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Chromogenic in situ hybridization (CISH) was used to detect amplification of the epidermal growth factor receptor (EGFR) gene in tissue microarrays of tumours derived from 287 patients with grade II-IV diffuse astrocytomas. Amplification was found in 32% of the tumours with a highly significant association with histological grade (4% in grade II, 21% in grade III and 39% in grade IV; P < 0.001). Amplification of the EGFR gene was more common in primary than in secondary glioblastomas (41%vs. 16%, P = 0.033). Overexpression of EGFR mRNA and protein (wild-type and vIII variant) was found to correlate with EGFR gene amplification (P = 0.028, P = 0.035 and P = 0.014 respectively), but wild-type EGFR protein was also frequently overexpressed in tumours without EGFR gene amplification. Patients with older age (P < 0.001) and tumours with lack of p53 overexpression (P = 0.03) and higher apoptosis rate (P < 0.001) had significantly more EGFR gene amplifications than their counterparts. No such correlation with apoptosis was found in glioblastomas. The survival of patients with EGFR gene-amplified grade III tumours was significantly shorter than in those with grade III non-amplified tumours (P = 0.03). No such difference was noted in glioblastomas (grade IV tumours). Our data verify the central role of EGFR in the pathobiology of astrocytic tumours, and highlight the advantages of CISH as a simple and practical assay to screen for EGFR gene amplification in astrocytic tumours.

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.

Genomic imbalances detected by comparative genomic hybridization are prognostic markers in invasive ductal breast carcinomas Aims: The aim of this work is the study of the prognostic significance of the chromosomal aberrations described in a series of invasive ductal breast carcinomas. Methods and results: We analysed by comparative genomic hybridization a group of 70 formalin-fixed paraffin-embedded invasive ductal breast carcinomas. Aberrations showed a frequency similar to previous studies using frozen tumours. Interestingly, we identified gains involving 6q16-q24 more frequently than in other series. We analysed the association among the chromosomal imbalances, 11 histopathological factors, relapse rate and overall survival of patients. Associations showed 16q losses as a potential marker of good prognosis, as they were more frequent in node-negative (P=0.025) and in oestrogen-positive tumours (P < 0.001). Furthermore, 100% of bcl-2+ tumours presented this aberration compared with 29.3% in bcl-2– (P=0.014). 1q, 11q, 17q and 20q gains were associated with poor prognosis: 95% of cases with 1q gains were bigger than 20 mm (P=0.041). Tumours with 1q and 11q gains showed a higher relapse rate (P=0.063; P=0.066). Within the good prognosis group of lymph node-negative patients, 17q and 20q gains identify a subgroup with increased relapse rate (P=0.039). Conclusions: Chromosomal imbalances, together with histopathological factors, may help to predict outcome in breast cancer patients.

The aim of this trial was to study the value of adding post-operative radiotherapy to lumpectomy in a subgroup of breast cancer patients with favourable patient-, tumour-, and treatment-related prognostic features. 152 women aged over 40 with unifocal breast cancer seen in preoperative mammography were randomly assigned to lumpectomy alone (no-XRT group) or to lumpectomy followed by radiotherapy to the ipsilateral breast (50 Gy given within 5 weeks, XRT group). All cancers were required to be invasive node-negative, smaller than 2 cm in diameter and well or moderately differentiated, to contain no extensive intraductal component, to be progesterone receptor-positive, DNA diploid, have S-phase fraction ≤7 and be excised with at least 1 cm margin. During a mean follow-up time of 6.7 years, 13 (18.1%) cancers recurred locally in the no-XRT and 6 (7.5%) in the XRT group (P = 0.03). There was no difference between the groups in the ultimate breast preservation rate (95.0% vs. 94.4% in XRT and no-XRT, respectively, P = 0.88), distant metastasis-free survival (P = 0.36), or 5-year cancer-specific survival (97.1% in XRT and 98.6 in no-XRT). Radiation therapy given after lumpectomy reduces the frequency of ipsilateral breast recurrences even in women with small breast cancer with several favourable clinical and biological features. However, the breast preservation rate may not increase due to more frequent use of salvage mastectomies in patients treated with postoperative radiotherapy. © 2001 Cancer Research Campaign http://www.bjcancer.com

Radiotherapy after conservative surgery causes fat necrosis, fibrosis, skin thickening and other parenchymal distortion of the breast. The interpretation of a mammogram of the irradiated breast may therefore be difficult. We studied the effect of radiotherapy on the interpretation of the routine mammography used in the follow-up of breast cancer patients. A total of 144 low-risk breast cancer patients were randomized to radiotherapy or to no further treatment after conservative surgery. The first routine follow-up mammography was performed 18 months after surgery and every 18 months after that. The number of mammography examinations was estimated per patient and per follow-up year. The number of extra diagnostic tests and the occurrence of positive findings were assessed per mammography session and per follow-up year. Further diagnostic tests prompted by difficulties in interpreting the mammogram were performed to an extent of 0.19 per mammography examination in the radiotherapy group and of 0.15 in the non-radiotherapy group, i.e. 1.3 times more often. Findings that turned out to be negative at confirmation were 2.0 times (P< 0.05) more common in the radiotherapy group. These false-positive findings were more common in the radiotherapy group than in the surgery group and only shortly after treatment. Mammography is more difficult to interpret after radiotherapy than after conservative surgery alone, especially shortly after treatment, and more often involves extra diagnostic tests and findings that will be negative at confirmation.

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.

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. Images Figure 1 Figure 2

• To evaluate the prognostic value of histopathological variables and immunostainings of biomarkers enhancer of zeste homologue 2 (EZH2), Ki-67 and minichromosome maintenance protein 7 (MCM7) from core biopsies of hormonally treated patients with prostate cancer.• Biopsies of 247 primarily endocrine-treated patients were analysed for histopathological characteristics and Gleason scores (GS) according to the revised guidelines of International Society of Urologic Pathology (ISUP) consensus conference 2005. • Immunohistochemical stainings were analysed with the aid of digital image analysis. • The prognostic value of the histopathological variables and the biomarkers was analysed with univariate and multivariate Cox regression analysis, with biochemical recurrence as an endpoint.• Biomarkers EZH2 (relative risk [RR] 2.0, 95% confidence interval 1.2-3.3), Ki-67 (3.4, 2.1-5.5) and MCM7 (2.4, 1.5-3.9) were significantly associated with progression-free survival in a univariate analysis. • Ki-67 immunostaining index detected high-risk patients with GS of 7 (9.1, 8.0-10.3). • In a multivariate analysis with non-conventional GS groups 5-7 (3 + 4), 7(4 + 3)-8, and 9-10, the independent prognostic markers were pretreatment GS (2.2, 1.5-3.2), prostate-specific antigen (PSA) level (2.1, 1.1-4.2), perineural invasion (PNI) (1.6, 1.2-2.2), and clinical T-stage (cT) (1.9, 1.0-3.7). • Combination of the independent markers (PSA level20 ng/mL or GS3 + 4 or PNI3 or cT2) yielded best risk stratification (RR 11.6, 10.4-12.7).• GS remains one of the most important prognostic factors in prostate cancer. However, the refined guidelines by ISUP 2005 might have shifted the threshold between low-grade and high-grade cancers from GS 6 vs 7 to GS 3 + 4 vs 4 + 3. • PNI is an independent prognostic marker superior to cT. • Ki-67 is the most useful biomarker in detecting patients with GS = 7 at high risk for progression.

We analysed ploidy and S-phase fraction (SPF) from 78 paraffin-embedded primary prostatic carcinomas by DNA flow cytometry. DNA aneuploidy and above median (4.2%) SPF were both associated with high tumour grade, large size of prostate and presence of distant metastases. Both aneuploidy and high SPF (greater than 4.2%) indicated short 10-year progression-free (P = 0.01 for ploidy and P = 0.0002 for SPF), overall (P = 0.004 and P less than 0.0001) as well as prostate cancer survival (P = 0.002 and P less than 0.0001). Ten-year overall survival rate was 45% in cases with SPF below 4.2% and 0% in those with higher values, whereas the corresponding prostate cancer-specific survival rates were 80% and 11%, respectively. None of the seven tumours with SPF above 12% showed an objective response to endocrine therapy, whereas 26/49 (52%) of those with lower SPF values responded (P = 0.01). DNA ploidy, tumour grade, T-stage or M-stage did not significantly correlate with endocrine responsiveness. SPF was also the best predictor of progression free survival in patients treated hormonally. In conclusion, detection of high SPF in prostate cancer may indicate lack of hormonal responsiveness and poor prognosis.

Summary In coeliac disease gluten induces an immunological reaction in genetically susceptible patients, and influences on epithelial cell proliferation and differentiation in the small-bowel mucosa. Our aim was to find novel genes which operate similarly in epithelial proliferation and differentiation in an epithelial cell differentiation model and in coeliac disease patient small-bowel mucosal biopsy samples. The combination of cDNA microarray data originating from a three-dimensional T84 epithelial cell differentiation model and small-bowel mucosal biopsy samples from untreated and treated coeliac disease patients and healthy controls resulted in 30 genes whose mRNA expression was similarly affected. Nine of 30 were located directly or indirectly in the receptor tyrosine kinase pathway starting from the epithelial growth factor receptor. Removal of gluten from the diet resulted in a reversion in the expression of 29 of the 30 genes in the small-bowel mucosal biopsy samples. Further characterization by blotting and labelling revealed increased epidermal growth factor receptor and beta-catenin protein expression in the small-bowel mucosal epithelium in untreated coeliac disease patients compared to healthy controls and treated coeliac patients. We found 30 genes whose mRNA expression was affected similarly in the epithelial cell differentiation model and in the coeliac disease patient small-bowel mucosal biopsy samples. In particular, those genes involved in the epithelial growth factor-mediated signalling pathways may be involved in epithelial cell differentiation and coeliac disease pathogenesis. The epithelial cell differentiation model is a useful tool for studying gene expression changes in the crypt–villus axis.

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.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.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.

It has been suggested that estrogens and their receptors (ERs) may be involved in the development and progression of prostate cancer. To elucidate the significance of these receptors, expression of both ERalpha and ERbeta was measured in benign and malignant prostate tumors, as well as in cell lines.Expression of ERalpha and ERbeta was measured in prostate hyperplasia (BPH, n = 7), androgen-dependent (n = 30) as well as hormone-refractory (n = 12) prostate carcinomas, and in four prostate cancer cell lines (LNCaP, DU145, PC-3, and 22Rv1) using real-time quantitative RT-PCR.Only low-level expression of ERalpha was found in all tumor types and cell lines. The level of expression was similar to that observed in breast carcinomas found to be negative for ERalpha by immunohistochemistry. All cell lines showed low, but detectable, levels of ERbeta expression. The mean expression of ERbeta in the hormone-refractory carcinomas was about half that seen in BPH or the androgen-dependent carcinomas; however, the difference was not statistically significant.The data suggest it is unlikely that alterations in the expression of either ER are commonly involved in the progression of prostate cancer.

The aim of this work is the study of the prognostic significance of the chromosomal aberrations described in a series of invasive ductal breast carcinomas.We analysed by comparative genomic hybridization a group of 70 formalin-fixed paraffin-embedded invasive ductal breast carcinomas. Aberrations showed a frequency similar to previous studies using frozen tumours. Interestingly, we identified gains involving 6q16-q24 more frequently than in other series. We analysed the association among the chromosomal imbalances, 11 histopathological factors, relapse rate and overall survival of patients. Associations showed 16q losses as a potential marker of good prognosis, as they were more frequent in node-negative (P=0.025) and in oestrogen-positive tumours (P0.001). Furthermore, 100% of bcl-2+ tumours presented this aberration compared with 29.3% in bcl-2- (P=0.014). 1q, 11q, 17q and 20q gains were associated with poor prognosis: 95% of cases with 1q gains were bigger than 20 mm (P=0.041). Tumours with 1q and 11q gains showed a higher relapse rate (P=0.063; P=0.066). Within the good prognosis group of lymph node-negative patients, 17q and 20q gains identify a subgroup with increased relapse rate (P=0.039).Chromosomal imbalances, together with histopathological factors, may help to predict outcome in breast cancer patients.

This study investigated the expression of C-KIT and HER-2 receptors and their correlation with histopathological markers of cell proliferation, differentiation and apoptosis in poor-prognosis breast cancer.Formalin-embedded histopathological samples from forty patients with progressive metastatic breast cancer were reanalyzed to determine HER-2 expression by immunohistochemistry (ICH) and chromogenic in situ hybridization (CISH). C-KIT receptor expression, p53, Bcl-2 and Ki-67 were determined by immunohistochemistry.The mean p53 score was 18.03 (SD 30.69), that of Bcl-2 was 38.13 (39.25) and Ki-67 was 5.8 (SD 9.23), respectively. HER-2 expression was positive in 35% of patients by ICH and in 25% by CISH. C-KIT receptor staining was positive in 82% of the patients. A significant association was observed between HER-2-positive score in ICH and poorly-differentiated histology (p=0.03), negative Er/Pr status (p=0.04) and Bcl-2 expression (p=0.003). With CISH-determined HER-2, the corresponding p values were 0.07, 0.053, and 0.002, respectively. No correlation was found between HER-2 or C-KIT expression (p=0.456).CISH and ICH determination of HER-2 correlate similarly to hormone receptor status and Bcl-2 expression in breast cancer. C-KIT expression was commonly present and did not correlate to other prognosticators in poor-prognosis breast cancer.

The high incidence of gliomas in Li-Fraumeni families and the high frequency of somatic p53 mutations in sporadic glial tumors have raised the possibility that germline p53 mutations could play an important role in familial aggregation of gliomas. In the present study, 18 families with two or more gliomas were screened for germline p53 mutation. The families were identified through questionnaires sent to 369 consecutive glioma patients operated at Tampere University Hospital during 1983-1994. In these families, a family history of cancer was verified through the Finnish Cancer Registry. Interestingly, the questionnaires reveled only 15 of 57 cancers (index gliomas excluded) retrieved through the Cancer Registry. None of the 18 families fufilled the criteria for classic Li-Fraumeni syndrome. Immunostaining analysis of p53 protein accumulation suggested that alterations of the p53 gene are as common in familial as in sporadic gliomas. Sequencing analysis of exons 4-10 of the p53 gene revealed no germline mutations in any of the 18 families. Thus, although occasional glioma families carrying germline p53 mutations have been identified in earlier studies, systematic evaluation of familial glioma patients suggests that the p53 gene is not a common susceptibility gene in case of familial gliomas. The p53 tumor suppressor gene seems to have a similar role in the tumorigenesis of most familial and sporadic gliomas.

Amplification of the HER-2/neu oncogene and amplification of the topoisomerase IIalpha gene are important determinators of the response to chemotherapy in advanced breast cancer. Assays of these genes are usually carried out using primary tumor samples, because biopsies from metastatic lesions are not usually taken. We studied the concordance of Her-2/neu and topoisomerase IIalpha amplification in primary breast tumors and their metastases by immunostaining and DNA in situ hybridization. HER-2/neu amplification, present in 28% of the primary tumors (n = 46), was always associated with amplification in its metastasis. Conversely, no metastases with HER-2/neu amplification were seen without amplification in the primary tumor. Topoisomerase IIalpha gene copy status (amplification/deletion/unaltered) remained generally unchanged in HER-2/neu-positive tumors, but in three cases, the predominant cell population in metastatic tissue was present only as a subpopulation in the primary tumor. We conclude that amplification of HER-2/neu measured in primary tumor reflects the status of metastases. Minor discrepancies between primary and metastatic tumors in topoisomerase IIalpha gene copy status may reflect evolvement of the amplicon structure in successive cell divisions.

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.

The role of molecular markers predicting the prognosis and the selection of patients for further adjuvant therapies is not well established in oligodendroglioma patients. A potential prognostic as well as a therapeutically predictive factor, topoisomerase IIalpha (topoIIalpha), is a molecular target for certain cytotoxic drugs. Its expression has been shown to correlate with the prognosis in a number of different cancers and with the chemosensitivity of cancer cells in vitro. The expression of topoIIalpha was evaluated immunohistochemically in 59 oligodendrogliomas and in 29 mixed gliomas with a predominating oligodendroglioma component by the use of a tissue microarray technique. In the gliomas, the percentage of topoIIalpha immunopositive cells protein expression varied from 0.0 to 49.1% (5.2 +/- 8.3%, mean+/- SD). In oligoastrocytomas, the mean topoIIalpha score was significantly higher in the oligodendroglioma than in the astrocytoma component of the tumour (5.37 +/- 5.58% vs. 1.89 +/- 2.49%, P = 0.018). A significant association was found between the high proportion of topoIIalpha positive cells and high grade of the tumour (P0.0001), high tumour proliferation rate (P0.0001), p53 overexpression (P = 0.01) and high expression of tumour suppressing retinoblastoma protein (P = 0.023). TopoIIalpha expression was not associated with the age or sex of patient, and the rate of apoptosis. TopoIIalpha expression associated highly significantly with patient prognosis; a significantly higher proportion of patients with low rather than with high topoIIalpha score was alive at the end of the 5-year follow-up (P = 0.03). Cox analysis was used to demonstrate that topoIIalpha had an independent prognostic value for survival (P = 0.034). In conclusion, high topoIIalpha expression characterizes oligodendrogliomas and oligoastrocytomas which are poorly differentiated, have high proliferation rate, and has prognostic value for overall survival of these patients. Therefore, topoIIalpha may be a useful marker for better targeted selection of poor prognosis oligodendroglioma patients for adjuvant therapy.

Several systems for 24-color fluorescence in situ hybridization (FISH) have been developed and applied to karyotyping and detection of chromosomal abnormalities. We have developed a 42-color multicolor FISH (mFISH) technique (armFISH), which permits the detection of chromosomal aberrations at the resolution of chromosome arms. The armFISH uses a commercially available mFISH reagent kit (24XCyte, MetaSystems GmbH) supplemented with a set of differentially labeled chromosome arm-specific painting probes (arm-kit, comprising either p- or q-arms of all human chromosomes, except the p-arm of the acrocentric- and Y chromosomes). The mFISH-probe cocktail and the arm-kit are combined and hybridized together to metaphase chromosomes. The armFISH is analyzed in two steps; first, the conventional mFISH image analysis is performed, followed by the arm-kit analysis to reveal the chromosome arms involved. The examples demonstrate the utility of armFISH in defining chromosomal rearrangements of human cancers.

Inactivation of the BRCA1 and BRCA2 breast cancer susceptibility genes has been reported to occur via a germ-line mutation of one allele and a somatic loss of the remaining wild-type allele. We investigated the genetic mechanisms behind the second event in breast tumors from 17 BRCA1 and eight BRCA2 germ-line mutation carriers, as compared with 21 sporadic breast tumors. Microsatellite markers intragenic or in close proximity to both genes were used to analyze imbalances between the mutant and wild-type alleles. The actual and relative gene copy numbers were scored by fluorescence in situ hybridization (FISH) analysis of tumor cells using locus and centromere specific probes. All but one of the informative BRCA1 and BRCA2 tumors exhibited allelic imbalance and loss of the corresponding wild type allele. In contrast to sporadic tumors, however, where allelic imbalance at the BRCA1 and BRCA2 loci correlated well with relative copy number losses by FISH, a simple reduction to a single copy (average copy number ratio 2:1) was found in only two BRCA1 (12%) and four BRCA2 (50%) tumors. The majority of BRCA1 and BRCA2 tumors showed a copy number reduction (relative to reference probe with ratios 4:2, 3:2, 4:3) at corresponding loci, suggesting that a specific physical deletion of the wild-type BRCA gene allele has been followed by a duplication of the remaining mutant allele via polyploidization. Several tumors contained multiple copies of BRCA1 and BRCA2 genes without relative copy number changes, implying that loss of wild-type alleles is executed by alternative mechanisms such as mitotic recombination, non-disjunctional chromosomal loss with or without reduplication, or by gene conversion. A paradoxical relative copy number gain of the mutant allele was evident in three BRCA1 tumors (18%), which could be of biological relevance if a dominant negative or gain-of-function model was ascribed for certain BRCA1 mutants. Our results indicate that complex genetic alterations are operational at the BRCA1 and BRCA2 loci in tumors from genetically predisposed individuals.

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.

DNA amplification at chromosomal region 20q12-q13, which is common in breast cancer, has recently been described also in ovarian tumors. We studied the amplification of the recently identified candidate oncogenes in this region in 24 sporadic, 3 familial and 4 hereditary ovarian carcinomas, and in 8 ovarian cancer cell lines. High-level amplification of at least one of the five nonsyntenic regions at 20q12-q13.2 was found in 13 sporadic (54%) and in all four hereditary tumors. Typically, two or more distinct amplicons (separated by nonamplified DNA) were found coamplified in various combinations. The regions defined by the AIB1 and PTPN1 genes (at 20q12 and 20q13.1, respectively) were amplified in 25% and 29% of the sporadic tumors, also without simultaneous coamplification of other regions. Amplification of AIB1 (a steroid receptor coactivator gene) was associated with estrogen receptor positivity in sporadic ovarian carcinomas (P = 0.01) and showed a tendency to correlate with poor survival of patients. Of the genes amplified in breast cancer, the BTAK gene was amplified in 21%, the MYBL2 gene in 17%, and the ZNF217 gene in 12.5% of the sporadic tumors. The high frequency of gene amplification at 20q12-q13.2 suggests that the genes amplified therein may play a central role in the pathogenesis of sporadic and hereditary ovarian carcinoma.

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.

Although cytotoxic chemotherapy is widely used in advanced breast cancer, there are no powerful predictors for the therapy response. Because topoisomerase IIalpha (Topo IIalpha) is the molecular target for the anthracycline class of anti-cancer drugs, we compared the immunocytochemical assay of Topo IIalpha with other biomarkers in the prediction of clinical response to Topo II inhibitor chemotherapy. Fifty-five patients with advanced breast cancer were treated with a single cytotoxic drug, Topo II-inhibitor, epirubicin (30 mg m(-2) weekly up to 1000 mg m(-2)), as first line cytotoxic chemotherapy. Objective response to treatment was analysed according to UICC criteria. The predictive value of Topo IIalpha expression, c-erbB2 oncoprotein, p53 tumour-suppressor protein, oestrogen (ER) and progesterone receptor (PR), S-phase fraction and DNA ploidy were analysed from representative formalin-fixed paraffin-embedded primary tumour samples. The proportion of Topo IIalpha-positive cells (Topo IIalpha index) failed to predict response to epirubicin therapy. Mean Topo IIalpha scores in 29 responding patients were similar when compared with those with no change in disease progression (n = 13) and those with progressive disease (n = 13) (14.9% +/- 11.4% vs 15.5% +/- 7.6% vs 17.3% +/- 13.2%, not significant). Among the other biomarkers tested, overexpression of c-erbB2 oncoprotein and hormone receptor negativity were significantly associated with poor response. Response rate in patients with c-erbB2-overexpressing tumours was 32% compared with 65% in patients with no c-erbB2 overexpression (P = 0.0058). Accordingly, the response rate for ER-positive patients was 67% compared with 26% in ER-negative patients (P = 0.0021). Although both negative ER status and c-erbB2 overexpression are associated with high Topo IIalpha expression in breast cancer, step-wise logistic regression analysis showed that ER and c-erbB2 were associated with therapy response independent of Topo IIalpha expression. Histological grade, p53, DNA-ploidy, tumour proliferation rate (S-phase fraction), stage of the disease at diagnosis, age of the patient, previous anti-oestrogen therapy or site of metastasis did not predict the response to epirubicin therapy. In conclusion, despite extensive in vitro evidence, expression of Topo IIalpha is unlikely to predict the response to Topo II inhibitor chemotherapy in advanced breast cancer. Among the prognostic biomarkers, overexpression of c-erbB2 oncogene and negative ER may have predictive value in epirubicin therapy in patients with advanced breast cancer. Images Figure 3

Infiltrating lobular carcinoma (ILC) and infiltrating ductal carcinoma (IDC) are distinguished by their histopathological appearance. However, little is known about the differences in genetic changes between lobular cancers and ductal cancers. We used comparative genomic hybridization (CGH) and compared aberrations in 19 ILCs and 46 IDCs. The total number of aberrations was lower in ILC than in IDC. While the average number of DNA copy number losses did not reach significance between them, copy number gains were significantly lower in ILCs. Fifteen of 19 ILCs (79%) showed increased copy number of 1q, and 12 cases (63%) revealed loss of 16q. The presence of these aberrations was independent of nodal status, histologic subtypes (pleomorphic or classic ILC), or BrdUrd-labeling index. ILCs had a higher frequency of 16q loss than did ductal cancers, and a lower frequency of 8q and 20q gains. Our data suggest that the altered growth pattern and clinical presentation which characterize infiltrating lobular cancers are correlated with distinct genetic alterations. Int. J. Cancer 74:513–517, 1997. © 1997 Wiley-Liss, Inc.

To understand the genetic basis and clonal evolution underlying metastatic progression of human breast cancer in vivo, we analyzed the genetic composition of 29 primary breast carcinomas and their paired asynchronous metastases by comparative genomic hybridization and fluorescence in situ hybridization. The mean number of genetic changes by comparative genomic hybridization was 8.7 +/- 5.3 in primary tumors and 9.0 +/- 5.7 in their metastases. Although most of the genetic changes occurred equally often in the two groups, gains of the Xq12-q22 region were enriched in the metastases. According to a statistical analysis of shared genetic changes and breakpoints in paired specimens, 20 of the metastases (69%) showed a high degree of clonal relationship with the corresponding primary tumor, whereas the genetic composition of 9 metastases (31%) differed almost completely from that of the paired primary tumors. In both groups, however, chromosome X inactivation patterns suggested that the metastatic lesions originated from the same clone as the primary tumor. Fluorescence in situ hybridization analysis with probes specific to metastatic clones usually failed to find such cells in the primary tumor sample. In conclusion, detailed characterization of the in vivo progression pathways of metastatic breast cancer indicates that a linear progression model is unlikely to account for the progression of primary tumors to metastases. An early stem line clone apparently evolves independently in the primary tumor and its metastasis, eventually leading to multiple, genetically almost completely different, clones in the various tumor locations in a given patient. The resulting heterogeneity of metastatic breast cancer may underlie its poor responsiveness to therapy and explain why biomarkers of prognosis or therapy responsiveness measured exclusively from primary tumors give a restricted view of the biological properties of metastatic breast cancer.

The alterations in deoxyribonucleic acid (DNA) ploidy and p53 expression during progression of bladder cancer were determined.p53 Expression and DNA ploidy were studied in 51 patients with transitional cell carcinoma of the bladder (mean followup 5 years). Of 29 primarily superficial tumors (stages Ta and T1) 17 became subsequently invasive (greater than stage T2) within an average of 4 years (group 2) and 12 recurred superficially with no sign of progression during a mean followup of 10.8 years (group 1). Of the patients 22 had metastatic disease (group 3). Samples of tumors at diagnosis and recurrence or progression were analyzed by immunohistochemistry and flow cytometry.p53 Accumulation was detected at diagnosis in 29 of the 51 patients (57%), including 3 of 12 (25%) in group 1, 5 of 17 (29%) in group 2 and 14 of 22 (64%) in group 3. The p values for the differences between groups 1 and 3, and 2 and 3 were 0.07 and 0.054, respectively. Abnormal DNA contents were noted in 3 of 12 (25%), 11 of 17 (65%) and 16 of 22 (73%) patients in groups 1 to 3, respectively, and the differences between groups 1 and 2, and 1 and 3 were statistically significant. Using these 2 genetic markers, we found genetic progression to be uncommon in groups 1 and 3, whereas in group 2 an initially negative p53 staining became positive at invasion into the muscle in 5 of 12 patients (42%).The tumors in patients with superficial recurrences are mostly diploid and negative for p53, and those with metastasis are nondiploid and positive for p53 from the beginning, while further genetic progression is uncommon. However, p53 tends to accumulate frequently when the tumor begins to invade the muscle. There seems to be a need for caution against under staging an apparent stage T1 tumor that is positive for p53.

The standard comparative genomic hybridization (CGH) protocol relies on availability of macroscopic tumor samples, which do not contain too much interfering normal cells. Recently, CGH after universal amplification of genomic DNA with degenerate oligonucleotide primed PCR (DOP-PCR) has been used to detect genetic aberrations in microdissected tumor specimens. However, owing to the technical difficulties, CGH results of only few microdissected samples have so far been published. We have developed an improved protocol for DOP-PCR, which includes direct incorporation of fluorochrome-conjugated nucleotides into the PCR product. Among the four polymerase enzymes tested. ThermoSequenase gave the best yield, with PCR products ranging from 100-4,000 bp. A two-step PCR-procedure was used, consisting of a preamplification with low stringency conditions followed by amplification in more stringent conditions. The method was first validated by hybridizing DOP-PCR-amplified normal DNA against nick-translated reference DNA, which showed uniform amd even hybridization result for all chromosomes. Comparison of DOP-PCR CGH to conventional CGH in MCF-7 breast cancer cell line further indicated that genetic aberrations can be reliable detected after DOP-PCR amplification. The sensitivity of the DOP-PCR-CGH was tested by serial dilution of MCF-7 DNA. Fifty picograms of sample DNA (corresponding roughly to two MCF-7 cells) was sufficient for high quality CGH. Experiments with cells microdissected from intraductal breast cancer demonstrated that carcinoma cells from 1 to 2 ducts were sufficient for a successful DOP-PCR CGH analysis. We conclude that the improved DOP-PCR-CGH protocol provides a powerful tool to study genetic aberrations in different histological subpopulations of malignant as well as precancerous lesions. DOP-PCR also improves the success rate of conventional paraffin-block CGH, because a poor quality or a too low yield of extracted DNA can be compensated by universal DNA amplification by DOP-PCR.

Inflammation precedes erosion and rupture of atherosclerotic atheromas and aneurysms. Inflammatory infiltrates of macrophages have been shown to secrete proteolytic enzymes, including matrix metalloproteinases (MMPs), that weaken the arterial wall. The effect of inflammation on arterial structure and remodeling can be studied in primary vascular inflammatory diseases such as in temporal arteritis. We examined the 72-kd gelatinase (MMP-2) and the 92-kd gelatinase (MMP-9) in inflamed and uninvolved temporal arteries from 10 patients with temporal arteritis and 5 controls by immunohistochemistry. The substrates of these enzymes, type IV collagen and elastin, were detected by immunohistochemistry and histochemical staining, respectively. Both diseased and normal artery specimens had moderate staining for immunoreactive MMP-2. Temporal arteritis specimens had clearly enhanced immunostaining for MMP-9 compared with normal arteries. MMP-9 was specifically localized to macrophages in regions of internal elastic lamina disruption, which may thus be of pathological significance.

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.

The molecular mechanisms and genetic changes that lead to the progression of prostate cancer during endocrine therapy are poorly characterized. Here, paired specimens from both untreated primary tumors and from local recurrences were collected from 10 prostate cancer patients treated by conventional androgen deprivation therapy. The genetic progression of the tumors was studied by using interphase fluorescence in situ hybridization and chromosome-specific probes. Six primary tumors (60%) and all ten recurrent tumors were aneuploid by fluorescence in situ hybridization. The recurrent tumors also showed a high degree of chromosome copy number variability from one cell to another. Increased copy number of chromosome X was particularly common in the recurrent tumors. In addition, specific high level amplification of the androgen receptor (AR) gene (Xq12) was detected in three highly aneuploid recurrent tumors. Our findings suggest that hormone-refractory prostate cancers are genetically very complex and show intratumor genetic heterogeneity. Increased copy number of chromosome X and the amplification of the androgen receptor (AR) gene may confer proliferative advantage during androgen deprivation and thus contribute to the development of recurrence.

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.

Breast cancer progression is determined by a complex pattern of multiple genetic aberrations the association of which with patient prognosis is unknown. In this study, we have undertaken a genome-wide screening to detect genetic changes associated with clinical outcome in node-negative breast cancer. Comparative genomic hybridization was used to screen for DNA sequence gains and losses across all human chromosomes in 23 tumors from node-negative breast cancer patients with no disease recurrence after at least 5 years of follow-up and in 25 node-negative patients with recurrence during the first 5 years of follow-up. The total number of genetic aberrations (copy number gains and losses) per tumor was significantly greater in the recurrence group (P = 0.019) and in the subgroup of these patients who died as a result of breast cancer (P = 0.0022). When copy number losses and gains were analyzed separately, only losses were significant (P = 0.013 for recurrence and P = 0.002 for overall survival). Of the individual loci involved, a high level gain of the long arm of chromosome 8 was significantly associated with recurrence (P = 0.01, Fisher's exact test). Furthermore, amplification of DNA sequences at chromosome 20q12-13 was found in 7 cases (15%), 6 of which had early recurrence within 32 months of diagnosis. This genome-wide overview by comparative genomic hybridization suggests that genetically advanced node-negative breast cancers having a high overall number of genetic aberrations may have a poor prognosis and that increased copy number of two specific regions, 8q and 20q13, may confer a more aggressive phenotype. Results of this pilot study suggest that determination of the total number of DNA sequence copy number aberrations may help therapeutic decision making. Specific probes should be developed to test the prognostic value of 8q and 20q12-13 amplifications in large numbers of patients.

There is evidence that tumor angiogenesis, as detected by immunohistochemical staining of endothelium, is of prognostic significance in breast cancer. However, little attention has been paid to possible differences between antibodies or to quantitation of the stained microvessels. We compared three endothelial cell antibodies [anti-human von Willebrand factor (anti-VWF, also termed factor VIII), anti-CD31, and anti-CD34] in archival paraffin-embedded specimens. Anti-CD34 and anti-VWF showed better staining performances than anti-CD31, although the staining results with different antibodies were comparable. Two different methods of microvessel quantitation (the highest microvessel count and percentage microvessel area) were evaluated and also showed significant correlation. From a retrospective database (n = 1000), 77 axillary node-negative invasive ductal breast carcinomas were selected on the basis of clinical outcome to maximize the prognostic power of the sample set (37 died due to a metastatic breast carcinoma, 40 showed no recurrence during 8-yr follow-up). Microvessel quantitations were related to flow cytometric DNA ploidy, c-erb-B-2 overexpression, and estrogen receptor status of the tumor. Surprisingly, neither highest microvessel counts nor microvessel area measurements quantitated with anti-CD34 or anti-VWF immunohistochemistry were able to discriminate between favorable and unfavorable outcome patients. Thus, our results suggest that further evidence is still needed on tumor angiogenesis immunohistochemistry before it can be adopted as a prognostic marker in routine, clinical practice.

It is not clear whether screening for breast cancer works as public health policy and whether early indicators of effect predict an ultimate reduction in mortality. The malignant potentials of 248 breast cancers detected by the screening service in Finland were compared with those of 490 control cancers diagnosed before the screening service was established. Aggressiveness was assessed by DNA flow cytometry and clinical status by cancer size and node involvement. After the first screening round, the results of DNA flow cytometry were the same in cancers diagnosed by screening and in controls; these findings are consistent with the hypothesis that the biological aggressiveness of breast cancer remains constant as the cancer progresses. The proportion of patients with node-negative and small T1 cancers after the first screening was higher among the screened population than among controls, indicating earliness of diagnosis among those screened. Cancers diagnosed in the first round had a low malignant potential, as indicated by the DNA flow-cytometry and by clinical stage. Lower aggressiveness of cancers found by screening than of control cancers would indicate overdiagnosis or length-biased sampling, but not earliness of diagnosis. Screening with mammography is practised as a public-health policy in Finland. The results predict that the mortality reduction found in randomised trials can be repeated with a screening service.

Genetic changes leading to the development of prostate cancer and factors that underlie the clinical progression of the disease are poorly characterized. Here, we used comparative genomic hybridization (CGH) to screen for DNA sequence copy number changes along all chromosomes in 31 primary and 9 recurrent uncultured prostate carcinomas. The aim of the study was to identify those chromosome regions that contain genes important for the development of prostate cancer and to identify genetic markers of tumor progression. CGH analysis indicated that 74% of primary prostate carcinoma showed DNA sequence copy number changes. Losses were 5 times more common than gains and most often involved 8p (32%), 13q (32%), 6q (22%), 16q (19%), 18q (19%), and 9p (16%). Allelic loss studies with 5 polymorphic microsatellite markers for 4 different chromosomes were done from 13 samples and showed a 76% concordance with CGH results. In local recurrences that developed during endocrine therapy, there were significantly more gains (P0.001) and losses (P0.05) of DNA sequences than in primary tumors, with gains of 8q (found in 89% of recurrences versus 6% of primary tumors), X (56% versus 0%), and 7 (56% versus 10%), as well as loss of 8p (78% versus 32%), being particularly often involved. In conclusion, our CGH results indicate that losses of several chromosomal regions are common genetic changes in primary tumors, suggesting that deletional inactivation of putative tumor suppressor genes in these chromosomal sites is likely to underlie development of prostate cancer. Furthermore, the pattern of genetic changes seen in recurrent tumors with the frequent gains of 7, 8q, and X suggests that the progression of prostate cancer and development of hormone-independent growth may have a distinct genetic basis. These chromosome aberrations may have diagnostic utility as markers of prostate cancer progression.

Recommendations are made for hardware and software capabilities that will permit a level of performance of comparative genomic hybridization (CGH) analysis on metaphase chromosomes that is comparable to the best current practice. Guidelines for interpreting the results of CGH analysis in terms of chromosomal gains or losses are also presented. © 1995 Wiley-Liss, Inc.

The effects of the timing of operation in relation to menstrual phase and hormone receptor protein positivity and concentration of the 5 year survival of 267 premenopausal women with operable breast cancer were evaluated. The patients were treated in the Tampere University Hospital Area in 1980-87, and information about menstrual cycle was recorded before the operation. Patients operated on during the luteal phase (days 15-32) had a trend towards a better survival rate (80.4%) than those treated in the follicular phase (days 1-14) (75.9%), but the difference did not reach statistical significance (P = 0.079). There was a small difference in the positivity and concentration of hormone receptor proteins, depending on the phase of the menstrual cycle. A more sensitive analysis found a statistically significant linear association between survival and day since last menstrual period (LMP) which was not totally accounted for by the variation in hormone receptor levels during the menstrual cycle or other main prognostic factors (P = 0.018 by Cox's multivariate regression analysis when LMP was used as a continuous variable). One possible mechanism for the effect of timing can be that physiological changes related to different phases of menstrual cycle unfavourably affect the quality of diagnostic and/or treatment procedures. Variation in the lag between the diagnostic confirmation and the operation of the patient affects the evaluation of such an effect and may account for the inconsistent results reported so far.

The pattern of chromosomal aberrations and their significance in prostate cancer are poorly understood. We studied 23 prostate cancer and 10 benign prostatic hyperplasia (BPH) specimens by fluorescence in situ hybridization (FISH) using pericentromeric repeat-specific probes for 10 different chromosomes. The aims of the study were: 1) to compare the sensitivity of FISH and DNA flow cytometry in aneuploidy detection, 2) to determine which chromosome copy number changes are most common, and 3) which probe combinations would be most effective in aneuploidy diagnosis. Disaggregated tumor cells from formalin-fixed, paraffin-embedded tissues were pretreated with our newly developed method based on hot glycerol solution to improve probe penetration. All BPH specimens were diploid by DNA flow cytometry and showed no numerical chromosome aberrations by FISH. In prostate cancer, flow cytometry showed abnormal DNA content in 35% of cases, whereas 74% were abnormal by FISH. Aberrant copy number of chromosomes 8 (48% of cases), X (43% of cases), and 7 (39% of cases) were most common. Ninety-four percent of all aneuploid cases would have been detected with these three probes alone. Simple chromosome losses were uncommon but in DNA tetraploid tumors relative losses (trisomy or disomy) of several chromosomes were often found, suggesting progression of prostate cancer through tetraploidization followed by losses of selected chromosomes. In conclusion, our results indicate that FISH using three selected chromosome-specific probes is two to three times more sensitive than flow cytometric DNA content analysis in aneuploidy detection.

The reproducibility in quantitation of proliferation activity, determined using the monoclonal antibody 19A2 to proliferating cell nuclear antigen (PCNA), was tested in visual and computer-assisted analyses of brain tumor material. The PCNA labeling index was scored using count (PCNA-LI, visual and computer analyses) and area (PCNA-LIa, computer analysis) estimates of immunopositivity. The quality of immunostaining was the most important reason for variation in the assessment results. Other significant variation sources in the assessment were experience in selecting microscopic fields and distinguishing immunopositive nuclei from immunonegative ones. Computer-assisted analysis improved the reproducibility of quantitation between different observers (visual rPCNA-LI = 0.624 versus computer assisted rPCNA-LI = 0.904). Also, the use of PCNA-LIa improved the intraobserver and interobserver reproducibility in different stainings (observer 1:rPCNA-LI = 0.857 versus rPCNA-LIa = 0.874; observers 1 and 2: rPCNA-LI = 0.904 versus rPCNA-LIa = 0.927; observers 3 and 4: rPCNA-LI = 0.848 versus rPCNA-LIa = 0.906). PCNA-LIa by computerized image analysis improves accuracy in the evaluation of the granularly expressed PCNA level. Furthermore, the effect of tumor heterogeneity on the assessment results can be diminished with the computerized method because large tissue areas can be analyzed faster.

British Journal of Cancer (2002) 86, 310–311. DOI: 10.1038/sj/bjc/6600005 www.bjcancer.com © 2002 The Cancer Research Campaign

NORs are loops of DNA that transcribe to ribosomal RNA, and their number reflects the rDNA transcription, and, in this way, cell proliferation.The effect of muscle immobilization and remobilization on the number of nucleolar organizer regions (NORs) in the muscle cells and fibroblasts of rat calf muscles was studied histologically using a silver-staining technique (AgNOR).In muscle-cell nuclei, immobilization of 3 weeks produced a significant decrease in the number of AgNORs per nucleus. This phenomenon was reversible since, free remobilization of 4 weeks and especially intensified remobilization (free mobilization of 1 week plus treadmill running of 3 weeks) produced significant increase in the AgNOR counts. The treadmill running also produced a significant increase in the number of AgNORs in the muscle cells of contralateral (nonimmobilized) limbs. In intramuscular fibroblast nuclei, immobilization caused, in turn, a steady increase in the AgNOR count, the change being significant after 3 weeks. Free remobilization returned the count towards controls, and intensified remobilization returned it to the control level.Previous studies have shown that in the muscle cells immobilization decreases and remobilization (training) increases intracellular protein synthesis and that in the fibroblasts the opposite happens (immobilization increases and remobilization decreases intramuscular connective tissue formation). Our study gives indirect evidence that the number of NORs is a marker of ribosomal protein synthesis activity in non-proliferating cells. The cell-proliferation activity was proved to be zero using a proliferating-cell-nuclear-antigen technique.

Aberrant expression of the p53 suppressor gene was evaluated in 102 cases of astrocytic neoplasms. Immunohistochemical staining with a monoclonal antibody (DO-7) and a polyclonal antibody (CM-1) to p53 protein (both wild type and mutant) on formalin-fixed paraffin sections showed a strong correlation with malignancy grade. The staining was positive in 49% of malignant neoplasms (grades III and IV) and in 19 to 29% of grade II astrocytomas, whereas none of the grade I tumors were positive. p53 expression was significantly associated with proliferation rate determined by immunohistochemical proliferating cell nuclear antigen (PCNA)-staining (median PCNA-labeling index (%): 4.22 (DO-7-positive) versus 1.18 (DO-7-negative), P < 0.0001; 4.02 (CM-1-positive) versus 1.18 (CM-1-negative), P < 0.001). Interestingly, in the glioblastoma group (n = 44), p53-positive tumors had higher proliferation indices, suggesting that histologically similar tumors could be divided into prognostically different subgroups by immunohistochemical demonstration of aberrant p53 expression.

We evaluated whether proliferating cell nuclear antigen (PCNA) immunohistochemistry with antigen retrieval could be used as a measure of cell proliferation in archival, formalin-fixed, paraffin-embedded tissues and whether the staining results have long-term prognostic significance in axillary node-negative breast cancer. Primary tumor samples obtained from 109 axillary-node-negative breast cancer cases were used for the study. The best staining results were obtained with the 19A2 antibody after microwave heating in a solution of saturated lead thiocyanate. Using this method, there was a significant correlation (linear regression, r = 0.580, P < 0.001) between the proportion of PCNA19A2-positive carcinoma cells (PCNA19A2 score) and DNA flow cytometric S phase fraction. A high PCNA19A2 score was associated with high mitotic count, DNA aneuploidy, and absence of estrogen receptors. Axillary-node-negative patients with a high PCNA19A2 score (cut-point 8%) had significantly worse prognosis than those with a low PCNA19A2 score (P = 0.008). According to a Cox multivariate analysis, PCNA19A2 score had independent prognostic value but only if S phase fraction was excluded from the analysis. In our study, the PCNAPC10 score correlated weakly only with primary tumor size (analysis of variance) and prognosis (5-year univariate survival analysis), but the significance of these findings needs further evaluation. In conclusion, PCNA immunohistochemistry with the 19A2 antibody after an appropriate antigen retrieval treatment may offer a useful alternative to DNA flow cytometry for the analysis of cell proliferation activity from formalin-fixed, paraffin-embedded breast carcinomas.

We analyzed the predictive value of a number of demographic and anesthesiological variables with respect to the three most common complications during spinal anesthesia: hypotension, bradycardia, and nausea. A stepwise logistic regression model was created, using data from a prospective study of 1752 patients to combine the predictive value of all entry variables. The highest risk factors for hypotension were: age greater than or equal to 50, a sensory level above Th6, receiving bupivacaine as a local anesthetic, body mass index greater than or equal to 30, and receiving opiate as a premedication. An anesthetic level above Th6 and age below 50 were primarily associated with bradycardia. Females and those with a high sensory level or receiving opiate as a premedication were at significant risk of nausea. The model was also reliably predictive for a separate group of 200 consecutive spinal anesthesia patients. Thus, the risk model may be clinically useful in identifying high-risk patients requiring additional attention.

The macromolecular composition and morphometry of the myotendineal junction (MTJ) of slow-twitch (type 1) and fast-twitch (type 2) muscle fibers were studied in gastrocnemius-soleus-Achilles unit of the rat. Proteoglycans and glycosaminoglycans, type III collagen, fibronectin and laminin could be detected at the myotendineal junction. Due to the membrane folding finger-like processes were seen at the MTJ. The processes of type 1 fibers were greater in size. However, due to the subdivisions the processes of type 2 muscle fibers had a significantly greater surface length per muscle cell diameter than type 1 fibers. The myotendineal endings of both fiber types had a characteristic basal lamina, which was about three times thicker than in the longitudinal site of the same muscle cells. The basal lamina of type 1 fibers at the MTJ was significantly thicker than that of type 2 fibers.