WO1999047710A1

WO1999047710A1 - Method of detecting tumor cell metastases

Info

Publication number: WO1999047710A1
Application number: PCT/US1999/006154
Authority: WO
Inventors: Jeffrey Sklar
Original assignee: The Brigham And Women's Hospital, Inc.
Priority date: 1998-03-20
Filing date: 1999-03-16
Publication date: 1999-09-23

Abstract

The present invention is directed to a method for detecting metastatic cells in samples taken from cancer patients. The method relies upon PCR amplification of gene sequences known to harbor point mutations in the particular type of cancer present in the patient.

Description

Method of Detecting Tumor Cell Metastases

Field of the Invention

The present invention is directed to sensitive methods for detecting the presence of tumor cell metastases in biological samples obtained from cancer patients.

Background of the Invention

The ability to detect a small number of tumor cells in biopsy materials is important in assessing the prognosis of cancer patients and in determining whether a relapse has occurred. Conventional methods rely almost exclusively on light microscopy for this assessment, but the sensitivity of this method is very low (perhaps no greater than 1 tumor cell in 10 total cells within biopsy material). Attempts have been made to improve the sensitivity of diagnostic methods using techniques for assessing tumor-specific nucleic acid sequences, but thus far these procedures have proven labor-intensive, expensive, and of limited general applicability. The development of improved procedures would represent a significant advance in clinical oncology.

Summary of the Invention

The present invention is directed to a method of detecting cancerous cells in a sample of tissue or cells derived from a cancer patient. The first step in the method involves obtaining a biological sample from the patient known to contain a large number of cancerous cells, e.g., obtaining a tumor sample. PCR amplification is performed on the nucleic acid of this sample using primers designed to amplify a target gene sequence known to harbor one or more point mutations associated with the type of cancer that the patient has. Denatured DNA high performance liquid chromatography (HPLC) is performed on the amplification product to determine the retention time of heteroduplexes of the amplified gene sequence. The heteroduplexes are indicative of the presence of a point mutation, and this HPLC step serves to define the chromatographic properties associated with mutations in the particular transformed cells present in the patient.

In order to determine whether the cancer has spread to other sites in the patient's body, one or more additional biological samples are obtained from sites suspected of containing metastatic cells. The amplification and HPLC separation steps are then repeated on these biological samples and a determination is made as to whether any heteroduplexes are present 2 in the column eluate. These heteroduplexes should elute with the same retention time previously determined and their presence is an indication that the tumor has spread to the site undergoing examination.

In certain cases, metastatic tumor cells may be present in biopsy samples in too low of a concentration for their DNA to be directly detected by monitoring the eluate from a HPLC column. In order to improve sensitivity further, fractions are collected from the column corresponding to the retention time that has been determined to be characteristic of a patient's cancer. PCR amplification may then be repeated using the same primers as previously in order to increase the number of heteroduplexes that are present. The amplification product may be again separated on the HPLC column and the eluate monitored for the presence of DNA emerging from the column at a retention time characteristic of tumor-associated heteroduplexes. Any method for monitoring columns may be used, but, in general, it is expected that UV absorbance or fluorescence will be employed.

Detailed Description of the Invention The purpose of the present invention is to provide a simple, rapid and inexpensive way of detecting very small numbers of tumor cells in larger populations of normal cells using point mutations within a variety of genes as markers of malignancy. The method utilizes denatured DNA HPLC, which separates heteroduplex fragments of DNA mismatched at a single base pair from homoduplexes containing perfectly matched strands. Heteroduplexes are produced when DNA is PCR amplified from cells that are heterozygous for a mutation or from mixtures of mutant and normal cells. The heteroduplexes elute from the HPLC matrix ahead of the homoduplex peak and appear in the eluate after a characteristic retention time.

The procedure involves performing a PCR amplification of a particular target gene suspected of harboring a point mutation in an individual tumor under analysis (e.g., the APC tumor suppressor gene in colon cancer). Methods are well known for choosing and synthesizing appropriate PCR primers. The amplified material is analyzed by HPLC and the retention time of the heteroduplex, signifying the presence of a mutation in the product, is determined. A second similar analysis is then performed on DNA amplified from a specimen obtained at a second site or in a subsequent biopsy of the same patient. In these cases, only very small quantities of tumor cells may be present (e.g. , possible micrometastases to regional lymph nodes or tumor cells shed into a body fluid during early relapse). Eluate from the HPLC column is collected in fractions corresponding to a retention time similar to that observed for the heteroduplex amplified earlier from the tumor DNA of the same patient. DNA may not be detected by ultraviolet absorbance or fluorescence within the eluate of this material because the amount of heteroduplex may be very small. However, if tumor cells are present in the tissue tested, heteroduplexes should be located in these fractions, and this DNA may be amplified using the same primers as used in the PCR reaction performed to generate the product applied initially to the column. Possible amplified product from the selected fractions may be reapplied to the same HPLC column, and if a heteroduplex peak is observed, the results would indicate the presence of the tumor-specific mutation within the material from which the DNA was amplified. Other sensitive methods for detecting the presence of DNA in fractions may be used as well.

The procedure described above utilizes what is probably the most common type of tumor marker (point mutations within oncogenes or tumor suppressor genes) and a technique that does not require detailed sequence analysis or the synthesis of special tumor-specific probes or oligonucleotides primers. In principle, this method may be sensitive to greater than 1 tumor cell in 100,000 total cells, provide cheap and rapid results, be applicable to a wide variety of solid tumors and utilize tissues which have been obtained or preserved in many different forms.

Claims

What is Claimed is:

1. A method of detecting metastatic cells in a sample of tissue or cells obtained from a cancer patient, comprising: a) PCR amplifying a target gene sequence suspected of harboring one or more point mutations in a tumor sample obtained from said patient; b) performing denatured DNA HPLC on the product of step a) to determine the retention time of heteroduplexes of the amplified gene sequence; c) repeating the amplification and HPLC separation of steps a) and b) on a second sample obtained from said patient, wherein said second sample is suspected of containing metastatic cells; d) determining whether heteroduplexes are present in the eluate of the HPLC column used for the separation performed in step c) and concluding that metastatic cells are present if said heteroduplexes are detected.

2. The method of claim 1, wherein samples corresponding to said retention time of heteroduplexes determined in step b) are collected and used in a third PCR amplification performed using the same primers as those used in the amplifications of steps a) and c).

3. The method of claim 2, wherein the presence of heteroduplexes in the product of said third PCR amplification is determined by HPLC performed under the same conditions as in steps b) and c).