Chemistry: molecular biology and microbiology – Treatment of micro-organisms or enzymes with electrical or... – Cell membrane or cell surface is target
Reexamination Certificate
1999-10-28
2001-11-13
Tate, Christopher R. (Department: 1651)
Chemistry: molecular biology and microbiology
Treatment of micro-organisms or enzymes with electrical or...
Cell membrane or cell surface is target
C435S173100, C435S286200, C435S173900, C435S001100, C435S325000, C435S284100
Reexamination Certificate
active
06316234
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention involves a novel method and apparatus for obtaining pure cell populations or cell constituents such as DNA, RNA or proteins from target cells in tissue sections using ultraviolet (UV) laser-assisted ablation of non-target cells.
2. Description of the Background Art
Cancer is a leading cause of death in the United States. Treatments for cancers include surgery, chemotherapy, and radiation therapy, which cause considerable morbidity and often are ineffective. Standard pathological grading and staging cannot predict the susceptibility of a particular tumor to eradication by chemotherapy, radiation therapy, or other therapy, and thus many patients with solid tumors receive ineffective toxic therapy. Better prognostic indicators and therapeutic targets are needed for cancer treatment.
A large worldwide effort is underway to develop improved prognostic and therapeutic tools for cancer. New molecular biology techniques permit investigation of specific genetic alterations in cancers. Evidence is accumulating that information about specific DNA alterations in tumors, which predict cancer behavior, may provide important new tools for cancer diagnosis, prognosis, and therapy.
For example, a recent study found that in one of the most common cancers in children (Wilms' tumor), tumor-specific loss of heterozygosity of chromosome 16q predicts adverse outcome independent of histological type. Based on this knowledge, the subgroup of Wilms' tumor patients with favorable histology and loss of heterozygosity for chromosome 16q in their tumors may now benefit from earlier, more aggressive therapy. Further, in colon cancer, the status of chromosome 18q has recently been shown to have strong prognostic value in patients with cancer extending through the bowel without lymph node metastasis (stage II). Thus, stage II colon cancer patients with tumor specific loss of heterozygosity on chromosome 18q are a newly defined subset of patients that may benefit from more aggressive adjuvant therapy at the time of their initial diagnosis.
Other types of tumor-specific genetic alterations, including amplification of specific alleles and inactivation of specific genes or alleles by cytidine methylation have shown promise for providing important new prognostic information. It may be possible to define the “signature” of genetic lesions in an individual patient's tumor, permitting therapy tailored specifically to the genetic defects of the tumor.
Current molecular biologic techniques allow the study of DNA, RNA, and protein contained within cells. Some techniques allow cells to be studied in situ, with labelled molecular probes visualized under a microscope. These techniques are very useful, but currently are quite limited in their resolution and consistency. Other more powerful techniques for studying cellular DNA, RNA or protein depend on pooling of material from one or several cells. Studies based on such pooling have identified the first known gene-specific changes associated with cancer and other diseases, and have provided insight into the molecular processes involved in the transformation of cells from normal to abnormal.
Understanding molecular genetic changes involved in the pathogenesis of organ dysfunction requires studying groups of diseased cells in isolation and comparing them to phenotypically normal cells. The difficulty is that diseased cells in any tissue are usually accompanied by many phenotypically normal cells. Thus molecular studies reported to date have been limited to tissues in which the concentration of diseased cells is relatively high, such as in large, concentrated tumor masses. Various researchers have attempted to obtain purer samples of DNA, RNA, or protein from diseased cells by scraping portions of tissue sections away with a cutting instrument or by inking target areas of tissue sections and later exposing the section to UV light to destroy non-inked DNA and RNA.
Ultraviolet irradiation of such tissue has been found to cause single and double stranded DNA breaks, DNA crosslinks, generation of local denatured sites in DNA and DNA base destruction. Thus, it is known that ultraviolet irradiation of a tissue section can massively disrupt the DNA strand (as well as RNA and protein) contained within that tissue section. For example, selective UV irradiation (non-laser) exposure of portions of tissue sections was achieved by Shibata et al. (Amer. J. Pathol. 141(3):539-543, 1992) by covering target areas of stained tissue sections with black ink and UV irradiating the entire tissue sample with a standard broad spectrum UV light bulb. Shibata demonstrated that DNA within cells covered with the black ink is preserved, while DNA in UV exposed adjacent portions of the tissue was destroyed. This crude technique is markedly limited by the width of the black marking pen used, by difficulty in directing the pen to the desired location, and by the need to continually replace the pen in order to avoid contamination of inked areas by cellular material from areas previously inked. Another limitation is that inking must be performed while no cover slip is in place, markedly reducing optical resolution and making visual identification of cells nearly impossible in many cases.
Formalin fixed, paraffin embedded (FFPE) tissues are the basis for current pathology practice. They are readily available to most pathologists and cancer researchers and provide histological detail that remains the benchmark for pathology. FFPE tissue is not ideal for many molecular methods because DNA and RNA contained within this tissue is partially degraded. Although it is more difficult to isolate DNA of adequate quality for analysis from FFPE tissue sections than from unfixed, unembedded tissue, a number of studies have demonstrated that amplification of DNA fragments as long as 536 base pairs can be accomplished with tissue fixed in buffered formalin. However, the duration of storage, fixative used, fixation time, fixation temperature, and extraction procedures all affect the quality of DNA that can be isolated from paraffin. Recent molecular techniques have allowed a wide range of genetic alterations to be detected in DNA and RNA isolated from archival tissues. Most of these techniques are based on Polymerase Chain Reaction (“PCR”).
PCR based genetic analysis of single cells or groups of cells has been used to discover molecular alterations in cells. For example, PCR techniques have been used to detect loss of heterozygosity, genomic DNA mutation, mitochondrial DNA mutation, DNA methylation, gene dosage, gene rearrangements, clonality and detection of DNA adducts. However, because cancer cells grow in close relation to noncancerous cells in all tissues, it is nearly impossible using heretofore known techniques to obtain pure tumor DNA. Hence, background signals from noncancerous cells often distort the analysis of genetic changes in tumors. For example, when a mutation is not detected in a particular gene in DNA isolated from a tumor, it is quite possible that the nonmutated sequence came from noncancerous cells' DNA contaminating the sample. This contamination problem was demonstrated in a controversy concerning the importance of the recently identified gene p16/MTS1. One of the gene's discoverers cast doubt on analyses of certain DNA samples which did not show p16 mutations because of contamination by noncancerous DNA.
The problem of background noise created by contaminating noncancerous cells was again emphasized by difficulty in identifying mutations in the breast cancer associated gene BRCA1 in sporadic and hereditary tumors. In cases of hereditary tumors, the individual inherits one mutated copy of the gene. Researchers have had difficulty studying the remaining copy of the gene in hereditary tumor samples because of background noise from contaminating normal cells, making it difficult to ascertain the frequency of specific BRCA1genetic alterations. Because of the difficulty of obtaining pure breast cancer DNA samples in g
Rothwell Figg Ernst & Manbeck
Tate Christopher R.
Winston Randall
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