Purpose: The study aim to identify novel molecular subtypes of ovarian cancer by gene expression profiling with linkage to clinical and pathologic features. Experimental Design: Microarray gene expression profiling was done on 285 serous and endometrioid tumors of the ovary, peritoneum, and fallopian tube. K-means clustering was applied to identify robust molecular subtypes. Statistical analysis identified differentially expressed genes, pathways, and gene ontologies. Laser capture microdissection, pathology review, and immunohistochemistry validated the array-based findings. Patient survival within k-means groups was evaluated using Cox proportional hazards models. Class prediction validated k-means groups in an independent dataset. A semisupervised survival analysis of the array data was used to compare against unsupervised clustering results. Results: Optimal clustering of array data identified six molecular subtypes. Two subtypes represented predominantly serous low malignant potential and low-grade endometrioid subtypes, respectively. The remaining four subtypes represented higher grade and advanced stage cancers of serous and endometrioid morphology. A novel subtype of high-grade serous cancers reflected a mesenchymal cell type, characterized by overexpression of N-cadherin and P-cadherin and low expression of differentiation markers, including CA125 and MUC1. A poor prognosis subtype was defined by a reactive stroma gene expression signature, correlating with extensive desmoplasia in such samples. A similar poor prognosis signature could be found using a semisupervised analysis. Each subtype displayed distinct levels and patterns of immune cell infiltration. Class prediction identified similar subtypes in an independent ovarian dataset with similar prognostic trends. Conclusion: Gene expression profiling identified molecular subtypes of ovarian cancer of biological and clinical importance.

Tumor IHC analysis of four MMR proteins and MSI testing provide a highly sensitive strategy for identifying MMR gene mutation-carrying, early-onset colorectal cancer patients, half of whom would have been missed using Amsterdam Criteria alone. Tumor-based approaches for triaging early-onset colorectal cancer patients for MMR gene mutation testing, irrespective of family history, appear to be an efficient screening strategy for HNPCC.

Wnt signaling increases bone mass by stimulating osteoblast lineage commitment and expansion and forms the basis for novel anabolic therapeutic strategies being developed for osteoporosis. These strategies include derepression of Wnt signaling by targeting secreted Wnt pathway antagonists, such as sclerostin. However, such therapies are associated with safety concerns regarding an increased risk of osteosarcoma, the most common primary malignancy of bone. Here, we analyzed 5 human osteosarcoma cell lines in a high-throughput screen for epigenetically silenced tumor suppressor genes and identified Wnt inhibitory factor 1 (WIF1), which encodes an endogenous secreted Wnt pathway antagonist, as a candidate tumor suppressor gene. In vitro, WIF1 suppressed β-catenin levels in human osteosarcoma cell lines, induced differentiation of human and mouse primary osteoblasts, and suppressed the growth of mouse and human osteosarcoma cell lines. Wif1 was highly expressed in the developing and mature mouse skeleton, and, although it was dispensable for normal development, targeted deletion of mouse Wif1 accelerated development of radiation-induced osteosarcomas in vivo. In primary human osteosarcomas, silencing of WIF1 by promoter hypermethylation was associated with loss of differentiation, increased β-catenin levels, and increased proliferation. These data lead us to suggest that derepression of Wnt signaling by targeting secreted Wnt antagonists in osteoblasts may increase susceptibility to osteosarcoma.

There is increasing evidence showing that the stromal cells surrounding cancer epithelial cells, rather than being passive bystanders, might have a role in modifying tumor outgrowth. The molecular basis of this aspect of carcinoma etiology is controversial. Some studies have reported a high frequency of genetic aberrations in carcinoma-associated fibroblasts (CAFs), whereas other studies have reported very low or zero mutation rates. Resolution of this contentious area is of critical importance in terms of understanding both the basic biology of cancer as well as the potential clinical implications of CAF somatic alterations. We undertook genome-wide copy number and loss of heterozygosity (LOH) analysis of CAFs derived from breast and ovarian carcinomas using a 500K SNP array platform. Our data show conclusively that LOH and copy number alterations are extremely rare in CAFs and cannot be the basis of the carcinomapromoting phenotypes of breast and ovarian CAFs. © 2008 Nature Publishing GroupCorrespondence should be addressed to I.G.C. ian.campbell@petermac.org. AUTHOR CONTRIBUTIONS I.G.C., I.H. and W.Q. designed the study and wrote the paper. W.Q. undertook the bulk of the experimental work including tissue microdissection, SNP genotyping and microsatellite analysis. K.P. provided cell lines, academic support and assisted in manuscript preparation. A.S., E.R.T., M.R. and K.L.G. assisted in SNP genotyping. However, not all studies have identified genetic alterations in CAFs; for example, one study did not find any clonally selected somatic genetic alterations in CAFs separated from fresh breast cancer biopsies using array comparative genomic hybridization (CGH) and SNP array analysis 17 , although these CAFs were epigenetically distinct from those from normal breast tissue, as demonstrated by subsequent genome-wide DNA methylation studies 18 .The evidence for somatic genetic alterations as important mediators of the CAF phenotype is controversial and conflicting. We hypothesized that the contradictory data may in part be a reflection of inherent technical limitations of the various methodologies used. Therefore, we took advantage of innovative SNP array-based technologies 19 to investigate in detail the genomic integrity of CAFs microdissected from fresh frozen primary human ovarian and breast cancers as well as short-term cultures of primary breast CAFs.We assessed the sensitivity of the Affymetrix 500K SNP array platform to detect copy number and LOH in the context of normal DNA contamination in a mixing experiment using tumor epithelial cell DNA from a microdissected primary ovarian cancer that was mixed with various ratios of matched normal DNA. This tumor harbors a complex copy number profile on chromosome 17, including regions of high level copy number gain and regions of LOH with and without associated copy number loss. As shown in Supplementary Figure 1a online, the single copy number gain was clearly visible at 70% tumor DNA, and the high level gain was still discernible at 25% tumor DNA. LOH w...

The molecular basis for the inverse relationship between differentiation and tumorigenesis is unknown. The function of runx2, a master regulator of osteoblast differentiation belonging to the runt family of tumor suppressor genes, is consistently disrupted in osteosarcoma cell lines. Ectopic expression of runx2 induces p27KIP1, thereby inhibiting the activity of S-phase cyclin complexes and leading to the dephosphorylation of the retinoblastoma tumor suppressor protein (pRb) and a G1 cell cycle arrest. Runx2 physically interacts with the hypophosphorylated form of pRb, a known coactivator of runx2, thereby completing a feed-forward loop in which progressive cell cycle exit promotes increased expression of the osteoblast phenotype. Loss of p27KIP1 perturbs transient and terminal cell cycle exit in osteoblasts. Consistent with the incompatibility of malignant transformation and permanent cell cycle exit, loss of p27KIP1 expression correlates with dedifferentiation in high-grade human osteosarcomas. Physiologic coupling of osteoblast differentiation to cell cycle withdrawal is mediated through runx2 and p27KIP1, and these processes are disrupted in osteosarcoma.

The BRAF(T1799A) mutation encodes BRAF(V600E) that leads to activation of the mitogen-activated protein kinase pathway. This study aimed to assess the clinico-pathological features of primary invasive melanomas containing the BRAF(T1799A) mutation. Patients (n=251) with invasive primary melanomas from Australia were interviewed and examined with respect to their melanoma characteristics and risk factors. Independent review of pathology, allele-specific PCR for the BRAF(T1799A) mutation, immunohistochemical staining with Ki67, and phospho-histone-H3 (PH3) were performed. The BRAF(T1799A) mutation was found in 112 (45%) of the primary melanomas. Associations with the BRAF(T1799A) mutation (P<0.05) were as follows: low tumor thickness (odds ratio (OR)=3.3); low mitotic rate (OR=2.0); low Ki67 score (OR=5.0); low PH3 score (OR=3.3); superficial spreading melanoma (OR=10.0); pigmented melanoma (OR=3.7); a lack of history of solar keratoses (OR=2.7); a location on the trunk (OR=3.4) or extremity (OR=2.0); a high level of self-reported childhood sun exposure (OR=2.0); < or =50 years of age (OR=2.5); and fewer freckles (OR=2.5). We conclude that the BRAF(T1799A) mutation has associations with host phenotype, tumor location, and pigmentation. Although implicated in the control of the cell cycle, the BRAF(T1799A) mutation is associated with a lower rate of tumor proliferation.

Patients suspected on clinical grounds to have hereditary non-polyposis colorectal cancer (HNPCC) may be offered laboratory testing in order to confirm the diagnosis and to facilitate screening of pre-symptomatic family members. Tumours from an affected family member are usually pre-screened for microsatellite instability (MSI) and/or loss of immunohistochemical expression of mismatch repair (MMR) genes prior to germline MMR gene mutation testing. The efficiency of this triage process is compromised by the more frequent occurrence of sporadic colorectal cancer (CRC) showing high levels of MSI (MSI-H) due to epigenetic loss of MLH1 expression. Somatic BRAF mutations, most frequently V600E, have been described in a significant proportion of sporadic MSI-H CRC but not in HNPCC-associated cancers. BRAF mutation testing has therefore been proposed as a means to more definitively identify and exclude sporadic MSI-H CRC cases from germline MMR gene testing. However, the clinical validity and utility of this approach have not been previously evaluated in a familial cancer clinic setting. Testing for the V600E mutation was performed on MSI-H CRC samples from 68 individuals referred for laboratory investigation of suspected HNPCC. The V600E mutation was identified in 17 of 40 (42%) tumours showing loss of MLH1 protein expression by immunohistochemistry but in none of the 28 tumours that exhibited loss of MSH2 expression (P < 0.001). The assay was negative in all patients with an identified germline MMR gene mutation. Although biased by the fact that germline testing was not pursued beyond direct sequencing in many cases lacking a high clinical index of suspicion of HNPCC, BRAF V600E detection was therefore considered to be 100% specific and 48% sensitive in detecting sporadic MSI-H CRC amongst those cases showing loss of MLH1 protein expression, in a population of patients with MSI-H CRC and clinical features suggestive of HNPCC. Accordingly, we recommend the incorporation of BRAF V600E mutation testing into the laboratory algorithm for pre-screening patients with suspected HNPCC, whose CRCs show loss of expression of MLH1. In such tumours, the presence of a BRAF V600E mutation indicates the tumour is not related to HNPCC and that germline testing of MLH1 in that individual is not warranted. We also recommend that in families where the clinical suspicion of HNPCC is high, germline testing should not be performed on an individual whose CRC harbours a somatic BRAF mutation, as this may compromise identification of the familial mutation.

Giant cell tumor of bone (GCT) is a generally benign, osteolytic neoplasm comprising stromal cells and osteoclast-like giant cells. The osteoclastic cells, which cause bony destruction, are thought to be recruited from normal monocytic pre-osteoclasts by stromal cell expression of the ligand for receptor activator of nuclear factor kappaB (RANKL). This model forms the foundation for clinical trials in GCTs of novel cancer therapeutics targeting RANKL. Using expression profiling, we identified both osteoblast and osteoclast signatures within GCTs, including key regulators of osteoclast differentiation and function such as RANKL, a C-type lectin, osteoprotegerin, and the wnt inhibitor SFRP4. After ex vivo generation of stromal- and osteoclast-enriched cultures, we unexpectedly found that RANKL mRNA and protein were more highly expressed in osteoclasts than in stromal cells, as determined by expression profiling, flow cytometry, immunohistochemistry, and reverse transcriptase-polymerase chain reaction. The expression patterns of molecules implicated in signaling between stromal cells and monocytic osteoclast precursors were analyzed in both primary and fractionated GCTs. Finally, using array-based comparative genomic hybridization, neither GCTs nor the derived stromal cells demonstrated significant genomic gains or losses. These data raise questions regarding the role of RANKL in GCTs that may be relevant to the development of molecularly targeted therapeutics for this disease.