Juha Kononen

Gene-expression profiles in hereditary breast cancer.

BACKGROUND Many cases of hereditary breast cancer are due to mutations in either the BRCA1 or the BRCA2 gene. The histopathological changes in these cancers are often characteristic of the mutant gene. We hypothesized that the genes expressed by these two types of tumors are also distinctive, perhaps allowing us to identify cases of hereditary breast cancer on the basis of gene-expression profiles. METHODS RNA from samples of primary tumor from seven carriers of the BRCA1 mutation, seven carriers of the BRCA2 mutation, and seven patients with sporadic cases of breast cancer was compared with a microarray of 6512 complementary DNA clones of 5361 genes. Statistical analyses were used to identify a set of genes that could distinguish the BRCA1 genotype from the BRCA2 genotype. RESULTS Permutation analysis of multivariate classification functions established that the gene-expression profiles of tumors with BRCA1 mutations, tumors with BRCA2 mutations, and sporadic tumors differed significantly from each other. An analysis of variance between the levels of gene expression and the genotype of the samples identified 176 genes that were differentially expressed in tumors with BRCA1 mutations and tumors with BRCA2 mutations. Given the known properties of some of the genes in this panel, our findings indicate that there are functional differences between breast tumors with BRCA1 mutations and those with BRCA2 mutations. CONCLUSIONS Significantly different groups of genes are expressed by breast cancers with BRCA1 mutations and breast cancers with BRCA2 mutations. Our results suggest that a heritable mutation influences the gene-expression profile of the cancer.

Expression of Cytokeratins 17 and 5 Identifies a Group of Breast Carcinomas with Poor Clinical Outcome

While several prognostic factors have been identified in breast carcinoma, the clinical outcome remains hard to predict for individual patients. Better predictive markers are needed to help guide difficult treatment decisions. In a previous study of 78 breast carcinoma specimens, we noted an association between poor clinical outcome and the expression of cytokeratin 17 and/or cytokeratin 5 mRNAs. Here we describe the results of immunohistochemistry studies using monoclonal antibodies against these markers to analyze more than 600 paraffin-embedded breast tumors in tissue microarrays. We found that expression of cytokeratin 17 and/or cytokeratin 5/6 in tumor cells was associated with a poor clinical outcome. Moreover, multivariate analysis showed that in node-negative breast carcinoma, expression of these cytokeratins was a prognostic factor independent of tumor size and tumor grade.

High-Throughput Tissue Microarray Analysis to Evaluate Genes Uncovered by cDNA Microarray Screening in Renal Cell Carcinoma

Many genes and signaling pathways are involved in renal cell carcinoma (RCC) development. However, genetic tumor markers have not gained use in RCC diagnostics and prognosis prediction. Identification and evaluation of new molecular parameters are of utmost importance in cancer research and cancer treatment. Here we present a novel approach to rapidly identify clinically relevant molecular changes in cancer. To identify genes with relevance to RCC, a cDNA array analysis was first performed on 5184 cDNA clones on a filter to screen for genes with differential expression between the renal cancer cell line CRL-1933 and normal kidney tissue. There were 89 differentially expressed genes in the cancer cell line, one of them coding for vimentin, a cytoplasmic intermediate filament. In a second step, a renal cancer tissue microarray containing 532 RCC specimen was used to determine vimentin expression by immunohistochemistry. Vimentin expression was seen frequently in clear cell (51%) and papillary RCC (61%), but rarely in chromophobe RCC (4%) and oncocytomas (12%). Furthermore, vimentin expression was significantly associated with poor patient prognosis (P < 0.007) independent of grade and stage. These results obtained from minute arrayed tumor samples match well with previous findings on vimentin expression in renal tumors. It is concluded that the combination of tumor arrays and cDNA arrays is a powerful approach to rapidly identify and further evaluate genes that play a role in tumor biology.

Genome screening by comparative genomic hybridization

Comparative genomic hybridization (CGH) provides a molecular cytogenetic approach for genome-wide scanning of differences in DNA sequence copy number. The technique is now attracting wide-spread interest, especially among cancer researchers. The rapidly expanding database of CGH publications already covers about 1500 tumors and is beginning to reveal genetic abnormalities that are characteristic of certain tumor types or stages of tumor progression. Six novel gene amplifications, as well as a locus for a cancer-predisposition syndrome, have been discovered based on CGH data. CGH has now been established as a first-line screening technique for cancer researchers and will serve as a basis for ongoing efforts to develop high-resolution next-generation genome scanning, such as the microarray technology.

Microarrays of bladder cancer tissue are highly representative of proliferation index and histological grade

The number of genes suggested to play a role in cancer biology is rapidly increasing. To be able to test a large number of molecular parameters in sufficiently large series of primary tumours, a tissue microarray (TMA) approach has been developed where samples from up to 1000 tumours can be simultaneously analysed on one glass slide. Because of the small size of the individual arrayed tissue samples (diameter 0.6 mm), the question arises of whether these specimens are representative of their donor tumours. To investigate how representative are the results obtained on TMAs, a set of 2317 bladder tumours that had been previously analysed for histological grade and Ki67 labelling index (LI) was used to construct four replica TMAs from different areas of each tumour. Clinical follow‐up information was available from 1092 patients. The histological grade and the Ki67 LI were determined for every arrayed tumour sample (4×2317 analyses each). Despite discrepancies in individual cases, the grade and Ki67 information obtained on minute arrayed samples were highly similar to the data obtained on large sections (p<0.0001). Most importantly, every individual association between grade or Ki67 LI and tumour stage or prognosis (recurrence, progression, tumour‐specific survival) that was observed in large section analysis could be fully reproduced on all four replica TMAs. These results show that intra‐tumour heterogeneity does not significantly affect the ability to detect clinico‐pathological correlations on TMAs, probably because of the large number of tumours that can be included in TMA studies. TMAs are a powerful tool for rapid identification of the biological or clinical significance of molecular alterations in bladder cancer and other tumour types. Copyright

Loss of estrogen receptor in recurrent breast cancer is associated with poor response to endocrine therapy.

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.

High-Throughput Tissue Microarray Analysis of Cyclin E Gene Amplification and Overexpression in Urinary Bladder Cancer

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

Prevalence of KIT Expression in Human Tumors

PURPOSE KIT is a target for imatinib mesylate (Gleevec; Novartis Pharma, Basel, Switzerland). Gastrointestinal stromal tumors (GISTs) express KIT and respond favorably to imatinib therapy. To determine other tumors in which such a molecular targeted therapy might be indicated, we investigated KIT expression in different human tumor types. Because recent studies in GISTs suggest that KIT-activating mutations predict response to imatinib therapy, we also sequenced a subset of positive tumors. MATERIALS AND METHODS More than 3,000 tumors from more than 120 different tumor categories were analyzed by immunohistochemistry in a tissue microarray format. Seven commercially available anti-KIT antibodies were initially evaluated. The antibody A4502 (DAKO) was selected for analysis because of a high frequency of positivity in GIST and low staining background in other tissues. To determine the frequency of KIT mutations in various tumor types, the exons 2, 8, 9, 11, 13, and 17 (where mutations previously were reported) were sequenced in 36 tumors with strong KIT expression. RESULTS KIT positivity was detected in 28 of 28 GISTs (100%), 42 of 50 seminomas (84%), 34 of 52 adenoid-cystic carcinomas (65%), 14 of 39 malignant melanomas (35%), and eight of 47 large-cell carcinomas of the lung (17%), as well as in 47 additional tumor types. KIT mutations were found in six of 12 analyzed GISTs, but only in one of 24 other tumors. CONCLUSION The results suggest that KIT expression occurs infrequently in most tumor types and that, with the exception of GISTs, KIT gene mutations are rare in immunohistochemically KIT-positive tumors.

Tissue microarrays (TMAs) for high-throughput molecular pathology research.

A rapidly increasing number of genes are being suspected to play a role in cancer biology. To evaluate the clinical significance of newly detected potential cancer genes, it is usually required to examine a high number of well‐characterized primary tumors. Using traditional methods of molecular pathology, this is a time consuming endeavor rapidly exhausting precious tissue resources. To allow for a high throughput tissue analysis we have developed a “tissue chip” approach (Kononen et al., Nat. Med. 1998;4:844–7). Using this tissue microarray (TMA) technology, samples from up to 1,000 different tumors are arrayed in one recipient paraffin block, sections of which can be used for all kind of in situ analyses. Section from TMA blocks can then be utilized for the simultaneous analysis of up to 1,000 different tumors on the DNA, RNA or protein level. TMAs allow a high throughput molecular analysis of thousands of tumors within a few hours. All currently available data have suggested that minute arrayed tissue specimens are highly representative of their donor tissues. There are multiple different types of TMAs that can be utilized in cancer research including multi tumor arrays (containing different tumor types), tumor progression arrays (tumors of different stages) and prognostic arrays (tumors with clinical endpoints). The combination of multiple different TMAs allows a very quick but comprehensive characterization of biomarkers of interest. We anticipate that the use of TMAs will greatly accelerate the transition of basic research findings to clinical applications.

A Nuclear Factor, ASC-2, as a Cancer-amplified Transcriptional Coactivator Essential for Ligand-dependent Transactivation by Nuclear Receptors in Vivo

Many transcription coactivators interact with nuclear receptors in a ligand- and C-terminal transactivation function (AF2)-dependent manner. We isolated a nuclear factor (designated ASC-2) with such properties by using the ligand-binding domain of retinoid X receptor as a bait in a yeast two-hybrid screening. ASC-2 also interacted with other nuclear receptors, including retinoic acid receptor, thyroid hormone receptor, estrogen receptor α, and glucocorticoid receptor, basal factors TFIIA and TBP, and transcription integrators CBP/p300 and SRC-1. In transient cotransfections, ASC-2, either alone or in conjunction with CBP/p300 and SRC-1, stimulated ligand-dependent transactivation by wild type nuclear receptors but not mutant receptors lacking the AF2 domain. Consistent with an idea that ASC-2 is essential for the nuclear receptor function in vivo, microinjection of anti-ASC-2 antibody abrogated the ligand-dependent transactivation of retinoic acid receptor, and this repression was fully relieved by coinjection of ASC-2-expression vector. Surprisingly, ASC-2 was identical to a gene previously identified during a search for genes amplified and overexpressed in breast and other human cancers. From these results, we concluded that ASC-2 is a bona fidetranscription coactivator molecule of nuclear receptors, and its altered expression may contribute to the development of cancers.