Johns Hopkins University

Genzyme Corporation

Saraswati Vaidyanathan Sukumar

Mary Jo Fackler

Nancy Davidson

Ella Evron

William C Dooley

Antonio Wolff

Theresa Swift-Scanlan

Nicoletta Sacchi

Wei Wen Teo

Zoila Areli Lopez Bujanda

Stephen L Madden

Christopher Umbricht

Leslie M Cope

Peng Huang

Zhe Zhang

Kala Visvanathan

Liangfeng Han

Kideok Jin

Mary Jo Steele Fackler

Scott L Kominsky

A cMethDNA method quantitatively detects tumor DNA (or other circulating DNAs) in fluids such as serum or plasma at the lowest copy number. Unique compared to any other PCR-based assay, a small number of copies of a synthetic polynucleotide standard (STDgene) is added to an aliquot of patient serum. A cocktail of standards for a plurality of genes of interest (TARGETgene) is added to a sample of serum. Once total DNA is purified and processed, a PCR (multiplex step) is performed wherein the STDgene and the TARGETgene are co-amplified with the same external primer set. In the second nested PCR step, amplicons present in a dilution of the first PCR reaction are subjected to real time PCR, and quantified for each gene in one well by two-color real-time PCR. Products are calculated by absolute quantitation with internal primer sets specific for the methylated TARGETgene and associated STDgene.

Quantitative multiplex methylation specific PCR method-CMethDNA, reagents, and its use

The cMethDNA method of the present invention is a novel modification of the QM-MSP method (U.S. Pat. No. 8,062,849), specifically intended to quantitatively detect tumor DNA (or other circulating DNAs) in fluids such as serum or plasma at the lowest copy number yet reported. Unique compared to any other PCR-based assay, a small number of copies of a synthetic polynucleotide standard (STDgene) is added to an aliquot of patient serum with standards for a plurality of genes of interest (TARGETgene). Once total DNA is purified PCR is performed wherein the STDgene and the TARGETgene are co-amplified with the same external primer set, and the amplicons present in a dilution of the first PCR reaction are subjected to real time PCR, and quantified for each gene. Methods of making the STDgene standards and the use of the cMethDNA methods and kits containing the same are disclosed.

Quantitative multiplex methylation specific PCR method&#x2014;cMethDNA, reagents, and its use

To better understand the biology of hormone receptor-positive and negative breast cancer and to identify methylated gene markers of disease progression, a genome-wide methylation array analysis was performed on 103 primary invasive breast cancers and 21 normal breast samples using the Illumina Infinium HumanMethylation27 array that queried 27,578 CpG loci. Forty 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 (TCGA). In addition, 100 methylated CpG loci were identified that were significantly associated with disease progression. Arrays containing the ER-subtype-specific loci and their use in methods of diagnosis and treatment of breast cancer are provided.

Genome-wide methylation analysis and use to identify genes specific to breast cancer hormone receptor status and risk of recurrence

The invention generally features compositions and methods that are useful for treating or diagnosing a neoplasia, in particular breast neoplasia. The invention is based in part on the observation that the basic helix loop helix transcription factor HEYL was found to be overexpressed in breast cancer cells. Accordingly, the invention provides therapeutic compositions and methods for altering the levels and expression of HEYL of the invention, thereby treating a neoplasia, as well as compositions and methods for diagnosing a neoplasia.

HEYL as a therapeutic target and a diagnostic marker for neoplasia and uses therefor

Methods are provided for diagnosing in a subject a condition, such as a carcinoma, sarcoma or leukemia, associated with hypermethylation of genes by isolating the genes from tissue containing as few as 50 to 1000 tumor cells. Using quantitative multiplex methylation specific PCR (QM-MSP), multiple genes can be quantitatively evaluated from samples usually yielding sufficient DNA for analyses of only 1 or 2 genes. DNA sequences isolated from the sample are simultaneously co-amplified in an initial multiplex round of PCR, and the methylation status of individual hypermethylation-prone gene promoter sequences is then determined separately or in multiplex using a real time PCR round that is methylation status-specific. Within genes of the panel, the level of promoter hypermethylation as well as the incidence of promoter hypermethylation can be determined and the level of genes in the panel can be scored cumulatively. The QM-MSP method is adaptable for high throughput automated technology.

Quantitative multiplex methylation-specific PCR

The inventors found that the gene, HOXB7, was frequently overexpressed in breast cancer, and is a major upstream regulator of events leading to tamoxifen resistance. The present invention provides double-stranded short interfering nucleic acid (siNA) molecules that targets the HOXB7 gene in cells, and also provides methods of use of this siNA molecule for methods of screening, diagnosis and prediction of treatment outcomes as well as treatment of cancer.

Compositions and methods for treatment of tamoxifen resistant breast cancer

Methods are provided for diagnosing in a subject a condition, such as a carcinoma, sarcoma or leukemia, associated with hypermethylation of genes by isolating the genes from tissue containing as few as 50 to 1000 tumor cells. Using quantitative multiplex methylation specific PCR (QM-MSP), multiple genes can be quantitatively evaluated from samples usually yielding sufficient DNA for analysis of only 1 or 2 genes. DNA sequences isolated from the sample are simultaneously co-amplified in an initial multiplex round of PCR, and the methylation status of individual hypermethylation-prone gene promoter sequences is then determined separately or in multiplex using a real time PCR round that is methylation status-specific. Within genes of the panel, the level of promoter hypermethylation as well as the incidence of promoter hypermethylation can be determined and the level of genes in the panel can be scored cumulatively. The QM-MSP method is adaptable for high throughput automated technology.

To gain a better understanding of breast tumor angiogenesis, breast endothelial cells (ECs) were isolated and evaluated for gene expression patterns. When transcripts from breast ECs derived from normal and malignant breast tissues were compared, genes that were specifically elevated in tumor-associated breast endothelium were revealed. These results confirm that neoplastic and normal endothelium in human breast are distinct at the molecular level, and have significant implications for the development of anti-angiogenic therapies in the future.

Breast endothelial cell expression patterns

Methods of diagnosis, prognosis, and treatment of breast cancer, and of metastatic brain cancer, are provided The diagnostic and prognostic methods involve the immunohistochemical detection of the level of expression of the proteins claudin 1, 3, 4, and 7 in tissue or cell samples. Claudins 1 and 7 are underexpressed in the majority of breast cancers, and claudins 3 and 4 are overexpressed. The methods of treatment involve the use ofenterotoxin (or a variant thereof) to lyse metastatic cancer cells in the brain and bone that overexpress claudins 3 and 4.

Claudins as markers for early detection, diagnosis, prognosis and as targets of therapy for breast and metastatic brain or bone cancer

The invention is directed to a method of diagnosing a cell proliferative disorder of breast tissue by determining the methylation status of nucleic acids obtained from a subject. Aberrant methylation of several genes including TWIST, HOXA5, NES-1, retinoic acid receptor beta (RAR&#x3b2;), estrogen receptor (ER), cyclin D2, WT-1, 14.3.3 sigma, HIN-1, RASSF1A, and combinations of such genes serve as markers of breast malignancy.

Aberrantly methylated genes as markers of breast malignancy

The present invention provides prophylactic and therapeutic methods of treating the ductal epithelium of an exocrine gland, in particular a mammary gland, for disease, in particular cancer. The methods comprise contacting the ductal epithelium of the exocrine gland with an epithelium-destroying gent, preferably by ductal cannulation, so as to realize a prophylactic or therapeutic effect.

Prophylactic and therapeutic treatment of the ductal epithelium of a mammary gland for cancer

The invention is directed to a method of diagnosing a cell proliferative disorder of breast tissue by determining the methylation status of nucleic acids obtained from a subject. Aberrant methylation of several genes including TWIST, HOXA5, NES-1, retinoic acid receptor beta (RAR&bgr;), estrogen receptor (ER), cyclin D2, WT-1, 14.3.3 sigma, HIN-1, RASSF1A, and combinations of such genes serve as markers of breast malignancy.

The invention is directed to a method of diagnosing a cell proliferative disorder of breast tissue by determining the methylation status of nucleic acids obtained from a subject. Aberrant methylation of several genes including TWIST, HOXA5, NES-1, retinoic acid receptor beta (RAR&bgr;), estrogen receptor (ER), cyclin D2, WT-1, 14.3.3 sigma, and combinations of such genes serve as markers of breast malignancy.

Growing community of inventors

Columbia, MD, United States of America

Saraswati Vaidyanathan Sukumar