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NPScreen™

NP Screen™ is a specially designed test kit containing a trans-oral brush and preserving solution is used to retrieve cells from the Nasopharynx for DNA collection, preservation and analyses. NP Screen™ was developed as a Laboratory Developed Test (LDT) and the results were validated by Primex Clinical Laboratory which is regulated under the Clinical Laboratory Improvement Amendments of 1988 (“CLIA”) as qualified to perform high complexity clinical testing.

It is a highly sensitive and specific genetic based screening test kit for the detection of Nasopharyngeal Cancer. Since it has been demonstrated that NPC tumor cells have copies of Epstein-Barr Virus DNA, NP Screen™ is designed to specifically measure the presence of EBV DNA as an indicator of the presence of NPC. Patients harboring the cancer without findings on endoscopy have also been found to be positive using this method.

More than 80% of Nasopharyngeal Cancer (NPC) diagnosed cases from around the world are presented in the late stage of the disease, often requiring treatments that may include serious side effects from treatment. NPC in its early stages is highly curable with a lower re-occurrence rate, and better prospects for long-term survival with just radiation therapy. Therefore, early diagnosis of the disease is critical for patient survival. Even with recent advancements in medical treatment, survival rates and quality of life have not significantly improved for NPC patients. The main reason is due to the late stage detection with no alerting recognizable symptoms. Early detection improves survival rates from 30-40% to over 90%.

NP Screen™ provides the only reliable and simple method for a regular screening program to help manage NPC for high and moderate risk patients. Combined with the latest genetic technology, and advancement in research. Using an innovative, trans-oral brush process for direct sampling. NP Screen™ bridges the gap between accuracy and non-invasiveness conventional methods such as endoscopy, biopsy and serology, etc. cannot provide.   

Existing Method for Detection of Nasopharyngeal Cancer

There are currently six known medical techniques that can detect NPC:

1.     Trans-oral mirror examination – This is the routine and traditional examination of the nasopharyngeal space by an ENT specialist/Otolaryngologist. However, this method is highly inaccurate and difficult to perform due to frequent patients gag reflex. The compliance rate is low and false negative rates are unacceptable.

2.     EBV Serology – This method is a blood laboratory test that also has a high rate of false positive results. Over 90% of the general population have past exposure to EBV infections and subsequently developed positive serology. Even a common cold that happened a few months ago can affect the result. In addition, patients with positive serology may already be in an advanced large tumor stage and may not benefit in detecting early stage disease.

3.     Circulating EBV DNA in plasma (Plasma DNA) – This method involves testing the amount of circulating plasma EBV DNA in the blood. The presence of the viral DNA in the blood is due to rapid tumor cell growth and release of DNA into the blood. Some of the DNA comes from dead tumor cells. Circulating DNA has a short half-life (10 to 15 min) which is removed by the liver. In order for the circulating EBV DNA to be detected, a significant tumor load needs to be present. This method has been shown to be better than the serology method with higher sensitivity and specificity in detecting NPC. At the present time, the value of plasma EBV DNA in screening for early NPC is not known.

4.     Nasoendoscopy – This is the current gold standard of identifying NPC. Local anesthetic is sometime applied to the nasal passage for fibre-optic nasopharyngoscopy by an Ear, Nose and Throat (ENT) Specialist/Otolaryngologist. However, a significant proportion of NPC patients are clinically inconspicuous (sub-mucosal disease). False negative rates can be as high as 30% to 40%. There is also a risk of contamination and cross-infection due to insufficient sterilization of the endoscope.

5.     Biopsy – A surgeon inserts an endoscope into the patient’s nose, passes it through to the nasopharynx to view the area and then uses a cutting tool to cut out tissue samples for subsequent examination under a microscope by a pathologist. This method is the ‘gold standard’ for detection, but it is still very subjective, since the sample area is approximate. Human inspection of the samples through a microscope is only 80+% effective (from meta-analysis studies). Sample collection is extremely important. If a tumor is prominently visible, then there is no issue about where to sample. However, if there is just a suspicion of NPC, or even a non-visible sub-mucosal tumor, then there is a high chance of missing the actual tumor when sampling. Current practice is to perform a blind biopsy where the nasopharynx is divided into 9 quadrants and samples are collected in all quadrants to minimize the risk of missing cancerous growths. Yet still, sub-mucosal tumor identification depends on whether or not the biopsy sample was collected deep enough or not.

6. NP Screen™ – A clinician takes a simple swab through the patient’s mouth, obtaining a cell sample, which is then analyzed for the presence of the Epstein Barr virus in the DNA of the cells. This screening test has a number of significant advantages, including: ease for the patient, ability to detect very tiny (early stage) tumours, as well as recurrent growths, extreme specificity and ability to detect sub-mucosal tumours which grow just underneath the surface and may be missed by both nasoendoscopic examination and biopsy.   

Current screening methods and their estimated values for early detection are provided below:   

	Sensitivity	Specificity	Value for Early Detection	Invasive Index (1-10)
Indirect Mirror	30%	30%	0%	3
EBV Serology	80%-90%	50%-80%	20%	4
Plasma DNA	75%-95%	87%-95%	unknown	4
Nasoendoscopy	80%	60%-70%	70%	7
Biopsy	100%	100%	95%	10
NP Screen™	99%	99%	99%	3

Medical Uses   

There are many different ways of detecting Nasopharyngeal Cancer (NPC) such as fiberoptic examination, diagnostic imaging, and tumor serology. However, even with these methods, only 10% of cases worldwide are presently diagnosed in their early stages. About 60% of patients are diagnosed for the first time with a neck lump, when cancer has already metastasized in the lymph nodes; 30% of NPC cases are sub-mucosal (under the skin) leaving naso-endoscopy or fiberoptic examination ineffective.

The most accurate way to test for the presence of NPC is the NP Screen™ genetic test. This test is 99% accurate and very sensitively detects the infectious presence of Epstein-Barr virus in the genetic materials of the patient.

NP Screen™
Positive Predictive Value (PPV)	Negative Predictive Value (NPV)
96.9%	99.7%

*Based on clinical trials

NP Screen™ has 99% accuracy for detection of NPC, a high accuracy rate that compares favorably to the other screening tests, including Mammograms (85-90%) and Pap Smears (71-81%). The test directly samples cells from the nasopharyngeal area. So, in addition to its high level of accuracy, the test is very easy for patients to tolerate, since it only requires a simple brushing at the back of the throat. The test is recommended to be repeated annually or semi-annually, especially for individuals from high or moderate-risk endemic areas of the world, or those with a family history of NPC. (Patients with a family history of NPC have 20 times risk than high-risk population)

Mechanism   

Step1: Brush

A Physician or a nurse practitioner performs the procedure in less than 1 minute. The process involves a simple throat swab that brushes the back of the throat in the nasopharynx for cell collection and subsequent DNA analysis. There is no need for anesthetic, surgery room time and painful recovery time from the conventional biopsy. The innovative trans-oral brush process for direct sampling means patients have minimal discomfort. It also eliminates uncertainties and the large tumor load presence necessary to diagnose with conventional blood tests.

Step 2: Ship

Once the preserved sample is secured in the provided vial, our proprietary preservation agent preserves the sample at room temperature for up to 30 days with no DNA degeneration. Unlike blood plasma, it will not degenerate and affect the test result over a matter of hours. Our samples are shipped all over the world using Fedex Clinical Pak where service is provided.

Step 3: Results

Once the designated laboratories receive the sample, it will be cataloged, sorted and prepared for Q-PCR (Quantitative PCR) processing under our proprietary lab assay protocol, using the real time advance Q-PCR (Quantitative polymerase chain reaction) process, to detect and count abnormal cells in the patient’s sample. Genetic analysis avoids human errors from conventional testing methods and accurately detect if there is sufficient sample and quantitation for normal or abnormal results. Results are made available within 7 calendar days after arrival of the sample.

Benefits

• Detects the earliest form of NPC using the integrated EBV genetic marker. It screens what cannot be detected visually through endoscopy or MRI, including sub-mucosal indicators.
• In conjunction with conventional fiberoptic examination, diagnostic imaging can significantly increase the chance for early detection versus the current 10% early detection rate
• Minimize Human Error
• High accuracy, sensitive and specific for detection of NPC
• Detect NPC in patients with sub-mucosal or inconspicuous lesions
• Helps identify local reoccurrences in post radiated patients
• Non-invasive, painless and highly tolerable
• Rapid out-patient and ambulatory procedure with negligible risk
• No equipment dependency
• Efficient result reporting

Technology

The NP Screen™ detects the earliest form of nasopharyngeal cancer by identifying and quantifying carcinoma in-situ cells. Our screening tool does this by using Quantitative polymerase chain reaction (Q-PCR), which pinpoints cells with EBV DNA integration, a marker for NPC tumors. This technology is so sensitive that it can detect the earliest stage of NPC when it is not even visible above or under the skin.

Collection – NP Screen™ directly samples from the nasopharynx where the cancer is first formed. In the nasopharynx, submucosal tumour cells will migrate outward to the surface, which allows NP Screen™’s mildly abrasive brush to pick up the necessary samples for analysis— a mere 2000 to 5000 cells. This contrasts past screening tools that analyze blood samples for DNA material and dead tumour cells from rapidly growing tumors.

Preservation – The cells collected can be preserved within a proprietary solution at room temperature for up to 30 days without degradation. This permits the most accurate analysis possible. In comparison, DNA analysis of blood samples can be hindered by DNA’s 15 minute half-life in the blood stream in which immediately fragments.

QPCR – NP Screen™ utilizes the quantitative result Q-PCR process to pinpoint the cell samples with NPC tumor markers, EBV DNA integration. By amplifying DNA samples collected, Q-PCR analyzes the sample with a clinically validated interpretation. This allows NP Screen™ to detect the earliest stage of NPC (carcinoma in-situ stage 0) with 99% accuracy.   

Evidence and Current Research

NP Screen™ has been in development since 2000 and approved in 2008 as a screening kit and laboratory protocol for the detection of nasopharyngeal carcinoma in high risk populations using EBV DNA amplification methodology under the Clinical Laboratory Improvement Amendments of 1988 (“CLIA”).

The initial 5 to 6 years was devoted particularly to a highly reproducible in-vitro DNA assay for detection of Epstein-Barr Nuclear Antigen-1 gene (EBNA1) in human specimens collected from the posterior nasopharyngeal epithelium using a developed trans-oral nasopharyngeal brush and brushing methodology. The test was developed in a single laboratory, using real-time polymerase chain reaction on an ABI Prism SDS Platform using NP Screen™ specifications. A proprietary transport buffer was ISO developed for the transport of epithelial brushings at room temperature and has been found to be stable for a prolonged time interval which enables the transport of specimens by mail carrier service.

Clinical trials in Canada and Hong Kong confirm that normal patients present negligible EBV DNA levels in the brush samples while test results from NPC patients indicate significantly higher EBV DNA levels. A statistically significant difference in EBV DNA counts between NPC positive and negative patients have been confirmed. The cutoff levels correlating between positive and negative NPC was established   

Clinical Studies

Study 1:

NP Screen™ is excited to announce supporting a study designed by Dr. Dora Kwong, Head of Department of Clinical Oncology at Hong Kong University. Dr. Kwong will perform a prospective study to follow post-radiation NPC patients to assess

NP Screen™’s ability to diagnose the recurrence of NPC as compared to clinical assessment and Plasma EBV DNA. The total study will involve 20 patients and is expected to take 3 years to complete.

Study 2:

NP Screen™ is excited to announce supporting a second study designed by

Dr. Dora Kwong, Head of Department of Clinical Oncology at The University of Hong Kong . Dr. Kwong will perform a prospective study on patients diagnosed histologically as having recurrent NPC. A NP Screen™ brushing will also be performed to assess the sensitivity and specificity of NP Screen™ for detection of recurrent disease.

Study 3:

NP Screen™ is excited to announce supporting a second study designed by

Dr. Raymond King-Yin Tsang, Clinical Assistant Professor, Department of Surgery at The University of Hong Kong. Dr. Tsang will perform a study on detecting local recurrence in nasopharyngeal carcinoma by EBV DNA level in nasopharynx obtained by using transoral brush biopsies. A NP Screen™ brushing will also be performed to assess the sensitivity and specificity of NP Screen™ for detection of recurrent disease.

Selected Criticism

Sensitivty and Specificity

Double blinded clinical studies were conducted at referral clinics in Chinese communities in Toronto and Hong Kong. Chinese patients over 20 years of age (n=438, 37% female avg. age 52 years, 63% male avg. age 52 years) not previously treated for NPC were enrolled in the studies. A sample was collected from each patient using the NP Screen™ trans-oral swab which was then dispatched via common courier to the laboratories, Inc. for real-time PCR analysis using proprietary chemistries specifically designed and optimized to detect NPC markers. After sample collection, the post-nasal space was visualized using fiberoptic nasopharyngoscope and rated by an experienced ENT surgeon/Otolaryngologist and patients with suspicious findings were referred for biopsy and histopathologic evaluation. Patients whose biopsy was negative or who presented no clinical suspicions for NPC were classified as normal. The results of the clinical trials showed that the NP Screen™ assay screening results closely correlated with the final clinical diagnosis of the patient. The clinical sensitivity for NP Screen™ (i.e. the probability the test is positive when the disease is present) =98.8% and the clinical specificity for NP Screen™ (i.e. the probability the test is negative when the disease is not present)=100%. These results demonstrate the utility of NP Screen™ as an effective screen for NPC (Table 1).   

Table 1, Screening Performance, NP Screen™ Result Compared to Final Clinical Status of Patient

	TP	FN	TN	FP	Clinical Sensitivity	Clinical Specificity
Patients	85	1	35	0	98.8% 95%CI[93.7to99.80%]	100% 95%CI[98.5to100%]

External Links

http://www.npscreen.com/

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