Variation of Blood Gene Expression in Healthy Subjects and Patients

To test the ability of gene arrays to identify the blood gene expression patterns caused by diseases and drug treatments, we decided first to characterize the inter-and intrasubject variation between samples. The variation of gene expression in human blood has been related to many factors, which would tend to create noise, or possible spurious results (type I error) when comparing disease cases to controls. Non-disease-related factors, including relative proportions of the different blood cell...

Integrins And Infertility

Infertility affects approximately 2 to 4 million couples annually and, despite a widening in the number of infertility diagnosis, many of the molecular defects associated with female infertility are still not understood. Recurrent spontaneous abortion and failure of implantation due to defects in uterine receptivity may contribute to 20 of these cases.43 One of the methods used earlier to monitor the defects in implantation is based on dating of the endometrial biopsy. The classic work of Noyes...

Studies Evaluating Extracellular Fluid In

NAF contains a variety of chemical substances either secreted from or which passively diffuse through the epithelial cells into the ductal lumen. These include substances of endogenous origin, including a-lactalbumin, immunoglobulins, lipids, fatty acids, proteins, cholesterol and cholesterol oxidation products, and hormones (Petrakis, 1986), as well as exogenous substances including nicotine and cotinine from cigarette smoking (Petrakis et al., 1978) and mutagenic agents of undetermined origin...

Methylation Profiles In Circulating Tumor Cells Isolated From Blood Of Patients With Cancer And Biological Implications

Hematogenous dissemination is presumably a major route of metastasis, and circulating tumor cells (CTCs) may remain dormant for long times before recurrence or the development of metastasis.23-27 After necrosis of some of these CTCs, tumor DNA may also possibly be released (Figure 14.1). To improve the current diagnostic procedures in identifying high-risk patients, we need to develop new approaches for early diagnosis of human cancers or premalignancies. DNA methylation profiling is a new...

Endogenous Substances Single Protein Analysis 9411 Hormones and Growth Factors

A variety of hormones have been measured in NAF, including estrogens, androgens, progesterone, dehydroepiandrosterone sulfate, prolactin, growth hormone, and the growth factors epidermal growth factor, transforming growth factor-a, vascular endothelial growth factor, and basic fibroblast growth factor (Chatterton et al., 2004 Hsiung et al., 2002 Petrakis, 1989 Sauter et al., 2002b). Elevated levels of estrogens, cholesterol, and cholesterol epoxides have been suggested to have etiologic...

Integrins And Reproductive Dysfunction

Studies have indicated that adhesion molecules play a critical role in the migration of PBLs and their recruitment into the endometrium.38 Through their binding to specific receptors, cytokines may activate molecular changes in the expression pattern of adhesion and anti-adhesion molecules (MUC-1) that are essential for the adhesion of endometrial epithelial cells (EECs). During the adhesion phases, direct contact occurs between the lateral borders of EECs and the trophectoderm.39 Finally,...

Role Of Peripheral Blood Lymphocytes In Endometrial Function

PBLs are resident in the spleen, liver, and uterus. Some of the signaling pathways transduced by integrins facilitate the recruitment of PBLs to the site of implantation.43 It is now believed that endometrial lymphocytes and PBLs play a vital role in implantation and maintenance of pregnancy.32 If the functional activities of these cells are suboptimal, the establishment of early pregnancy may be impaired. Consequently, utero-placental function may be associated not only with miscarriage but...

Summary And Conclusions

The multidimensional nature of integrins, as well as the redundancy in integrin expression on reproductive cell types and their ability to bind to more than one integrin, is likely to make them important participants in the process of reproduction. The embryo and endometrium use the language of integrins for the early stages of communication. As mediators of attachment and signal transduction, their expression offers clues to understanding the regulation of uterine receptivity. Aberrant...

Surrogate Tissue Profiling In Translational Medicine And Oncology Drug Development

Chemotherapeutics are undergoing a revolution in the field of cancer drug development. Whereas previous chemotherapeutic strategies typically included the identification of highly toxic agents designed to kill tumor cells in a nonspecific manner, more and more oncology drug development programs are focusing on agents that target specific molecular features of specific types of tumors. The recent development of Trastuzumab (Herceptin, Genentech, San Francisco, CA) and Imatinib (Gleevec,...

Relevance Of Peripheral Blood In Assessment Of Patients With Solid Tumors

Of the major tumors afflicting the worldwide population, more progress has been made in applying transcriptional profiling to breast cancer than any other tumor. An initial expression profiling study in breast cancer characterized the differences between breast carcinoma tissue and human mammary epithelial cells,1 while another examined transcriptional profiles in laser dissected normal, malignant, and metastatic breast cancer cell populations from the same patient.2 The latter study identified...

Transcript Survey Techniques

Early attempts to build gene expression databases relied on Northern blotting, RNase protection, RT-PCR, in situ hybridization, and nuclease protection strategies. For the most part, these methodologies are limited by low throughput and their laborintensive nature. More recent strategies have included the use of subtractive hybridization and differential display. However, these protocols are labor intensive and cost prohibitive. They also suffer from varied false positive rates and a general...

Initial Studies Of Naf Focus On Feasibility

George Papanicolaou was the first to design a large study evaluating fluid aspirated from the nipple rather than collecting fluid that came forth spontaneously. In his 1958 report evaluating NAF, he stated, The practicability of utilizing breast secretion smears in screening for mammary carcinoma is in great measure dependent upon obtaining secretion in a relatively large proportion of the female population (Papanicolaou et al., 1958). He cleansed the nipple and applied gentle massage toward...

Nuclear DNA Alterations

Both deletions in DNA, evidenced by loss of heterozygosity (LOH), and changes (either gains or losses) in the number of repeat units of DNA (de la Chapelle, 2003), termed microsatellite instability (MSI), had been identified in a variety of human physiological fluids from subjects with cancer, including sputum (Arvanitis et al., 2003), urine (Neves et al., 2002), stool (Koshiji et al., 2002), blood (Schwarzenbach et al., 2004), and SND (Miyazaki et al., 2000). To determine if LOH and or MSI...

Mutations in Mitochondrial DNA

While each cell contains one matched pair of nuclear DNA (nDNA), the same cell contains several hundred to thousands of mitochondria and each mitochondrion contains 1 to 10 mitochondrial genomes (Chen et al., 2002). Both because of the sheer abundance of mitochondrial DNA (mtDNA) per cell and the tendency for mtDNA mutations to be homoplastic, mtDNA may provide a distinct advantage in terms of feasibility and sensitivity over nDNA-based methods for cancer detection, especially when one is...

Endogenous Substances Proteomic Analysis

Recent advances in comprehensive molecular technologies have allowed the analysis of global gene expression or protein profiles in cancerous vs. normal tissues with the goal of identifying markers that are differentially expressed between benign and malignant tissue. One such study (Porter et al., 2001) used serial analysis of gene expression to identify molecular alterations involved in breast cancer progression. The authors concluded that many of the highly expressed genes encoded secreted...

Two Dimensional Polyacrylamide Gel Electrophoresis

The traditional method of proteomic analysis is one- or two-dimensional polyacrylamide gel electrophoresis (2-D-PAGE). Using two-dimensional rather than one-dimensional PAGE allows better separation of proteins of equal molecular weight based on charge. Once a protein of interest is found, it can be cut from the gel and identified. 2-D-PAGE has been used to screen NAF because it provides a convenient and rapid method for protein identification based on matrix-assisted laser...

Surface Enhanced Laser Desorption Ionization Timeof Flight Mass Spectrometry Selditofms

Although 2-D-PAGE is quite powerful, it has limitations in protein separation and sensitivity. Recent advances in comprehensive molecular technologies allow the simultaneous analysis of multiple protein expression targets. The SELDI-TOF technique can be performed with 1 ml of NAF, can detect components in the high femtomole range, and the chip surface, which allows the rapid evaluation of 8 to 24 samples, has high-throughput potential. Candidate breast cancer biomarkers can be identified using...

Naf As A Tool To Investigate The Presence Of Mutagens In The Breast

It is thought that environmental mutagens stored in the adipose tissue of the breast could affect carcinogenesis through direct exposure to the adjacent ductal epithelial cells, and that evaluating NAF would provide information on carcinogen exposure (Petrakis et al., 1980). A standard assay for the presence of mutagens is the Ames test using one of a variety of Salmonella strains to detect the mutagen. A number of studies using different Salmonella strains have been conducted (Klein et al.,...

Effect Of Botanicals On The Breast

The role of food in health and disease is of immense and ongoing interest. One of the most studied botanicals is soy. Soy has been reported to have protective effects against breast cancer in Asian women. At least two studies have evaluated the effect of soy isoflavones on the breast using NAF, one (Hargreaves et al., 1999) short term (2 weeks) and the other (Petrakis et al., 1996) for a longer duration (6 months). The short-term study administered 45 mg soy isoflavones to 84 healthy...

Highthroughput Methods For Methylation Profiling In Cancer Cells And The Selection Of Target Genes As Epigenetic

The combination of MSP with real-time PCR technology allows quantitative analyses of DNA methylation.22 Quantitative assessment by enzymatic regional methylation assay (ERMA)78 and microarray technologies, such as methylation-specific oligonucleotide microarray (MSO)79 and expressed CpG island sequence tag (ECIST) microarray,80 allow high-throughput analyses of the methylation profiles in different tumor types. ERMA is a novel method for quantifying the methylation density of CpG sites within a...

Biochemical Events in Target Tissues Subsequently Detected in Surrogate Tissues

These markers involve analytes that are formed and secreted, or lost, by the primary tissue of interest through a physiological, pathophysiological, toxicological, or pharmacological process. The chapter by Petricoin et al. (Chapter 7) demonstrates how a portion of the low-molecular-weight circulatory proteome may consist of aberrantly processed protein fragments produced in the tumor microenvironment and intimates that a suitably sensitive method may have diagnostic implications for early...

Data Interpretation

Perhaps the greatest challenge of all will be the interpretation and appropriate utilization of all the omic and other data obtained from target and surrogate tissues. Validating the relationship between gene expression or protein profiles and toxicant exposure or disease state has already begun. If and when these relationships have been fully verified in target tissues, then the relationship between gene or protein expression in target and surrogate tissues must be established. In doing so, it...

Image Analysis

While NAF cytologic evaluation is very specific in the diagnosis of breast cancer, it is not very sensitive (Krishnamurthy et al., 2003 Papanicolaou et al., 1958 Sauter et al., 1997). One approach that has been used to increase the sensitivity of NAF is to evaluate the DNA content of the cells. Normal cells contain 46 chromosomes, are called diploid, and have a DNA index (DI) of 1.0. An abnormal amount of cellular DNA is called aneuploidy and is associated with a high nuclear grade....

Tumor Antigens

A number of proteins present in NAF have previously been associated with cancer in the blood. Two of these are PSA and carcinoembryonic antigen (CEA). PSA, a chymotrypsin-like protease first found in seminal fluid and associated with prostate cancer (Soderdahl and Hernandez, 2002), is also found in breast tissue (Howarth et al., 1997 Sauter et al., 2002b) and in NAF. PSA levels in cancerous breast tissue are lower than in benign breast tissue (Sauter et al., 2002b). PSA is thought to cleave...

Breast Carcinoma

The majority of patients with mammary carcinoma (approximately 90 ) present with tumors that are clinically confined to the breast and neighboring axillary lymph nodes. Essentially, all these patients are rendered free of measurable disease after primary surgery.73 Despite this highly efficient locoregional therapy, 30 to 40 of these patients will develop clinically detectable metastases within 10 years if no further treatment is instituted.73 The chief reason for these relapses is that breast...

Activation in a Subgroup of Patients with Tourette Syndrome

To determine whether blood gene expression patterns distinguish heritable neurologic diseases for which no causative gene has been identified, we measured blood gene expression profiles in patients with familial Tourette syndrome (TS). TS is a chronic, childhood-onset disorder characterized by motor and vocal tics, which are VPA resistant VPA responsive Drug free Figure 3.6 Hierarchical cluster analysis of 461 genes regulated by chronic VPA monotherapy. A parametric f-test (BRB-Array Tools 2.0)...

Issues And Caveats With Pbmc Profiling In Oncology Studies

PBMC profiling represents a difficult task, both in terms of logistics and interpretation. Our own internal studies and more recent reports have documented significant alterations in select subsets of transcripts in PBMCs following overnight incubation of whole blood at room temperature under conditions that mimic those encountered in clinical trials, where blood samples are drawn at clinical sites and then shipped overnight to a central processing laboratory.3940 Debey et al.39 reevaluated the...

Ficoll Hypaque Method

So far, most gene expression analyses of peripheral blood have been carried out on PBMCs isolated from anticoagulated phlebotomy samples,25'294041 because PBMCs are the most transcriptionally active cells in blood along with granulocytes. During phlebotomy, whole blood is usually drawn in standard blood collection tubes, but different anticoagulants, e.g., EDTA, sodium citrate, and heparin, are used. The subsequent separation of PBMC from other blood cells, such as erythrocytes, can be...

Evaluation of Cell Morphology

As previously mentioned, Papanicolaou was the first to report the presence of breast epithelial cells in NAF, and found malignant cells in 1 of 438 asymptomatic women (Papanicolaou et al., 1958). NAF was found to contain not only epithelial cells, but also foam cells, a term used to describe the foamy appearance of the cytoplasm. He speculated, It thus appears possible that under the term foam cell we are dealing with a variety of cell types that, although morphologically indistinguishable ,...

Correlation Between Endometrial Cell And Peripheral Lymphocyte Integrins

Expression of integrins on endometrial cells has been compared with that on PBLs with an aim of identifying a marker for assessing implantation failure in infertile women. We have shown that changes in expression of a4p1 and avp3 integrins on PBLs mirror the changes in endometrial cells during implantation and that this may be helpful in assessing endometrial functional defects.32 Immunocy-tochemical and immunofluorescence studies revealed that the expression of both a4p1 and avp3 integrins was...

Specimen Suitability

Surrogate tissues vary in the types of analysis that can be carried out on them. For example, DNA can be obtained from nail and hair (Tanigawara et al., 2001), but these tissues do not yield RNA. Hair follicles, on the other hand, are a good source of RNA, and work published by Mitsui et al. (1997) indicates that as much as 900 ng of total RNA can be extracted from a single human hair follicle. Buccal cells yield both DNA and RNA. Unfortunately, since these particular cells, which are obtained...

Section I

Introduction to Surrogate Tissue Chapter 1 Introduction to Surrogate Tissue John C. Rockett and Michael E. Burczynski Genomic Chapter 2 Impact of Sample Handling and Preparation on Gene Signatures as Exemplified for Transcriptome Analysis of Peripheral Blood 15 Joachim L. Schultze, Svenja Debey, Raji Pillai, and Thomas Zander Chapter 3 Blood Genomic Fingerprints of Brain Yang Tang, Donald L. Gilbert, Tracy A. Glauser, Andrew D. Hershey, Aigang Lu, Ruiqiong Ran, Huichun Xu, and Frank R. Sharp...

Methylation Profiling Of Circulating Tumor Dna In Plasma And Serum From Patients With Cancer

Detection of methylation abnormalities may form a novel basis for noninvasive diagnosis of very small to large tumors among high-risk populations and for disease monitoring. Methylated DNA sequences can be successfully detected in plasma and serum of patients with cancer (Table 14.1). 12,29-31,34,35 Aberrant p16 methylation appears to be a common biomarker for the noninvasive diagnosis of multiple cancers among high-risk populations at early stages. Tumors that have not developed to metastasize...

Metabonomics

Metabonomics is defined as the study of metabolic responses to drugs, environmental changes, and diseases. In essence, the approach involves the quantitative measurement of changes in multiparametric metabolic response of living systems to internal or external stimuli, or as a consequence of genetic change.33 The term is often used in an interchangeable fashion with metabolomics, which more specifically relates to the analysis of all metabolites in a biological sample. Clearly, the emerging...