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Background: We recently showed that bone morphogenetic protein 7 (BMP7) is overexpressed in primary breast tumors. Here we explored the clinical significance of BMP7 expression in breast cancer. Materials and methods: This study included 483 breast cancer patients with complete clinicopathological information and up to 15 years of follow-up. Samples contained 241 lobular carcinomas, 242 ductal carcinomas, and 40 local recurrences. BMP7 protein expression was determined using immunohistochemistry. Results: BMP7 was expressed in 47% of the primary tumor samples and 13% of the local recurrences. The primary tumors expressed BMP7 more often than the corresponding local recurrences (P = 0.004). BMP7 expression was dependent on the tumor subtype; 57% of the lobular carcinomas but only 37% of the ductal carcinomas were BMP7 positive (P = 0.0001). BMP7 expression was associated with accelerated bone metastasis formation (P = 0.040), especially in ductal carcinomas (P = 0.033), and multivariate analysis confirmed that BMP7 is an independent prognostic indicator for early bone metastasis development (P = 0.032). Conclusion: BMP7 is clearly associated with bone metastasis formation and thus might have clinical utility in identification of patients with increased risk of bone metastasis. This is the first time that bone inducing factor BMP7 has been linked to the bone metastasis process in breast cancer. [ABSTRACT FROM AUTHOR]

The aim of the study was to evaluate the long-term survival of patients with invasive lobular carcinomas (ILC) and invasive ductal carcinomas (IDC) and the metastatic behavior of these two disease entities. Originally, all consecutive patients with pure lobular invasive breast cancers diagnosed between 1990 and 1999 in the area served by the Tampere University Hospital and their matched IDC controls were identified and re-evaluated histopathologically in this follow-up study, resulting in a total of 243 ILCs and 243 IDCs. Data on recurrences and survival were collected until the end of year 2009. Statistical analyses including Kaplan-Meier method, log-rank test, Fisher's exact test and Cox regression analysis were performed with the PASW Statistics 18.0 computer program. P-values of <0.05 were considered statistically significant. Within the mean follow-up time of 10.04 years, locoregional recurrences were significantly more common among the ILCs than IDCs (35 vs. 20, p = 0.04), but no differences in the total number of distant recurrences or bilaterality were observed. However, when the first distant recurrence sites were studied, ILC patients had significantly less lung metastases (p = 0.04), but more skin metastases (p = 0.04). During the whole follow-up period IDCs metastasized significantly more frequently to the lungs (p = 0.002), whereas gastrointestinal metastases were more common among ILCs (p = 0.02). Although the known favorable prognostic factors (hormone receptor positivity, low grade, low s-phase) were more common for the ILCs, the disease-free survival, the overall survival and the survival after recurrence did not differ between the groups. However, the Cox-regression model showed significantly worse survival for ILCs after adjusting for age, TNM-status, grade and ER-positivity (p = 0.004). In conclusion, ILC and IDC differ in respect for visceral metastases. Despite the known favorable prognostic factors and originally favorable survival, patients with lobular histology appear to have a worse survival in the multivariate analysis after a prolonged follow-up., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Bone morphogenetic proteins (BMPs) are extracellular signaling molecules that belong to the transforming growth factor β (TGFβ) superfamily and are known to regulate cell proliferation, differentiation and motility, especially during development. BMP4 has an indispensable role in vertebrate development while limited information on BMP4 expression and function exists in adult tissues. Nevertheless, its contribution to cancer development and progression has gained increasing interest in recent years. Functional studies, especially in breast cancer, have implicated BMP4 both in inhibition of cell proliferation and in promotion of cell migration and invasion. To gain an insight into the function of BMP4 in normal and cancer tissues, BMP4 protein expression levels were analyzed by immunohistochemistry in 34 different normal organs/tissues, 34 different tumor types and finally in 486 breast cancer samples where possible associations between BMP4 and clinicopathological parameters were statistically evaluated. In over 20% of normal and malignant tissues, BMP4 was expressed at high level. Strong expression was observed particularly in some normal epithelial cells, such as bladder and stomach, and in squamous cell carcinomas. In breast cancer, strong BMP4 expression was detected in 25% of patients, and was associated with low proliferation index and increased frequency of tumor recurrence. Taken together, BMP4 is expressed in a subset of normal adult tissues and is likely to contribute to tissue homeostasis. However, in tumors, BMP4 expression levels vary considerably, implying diverse roles in different tumor types. This role is biphasic in breast cancer as BMP4 expression is linked to reduced proliferation and increased recurrence, thus corroborating our previous in-vitro functional data.

Background: Dynamic infrared (IR) imaging is an emerging functional imaging modality for the detection of breast cancer without evidence of optimal imaging and diagnostic application., Purpose: To evaluate dynamic IR imaging in breast cancer diagnostics by comparing a stepwise diagnostic scheme to digital mammography and postoperative histopathology., Material and Methods: Dynamic IR imaging of breasts was undertaken preoperatively with a long-wave quantum well (QWIP) and two mid-wave photovoltaic (PV) IR cameras in 10 cases (age 34-80 years) with breast cancer size 6-45 mm on mammography. Image stabilization, two-phase frequency analysis, and two image-processing algorithms were applied., Results: Combining image processing with frequency analysis proved advantageous in detecting breast cancer. The IR imaging process recognized the cancer area independently of tissue density, cancer size, and cancer appearance on mammography. Compared to histopathology, all cancers yielded abnormal analysis results, including one case of ductal carcinoma in situ. Evidence of lymphatic invasion in postoperative histopathology, imaging with PV camera, and image processing with the Wiener filtering combination correlated with highest confidence between normal and cancer tissue measured by the calculated superiority value., Conclusion: Dynamic IR imaging with image-processing-guided frequency analysis is a promising modality for breast cancer detection and may not have the tissue-dependent limitations of mammography. Our results encourage further work on medical IR imaging and comparison to established breast-imaging modalities.

Five combinations of image-processing algorithms were applied to dynamic infrared (IR) images of six breast cancer patients preoperatively to establish optimal enhancement of cancer tissue before frequency analysis. mid-wave photovoltaic (PV) IR cameras with 320x254 and 640x512 pixels were used. The signal-to-noise ratio and the specificity for breast cancer were evaluated with the image-processing combinations from the image series of each patient. Before image processing and frequency analysis the effect of patient movement was minimized with a stabilization program developed and tested in the study by stabilizing image slices using surface markers set as measurement points on the skin of the imaged breast. A mathematical equation for superiority value was developed for comparison of the key ratios of the image-processing combinations. The ability of each combination to locate the mammography finding of breast cancer in each patient was compared. Our results show that data collected with a 640x512-pixel mid-wave PV camera applying image-processing methods optimizing signal-to-noise ratio, morphological image processing and linear image restoration before frequency analysis possess the greatest superiority value, showing the cancer area most clearly also in the match centre of the mammography estimation.

In this novel study the breasts of 15 women with palpable breast cancer were preoperatively imaged with three technically different infrared (IR) cameras - micro bolometer (MB), quantum well (QWIP) and photo voltaic (PV) - to compare their ability to differentiate breast cancer from normal tissue. The IR images were processed, the data for frequency analysis were collected from dynamic IR images by pixel-based analysis and from each image selectively windowed regional analysis was carried out, based on angiogenesis and nitric oxide production of cancer tissue causing vasomotor and cardiogenic frequency differences compared to normal tissue. Our results show that the GaAs QWIP camera and the InSb PV camera demonstrate the frequency difference between normal and cancerous breast tissue; the PV camera more clearly. With selected image processing operations more detailed frequency analyses could be applied to the suspicious area. The MB camera was not suitable for tissue differentiation, as the difference between noise and effective signal was unsatisfactory.

Background: We recently showed that bone morphogenetic protein 7 (BMP7) is overexpressed in primary breast tumors. Here we explored the clinical significance of BMP7 expression in breast cancer., Materials and Methods: This study included 483 breast cancer patients with complete clinicopathological information and up to 15 years of follow-up. Samples contained 241 lobular carcinomas, 242 ductal carcinomas, and 40 local recurrences. BMP7 protein expression was determined using immunohistochemistry., Results: BMP7 was expressed in 47% of the primary tumor samples and 13% of the local recurrences. The primary tumors expressed BMP7 more often than the corresponding local recurrences (P = 0.004). BMP7 expression was dependent on the tumor subtype; 57% of the lobular carcinomas but only 37% of the ductal carcinomas were BMP7 positive (P = 0.0001). BMP7 expression was associated with accelerated bone metastasis formation (P = 0.040), especially in ductal carcinomas (P = 0.033), and multivariate analysis confirmed that BMP7 is an independent prognostic indicator for early bone metastasis development (P = 0.032)., Conclusion: BMP7 is clearly associated with bone metastasis formation and thus might have clinical utility in identification of patients with increased risk of bone metastasis. This is the first time that bone inducing factor BMP7 has been linked to the bone metastasis process in breast cancer.

Bone morphogenetic proteins (BMPs) regulate diverse cellular processes, such as proliferation, differentiation, and apoptosis. The BMPs have been studied in several cancers, but thus far contradictory results have been obtained and, especially in breast cancer, information on BMPs is still limited. We performed a systematic expression survey of BMPs and their receptors in breast cancer. mRNA expression was studied of seven BMP ligands (BMP2-BMP8) and six receptors (ACVR1, BMPR1A, BMPR1B, BMPR2, ACVR2A, and ACVR2B) that specifically mediate BMP signals. Expression levels were determined in 22 breast cancer cell lines, 39 primary breast tumors, normal human mammary epithelial cell line, and normal mammary gland using semiquantitative RT-PCR. The expression frequencies and expression levels of different BMPs varied considerably in breast cancer with BMP4 and BMP7 being most frequently expressed and showing highest expression levels. The BMP specific receptors were more uniformly expressed and indicated that breast cancer is fully capable of transmitting BMP signals. Expression frequencies and levels for both the ligands and the receptors were in good concordance between the breast cancer cell lines and primary tumors. We can conclude that breast cancers possess functional BMP signaling machinery on the cell surface with distinct differences in the expression of various BMP ligands. Our survey focuses the attention particularly toward BMP4 and BMP7 and suggests their importance in breast cancer. Breast cancer cell lines and the data generated here serve as a good resource for further studies on BMP function in breast cancer.

Increased copy numbers of 17q23 chromosomal region have been shown to occur in different tumour types and to be associated with tumour progression and with poor prognosis. Several genes have earlier been proposed as potential oncogenes at this region largely on the grounds of cell lines studies. In this study, we performed a systematic gene expression survey on 26 primary breast tumours with known 17q23 amplification status by quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR). The 17q23 amplicon is restricted to an approximately 5 MB region in breast cancer and contains 29 known genes. Our survey revealed a statistically significant (P<0.01) difference between the high level and no amplification groups in a set of eleven genes whereas no difference between the moderate and the non-amplified tumour groups were observed. Interestingly, these 11 genes were located adjacent to one another within a 1.56 Mb core region in which all except one of the genes were overexpressed. These data suggest that only high-level amplification at the 17q23 amplicon core leads to elevated gene expression in breast cancer. Moreover, our results highlight the fact that 17q23 amplicon carries multiple candidate genes and that this may be a more common event in gene amplification than previously thought.

Mechanisms of prostate cancer progression during hormonal therapy and the pathobiologic consequences of androgen receptor (AR) gene amplification are inadequately known. To further investigate the hypothesis that AR gene amplification is associated with increased cell proliferation, we analyzed 123 paraffin-embedded prostate cancer specimens from men who experienced tumor relapse during androgen withdrawal therapy. We used fluorescence in situ hybridization to quantify AR gene copy number and Ki-67 immunohistochemistry to determine cell proliferation. One third of the tumors showed AR gene amplification. Among tumors with AR amplification, the mean cell proliferation rate was 19.8 (SD, 12.3; 95% confidence interval [CI], 15.4-24.1), whereas it was 13.0 (SD, 15.9; 95% CI, 9.1-16.8) in tumors without amplification (P = .032). In the best fitting logistic regression model, only proliferation remained significant (P = .040). When the median Ki-67 labeling index (6.7%) of all tumors was used as a cutoff point, the tumors with AR amplification were more frequently highly proliferating than tumors with no amplification (P = .010; odds ratio, 3.4; 95% CI, 1.4-8.3). Our results imply that progression of prostate cancer during androgen withdrawal therapy is associated with AR gene amplification and increased cell proliferation rate in one third of tumors. We suggest that AR gene amplification is an important molecular mechanism underlying the increase in proliferation rate of a substantial fraction of recurrent prostate carcinomas. However, efforts should be targeted to develop prostate cancer cell lines to study causal relationships between AR gene amplification and various biologic variables.

Bone morphogenetic proteins (BMP) make up a family of extracellular signaling molecules that play a critical role in vertebrate development and both inhibit and stimulate growth in cancer cells. BMP7 was recently identified in our genomewide copy number and expression survey as being activated through amplification in breast cancer cell lines. In the present study, we further explored BMP7 gene copy number and expression changes in 22 breast cancer cell lines and 146 primary breast tumors. FISH analysis revealed that BMP7 copy number varied greatly from one cell line to another, with three cell lines showing extremely high-level amplification. Among primary tumors, BMP7 copy number was increased in 16% of the cases. BMP7 mRNA expression was determined in the cell lines and in a subset of 44 tumor samples by RT-PCR or quantitative real-time RT-PCR, respectively. Despite elevated mRNA levels in cancer cells, there was no significant association between copy number increase and mRNA expression, even though the highest expression was seen in cell lines and tumors with increased BMP7 copy number. Most interestingly, immunohistochemical analysis revealed BMP7 protein staining in all 11 breast cancer cell lines examined and strongly elevated BMP7 protein expression in 71.4% of the tumor samples as compared to normal mammary epithelium. Our results illustrate the frequent involvement of BMP7 alterations in breast cancer and especially highlight overexpression of the BMP7 protein in a very large fraction of primary breast tumors, thus suggesting a possible functional role for BMP7 in breast cancer development.

The serine-threonine protein phosphatase PPM1D is likely to play an important role in tumorigenesis. Through inactivation of p38 MAPK, PPM1D acts as a negative feedback regulator of p53 tumour suppressor gene and controls the expression of other cell cycle regulatory proteins, such as CCND1. In addition, recent knock-out mouse studies implicated PPM1D in the regulation of p16 expression and the RB tumour suppressor pathway. Here we explored the role of PPM1D aberrations in primary breast cancer. PPM1D copy number analysis showed amplification in 11% (13/117) of the tumours and quantitative real-time RT-PCR revealed a significant correlation (p = 0.0148) between PPM1D amplification and increased expression. PPM1D amplification occurred almost exclusively in tumours with wild-type p53 suggesting that these events are mutually exclusive and further confirming the role of PPM1D as a negative regulator of p53. Interestingly, PPM1D amplification was associated with ERBB2 expression (p = 0.0001) thus implying that PPM1D aberrations occurs in tumours with poor prognosis. We also explored the expression levels of two possible downstream targets of PPM1D. However, immunohistochemical analyses revealed no differences in the staining patterns of CCND1 and p16 proteins in tumours with or without PPM1D aberrations, thus suggesting that previous data from animal model experiments is not directly transferable to primary human tumours. On the other hand, these key cellular proteins are likely to be regulated through a complex fashion in breast cancer and apparently PPM1D represents only one of these mechanisms. Taken together, our findings substantiate an important role for PPM1D in breast cancer.

Hereditary hemochromatosis (HH), the most common genetic disease in northern Europeans, is an autosomal recessive disorder of iron metabolism. The association between hepatocellular carcinoma and HFE homozygosity is well documented, but recently HFE hetero- and homozygosity has also been linked to nonhepatocellular malignancies, including female breast cancer. We hypothesized that C282Y and H63D mutations in the HFE gene could contribute to male breast cancer (MBC) and prostate cancer (PC) susceptibility at the population level in Finland. We screened the 2 major HFE mutations, H63D and C282Y, from 116 MBC cases diagnosed in Finland between 1967 and 1996, 843 consecutive unselected PC cases diagnosed at the Pirkanmaa Hospital District between 1999 and 2001 and 480 anonymous blood donor controls by minisequencing. Our results indicate that the frequencies of the HFE mutations do not significantly differ between MBC and PC patients and the population-based controls. No significantly altered risks for MBC or PC among carriers of the 2 variants were observed. However, HFE mutations were seen twice as often among carriers of a common BRCA2 mutation 9346(-2)A-->G compared with the rest of the MBC cases, indicating that HFE may be an MBC risk modifier gene among BRCA2 mutation carriers. In conclusion, our results indicate a minor role for the HFE mutations C282Y and H63D in the causation of MBC and PC, but carriers of both BRCA2 9346(-2)A-->G and an HFE mutation may be at an increased risk., (Copyright 2005 Wiley-Liss, Inc.)

Genetic events underlying the pathogenesis of breast cancer have been studied extensively and several clinically significant markers have been identified. For example, amplification and overexpression of the ERBB2 oncogene is associated with poor prognosis in breast cancer and ERBB2 serves as a target for antibody-based therapy. Current knowledge on the pathogenesis of male breast cancer (MBC) is limited. The purpose of our study was to investigate the potential relevance of a series of genes known to be amplified in female breast cancer (FBC) in a the development and pathogenesis of MBC. To this end, we applied fluorescence in situ hybridization and immunohistochemistry to the analysis of 128 breast tumors from males. Amplification of ERBB2, MYC, PPM1D and ZNF217 was detected rarely (1-2% of tumors) indicating a considerably lower amplification frequency than in FBC. CCND1 amplification was observed in 12% of cases, being in good concordance with findings from FBC. In addition, CCND1 overexpression was detected in 63% of tumors and was associated with ER positivity (p < 0.0001). Our results indicate distinct differences in the genetic basis of MBC and FBC and suggest that marked differences exist in the pathogenesis of these diseases. The lack of ERBB2 involvement was especially unexpected and implies that ERBB2-targeted therapies are unlikely to be beneficial in MBC. Furthermore, the high frequency of hormone receptor positivity and the association between ER positivity and CCND1 overexpression supports the notion that hormonal regulation is likely to be essential for the development of MBC.

The etiology and pathogenesis of male breast cancer (MBC) are poorly known. This is due to the fact that the disease is rare, and large-scale genetic epidemiologic studies have been difficult to carry out. Here, we studied the frequency of eight recurrent Finnish BRCA2 founder mutations in a large cohort of 154 MBC patients (65% diagnosed in Finland from 1967 to 1996). Founder mutations were detected in 10 patients (6.5%), eight of whom carried the 9346(-2) A>G mutation. Two novel mutations (4075 delGT and 5808 del5) were discovered in a screening of the entire BRCA2 coding region in 34 samples. However, these mutations were not found in the rest of the 120 patients studied. Patients with positive family history of breast and/or ovarian cancer were often BRCA2 mutation carriers (44%), whereas those with no family history showed a low frequency of involvement (3.6%; P < .0001). Finally, we found only one Finnish MBC patient with 999 del5, the most common founder mutation in Finnish female breast cancer (FBC) patients, and one that explains most of the hereditary FBC and MBC cases in Iceland. The variation in BRCA2 mutation spectrum between Finnish MBC patients and FBC patients in Finland and breast cancer patients in Iceland suggests that modifying genetic and environmental factors may significantly influence the penetrance of MBC and FBC in individuals carrying germline BRCA2 mutations in some populations.

Genetic risk factors for male breast cancer (MBC) are poorly understood. High penetrance genes such as BRCA1 or BRCA2 account for only a small proportion of the disease. A 1100delC mutation in CHEK2 (previously known as CHK2), a cell-cycle checkpoint kinase, has been implicated in predisposition of Li-Fraumeni syndrome (LFS) and breast cancer in families suggestive of LFS. This 1100delC mutation has also been shown to confer a 2-fold increase of breast cancer risk in women and a 10-fold increase of risk in men. It was estimated to account for 1% of breast cancers in women and as much as 9% of breast cancers in men at the population level based on analysis of breast cancer families without BRCA1 or BRCA2 mutations. We wanted to evaluate the significance of CHEK2 1100delC in predisposition to MBC by assessing its frequency in a population-based material of 114 Finnish MBC patients. Two patients (1.8%) carried the 1100delC mutation. The mutation frequency among MBC cases was similar to that seen in population controls (26/1885, 1.4%). Our results indicate that CHEK2 1100delC variant does not substantially increase the risk of male breast cancer at the population level. We cannot exclude the fact that a small fraction of hereditary, family-positive male breast cancers could be attributable to CHEK2 mutations., (Copyright 2003 Wiley-Liss, Inc.)

Amplification of the ERBB2 oncogene at 17q12 is clinically the most relevant genetic aberration in breast cancer and several studies have linked ERBB2 activation to poor clinical outcome. The development of targeted antibody-based therapy for ERBB2-overexpressing tumors and the possible role of ERBB2 as a predictor of chemotherapy treatment response have further emphasized the essential role of ERBB2 in breast cancer. Here, we performed a detailed characterization of the molecular events occurring at the ERBB2 amplicon in primary breast tumors. Analysis of the amplicon structure in 330 breast tumors by fluorescence in situ hybridization to a tissue microarray revealed a 280-kb common region of amplification that contains 10 transcribed sequences, including eight known genes. The expression levels of these 10 transcripts were determined in 36 frozen samples of grade-matched ERBB2-amplified and -nonamplified (as determined by fluorescence in situ hybridization) primary breast tumors by using quantitativereal-time reverse transcriptase-polymerase chain reaction. A highly significant association between amplification and expression levels was observed for six of these genes, including ERBB2 and two uncharacterized hypothetical proteins, MGC9753 and MGC14832. These results support the recent findings on the influence of copy number on gene expression levels and highlight novel genes that might contribute to the clinical behavior of ERBB2-amplified breast tumors.

Mutations in the androgen receptor (AR) gene have been suggested to predispose to male breast cancer (MBC). Studies on MBC patients have not been based on the mutation screening of the entire coding region of the AR and the number of subjects has been small. Therefore, some AR gene alterations may have remained undetected. In the present study, we have comprehensively screened the entire coding region of the AR gene for mutations and also studied the role of AR CAG and GGC repeat lengths as risk factors for MBC in a cohort of 32 Finnish MBC patients. To estimate the possible involvement of the prostate cancer predisposing AR Arg726Leu germ-line mutation in MBC, this mutation was tested in 117 MBC patients. No germ-line mutations were found and the CAG and GGC repeat lengths were similar among MBC cases as among Scandinavian population. Our data indicate that the AR gene does not substantially contribute to MBC predisposition.

Background: Germline mutations in recessive cancer predisposition genes are uncovered by somatic genetic deletions during tumor development. Analysis of genetic changes in tumor tissues from patients with an inherited predisposition may therefore highlight regions of the genome containing susceptibility or modifier genes. Our aim was to characterize genetic changes in familial prostate cancer, Methods: Twenty-one primary prostate cancers from 19 Finnish prostate cancer families were analyzed for somatic genetic changes by comparative genomic hybridization (CGH)., Results: The average number of genetic alterations per tumor was 4.0 +/- 1.9, distributed equally among losses and gains. The most common losses were found at chromosomal regions 13q14-q22 (29%), 8p12-pter (24%), and 6q13-q16 (14%), and the most common gains at 19p (25%), 19q (14%) and 7q (14%)., Conclusions: These results suggest that prostate cancers in genetically predisposed individuals arise for the most part through similar somatic genetic progression pathways as sporadic prostate cancers. This also implies that the biological properties of tumors from the two groups may not be different from one another., (Copyright 2001 Wiley-Liss, Inc.)

A significant proportion of familial breast cancers cannot be explained by mutations in the BRCA1 or BRCA2 genes. We applied a strategy to identify predisposition loci for breast cancer by using mathematical models to identify early somatic genetic deletions in tumor tissues followed by targeted linkage analysis. Comparative genomic hybridization was used to study 61 breast tumors from 37 breast cancer families with no identified BRCA1 or BRCA2 mutations. Branching and phylogenetic tree models predicted that loss of 13q was one of the earliest genetic events in hereditary cancers. In a Swedish family with five breast cancer cases, all analyzed tumors showed distinct 13q deletions, with the minimal region of loss at 13q21-q22. Genotyping revealed segregation of a shared 13q21 germ-line haplotype in the family. Targeted linkage analysis was carried out in a set of 77 Finnish, Icelandic, and Swedish breast cancer families with no detected BRCA1 and BRCA2 mutations. A maximum parametric two-point logarithm of odds score of 2.76 was obtained for a marker at 13q21 (D13S1308, theta = 0.10). The multipoint logarithm of odds score under heterogeneity was 3.46. The results were further evaluated by simulation to assess the probability of obtaining significant evidence in favor of linkage by chance as well as to take into account the possible influence of the BRCA2 locus, located at a recombination fraction of 0.25 from the new locus. The simulation substantiated the evidence of linkage at D13S1308 (P < 0.0017). The results warrant studies of this putative breast cancer predisposition locus in other populations.

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.

With the recent rapid expansion in the use of the comparative genomic hybridization (CGH) technique, increased attention to quality control is essential. In the present study, we show that despite optimization and standardization of metaphase preparation techniques and the commercial availability of metaphase spreads, batch-to-batch variability of the preparations remains a significant problem. To facilitate reliable CGH analysis despite this variability, we have developed a rapid denaturation test to assess the quality of the preparations without hybridization and quantitative image analysis criteria for assuring the day-to-day quality of CGH experiments, including sensitivity, specificity, and dynamic range. Monitoring the dynamic range of the hybridizations was found to be particularly critical for achieving sensitive and reliable CGH results. This reliability can be achieved, for example, by hybridization of a green-labeled normal male DNA against red-labeled female DNA and monitoring of the green:red ratio of the X chromosome in relation to that of the autosomes.

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.

Ductal carcinoma in situ (DCIS) is considered a direct precursor of invasive ductal breast cancer (IDC). We combined tissue microdissection and comparative genomic hybridization to identify genetic changes in five DCIS lesions with no invasion and in two that were adjacent to IDC. Extensive genetic changes characterized pure DCIS cases with gains of 1q, 6q, 8q, and Xq as well as losses of 17p and chromosome 22 being most often involved. Except for the Xq gain, these changes are also common to IDC. Separate analysis of DCIS and IDC components in the same tumor revealed an almost identical pattern of genetic changes in one case, whereas substantial differences were found in another. We conclude that many of the common genetic changes in IDC may take place before development of invasive growth. However, a simple linear progression model may not always account for the DCIS-IDC transition.

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.

Purpose: Up to 30% to 40% of metastases from hormone receptor-positive primary breast cancer do not respond to endocrine therapy. We studied how often hormone receptor status changes between primary and recurrent tumors and whether such a change might explain unresponsiveness to endocrine therapy., Patients and Methods: Primary breast cancer samples and matched asynchronous recurrences were studied from 50 patients who had not received any adjuvant therapy. Estrogen receptor (ER) and progesterone receptor (PR) status was determined immunohistochemically from histologically representative formalin-fixed paraffin-embedded tumor samples. ER status was ascertained by mRNA in situ hybridization., Results: Thirty-five (70%) of 50 primary tumors were positive for ER and 30 (60%) for PR. Hormone receptor status of the recurrent tumor differed from that of the primary tumor in 18 cases (36%). Discordant cases were due to the loss of ER (n = 6), loss of PR (n = 6), or loss of both receptors (n = 6). Receptor-negative primary tumors were always accompanied by receptor-negative recurrences. Among 27 patients with ER-positive primary tumors, loss of ER was a significant predictor (P = .0085) of poor response to subsequent endocrine therapy. Only one of eight patients (12.5%) with lost ER expression responded to tamoxifen therapy, whereas the response rate was 74% (14 of 19) for patients whose recurrent tumors retained ER expression., Conclusion: Loss of ER expression in recurrent breast cancer should be considered as a cause for poor response to endocrine therapy in primarily ER-positive patients. We conclude that analysis of recurrent tumor samples may improve the predictive value of ER and PR assays.

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.