An integrated genomic approach identifies ARID1A as a candidate tumor-suppressor gene in breast cancer.

Tumor-suppressor genes (TSGs) have been classically defined as genes whose loss of function in tumor cells contributes to the formation and/or maintenance of the tumor phenotype. TSGs containing nonsense mutations may not be expressed because of nonsense-mediated RNA decay (NMD). We combined inhibition of the NMD process, which clears transcripts that contain nonsense mutations, with the application of high-density single-nucleotide polymorphism arrays analysis to discriminate allelic content in order to identify candidate TSGs in five breast cancer cell lines. We identified ARID1A as a target of NMD in the T47D breast cancer cell line, likely as a consequence of a mutation in exon-9, which introduces a premature stop codon at position Q944. ARID1A encodes a human homolog of yeast SWI1, which is an integral member of the hSWI/SNF complex, an ATP-dependent, chromatin-remodeling, multiple-subunit enzyme. Although we did not find any somatic mutations in 11 breast tumors, which show DNA copy-number loss at the 1p36 locus adjacent to ARID1A, we show that low ARID1A RNA or nuclear protein expression is associated with more aggressive breast cancer phenotypes, such as high tumor grade, in two independent cohorts of over 200 human breast cancer cases each. We also found that low ARID1A nuclear expression becomes more prevalent during the later stages of breast tumor progression. Finally, we found that ARID1A re-expression in the T47D cell line results in significant inhibition of colony formation in soft agar. These results suggest that ARID1A may be a candidate TSG in breast cancer.

Using biointelligence to search the cancer genome: an epistemological perspective on knowledge recovery strategies to enable precision medical genomics.

Genomic profiling is beginning to extend beyond the many applications in discovery research toward direct medical applications that hold the promise of more precise and individualized health-care delivery. There are many barriers and challenges that still need to be overcome before 'Precision Medical Genomics' can deliver the promise of more informed patient care, not the least of which is the unmet need for a new conceptual framework for recovering, understanding and translating potentially useful information from a single genome. Although a wide spectrum of scientific strategies, bioinformatic approaches, IT tools and knowledge resources have been developed to support discovery research, the interpretive requirements for recovering clinically useful insights from an individual's genome are different in many ways from those of traditional research goals. In this study, we compare and contrast the fundamental conceptual differences that distinguish 'research' to discover generalized knowledge from 'search' to recover individualized knowledge. We also consider the merits of applying evidence-based medicine and traditional scientific methods when n=1, and consider an alternative perspective based on a translational engineering approach and intelligence for interpreting genomic information from an individual case. Although the general idea of biological intelligence-based knowledge recovery that we introduce here can be broadly applied for personal genomics across many indications in medicine, we make a case that the need for adopting such a paradigm is greatest for supporting the management of complex diseases, and particularly suited for supporting therapeutic decisions in medical oncology. Early concepts for designing and implementing this kind of 'BioIntelligence' solution will be discussed. We also review the anticipated challenges of implementing genomic analysis and biological intelligence-based solutions in the practice of medical oncology by discussing some of the related pragmatic considerations for deploying the first generation of a 'Precision Medical Genomics' solution that can evolve and improve over time.

A common nonsense mutation in EphB2 is associated with prostate cancer risk in African American men with a positive family history.

BACKGROUND: The EphB2 gene was recently implicated as a prostate cancer (PC) tumour suppressor gene, with somatic inactivating mutations occurring in approximately 10% of sporadic tumours. We evaluated the contribution of EphB2 to inherited PC susceptibility in African Americans (AA) by screening the gene for germline polymorphisms. METHODS: Direct sequencing of the coding region of EphB2 was performed on 72 probands from the African American Hereditary Prostate Cancer Study (AAHPC). A case-control association analysis was then carried out using the AAHPC probands and an additional 183 cases of sporadic PC compared with 329 healthy AA male controls. In addition, we performed an ancestry adjusted association study where we adjusted for individual ancestry among all subjects, in order to rule out a spurious association due to population stratification. RESULTS: Ten coding sequence variants were identified, including the K1019X (3055A-->T) nonsense mutation which was present in 15.3% of the AAHPC probands but only 1.7% of 231 European American (EA) control samples. We observed that the 3055A-->T mutation significantly increased risk for prostate cancer over twofold (Fisher's two sided test, p = 0.003). The T allele was significantly more common among AAHPC probands (15.3%) than among healthy AA male controls (5.2%) (odds ratio 3.31; 95% confidence interval 1.5 to 7.4; p = 0.008). The ancestry adjusted analyses confirmed the association. CONCLUSIONS: Our data show that the K1019X mutation in the EphB2 gene differs in frequency between AA and EA, is associated with increased risk for PC in AA men with a positive family history, and may be an important genetic risk factor for prostate cancer in AA.

Failure of hormone therapy in prostate cancer involves systematic restoration of androgen responsive genes and activation of rapamycin sensitive signaling.

Androgen deprivation therapy for advanced prostate cancer is often effective, but not curative. Molecular pathways mediating the therapeutic response and those contributing to the subsequent hormone-refractory cell growth remain poorly understood. Here, cDNA microarray analysis of human CWR22 prostate cancer xenografts during the course of androgen deprivation therapy revealed distinct global gene expression profiles in primary, regressing and recurrent tumors. Elucidation of the genes involved in the transition between these states implicated specific molecular mechanisms in therapy failure and tumor progression. First, we identified a set of androgen-responsive genes whose expression decreased during the therapy response, but was then systematically restored in the recurrent tumors. In addition, altered expression of genes that encode known targets of rapamycin or that converge on the PI3K/AKT/FRAP pathway was observed in the recurrent tumors. Further suggestion for the involvement of these genes in hormone-refractory prostate cancer came from the observation that cells established from the recurrent xenografts were strongly inhibited in vitro by rapamycin. The results of this functional genomic analysis suggest that the combined effect of re-expression of androgen-responsive genes as well as the activation of rapamycin-sensitive signaling may drive prostate cancer progression, and contribute to the failure of androgen-deprivation therapy.

Comparison of p53 mutations in patients with localized osteosarcoma and metastatic osteosarcoma.

BACKGROUND: In some malignancies, p53 mutations are associated with tumor progression. To address the role of p53 mutations in the development and progression of osteosarcoma, the authors analyzed specimens from 247 patients with primary localized osteosarcomas and 25 patients with osteosarcomas that were metastatic at the time of diagnosis. The group included 27 matched biopsy-resection specimens and 21 biopsy-metastasis paired specimens. METHODS: The authors examined the nature and location of p53 mutations (exons 4-10) by polymerase chain reaction-single-strand conformation polymorphism and confirmed mutations by direct DNA sequencing. RESULTS: The overall frequency of p53 mutations was 22% (60 of 272 specimens), with 13 of 60 mutations located in exons 4 or 10. A similar proportion of localized osteosarcomas had alterations of the p53 gene (55 of 247 specimens; 22.3%) compared with tumors from patients who had metastases at the time of diagnosis (5 of 25 specimens; 20%; P = 0.96). Patients who had p53 missense mutations were older compared with patients who had nonsense alterations or a wild type gene (P = 0.01). Examination of paired biopsy-resection and biopsy-metastasis specimens revealed that the p53 status was concordant between the biopsy and later tumor specimens in all patients. CONCLUSIONS: The p53 mutation status did not differentiate between patients who presented with a localized osteosarcoma and those who presented with metastases at the time of diagnosis. The current data indicate that p53 mutations are not late events in osteosarcoma tumor progression, because they are evident before the development of metastases. The inclusion of exons 4 and 10 increased the sensitivity of the analysis.

From chromosomal alterations to target genes for therapy: integrating cytogenetic and functional genomic views of the breast cancer genome.

A vast number of recurrent chromosomal alterations have been implicated in cancer development and progression. However, most of the genes involved in recurrent chromosomal alterations in solid tumors remain unknown, despite the recent substantial progress in genomic research and availability of high-throughput technologies. For example, it is now possible to quickly identify large numbers of differentially expressed genes in cancer specimens using cDNA microarrays. Integration of this "functional genomic view" of the cancer genome with the "cytogenetic view" could lead to the identification of genes playing a critical role in cancer development and progression. In this review, we illustrate how the combination of three different microarray technologies, cDNA, CGH, and tissue microarrays, makes it possible to directly identify genes involved in chromosomal rearrangements in cell line model systems and then rapidly explore their significance as potential diagnostic and therapeutic targets in human primary breast cancer progression.

p53 missense but not truncation mutations are associated with low levels of p21(CIP1/WAF1) mRNA expression in primary human sarcomas.

Many growth-suppressing signals converge to control the levels of the CDK inhibitor p21(CIP1/WAF1). Some human cancers exhibit low levels of expression of p21(CIP1/WAF1) and mutations in p53 have been implicated in this down-regulation. To evaluate whether the presence of p53 mutations was related to the in vivo expression of p21(CIP1/WAF1) mRNA in sarcomas we measured the p21(CIP1/WAF1) mRNA levels for a group of 71 primary bone and soft tissue tumours with known p53 status. As expected, most tumours with p53 mutations expressed low levels of p21(CIP1/WAF1)mRNA. However, we identified a group of tumours with p53 gene mutations that exhibited normal or higher levels of p21(CIP1/WAF1) mRNA. The p53 mutations in the latter group were not the common missense mutations in exons 4-9, but were predominantly nonsense mutations predicted to result in truncation of the p53 protein. The results of this study suggest that different types of p53 mutations can have different effects on the expression of downstream genes such as p21(CIP1/WAF1) in human sarcomas.

Comprehensive copy number and gene expression profiling of the 17q23 amplicon in human breast cancer.

The biological significance of DNA amplification in cancer is thought to be due to the selection of increased expression of a single or few important genes. However, systematic surveys of the copy number and expression of all genes within an amplified region of the genome have not been performed. Here we have used a combination of molecular, genomic, and microarray technologies to identify target genes for 17q23, a common region of amplification in breast cancers with poor prognosis. Construction of a 4-Mb genomic contig made it possible to define two common regions of amplification in breast cancer cell lines. Analysis of 184 primary breast tumors by fluorescence in situ hybridization on tissue microarrays validated these results with the highest amplification frequency (12.5%) observed for the distal region. Based on GeneMap'99 information, 17 known genes and 26 expressed sequence tags were localized to the contig. Analysis of genomic sequence identified 77 additional transcripts. A comprehensive analysis of expression levels of these transcripts in six breast cancer cell lines was carried out by using complementary DNA microarrays. The expression patterns varied from one cell line to another, and several overexpressed genes were identified. Of these, RPS6KB1, MUL, APPBP2, and TRAP240 as well as one uncharacterized expressed sequence tag were located in the two common amplified regions. In summary, comprehensive analysis of the 17q23 amplicon revealed a limited number of highly expressed genes that may contribute to the more aggressive clinical course observed in breast cancer patients with 17q23-amplified tumors.

Hormone therapy failure in human prostate cancer: analysis by complementary DNA and tissue microarrays.

BACKGROUND: The molecular mechanisms underlying the progression of prostate cancer during hormonal therapy have remained poorly understood. In this study, we developed a new strategy for the identification of differentially expressed genes in hormone-refractory human prostate cancer by use of a combination of complementary DNA (cDNA) and tissue microarray technologies. METHODS: Differences in gene expression between hormone-refractory CWR22R prostate cancer xenografts (human prostate cancer transplanted into nude mice) and a xenograft of the parental, hormone-sensitive CWR22 strain were analyzed by use of cDNA microarray technology. To validate the data from cDNA microarrays on clinical prostate cancer specimens, a tissue microarray of specimens from 26 prostates with benign prostatic hyperplasia, 208 primary prostate cancers, and 30 hormone-refractory local recurrences was constructed and used for immunohistochemical detection of protein expression. RESULTS: Among 5184 genes surveyed with cDNA microarray technology, expression of 37 (0.7%) was increased more than twofold in the hormone-refractory CWR22R xenografts compared with the CWR22 xenograft; expression of 135 (2.6%) genes was reduced by more than 50%. The genes encoding insulin-like growth factor-binding protein 2 (IGFBP2) and 27-kd heat-shock protein (HSP27) were among the most consistently overexpressed genes in the CWR22R tumors. Immunohistochemical analysis of tissue microarrays demonstrated high expression of IGFBP2 protein in 100% of the hormone-refractory clinical tumors, in 36% of the primary tumors, and in 0% of the benign prostatic specimens (two-sided P =.0001). Overexpression of HSP27 protein was demonstrated in 31% of the hormone-refractory tumors, in 5% of the primary tumors, and in 0% of the benign prostatic specimens (two-sided P =.0001). CONCLUSIONS: The combination of cDNA and tissue microarray technologies enables rapid identification of genes associated with progression of prostate cancer to the hormone-refractory state and may facilitate analysis of the role of the encoded gene products in the pathogenesis of human prostate cancer.

Molecular and immunohistochemical identification of p53 alterations in bone and soft tissue sarcomas.

p53 has been shown to suppress tumor growth by regulating the cell cycle and by triggering apoptosis. Acquired somatic mutations of the p53 gene have been observed in a variety of human malignancies, and these result in a loss of its tumor suppressor function. To examine the occurrence of p53 abnormalities in bone and soft tissue sarcomas, 113 tumors were subjected to molecular analysis and mutations were confirmed in 16 tumors. The frequency of p53 alterations varied among the different subtypes of bone and soft tissue sarcomas, being observed predominantly in osteosarcomas (8/34 cases), rhabdomyosarcomas (2/3 cases), Ewing's sarcomas (1/5 cases), and liposarcomas (3/21 cases). In contrast, p53 gene mutations were detected at a lower frequency in malignant fibrous histiocytomas (2/34 cases) and not at all in nine chondrosarcomas and five leiomyosarcomas. Immunohistochemical staining of p53 protein was performed on 69 cases and compared to the DNA results. For 64 cases the results were concordant: 56 sarcomas were considered to have wild-type p53 by both techniques. As well, increased p53 protein expression was observed in eight of the nine tumors with p53 gene mutations. However, positive p53 staining was also seen in four sarcomas which had no detectable p53 mutations in exons 5 through 9. Because some sarcomas exhibit amplification and overexpression of MDM-2, which may interact with p53 and cause stabilization of wild-type p53 protein, we examined these tumors for MDM-2 amplification. None of the tumors with MDM-2 amplification exhibited p53 immunopositivity. Very weak p53 reactivity was detected in four malignant fibrous histiocytomas that had received either chemotherapy or radiotherapy. Of 16 metastatic lesions examined, only one contained a p53 mutation. In addition, for five cases in which both the original lesion and its metastases were analyzed, p53 alterations were not observed in the metastases if the tumor was wild-type at presentation. These data suggest that p53 alterations occur at different frequencies in various subtypes of sarcoma and, although detected in metastatic lesions, are not associated more frequently with progression.

Low levels of expression of an inhibitor of cyclin-dependent kinases (CIP1/WAF1) in primary breast carcinomas with p53 mutations.

Recently, several groups have isolated a cell cycle inhibitor gene (CIP1/WAF1) that is highly induced by wild-type, but not mutant forms of the p53 tumor suppressor. To test the hypothesis that p53 regulates CIP1/WAF1 expression in vivo, we evaluated CIP1/WAF1 mRNA expression levels in breast carcinomas from individuals with axillary node-negative disease with and without p53 mutations using quantitative reverse transcription-PCR. The data demonstrate that there is a strong negative correlation between the presence of p53 mutations and CIP1/WAF1 expression, suggesting that p53 mutations may reduce its ability to induce CIP1/WAF1 in vivo. In this study we observed tumors with low levels of CIP1/WAF1 mRNA in which there were no detectable p53 mutations. Determination of the CIP1/WAF1 levels in such specimens may provide a complementary strategy for analyzing the effects of p53 defects and/or may suggest the presence of alterations (such as coding mutations outside the conserved regions, promoter mutations, etc.) that may be missed by standard techniques.

Two variants of the CIP1/WAF1 gene occur together and are associated with human cancer.

Several groups have recently isolated and characterized an inhibitor of cyclin-dependent kinases, p21CIP1/WAF1 which is transcriptionally induced by wild-type but not mutant p53. It is likely that p21CIP1/WAF1 mediates the growth suppression effects of p53 by arresting the cell cycle at the G1/S checkpoint, and by inducing apoptosis. To test the hypothesis that primary human tumors have mutations in the CIP1/WAF1 gene which propagates the carcinogenic process, we examined primary breast and sarcoma tumor specimens for alterations in the CIP1/WAF1 gene. Unique, or acquired somatic mutations were not observed indicating that they are not selected for during the carcinogenic process; however, two common variants were identified. The variants were not unique to tumors as 10.7% of normal individuals exhibited the variants. Nonetheless, the frequency of the variants in tumors with wild-type p53 (20.4%) was significantly greater (p = 0.05) than in normal DNAs. In contrast, the frequency of the variants (4.1%) was found to be significantly lower in tumors with p53 mutations (p = 0.006). These data suggest that the occurrence of the variants may have a direct effect on tumor development and may, in some cases, be incompatible with p53 mutations.