Challenges of single-cell diagnostics: analysis of gene expression.

Analysis of single-cell gene expression promises a more precise understanding of human disease pathogenesis and important diagnostic applications. Here, we review the rationale for the study of gene expression at the single-cell level, practical methods to isolate homogeneous or single-cell samples, and current approaches to the analysis of single-cell gene expression. Finally, we highlight applications of laser microdissection-based gene expression analysis to the study of human disease and clinical diagnosis.

Gene expression profiling using laser capture microdissection.

Human disease is governed by a complex array of cellular populations and sub-population. Gene expression profiling is proving an important means of understanding and classifying pathophysiologic processes by identifying genes, gene pathways and pathway networks not previously known to be associated with particular diseases. However, disease-associated gene expression can be obscured by surrounding 'normal' tissue. Laser capture microdissection allows gene expression analysis of pooled single cells, cell subpopulations and cell populations. Analysis of laser capture microdissection-procured cells will allow a better understanding of the cellular components of disease.

Odontogenic carcinoma: a functional genomic comparison with oral mucosal squamous cell carcinoma.

Intraosseous squamous cell carcinomas of the mandible arise de novo or secondary to a tumor or transformed cyst epithelium. Current diagnostic tests frequently fail to distinguish between these tumors, leading to confusing classification schemes. We report the functional genomic analysis of a mandibular odontogenic carcinoma. Malignant keratinocytes from the lesion were isolated using laser capture microdissection. Target sample generated from the total RNA of the LCM-procured cells was used to hybridize high-density oligonucleotide arrays. Functional genomic analysis of the odontogenic carcinoma database compared with four oral mucosal squamous cell carcinoma gene expression databases was performed. Preliminary results suggest a small subset of genes distinguish this odontogenic carcinoma from oral mucosal epidermoid carcinomas.

Apoptosis, proliferation and p12(doc-1) profiles in normal, dysplastic and malignant squamous epithelium of the Syrian hamster cheek pouch model.

Disruption of the homeostatic balance between proliferation and apoptosis is widely believed to contribute to human oral carcinogenesis. Using the Syrian hamster oral cancer model, we examined normal, hyperplastic, dysplastic and malignant oral epithelium for the fraction of apoptotic, proliferating and p12(doc-1) expressing keratinocytes using the TUNEL assay, as well as PCNA and p12(doc-1) immunostaining, respectively. The percentage of TUNEL positive cells progressively increased from normal to dysplastic epithelium (P<0.0019), but returned to normal keratinocyte levels in the malignant epithelium (P<0.20). However, PCNA positive cells increased progressively through hamster oral malignant progression (P<0.0012). The overall ratio of apoptotic to proliferating keratinocytes remains similar until the transition between dysplastic and malignant epithelium, where the ratio is markedly reduced (P<0.05). p12(doc-1) labeling demonstrated a similar expression pattern (P<0.008). This study demonstrates that apoptosis, proliferation and the expression of p12(doc-1) reflects alterations reported during human oral carcinogenesis and supports the use of the Syrian hamster model for the further examination of these pathways.

Genomic dissection for characterization of cancerous oral epithelium tissues using transcription profiling.

Genome-wide and high-throughput functional genomic tools offer the potential of identifying disease-associated genes and dissecting disease regulatory patterns. There is a need for a set of systematic bioinformatic tools that handles efficiently a large number of variables for extracting biological meaning from experimental outputs. We present well-characterized statistical tools to discover genes that are differentially expressed between malignant oral epithelial and normal tissues in microarray experiments and to construct a robust classifier using the identified discriminatory genes. Those tools include Wilks' lambda score, error rate estimated from leave-one out cross-validation (LOOCV) and Fisher Discriminant Analysis (FDA). High Density DNA microarrays and Real Time Quantitative PCR were employed for the generation and validation of the transcription profile of the oral cancer and normal samples. We identified 45 genes that are strongly correlated with malignancy. Of the 45 genes identified, six have been previously implicated in the disease, and two are uncharacterized clones.

Microarrays and clinical dentistry.

BACKGROUND: The Human Genome Project, or HGP, has inspired a great deal of exciting biology recently by enabling the development of new technologies that will be essential for understanding the different types of abnormalities in diseases related to the oral cavity. LITERATURE REVIEWED: The authors review current literature pertaining to the advanced microarray technologies arising from the HGP and how they can contribute to dentistry. This technology has become a standard tool for monitoring activities of genes at both academic and pharmaceutical research institutions. RESULTS: With the availability of the DNA sequences for the entire human genome, attention now is focused on understanding various diseases at the genome level. Deciphering the molecular behavior of genetically encoded proteins is crucial to obtaining a more comprehensive picture of disease processes. Important progress has been made using microarrays, which have been shown to be effective in identifying gene expression patterns and variations that correlate with cellular development, physiology and function. Arrays can be used to classify tissue samples accurately based on molecular profiles and to select candidate genes related to a number of cancers, including oral cancer. This type of oral genetic approach will aid in the understanding of disease progression, thus improving diagnosis and treatment for patients. CLINICAL IMPLICATIONS: Microarrays hold much promise for the analysis of diseases in the oral cavity. As the technology evolves, dentists may see these tools as screening tests for better managing patients' dental care.

The nucleotide: DNA sequencing and its clinical application.

Information determining cellular structure and function is contained in chromosomal DNA. Genes, regions of DNA encoding this information, are composed of specific sequences of nucleotides. DNA sequencing methods have been developed to identify these sequences. Even subtle alteration (or mutation) of these sequences can lead to many human syndromes and diseases. This article reviews 1) the structure of the nucleotide, 2) the methods of DNA sequencing, and 3) its recent clinical application in analysis of the nevoid basal cell carcinoma syndrome.

The gene: the polymerase chain reaction and its clinical application.

Chromosomal DNA transfers and stores information regarding the structure and function of the cell. Genetic information, encoded within sequences of nucleotides that compose DNA, is grouped into functional units called genes. Genetic diseases are caused by changes in the chromosomal DNA, leading to a change in the quantity or function of the protein gene product. In the past, genetic diagnosis was limited by the availability of sufficient quantity and quality of DNA and the absence of an efficient amplification procedure. The polymerase chain reaction (PCR), an inexpensive, rapid, and accurate means of amplifying DNA, is already making a major contribution to the diagnostic sciences. PCR techniques have been widely used in diverse applications, including molecular analysis of microbial pathogens, inheritable diseases and syndromes, and neoplasms. The purpose of this article is to 1) Review gene structure and function, 2) review principles of PCR technology and its applications in molecular biology, and 3) discuss an experimental clinical application of PCR to identify novel infectious agents responsible for odontogenic infections.