Histologic grading of breast cancer defines morphologic subtypes informative of metastatic potential, although not without considerable interobserver disagreement and clinical heterogeneity particularly among the moderately differentiated grade 2 (G2) tumors. We posited that a gene expression signature capable of discerning tumors of grade 1 (G1) and grade 3 (G3) histology might provide a more objective measure of grade with prognostic benefit for patients with G2 disease. To this end, we studied the expression profiles of 347 primary invasive breast tumors analyzed on Affymetrix microarrays. Using class prediction algorithms, we identified 264 robust grade-associated markers, six of which could accurately classify G1 and G3 tumors, and separate G2 tumors into two highly discriminant classes (termed G2a and G2b genetic grades) with patient survival outcomes highly similar to those with G1 and G3 histology, respectively. Statistical analysis of conventional clinical variables further distinguished G2a and G2b subtypes from each other, but also from histologic G1 and G3 tumors. In multivariate analyses, genetic grade was consistently found to be an independent prognostic indicator of disease recurrence comparable with that of lymph node status and tumor size. When incorporated into the Nottingham prognostic index, genetic grade enhanced detection of patients with less harmful tumors, likely to benefit little from adjuvant therapy. Our findings show that a genetic grade signature can improve prognosis and therapeutic planning for breast cancer patients, and support the view that low-and high-grade disease, as defined genetically, reflect independent pathobiological entities rather than a continuum of cancer progression.

The aim of this study was to assess the morphological characteristics and immunohistochemical profile of breast carcinomas with basal and myoepithelial phenotypes to obtain a better understanding of their biological behaviour and nature. One thousand nine hundred and forty-four invasive breast carcinomas were examined, using tissue microarray (TMA) technology and immunohistochemistry, to identify those tumours that showed basal and myoepithelial phenotypes, and their immunophenotype profile was characterized using a variety of markers. In addition, haematoxylin and eosin-stained sections of these tumours were studied for several morphological parameters. The findings were correlated with patient and tumour characteristics and outcome data. Tumours were classified into two groups: (1) tumours with basal phenotype [expressing one or both basal markers (CK5/6 and/or CK14)] and (2) tumours with myoepithelial phenotype (expressing SMA and/or p63). Group 1 was further subdivided into two subgroups: (A) dominant basal pattern (more than 50% of cells positive) and (B) basal characteristics (10-50% of cells positive). Group 1 tumours constituted 18.6% (8.6% and 10% for groups 1A and 1B, respectively) and group 2 constituted 13.7% of the cases. In both groups, the most common histological types were ductal/no specific type, tubular mixed and medullary-like carcinomas; the majority of these tumours were grade 3. There were positive associations with adenoid cystic growth pattern, loss of tubule formation, marked cellular pleomorphism, poorer Nottingham prognostic index, and development of distant metastasis. In addition, associations were found with loss of expression of steroid hormone receptors and FHIT proteins and positive expression of p53 and EGFR. The most common characteristics in group 1 were larger size, high-grade comedo-type necrosis, development of tumour recurrence, and absence of lymph node disease. Group 2 tumours were more common in younger patients and were associated with central acellular zones, basaloid change, and positive E-cadherin protein expression. Group 1 characteristics were associated with both reduced overall survival (OS) [log rank (LR) = 22.5, p < 0.001] and reduced disease-free interval (DFI) (LR = 30.1, p < 0.001), while group 2 characteristics showed an association with OS (LR = 5, p = 0.02) but not with DFI. Multivariate analysis showed that basal, but not myoepithelial, phenotype has an independent value in predicting outcome. Breast cancers with basal and myoepithelial phenotypes are distinct groups of tumours that share some common morphological features and an association with poor prognosis. The basal rather than the myoepithelial phenotype has the strongest relationship with patient outcome.

Transplantation of human embryonic stem cells (hESC) into immune-deficient mice leads to the formation of differentiated tumors comprising all three germ layers, resembling spontaneous human teratomas. Teratoma assays are considered the gold standard for demonstrating differentiation potential of pluripotent hESC and hold promise as a standard for assessing safety among hESC-derived cell populations intended for therapeutic applications. We tested the potency of teratoma formation in seven anatomical transplantation locations (kidney capsule, muscle, subcutaneous space, peritoneal cavity, testis, liver, epididymal fat pad) in SCID mice with and without addition of Matrigel, and found that intramuscular teratoma formation was the most experimentally convenient, reproducible, and quantifiable. In the same experimental setting, we compared undifferentiated hESC and differentiated populations enriched for either beating cardiomyocytes or definitive endoderm derivatives (insulin-secreting beta cells), and showed that all cell preparations rapidly formed teratomas with varying percentages of mesoderm, ectoderm, and endoderm. In limiting dilution experiments, we found that as little as two hESC colonies spiked into feeder fibroblasts produced a teratoma, while a more rigorous single-cell titration achieved a detection limit of 1/4000. In summary, we established core parameters essential for facilitating safety profiling of hESC-derived products for future therapeutic applications.

Protocadherins constitute the largest subgroup in the cadherin superfamily of cell adhesion molecules. Their major functions are poorly understood, although some are implicated in nervous system development. As tumorspecific promoter methylation is a marker for tumor suppressor genes (TSG), we searched for epigenetically inactivated TSGs using methylation-subtraction combined with pharmacologic demethylation, and identified the PCDH10 CpG island as a methylated sequence in nasopharyngeal carcinoma (NPC). PCDH10 is broadly expressed in all normal adult and fetal tissues including the epithelia, though at different levels. It resides at 4q28.3 -a region with hemizygous deletion detected by array-CGH in NPC cell lines; however, PCDH10 itself is not located within the deletion. In contrast, its transcriptional silencing and promoter methylation were frequently detected in multiple carcinoma cell lines in a biallelic way, including 12/12 nasopharyngeal, 13/16 esophageal, 3/4 breast, 5/5 colorectal, 3/4 cervical, 2/5 lung and 2/8 hepatocellular carcinoma cell lines, but not in any immortalized normal epithelial cell line. Aberrant methylation was further frequently detected in multiple primary carcinomas (82% in NPC, 42-51% for other carcinomas), but not normal tissues. The transcriptional silencing of PCDH10 could be reversed by pharmacologic demethylation with 5-aza-2 0 -deoxycytidine or genetic demethylation with double knockout of DNMT1 and DNMT3B, indicating a direct epigenetic mechanism. Ectopic expression of PCDH10 strongly suppressed tumor cell growth, migration, invasion and colony formation. Although the epigenetic and genetic disruptions of several classical cadherins as TSGs have been well documented in tumors, this is the first report that a widely expressed protocadherin can also function as a TSG that is frequently inactivated epigenetically in multiple carcinomas.

Breast fibroepithelial tumors comprise a heterogeneous spectrum of pathological entities, from benign fibroadenomas to malignant phyllodes tumors. Although MED12 mutations have been frequently found in fibroadenomas and phyllodes tumors, the landscapes of genetic alterations across the fibroepithelial tumor spectrum remain unclear. Here, by performing exome sequencing of 22 phyllodes tumors followed by targeted sequencing of 100 breast fibroepithelial tumors, we observed three distinct somatic mutation patterns. First, we frequently observed MED12 and RARA mutations in both fibroadenomas and phyllodes tumors, emphasizing the importance of these mutations in fibroepithelial tumorigenesis. Second, phyllodes tumors exhibited mutations in FLNA, SETD2 and KMT2D, suggesting a role in driving phyllodes tumor development. Third, borderline and malignant phyllodes tumors harbored additional mutations in cancer-associated genes. RARA mutations exhibited clustering in the portion of the gene encoding the ligand-binding domain, functionally suppressed RARA-mediated transcriptional activation and enhanced RARA interactions with transcriptional co-repressors. This study provides insights into the molecular pathogenesis of breast fibroepithelial tumors, with potential clinical implications.