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  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
  • C12Q1/701—Specific hybridization probes
  • C12Q1/705—Specific hybridization probes for herpetoviridae, e.g. herpes simplex, varicella zoster
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes

Definitions

• the present invention relates to a system for screening nasopharyngeal carcinoma (NPC). In another of its aspects, the present invention relates to a method for screening for NPC.
• NPC nasopharyngeal carcinoma
• NPC BACKGROUND ART Nasopharyngeal carcinoma
• nasopharynx a portion of the pharynx which lies posterior to the nose and is bounded superiorly by the skull base and sphenoid and laterally by the paired tori of the Eustachian tubes and the RosenmuUer's fossae.
• Anteriorly the posterior choanae form the limit of the space and inferiorly an artificial line drawn at the level of the hard palate delimits the nasopharynx from the oropharynx.
• NPC is rare among North American and European Caucasians with age adjusted incidence rates of less than 1 per 100,000. In contrast, the incidence of NPC in Southern China is in the order of 60 to 80 per 100,000. People who have moved out of the endemic regions maintain their high-risk status and their successive generations also maintain a relatively high-risk status. With an increasing number of Asians immigrating to the United States and Canada, NPC has become an important social and medical issue.
• NPC has been detected either by visual examination of the nasopharynx or when the patient becomes symptomatic with satellite lesions that are clinically visible. Diagnosis of the lesion is then confirmed by performing a random, mapped or targeted punch biopsy in the nasopharynx after anaesthetizing the patient with a local or general anesthetic. The biopsy samples are then submitted for histologic analysis for the confirmation of diagnosis.
• NPC originates in an area that is not routinely examined on general physical examination, due to its difficult access and visualization without proper training and/or the availability of special medical instruments such as an endoscope. Thus, NPC is often left undetected for a long time without any signs and symptoms until the mass effects of the tumor are apparent.
• Epstein Barr virus is a human DNA tumor virus with an extraordinary diverse oncogenic potential.
• the association of EBV with NPC was first suggested based on serological evidence. It is well established utilizing Polymerase Chain Reaction (“PCR”) technique that EBV encoded deoxyribonucleic acid is present in virtually every biopsied NPC tumor and precancerous epithelial cells, irrespective of histological differentiation. There is widespread acceptance that EBV DNA has not been detected in healthy tissues.
• PCR Polymerase Chain Reaction
• EBV is ubiquitous, being found in every population in which it is sought. EBV is carried by 90% of the world adult population. It is exclusively harbored in a small subset of B-lymphocytes and is excreted in saliva and in the urogenital tract. Considerable concentrations of infectious virus particles are released at random intervals several times a month. EBV has also been isolated from the cervix and circulates through the blood contained in B-lymphocytes. EBV infections are much more common than the cancers that they produce. Infection is usually asymptomatic and malignancy is a relatively rare outcome. The strength of immune response to EBV has been studied for use as a possible screening tool to predict NPC.
• the specificity of a test screen is an indicator of the ability of the test to classify healthy individuals as having no abnormalities.
• EBV infection is a cofactor and a precursor to NPC.
• EBV is believed to infect the basal cells, Stratum Basale, of the stratified epithelium through micro lesions of the epithelia. The generation of infected daughter cells begins and continues at the basal layer. These progeny exit the basal layer and by ordinary cell movement migrate to the stratum corneum, which is the uppermost layer of the stratified epithelium. While it may take years for a lesion to be visualized, the EBV infected and cancerous cells are omnipresent in the nasopharynx almost from the first day they begin to migrate from the Stratum Basale. Collection of these cells to screen for the presence of the EBV genome; which is a surrogate marker of malignancy can be performed and the presence of NPC can be predicted.
• the nasopharynx is situated deep behind both nasal cavities and samples can be obtained either transnasally or transorally.
• the transnasal route is uncomfortable and can be difficult to perform in patients with anatomical abnormalities such as a deviated septum.
• bleeding can be a problem as the biopsy apparatus transverses the nasal cavities can cause injury to the nasal mucosal surface.
• the transoral route is ideal as this is a relatively comfortable and a non-traumatic mean of access to the nasopharynx with minimal or no bleeding.
• NPC cells contain substantially greater numbers of vial genome copies than the aggregate of all possible viral contamination from saliva.
• it would also be desirous to have a NPC screen with maximum sensitivity by designing the biopsy apparatus such that it minimizes or excludes contact of the sample collection area with saliva or tissue which is not the nasopharynx.
• Ng Device represents a major advance in the art of biopsy sample collection for screening for NPC.
• the principal manner by which NPC was detected involved relatively invasive transnasal and transoral approaches to obtain a punch biopsy sample.
• the punch biopsy sample would then be sent to a pathology lab for diagnosis of NPC in the patient.
• the advent of the Ng Device allowed for, in the preferred embodiment, collection of a brush biopsy for screening for NPC.
• the biopsy sample would be collected and then be assayed for the presence of Epstein Barr virus ("EBV"). Since, during proper us of the Ng device, the collected sample is retained in a relatively sterile form (i.e., substantially free from contamination), the assaying done was simply to detect the presence or absence EBV in the sample. The presence of EBV in the sample was indicative of NPC in the patient. Given the prior biopsy collection techniques, it is not been known or suggested or even desired in the art to quantify the amount of EBV in the sample.
• the present invention provides a method for screening for nasopharyngeal carcinoma comprising the steps of:
• the present invention provides a diagnostic method for predicting the presence of nasopharyngeal carcinoma in a patient, the method comprising the steps of: (i) obtaining a sample of epithelial cells from the nasopharynx of the patient;
• the present invention provides a diagnostic method for predicting the presence of nasopharyngeal carcinoma in a patient, the method comprising the steps of:
• the inventors have discovered a novel approach for screening for NPC for diagnosis of NPC and a system therefor.
• This approach is applicable with any method which, for a sample from the nasopharynx of a patient, can provide differentiation between cellular EBV genome and all EBV genome (e.g., a method in which the cells in the sample are washed away from non-cellular material such that cellular EBV genome specifically can be assayed or quantified).
• One aspect of this novel approach is based on obtaining a sample from the nasopharynx of a patient and quantifying the amount of EBV present in that sample (this could include cellular and non-cellular EBV). Thereafter, the amount of EBV detected in the sample is compared to a predetermined threshold.
• the predetermined threshold will be a measure of the amount of EBV in an NPC-free patient - e.g., the amount of non-cellular EBV in the patient (typically in the saliva, mouth and other regions of the nasopharnyx). If the amount of EBV in the sample is above the threshold, this is indicative of NPC in the patient. If the amount of EBV in the sample is less than the threshold, this is indicative of the absence of NPC in the patient.
• the invention allows for use of a simplified or modified form of the Ng Device since contamination of the sample during biopsy collection is rendered moot by establishing the threshold quantity of EBV to be substantially the same as the maximum aggregate amount of EBV present in the mouth and other areas of the patient which are not directly at the nasopharynx (i.e., at the particular site of interest where the amount of EBV should be detected).
• Another aspect of this novel approach involves treating (e.g., by washing or similar means) a sample comprising epithelial cells obtained from the nasopharnyx of the patient to separate epithelial cells from non-cellular material and thereafter assaying for the presence of EBV in the epithelial cells (i.e., epithelial EBV).
• This separation scheme results in removal of non-cellular EBV from the sample thereby obviating the occurrence of a false positive result.
• the Ng Device represents a significant advance in the art of biopsy sample collection devices, particularly for screening for NPC.
• the Ng Device is in the form of a "sheathed" apparatus.
• the sample collecting brush of the Ng Device is sheathed until the device is correctly positioned. At that point, the brush is exposed, a sample is taken and the brush is restowed in the sheath.
• This sheathing arrangement maintains the integrity of the sample epithelial cells and helps avoid the occurrence of contamination by, inter alia. EBV located in the mouth and other areas of the patient away from the nasopharynx.
• EBV located in the mouth and other areas of the patient away from the nasopharynx.
• the risk of contamination would be greatly enhanced since the brush would be exposed to the entire inner cavity of the patient's mouth and other areas away from the area of interest - i.e., away from the nasopharynx.
• the present inventors have discovered that it is possible to obviate these contamination-related problems by quantifying the amount of EBV in the sample and comparing this to a predetermined threshold. If the amount of EBV in the sample is above the threshold, this is a positive indication for NPC in the patient. If the amount of EBV in the sample is less than the threshold, this a negative indication for the presence of NPC in the patient.
• the predetermined threshold can be determined from control subjects - i.e., NPC-negative individuals. Thus, the predetermined threshold can be incorporated into a control database which is used for comparative purposes in a screening protocol for NPC - i.e., it is not necessary to quantify the amount of EBV in an NPC-negative patient each time the screen is used.
• the sample is pretreated to separate the epithelial cells from non-cellular material after which the epithelial cells portion of the sample may be assayed for the presence of EBV. In this case, it may not be necessary to quantify the amount of EBV in NPC-negative patients.
• the particular mode of quantifying the amount of EBV in the sample is not particularly restricted.
• the amount of EBV in the sample is determined using a Polymerase Chain Reaction ("PCR") technique. While PCR techniques are conventional, the use thereof to quantify the amount of EBV in a sample, particularly a sample comprising epithelial cells, for the purpose of screening for NPC has, heretofore, not been known. If the sample is one comprising epithelial cells, it is preferred that the sample be obtained using a transnasal or transoral approach.
• PCR Polymerase Chain Reaction
• PCR is a biochemical process in which a specific region of DNA (deoxyribonucleic acid) is repeatedly copied (i.e., amplified in a chain reaction manner) such that a large quantity of the DNA copies of interest can be obtained for biochemical studies.
• the advantage of the PCR process is that it allows a minute quantity of DNA to be detected and studied.
• DNA is a biochemical structure that contains genetic code of human cells.
• the basic building blocks of DNA are the four nucleotide basis: Adenine (A), cytosine (C), guanine (G) and thymine (T).
• A Adenine
• C cytosine
• G guanine
• T thymine
• DNA is a double helical structure with two strands pairing with one another where A pairs with T and C pairs with G. DNA resides in the nucleus of every cell.
• an enzyme is needed to catalyze the process of pairing A to T and C to G.
• the enzyme is Polymerase. Polymerization occurs when the DNA strand is lengthened as the base pairs are added one by one.
• a “primer” is a short sequence of DNA containing bases of nucleotides.
• a primer is needed at the beginning of the PCR process to initiate the amplification.
• the primer will hybridize or anneal to the beginning of the DNA sequence of interest and the polymerase will then start adding bases to the end of the primer sequence to lengthen the DNA sequence.
• primers are a specific sequence of complementary DNA that can recognize regions of the DNA sequence of interest.
• Various primers suitable for use in the present invention are set out below in the Sequence Listing section of this specification.
• the PCR reaction is carried out in three general parts.
• the two DNA strands in a double helix are separated (also known as “unzipping” or “denaturing”). This is usually done by heating the DNA sample in the presence of a biochemical.
• the temperature of the sample is usually lowered and the primer is added and allowed to bind or "anneal" to the specific region of the DNA strand of interest.
• the polymerase and the nucleotides are added, and the temperature of the sample is once again raised to allow the biochemical process of base pairing to occur.
• the primer which is used typically has anchored to it a fluorescent-dye.
• the final product containing the newly synthesized full sequence of DNA with the fluorescent- labelled primer is then induced to fluoresce using, for example, a laser device.
• the amount of laser-induced fluorescence signals are then analyzed and compared with a standard control containing a known amount of fluorescent- labelled PCR product as calibration.
• a calibration curve showing increasing fluorescence signals with increasing amplification cycles serves as a baseline level.
• a particularly preferred manner of detecting and quantifying the amount of DNA produced involves the use of a labeled oligonucleotide probe as desribed in Gelfand.
• a labeled oligonucleotide probe as desribed in Gelfand.
• the 5' to 3' nuclease activity of a nucleic acid polymerase is advantageously used to cleave annealed labeled oligonucleotide from hybridized duplexes thereby releasing labeled oligonucleotide fragments for detection.
• Various such probes suitable for use in the present invention are set out below in the Sequence Listing section of this specification.
• Samples containing the cells with DNA of interest are usually treated mechanically (e.g., grinding into powder under liquid-nitrogen frozen conditions) and biochemically (e.g., through the use of a lysis buffer) to release the nuclear content, including the DNA from the cells.
• the standard acid quanidinium thiocyanate-phenol method is then used to treat the isolated DNA.
• the PCR quantification method described herein is particularly advantageous to remove the "background” or contamination level thereby obviated the occurrence of false positives. Since NPC tumor cells have many more copies of EBV genome than the aggregate possible from contamination sources, further amplification of the EBV genome level from NPC will therefore magnify these differences. Thus, a "threshold background contamination" level can be readily established above which, for given a sample, there is a positive indication for the presence of NPC.
• biopsy sample it is possible to treat the biopsy sample to separate non- cellular EBV genome (not of interest in screening for NPC) from cellular EBV genome (of interest in screening for NPC). This can be achieved conventionally by using buffers and other known reagents to wash the sample.
• the ability to quantify the amount of EBV genome in a sample may lead to establishment of various predictive factors in how NPC may manifest itself in a patient who does not exhibit symptoms of NPC.
• a level of EBV genome determined using quantitative PCR can lead to a reference number for screening of NPC in high risk individuals. This allows for relatively early detection of NPC which leads to a much better prognosis for the patient - this is one of the significant advantages of the invention.

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• 2000
  • 2000-04-28 AU AU42795/00A patent/AU4279500A/en not_active Abandoned
  • 2000-04-28 WO PCT/CA2000/000456 patent/WO2000066769A2/en not_active Application Discontinuation
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