Tuula Kuukasjärvi

Optimizing DOP‐PCR for universal amplification of small DNA samples in comparative genomic hybridization

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 and 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 reliably 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. Genes Chromosom. Cancer 18:94–101, 1997.

A comprehensive expression survey of bone morphogenetic proteins in breast cancer highlights the importance of BMP4 and BMP7

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.

Amplification of a 280-Kilobase Core Region at the ERBB2 Locus Leads to Activation of Two Hypothetical Proteins in Breast Cancer

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.

Quality control of CGH: impact of metaphase chromosomes and the dynamic range of hybridization.

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.

The serine-threonine protein phosphatase PPM1D is frequently activated through amplification in aggressive primary breast tumours

SummaryThe 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.

Bone morphogenetic protein 7 is widely overexpressed in primary breast cancer

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.

CHEK2 1100delC is not a risk factor for male breast cancer population

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.

Bone morphogenetic protein 4 expression in multiple normal and tumor tissues reveals its importance beyond development

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.

Androgen receptor gene alterations in Finnish male breast cancer.

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.

Frequent amplification and overexpression of CCND1 in male breast cancer

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.