Christopher B. Umbricht

Extensive and coordinated transcription of noncoding RNAs within cell-cycle promoters

Transcription of long noncoding RNAs (lncRNAs) within gene regulatory elements can modulate gene activity in response to external stimuli, but the scope and functions of such activity are not known. Here we use an ultrahigh-density array that tiles the promoters of 56 cell-cycle genes to interrogate 108 samples representing diverse perturbations. We identify 216 transcribed regions that encode putative lncRNAs, many with RT-PCR–validated periodic expression during the cell cycle, show altered expression in human cancers and are regulated in expression by specific oncogenic stimuli, stem cell differentiation or DNA damage. DNA damage induces five lncRNAs from the CDKN1A promoter, and one such lncRNA, named PANDA, is induced in a p53-dependent manner. PANDA interacts with the transcription factor NF-YA to limit expression of pro-apoptotic genes; PANDA depletion markedly sensitized human fibroblasts to apoptosis by doxorubicin. These findings suggest potentially widespread roles for promoter lncRNAs in cell-growth control.

Detection of breast cancer cells in ductal lavage fluid by methylation-specific PCR

If detected early, breast cancer is curable. We tested cells collected from the breast ducts by methylation-specific PCR (MSP). Methylated alleles of Cyclin D2, RAR-beta, and Twist genes were frequently detected in fluid from mammary ducts containing endoscopically visualised carcinomas (17 cases of 20), and ductal carcinoma in situ (two of seven), but rarely in ductal lavage fluid from healthy ducts (five of 45). Two of the women with healthy mammograms whose ductal lavage fluid contained methylated markers and cytologically abnormal cells were subsequently diagnosed with breast cancer. Carrying out MSP in these fluid samples may provide a sensitive and powerful addition to mammographic screening for early detection of breast cancer.

Hypermethylation of 14-3-3 σ (stratifin) is an early event in breast cancer

We have identified 14-3-3 σ (σ) as a gene whose expression is lost in breast carcinomas, primarily by methylation-mediated silencing. In this report, we investigated the timing of loss of σ gene expression during breast tumorigenesis in vivo. We analysed the methylation status of σ in breast cancer precursor lesions using microdissection for selective tissue sampling. We found hypermethylation of σ in 24 of 25 carcinomas (96%), 15 of 18 (83%) of ductal carcinoma in situ, and three of eight (38%) of atypical hyperplasias. None of the five hyperplasias without atypia showed σ-hypermethylation. Unexpectedly, patients with breast cancer showed σ hypermethylation in adjacent histologically normal breast epithelium, while this was never observed in individuals without evidence of breast cancer. Also, samples of periductal stromal breast tissue were consistently hypermethylated, underscoring the importance of selective tissue sampling for accurate assessment of 14-3-3-σ methylation in breast epithelium. These results suggest that hypermethylation of 14-3-3-σ occurs at an early stage in the progression to invasive breast cancer, and may occur in apparently normal epithelium adjacent to breast cancer. These results provide evidence that loss of expression of σ is an early event in neoplastic transformation.

Mutational analysis of BRAF in Fine needle aspiration biopsies of the thyroid: A potential application for the preoperative assessment of thyroid nodules

Background: Fine needle aspiration (FNA) is routinely used in the preoperative evaluation of thyroid nodules, but subsequent patient management is often complicated by the inability to decisively recognize malignancy on cytologic grounds alone. Activating mutations of the BRAF oncogene commonly occur in papillary thyroid carcinomas (PTCs) but not in other types of benign and malignant thyroid lesions. Mutational analysis of FNAs could enhance selection of thyroid nodules for surgical removal. Methods: Ninety-five excised PTCs along with 49 corresponding FNAs were evaluated for BRAF mutations by a newly developed assay that uses a novel primer extension method (MutectorR assay) and by direct sequencing. An additional 42 FNAs from thyroid nodules that were excised based on a suspicion of malignancy were also evaluated. Results:BRAF mutations were identified in 36 of the 95 (38%) excised PTCs. By histological subtype, BRAF mutations were more common in conventional PTCs than in the follicular variant (67% versus 12%; P < 0.0001; χ2). Analysis of the preoperative FNAs accurately reflected BRAF status of the resected PTC in 46 of the 49 paired samples (94% concordance). In FNA samples grouped according to the preoperative cytologic findings (malignant, n = 25; benign, n = 11; and indeterminate, n = 55), a BRAF mutation confirmed the diagnosis of PTC in 72% of carcinomas within the malignant group, and it established the diagnosis of PTC in 16% of carcinomas within the indeterminate group. BRAF mutations were not detected in FNAs from 32 benign thyroid lesions. Direct sequencing and the MutectorR assay yielded completely concordant results. Conclusions:BRAF mutations are common in conventional PTCs, and they are specific for PTC. A BRAF mutation can be reliably detected in cells aspirated from a thyroid nodule suggesting a role for this marker in the preoperative evaluation of thyroid nodules.

Using Gene Expression Profiling to Differentiate Benign versus Malignant Thyroid Tumors

DNA microarrays allow quick and complete evaluation of a cells transcriptional activity. Expression genomics is very powerful in that it can generate expression data for a large number of genes simultaneously across multiple samples. In cancer research, an intriguing application of expression arrays includes assessing the molecular components of the neoplastic process and utilizing the data for cancer classification (Miller LD, et al. Cancer Cell 2002;2:353-61). Classification of human cancers into distinct groups based on their molecular profile rather than their histological appearance may prove to be more relevant to specific cancer diagnoses and cancer treatment regimes. Several attempts to formulate a consensus about classification and treatment of thyroid carcinoma based on standard histopathological analysis have resulted in published guidelines for diagnosis and initial disease management (Sherman SI. Lancet 2003;361:501-11). In the past few decades, no improvement has been made in the differential diagnosis of thyroid tumors by fine needle aspiration biopsy, specifically suspicious or indeterminate thyroid lesions, suggesting that a new approach to this should be explored. Therefore, in this study, we developed a gene expression approach to diagnose benign versus malignant thyroid lesions in 73 patients with thyroid tumors. We successfully built a 10 and 6 gene model able to differentiate benign versus malignant thyroid tumors. Our results support the premise that a molecular classification system for thyroid tumors is possible, and this in turn may provide a more accurate diagnostic tool for the clinician managing patients with suspicious thyroid lesions.

Genome-wide Methylation Analysis Identifies Genes Specific to Breast Cancer Hormone Receptor Status and Risk of Recurrence

To better understand the biology of hormone receptor-positive and-negative breast cancer and to identify methylated gene markers of disease progression, we carried out a genome-wide methylation array analysis on 103 primary invasive breast cancers and 21 normal breast samples, using the Illumina Infinium HumanMethylation27 array that queried 27,578 CpG loci. Estrogen and/or progesterone receptor-positive tumors displayed more hypermethylated loci than estrogen receptor (ER)-negative tumors. However, the hypermethylated loci in ER-negative tumors were clustered closer to the transcriptional start site compared with ER-positive tumors. An ER-classifier set of CpG loci was identified, which independently partitioned primary tumors into ER subtypes. A total of 40 (32 novel and 8 previously known) CpG loci showed differential methylation specific to either ER-positive or ER-negative tumors. Each of the 40 ER subtype-specific loci was validated in silico, using an independent, publicly available methylome dataset from the Cancer Genome Atlas. In addition, we identified 100 methylated CpG loci that were significantly associated with disease progression; the majority of these loci were informative particularly in ER-negative breast cancer. Overall, the set was highly enriched in homeobox containing genes. This pilot study shows the robustness of the breast cancer methylome and illustrates its potential to stratify and reveal biological differences between ER subtypes of breast cancer. Furthermore, it defines candidate ER-specific markers and identifies potential markers predictive of outcome within ER subgroups.

Association of aberrant methylation of tumor suppressor genes with tumor aggressiveness and BRAF mutation in papillary thyroid cancer

The role of aberrant tumor suppressor gene methylation in the aggressiveness of papillary thyroid cancer (PTC) has not been documented. By showing promoter methylation‐induced gene silencing in PTC‐derived cell lines, we first demonstrated the functional consequence of methylation of several recently identified tumor suppressor genes, including those for tissue inhibitor of metalloproteinase‐3 (TIMP3), SLC5A8, death‐associated protein kinase (DAPK) and retinoic acid receptor β2 (RARβ2). We then investigated the role of methylation of these genes in the aggressiveness of PTC by examining the relationship of their aberrant methylation to clinicopathological characteristics and BRAF mutation in 231 primary PTC tumors. Methylation of TIMP3, SLC5A8 and DAPK was significantly associated with several aggressive features of PTC, including extrathyroidal invasion, lymph node metastasis, multifocality and advanced tumor stages. Methylation of these genes was also significantly associated with BRAF mutation in PTC, either individually or collectively in various combinations. Methylation of these genes, either individually or collectively, occurred more frequently in more aggressive classical and tall‐cell PTC subtypes than in less aggressive follicular‐variant PTC, with the latter known to infrequently harbor BRAF mutation. Several other tumor suppressor genes investigated were not methylated. These results suggest that aberrant methylation and hence silencing of TIMP3, SLC5A8, DAPK and RARβ2, in association with BRAF mutation, may be an important step in PTC tumorigenesis and progression.

Modeling precision treatment of breast cancer

BackgroundFirst-generation molecular profiles for human breast cancers have enabled the identification of features that can predict therapeutic response; however, little is known about how the various data types can best be combined to yield optimal predictors. Collections of breast cancer cell lines mirror many aspects of breast cancer molecular pathobiology, and measurements of their omic and biological therapeutic responses are well-suited for development of strategies to identify the most predictive molecular feature sets.ResultsWe used least squares-support vector machines and random forest algorithms to identify molecular features associated with responses of a collection of 70 breast cancer cell lines to 90 experimental or approved therapeutic agents. The datasets analyzed included measurements of copy number aberrations, mutations, gene and isoform expression, promoter methylation and protein expression. Transcriptional subtype contributed strongly to response predictors for 25% of compounds, and adding other molecular data types improved prediction for 65%. No single molecular dataset consistently out-performed the others, suggesting that therapeutic response is mediated at multiple levels in the genome. Response predictors were developed and applied to TCGA data, and were found to be present in subsets of those patient samples.ConclusionsThese results suggest that matching patients to treatments based on transcriptional subtype will improve response rates, and inclusion of additional features from other profiling data types may provide additional benefit. Further, we suggest a systems biology strategy for guiding clinical trials so that patient cohorts most likely to respond to new therapies may be more efficiently identified.

A Diagnostic Predictor Model for Indeterminate or Suspicious Thyroid FNA Samples

BACKGROUND The management of patients with thyroid fine-needle aspiration (FNA) specimens that are neither benign nor malignant still remains problematic. Efforts to improve their management have focused on identifying risk factors that predict malignancy. This study seeks to identify clinical and tumor characteristics that predict thyroid malignancy among patients with indeterminate or suspicious FNA and to develop a diagnostic predictor model. METHODS The records of 639 patients with an indeterminate or suspicious thyroid FNA between January 1995 and April 2005 were reviewed. Patient and tumor characteristics were evaluated for their potential to predict malignancy in the final surgical histopathology. A diagnostic predictor model was designed based on statistically significant predictors. Patients seen between April 2005 and April 2007 were used to validate the model. RESULTS Patient age, nodule size, and FNA cytopathology were identified as risk factors. Patients at extremes of age were at increased risk. Patients 50 years of age had the lowest risk of malignancy. For patients less than age 50, the risk increased 3% for each year decrease in age (p = 0.001). After 50, the risk increased 3.4% for each year increase in age (p = 0.016). Nodules 2.5 cm had the lowest likelihood of malignancy. For smaller nodules, the risk increased 53% per cm decrease in size (p < 0.001). For larger nodules, the risk increased 39% per cm increase (p < 0.001). Patients with FNA cytology suspicious for papillary thyroid carcinoma had the greatest risk of malignancy (p < 0.001). CONCLUSIONS A predictor model was created using the variables age, nodule size, and FNA cytology to predict thyroid malignancy.

Beyond the Suspicious Thyroid Fine Needle Aspirate

Fine needle aspiration (FNA) is currently the best diagnostic tool for thyroid nodules. However, the cytologic category of indeterminate or suspicious lesion, which is found in 10-15% of cases, remains a challenge. Since neither clinical presentation nor intraoperative frozen section is often helpful in differentiating these lesions and since surgical procedures for benign and malignant lesions differ, there is a clear need to develop ancillary tests. In this review we identify 12 potential markers of thyroid malignancy that have been examined in thyroid cytologic samples. Although many of these markers hold promise as adjuncts to FNA cytology, multicenter studies have often shown limitations in the predictive value of these assays due to lack of specificity, sensitivity or both. The recent development, however, of tissue microarray techniques to validate promising new markers suggests that improvements in the approach to indeterminate thyroid FNA samples may soon be at hand. This review presents a summary of the issues facing the development of a clinically useful diagnostic test in the differential diagnosis of thyroid nodules.