy, and high cyclin E expression. MCM2-LI was not shown to be predictive of disease recurrence during the median follow-up of 5.3 years but was shown to be useful to distinguish aggressive-type HER2-amplified breast carcinomas with high malignancy grade and hormone receptor negativity. The fluoro-chromogenic double-labelling immunohistochemistry accompanied with digital image analysis provides an accurate carcinoma-specific determination of MCM2-LI on a single tumor section.

Aurora-A is a serine-threonine kinase having vital cellular functions in mitosis and is a promising target for therapy in treating patients with cancer. This study assesses the clinicopathological associations of AURKA gene amplification in clinical breast tumors and association of gene copy number with its mRNA and protein expression in breast cancer cell lines. In this pilot study, we examined Aurora-A gene (AURKA) amplification in 126 clinical breast tumors by chromogenic in situ hybridisation (CISH). AURKA amplification (found in 21%) showed an association with basal-like tumor phenotype (p=0.046). A separate series of basal-like breast tumors (n=26) provided further evidence of the importance of AURKA in these tumors. AURKA amplification status was associated with immunohistochemically detectable Aurora-A protein expression (p<0.0001). In breast cancer cell lines, gene amplification was strongly associated with high mRNA expression (p<0.0001). JIMT-1 cell line was found as a possible in vitro model system for testing Aurora-A inhibitors, since it has been classified as basal-like breast cancer and here it showed both AURKA gene amplification and elevated mRNA expression. AURKA gene amplification is a common genetic aberration in breast cancer, especially in tumors displaying basal-like phenotype. Thus, these patients might be suitable candidates for future targeted therapies with Aurora-A inhibitors.

Receptor tyrosine kinase aberrations are implicated in the genesis of gliomas. We investigated expression and amplification of KIT, PDGFRA, VEGFR2, and EGFR in 87 gliomas consisting of astrocytomas, anaplastic astrocytomas, oligodendrogliomas, or oligoastrocytomas in tumor samples collected at the time of the diagnosis and in samples of the same tumors at tumor recurrence. Gene amplifications were investigated using either chromogenic in situ hybridization or fluorescence in situ hybridization, and protein expression using immunohistochemistry. In samples collected at glioma diagnosis, KIT and PDGFRA amplifications were more frequent in anaplastic astrocytomas than in astrocytomas, oligodendrogliomas, and oligoastrocytomas [28% versus 5% (P = 0.012) and 33% versus 2% (P = 0.0008), respectively]. VEGFR2 amplifications occurred in 6% to 17% of the gliomas at diagnosis, and EGFR amplifications in 0% to 12%. Amplified KIT was more frequently present in recurrent gliomas than in newly diagnosed gliomas (P = 0.0066). KIT amplification was associated with KIT protein expression and with presence of PDGFRA and EGFR amplifications both at the time of the first glioma diagnosis and at tumor recurrence, and with VEGFR2 amplification at tumor recurrence. Three (4%) primary gliomas and 10 (14%) recurrent gliomas that were evaluable for coamplification of KIT, PDGFRA, and VEGFR2 showed amplification of at least two of these genes; the amplicon contained amplified KIT in all 13 cases. In conclusion, besides glioblastoma, amplified KIT, PDGFRA, and VEGFR may also occur in lower-grade gliomas and in their recurrent tumors. It is currently not known whether specific tyrosine kinase inhibitors are effective in the treatment of such gliomas.

Purpose: Amplification of the HER-2/neu and topoisomerase IIalpha (TOP2A) genes has been linked to the effects of anthracyclines. Their role in predicting the outcome of anthracycline-based adjuvant chemotherapy for breast cancer patients has remained controversial., Patients and Methods: The present substudy of the Scandinavian Breast Group trial 9401, in which an epirubicin-based regimen (nine courses of tailored and dose-escalated fluorouracil, epirubicin, and cyclophosphamide [FEC]) was compared with three or four courses of standard FEC followed by bone marrow-supported high-dose chemotherapy (cyclophosphamide, thiotepa, and carboplatin), included high-risk breast cancer patients (with eight or more positive axillary lymph nodes or at least five nodes with additional poor prognostic indicators). Amplification of HER-2/neu was determined retrospectively in paraffin-embedded tumor tissue sections by chromogenic in situ hybridization. TOP2A was tested only in HER-2/neu-amplified tumors., Results: HER-2/neu amplification alone, which was present in 32.7% of the tumors, was a strong prognostic factor for short relapse-free (P = .0034) and overall survival (P = .0008) but showed no direct association with treatment assignment. TOP2A coamplification, which was present in 37% of HER-2/neu-amplified tumors, was associated with better relapse-free survival in patients treated with tailored and dose-escalated FEC (hazard ratio [HR] = 0.45; P = .049). A statistical multivariate Cox's regression analysis confirmed the predictive significance of TOP2A coamplification (HR = 0.30; P = .020) in HER-2/neu-amplified tumors. There was no such association in patients with HER-2/neu-amplified tumors without TOP2A gene amplification., Conclusion: Coamplification of HER-2/neu and TOP2A may define a subgroup of high-risk breast cancer patients who benefit from individually tailored and dose-escalated adjuvant anthracyclines.

Since neuroblastomas are intratumorally heterogeneous, the analysis of genetic and biologic features of randomly selected tumor specimen spots may lead to erroneous conclusions. Our purpose was therefore to construct an easily assessable and strictly defined strategy to unify the detection of various molecular markers in paraffin-embedded neuroblastoma samples. We selected tumor specimen spots of highest proliferation activity, that is, hotspots, for the analysis of MYCN amplification status and proliferation-associated molecular markers, such as nestin, which role in neuroblastoma specimens was evaluated for the first time. Using a chromogenic in situ hybridization (CISH) technique, we showed that patients with a MYCN copy number higher than six in anti-Ki-67-detected hotspots have significantly worse overall survival prognosis than patients with low MYCN copy numbers (P = 0.0006). The chosen cutoff value of six was shown to dichotomize MYCN-amplified neuroblastomas at least as specifically as Southern blot hybridization, in which amplification was defined by a copy number of > or = 10. Interestingly, we also detected without difficulty MYCN-amplified neuroblastic cells in bone marrow samples using the CISH technique. The proliferation activity, assessed with an anti-Ki-67-based proliferation index, was significantly higher in MYCN-amplified than in nonamplified hotspots. The proliferation indices of the hotspots had also a significant correlation with the prognosis (International Classification) and histological type, whereas the proliferation accelerator Id2 did not associate with any of the mentioned parameters. The expression of nestin associated inversely with MYCN amplification (P = 0.018), which challenges a previously suggested role of nestin in neuroblastomas. In summary, hotspot focusing provides a means of analyzing proliferation-associated markers in neuroblastomas, and together with the CISH detection of the MYCN copy number enables an easy and reliable examination of MYCN status in neuroblastomas.

Background: HER-2/neu gene amplification has predictive value in breast cancer patients responding to trastuzumab. We wanted to investigate the frequency and clinical significance of HER-2/neu amplification in gastric carcinoma., Patients and Methods: The frequency of HER-2/neu and Topoisomerase IIalpha gene amplification was studied in adenocarcinomas of the stomach (n=131) and the gastroesophageal junction (n=100) by chromogenic in situ hybridization (CISH). Sensitivity of a gastric cancer cell line N87 with HER-2/neu amplification to trastuzumab was studied by a cell viability assay and compared with that of a HER-2 amplified breast cancer cell line SKBR-3. Growth inhibition of N87 cells was also verified in vivo in N87 xenograft tumors., Results: HER-2/neu amplification was present in 16 (12.2%) of the 131 gastric and in 24 (24.0%) of the 100 gastroesophageal adenocarcinomas. Co-amplification of Topoisomerase IIalpha was present in the majority of gastric (63%) and esophagogastric junction cancers (68%) with HER-2/neu amplification. HER-2/neu amplification was more common in the intestinal histologic type of gastric cancer (21.5%) than in the diffuse (2%) or the mixed/anaplastic type (5%, P=0.0051), but it was not associated with gender, age at diagnosis or clinical stage. Presence of HER-2/neu amplification was associated with poor carcinoma-specific survival (P=0.0089). HER-2/neu targeting antibody trastuzumab inhibited the growth of a p185(HER-2/neu) overexpressing gastric and breast carcinoma cell lines (N87 and SKBR-3) with equal efficacy., Conclusions: HER-2/neu amplification is common in the intestinal type of gastric carcinoma, and it is associated with a poor outcome. HER-2 might be a useful target in this disease, and this hypothesis deserves to be investigated in clinical trials.

Overexpression of the epidermal growth factor receptor (EGFR) is a common finding in invasive breast cancer and represents a potential target for new treatment options. However, little is known about the parameters that might indicate a potential clinical response for these anti-EGFR-based therapies. In order to gain further insights into the interplay between the length of a CA-SSR I repeat in intron 1 of egfr, copy numbers of this untranslated regulatory sequence, and protein expression, the present study investigated breast cancers from Germans and Japanese patients by microsatellite analysis, quantitative 5' nuclease assay by egfr enzyme-linked immunosorbent assay (ELISA), and comparative genomic hybridization (CGH). Japanese breast cancer patients displayed significantly longer alleles for the CA-SSR I repeat (p < 0.001), associated with significantly lower EGFR expression (mean 65 versus 36 fmol/mg membrane protein). Allelic imbalance (restricted to CA-SSR I) was observed in 55% of the informative Japanese breast cancers compared with only 34% of the German breast cancer reference group. Using a quantitative 5' nuclease assay for egfr, a significantly higher percentage of Japanese breast cancer patients revealed amplifications of the CA-SSR I repeat (p < 0.01). Japanese patients with these amplifications were characterized by a significantly higher EGFR content compared with the German breast cancer patients (p < 0.05). These data show, on the one hand, that the correlation of EGFR overexpression and an inherited CA repeat polymorphism within intron 1 of egfr is a general finding in breast cancer, as has been shown previously. On the other hand, the data demonstrate clearly for the first time an interaction between the length of a polymorphism in intron 1 of egfr as an inherited genetic factor and the frequency of egfr amplification, as an acquired genetic factor, both factors contributing to EGFR overexpression in breast cancer. This new knowledge about mechanisms of regulation of EGFR expression might serve as an additional basis for evaluating anti-EGFR-based therapies., (Copyright 2004 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Aims: To study HER2 oncogene amplification and over-expression in skin samples of 23 patients with extramammary Paget's disease (EMP). EMP is a rare intra-epidermal adenocarcinoma, which has been reported to over-express the HER2 oncoprotein., Methods and Results: HER2 gene amplification, detected by chromogenic in-situ hybridization, was found in 43% (10/23) of the lesions. HER2 protein over-expression (3+ immunostaining intensity) was found in 12 tumours (52%), including all 10 tumours with gene amplification. Two tumours showed low-level (2+) HER2 immunostaining. Mammary Paget's lesions, which were used as controls, showed HER2 amplification and over-expression in all 10 cases studied., Conclusions: These results indicate that HER-2 protein over-expression in EMP is common and due exclusively to gene amplification. They open up the possibility of HER2-targetted immunotherapy for patients with HER2+ disease.

Breast carcinoma is thought to arise because of multiple successive changes in the genome of the normal epithelial cells. However, little is known of the order of appearance of different types of genetic aberrations We studied the ERBB2 (Her-2/neu) and CCND1 (cyclin D1) oncogene amplification in flow cytometrically sorted diploid and nondiploid tumor cell populations by fluorescence in situ hybridization (FISH). The purity of the cell sorting was confirmed by static DNA image cytometry. Spectral karyotyping was used to define differences in a genome-wide manner between two distinctly different aneuploid cell clones found in each of two breast cancer cell lines. FISH indicated the presence of gene amplification both in diploid and nondiploid cell clones in 17 of the 21 amplification-containing tumors analyzed. The oncogene copy numbers remained unchanged throughout aneuploidization in 11 of 17 tumors. The remaining six tumors showed an increase in oncogene copy number as well as the number of chromosome 11 or 17 centromeres (the original location of CCNDI and ERBB2, respectively). Breast carcinoma cell lines MDA-157 and MDA-436 showed a significant number of chromosomal rearrangements in the near-diploid clones, which were present in duplicate in the corresponding aneuploid (polyploid) clones. These results indicate that ploidy shift, ie., aneuploidization, in breast cancer is a late genetic event which is preceded by both oncogene amplifications as well as many chromosomal rearrangements.

Determination of HER-2/neu oncogene amplification has become necessary for selection of breast cancer patients for trastuzumab (Herceptin) therapy. Fluorescence in situ hybridization (FISH) is currently regarded as a gold standard method for detecting HER-2/neu amplification, but it is not very practical for routine histopathological laboratories. We evaluated a new modification of in situ hybridization, the chromogenic in situ hybridization (CISH), which enables detection of HER-2/neu gene copies with conventional peroxidase reaction. Archival formalin-fixed paraffin-embedded tumor tissue sections were pretreated (by heating in a microwave oven and using enzyme digestion) and hybridized with a digoxigenin-labeled DNA probe. The probe was detected with anti-digoxigenin fluorescein, anti-fluorescein peroxidase, and diaminobenzidine. Gene copies visualized by CISH could be easily distinguished with a x40 objective in hematoxylin-stained tissue sections. HER-2/neu amplification typically appeared as large peroxidase-positive intranuclear gene copy clusters. CISH and FISH (according to Vysis, made from frozen pulverized tumor samples) correlated well in a series of 157 breast cancers (kappa coefficient, 0.81). The few different classifications were mostly because of low-level amplifications by FISH that were negative by CISH and immunohistochemistry with monoclonal antibody CB-11. We conclude that CISH, using conventional bright-field microscopy in evaluation, is a useful alternative for determination of HER-2/neu amplification in paraffin-embedded tumor samples, especially for confirming the immunohistochemical staining results.

Gain of chromosome arm 8q is a frequent genetic alteration in breast and prostate cancer. Two amplified subregions, 8q21 and 8q23-24, have been identified with comparative genomic hybridization (CGH). We have recently demonstrated that the EIF3S3 (eIF3-p40) gene, located at 8q23, is often amplified and overexpressed in both breast and prostate cancer. Here, we used fluorescence in situ hybridization (FISH) to map the amplified region around EIF3S3 in primary breast cancers and cell lines. The size of the common highly amplified region was about 2.5 Mb between the markers D8S1668 and WI-7959. Next, we analyzed the expression of all expressed sequence tags (ESTs) located within and near this region by RNA slot blot hybridization. In addition to EIF3S3, three anonymous ESTs and EXT1 were found to be highly expressed in cancer cell lines with the amplification at 8q23-q24. However, the anonymous ESTs were located outside the minimal highly amplified region and EXT1 was overexpressed only in one of the cancer cell lines with 8q amplification. Since EIF3S3 was the only consistently overexpressed gene located in the minimal highly amplified region, it is the strongest candidate target gene for 8q23-q24 amplification.

Topoisomerase IIalpha (topoIIalpha) is a key enzyme in DNA replication and a molecular target for many anti-cancer drugs called topoII inhibitors. The topoIIalpha gene is located at chromosome band 17q12-q21, close to the ErbB-2 oncogene (HER-2/neu), which is the most commonly amplified oncogene in breast cancer. Because of the physical proximity to ErbB-2, copy number aberrations may also occur in the topoIIalpha gene. These topoIIalpha gene copy number aberrations may be related to the altered chemosensitivity to topoII inhibitors that breast cancers with ErbB-2 amplification are known to have. We used fluorescence in situ hybridization to study copy number aberrations of both topoIIalpha and ErbB-2 in nine breast cancer cell lines and in 97 clinical breast tumors, which were selected for the study according to their ErbB-2 status by Southern blotting. TopoIIalpha-protein expression was studied with Western blot and sensitivity to doxorubicin (a topoII inhibitor) with a 96-well clonogenic in vitro assay. Two of the five cell lines with ErbB-2 gene amplification (SK-BR-3 and UACC-812) showed amplification of topoIIalpha. In MDA-361 cells, ErbB-2 amplification (14 copies/cell) was associated with a physical deletion of topoIIalpha (four copies of chromosome 17 centromere and two copies of topoIIalpha). The topoIIalpha amplification in UACC-812 cells was associated with 5.9-fold-increased topoIIalpha protein expression and 2.5-fold-increased sensitivity to the topoII inhibitor, doxorubicin, whereas the deletion in MDA-361 leads to decreased protein expression (45% of control) and a 2.4-fold-increased chemoresistance in vitro. Of 57 ErbB-2-amplified primary breast carcinomas, 25 (44%) showed ErbB-2-topoIIalpha coamplification and 24 (42%) showed a physical deletion of the topoIIalpha gene. No topoIIalpha copy number aberrations were found in 40 primary tumors without ErbB-2 amplification. TopoIIalpha gene amplification and deletion are common in ErbB-2-amplified breast cancer and are associated with increased or decreased sensitivity to topoII inhibitors in vitro, respectively. These findings may explain the altered chemosensitivity to topoII inhibitors reported in ErbB-2-amplified breast cancers.

Amplification of the ERBB2 oncogene has recently received attention as a target for antibody-based therapies and as a predictor of response to adjuvant chemotherapy. Modification of treatment strategies based on ERBB2 status has led to further interest in the genetic alterations that accompany ERBB2 gene amplification or overexpression. In this study, chromosome alterations that are associated with ERBB2 amplification were defined by comparative genomic hybridization (CGH). Additionally, fluorescence in situ hybridization (FISH) was used to validate gene amplification, and protein expression was detected immunohistochemically. ERBB2-amplified tumors as detected by FISH, immunohistochemistry (IHC), or CGH had twice as many CGH-defined chromosomal alterations (means of 11.8, 11.0, and 12.7, respectively) as the nonamplified tumors (means of 6.8, 7.0, and 5.6, respectively). ERBB2 positivity correlated with the total number of genetic events. A wide spectrum of copy number gains and losses was seen by CGH in all of the tumors. An increased number of losses of 18q and gains of 20q was found in ERBB2-positive tumors. Other common aberrations for all of the tumors were copy number gains of 1q (58%), 8q (52%), 20q (30%), and losses of 18q (39%), 13q (39%), and 3p (33%). A high degree of concordance was observed among the three methods in 33 primary breast cancers. The concurrence for ERBB2 detection between FISH and IHC was 90%, between FISH and CGH was 82%, and between IHC and CGH was 84%. This study shows that breast tumors showing erbB2 overexpression or gene amplification are genetically distinct from erbB2-negative tumors. These differences may relate to the mechanisms underlying altered response to adjuvant therapies and may define the responsiveness to erbB2-directed immunotherapy.

Topoisomerase IIalpha (TOP2A) is a key enzyme in DNA replication and a molecular target for many important anticancer drugs. TOP2A is amplified or deleted together with amplification of the closely located ERBB2/HER-2/neu oncogene in breast cancer. We characterized the copy number aberrations of TOP2A and ERBB2 in 136 primary breast tumors by FISH. Among the 70 primary tumors with ERBB2 amplification, amplification of TOP2A was found in 29 (41%); 30 tumors (43%) showed a physical deletion of TOP2A; and the copy number for TOP2A was not altered in 11 tumors with ERBB2 amplification (16%). No TOP2A gene aberrations were identified in 65 primary tumors without ERBB2 amplification. Fiber FISH revealed that simultaneously amplified ERBB2 and TOP2A were not present in the same amplicon, because repetitive tandem repeat-like signals of ERBB2 and TOP2A were in separate DNA fibers. The deletion of TOP2A (seen in the MDA-361 cell line and in 31 primary tumors) was interstitial, spanning less than two megabases of DNA. Mean copy numbers of TOP2A (2.4 +/- 0.6 for TOP2A vs. 4.9 +/- 1.1 for chromosome 17 centromere) suggest that the deletion of TOP2A occurs before polyploidization of the genome. Eight primary tumors with high-level ERBB2 amplification showed a new type of intratumoral heterogeneity; two different cell clones with either high-level amplification or deletion of TOP2A were found adjacent to each other in the same tumor. These results indicate that amplification of the ERBB2 oncogene is followed by complex secondary genetic aberrations, which lead to amplification or deletion of the TOP2A gene in a majority of tumors. Genes Chromosomes Cancer 26:142-150, 1999., (Copyright 1999 Wiley-Liss, Inc.)

We report here the molecular cloning of an approximately 1-Mb region of recurrent amplification at 20q13.2 in breast cancer and other tumors and the delineation of a 260-kb common region of amplification. Analysis of the 1-Mb region produced evidence for five genes, ZNF217, ZNF218, and NABC1, PIC1L (PIC1-like), CYP24, and a pseudogene CRP (Cyclophillin Related Pseudogene). ZNF217 and NABC1 emerged as strong candidate oncogenes and were characterized in detail. NABC1 is predicted to encode a 585-aa protein of unknown function and is overexpressed in most but not all breast cancer cell lines in which it was amplified. ZNF217 is centrally located in the 260-kb common region of amplification, transcribed in multiple normal tissues, and overexpressed in all cell lines and tumors in which it is amplified and in two in which it is not. ZNF217 is predicted to encode alternately spliced, Kruppel-like transcription factors of 1,062 and 1,108 aa, each having a DNA-binding domain (eight C2H2 zinc fingers) and a proline-rich transcription activation domain.

Progression of prostate cancer during endocrine therapy is a major clinical problem, the molecular mechanisms of which remain poorly understood. Amplification of the androgen receptor (AR) gene was recently described in recurrent prostate carcinomas from patients who had failed androgen deprivation therapy. To evaluate the hypothesis that amplification of the AR gene is a cause for the failure of androgen deprivation therapy in prostate cancer, we studied whether AR amplification leads to gene overexpression, whether the amplified AR gene is structurally intact, and whether tumors with AR amplification have distinct biological and clinical characteristics. Tumor specimens were collected from 54 prostate cancer patients at the time of a local recurrence following therapy failure. In 26 cases, paired primary tumor specimens from the same patients prior to therapy were also available. Fifteen (28%) of the recurrent therapy-resistant tumors, but none of the untreated primary tumors, contained AR gene amplification as determined by fluorescence in situ hybridization. According to single-stranded conformation polymorphism analysis, the AR gene was wild type in all but one of the 13 AR amplified cases studied. In one tumor, a presumed mutation in the hormone-binding domain at codon 674 leading to a Gly --> Ala substitution was found, but functional studies indicated that this mutation did not change the transactivational properties of the receptor. AR amplification was associated with a substantially increased level of mRNA expression of the gene by in situ hybridization. Clinicopathological correlations indicated that AR amplification was most likely to occur in tumors that had initially responded well to endocrine therapy and whose response duration was more than 12 months. Tumors that recurred earlier or those that showed no initial therapy response did not contain AR amplification. The median survival time after recurrence was two times longer for patients with AR amplification in comparison to those with no amplification (P = 0.03, Willcoxon-Breslow test). In conclusion, failure of conventional androgen deprivation therapy in prostate cancer may be caused by a clonal expansion of tumor cells that are able to continue androgen-dependent growth despite of the low concentrations of serum androgens. Amplification and the increased expression of a wild-type AR gene may play a key role in this process.

Objective: We recently found amplification of the androgen receptor (AR) gene in approximately 30% of locally recurrent prostate carcinomas from patients treated by conventional androgen deprivation (castration) therapy, whereas none of the untreated primary prostate tumors showed this amplification. This suggests that AR gene amplification was selected during androgen deprivation therapy. The present case study represents our initial approach to evaluate the role that AR amplification may play in therapy resistance after other forms of endocrine therapy., Material and Methods: Specimens from both a primary and a subsequent locally recurrent tumor were studied for amplification of the AR gene by fluorescence in situ hybridization from a prostate cancer patient who experienced tumor progression after monotherapy with the potent antiandrogen bicalutamide (Casodex, a trade mark, the property of Zeneca Ltd)., Results and Conclusions: High-level amplification of the AR gene was found in the recurrent tumor, whereas no evidence of amplification was found in the primary tumor. After recurrence, the patient first received chemotherapy (ifosfamide) for 15 weeks with no response, followed by maximal androgen blockade (MAB). The latter therapy resulted in a favorable short-term response. This case study has the following implications which warrant further research: (1) AR amplification may be selected not only by castration but also by therapy with a competitive peripheral androgen-receptor-blocking agent, and (2) recurrent tumors with AR amplification may be particularly likely to benefit from MAB as a second-line therapy.

DNA amplification at 20q13.2 is common in breast cancer, correlates with poor prognosis, and may reflect location of an important oncogene. Recently, other regions along 20q were also found to undergo amplification. Here, amplification levels and patterns of co-amplification were analyzed by interphase fluorescence in situ hybridization at 14 loci along 20q in 146 uncultured breast carcinomas and 14 cell lines. Three regions were independently amplified in uncultured tumors: RMC20C001 region at 20q13.2 (highly amplified in 9.6% of the cases), PTPN1 region 3 Mb proximal (6.2%), and AIB3 region at 20q11 (6.2%). Co-amplifications involving two or three of these regions were seen in 11 of the 19 highly amplified tumors. The results suggest that three distinct nonsyntenic regions along 20q may be important and that complex chromosomal rearrangements underlie their frequent co-amplification in breast cancer.

Recent studies suggest that aberrations of c-erbB-2 may be involved in astrocytic brain tumours. We screened immunohistochemically c-erbB2 protein (p185) expression in 94 astrocytic grade 1-4 neoplasms of the brain. The amplification of the c-erbB-2 oncogene was investigated in protein overexpression cases by dual colour fluorescence in situ hybridisation (FISH). p185 overexpression was correlated with p53 and epidermal growth factor receptor (EGFR) expression, as well as with clinicopathological features. Only two anaplastic (grade 3) astrocytomas and one glioblastoma (grade 4) showed overexpression of p185 protein by immunohistochemistry (monoclonal MAb1 antibody TA250), whereas none of the grade 1-2 astrocytomas was positive. Interestingly, the expression of p185 was confined solely to the cytoplasm of neoplastic astrocytic cells and not to the cell membranes as found in malignancies with amplification of the c-erbB-2 oncogene. Two of the three overexpression cases were also positive by EGFR. No amplification of the c-erbB-2 gene was observed by FISH in the three tumours with immunohistochemical p185 overexpression or seven weakly positive/negative tumours. In conclusion, our results suggest that p185 overexpression is infrequent in astrocytomas, that it is of no important diagnostic or prognostic value and that c-erbB-2 oncogene amplification is not seen in the few cases in which there is overexpression.

Amplification of the chromosome 20q13 region was recently discovered in breast cancer by comparative genomic hybridization and subsequently further defined by fluorescence in situ hybridization with specific probes. The target gene of the amplification remains unknown. Here, fluorescence in situ hybridization with a cosmid probe for the minimal region of amplification (RMC20C001) was used to study 20q13 amplification in 132 primary breast carcinomas and 11 metastases. The size of the amplicon was studied with four flanking probes. Thirty-eight (29%) primary tumors and 3 (27%) metastases showed increased copy number of the RMC20C001 probe (>1.5-fold relative to the p-arm control). Nine (6.8%) of the primary tumors were highly (>3-fold) amplified. Although the size and location of the amplified region varied from one tumor to another, only the RMC20C001 probe was consistently amplified. 20q13 amplification was significantly associated with a high histological grade (P = 0.01), DNA aneuploidy (P = 0.01), and high S-phase fraction (P = 0.0085). High-level amplification was also associated with short disease-free survival of patients with node-negative breast cancer (P = 0.002). We conclude that high-level 20q13 amplification may be an indicator of poor clinical outcome in node-negative breast cancer and that this chromosomal region is likely to contain a gene with an important role in breast cancer progression. A large definitive study is warranted to assess the independent prognostic value of 20q13 amplification.

Overexpression of amplified genes is often associated with the acquisition of resistance to cancer therapeutic agents in vitro. We have identified a similar molecular mechanism in vivo for endocrine treatment failure in human prostate cancer which involves amplification of the androgen receptor (AR) gene. Comparative genomic hybridization shows that amplification of the Xq11-q13 region (the location), is common in tumours recurring during androgen deprivation therapy. We found high-level AR amplification in seven of 23 (30%) recurrent tumours, but in none of the specimens taken from the same patients prior to therapy. Our results suggest that AR amplification emerges during androgen deprivation therapy by facilitating tumour cell growth in low androgen concentrations.