Human papillomavirus (HPV) infection has been convincingly proven to be a major risk factor for the development of pre-invasive or invasive carcinoma in epidemiologic studies. In women, HPV infection is considered as a necessary, yet not sufficient precursor of cervical cancer. An estimated 5% of the HPV-positive women will develop cervical cancer without medical intervention. Cervical cancer development goes through HPV infection, low-grade cervical dysplasia, high-grade cervical dysplasia, and invasive cancer stages. Seventy to ninety percent of HPV-infected women are undetectable within two years (transient infection), especially if < 30-year old, while others advance into low-grade cervical dysplasia (persistent infection). About 15% of low-grade cervical dysplasia patients will advance to high-grade cervical dysplasia while others will regress spontaneously. One third of the high-grade cervical dysplasia patients will develop invasive cancer during a ten-year period. Therefore, it is critical to accurately monitor HPV infection so that transient infections can be distinguished from persistent ones and risks for development of cervical cancer can be assessed in HPV-infected women.
HPVs are classified by genotype, and at least 130 types have been identified by sequencing the gene encoding the major capsid protein L1. High-risk genotypes 16 and 18 are associated with 70% of cervical, and about 80% HPV positive vulval and vaginal carcinoma.
Targeting high-risk HPV mRNA from the E6/E7 oncogenes, which are essential for development of cervical cancer, DiaCarta’s QuantiVirus™ HPV mRNA Test detects HPV oncogenes E6/E7 mRNA from 14 high-risk type (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68) and genotypes HPV 16 and HPV 18 directly from cervical samples (PreservCyt® and SurePath® preservation fluid) for cervical cancer screening, and directly from saliva for head & neck cancer screening in men. Unlike HPV DNA tests, the test focuses on persistent infections, rather than transient ones, thus proving a better predictor of CIN2+ lesions. It may be used as triage of HPV DNA+ samples as an alternative to cytology, that needs extensive experience and is operator-dependent.
QuantiVirus™ HPV E6/E7 mRNA Test is a CE/IVD-certified, highly sensitive and specific signal amplification nucleic acid probe assay.
Advantages of QuantiVirus™ HPV E6/E7 mRNA Test
No DNA/RNA purification, reverse transcription polymerase chain reaction (RT-PCR) or target RNA amplification needed
Higher Specificity and Sensitivity
mRNA assays provide higher specificity than DNA-based assays and more accurate correlation of HPV infection and potential cancer development. Higher sensitivity (>90%) and superior specificity (>90%) compared to Qiagen’s HC2 test
Only assaying E6/E7 mRNA can differentiate between persistent and transient infection
Increased Colposcopy Concordance
The combination of mRNA E6/E7 with colposcopic evaluation increases the colposcopy concordance with final histologic findings
Sample types include cervical samples (PreservCyt® and SurePath® preservation fluid), saliva, FFPE and head-neck samples
Most Cervical Cancers and Head & Neck Cancers are Caused by HPV Infection
Cervical cancer, occurring in cervix, the lower part of the uterus that connects to the vagina, is one of the most common cancers in women, with about 450,000 new cases diagnosed each year. Human Papillomavirus (HPV) infection, a sexually transmitted disease, plays an important role in the development of cervical cancer in a small group of infected women. Their compromised immune system allows the virus to survive for years and eventually transform normal cells on the surface of cervix to cancer cells, causing cervical cancer.
IFor both men and women, HPV, especially subtype 16, may also cause head and neck cancer, although it is unclear how. The most common location of cancer is in the region of the throat including the base of the tongue and the tonsils.
How Does HPV L
ead to Cervical Cancer?
Cervical Cancer is Caused by HPV Infection
Upon infection, the HPV genome persists in an episomal state. However, in many high-grade lesions and carcinomas, HPV genomes are found integrated into the host chromosome. Once integrated, oncogenes start to express in host cells. Two viral genes, E6 and E7, are invariably expressed in HPV-positive cancer cells. Their gene products inactivate tumor suppressors, p53 and retinoblastoma protein (pRB), respectively, and promotes cancerization of primary epithelial cells.
HPVs are classified by genotype into low-risk (non-oncogenic) and high-risk subtypes based on their ability to cause cellular carcinogenesis. High-risk genotypes 16 and 18 are associated with 70% of cervical carcinoma, and ~80% HPV positive vulval and vaginal carcinoma.
HPV E6/E7 as a Biomarker for Prognosis Assessment and Specific Therapy
E6E6 and E7 DNA are used as biomarkers for molecular diagnostics for HPV. It has been demonstrated that HPV E6/E7 expression level plays a key role in the progression of invasive carcinoma of the uterine cervix. Detection of the HPV E6/E7 mRNA level provides a more accurate assessment for the prognosis for cervical cancer compared to E6/E7 DNA measurement. The latter fails to distinguish transient infection from persistent infection, therefore generates false positive results (low clinical specificity). In addition, diagnostic analysis based on DNA amplification also needs clean room to avoid cross-contamination, which is not a concern for the signal amplification method we use.
Detection of the HPV E6/E7 mRNA provides more accurate prognosis assessment
Technologies Used for HPV Testing
Multiple types of technologies are used for molecular diagnostics of HPV
Signal Amplification-based Methods
Signal amplification-based methods use a compound-probe or branched-DNA technology to amplify the signals through DNA:DNA or DNA:RNA hybridization while maintaining the same number of the target DNA or RNA molecules in the samples. These methods can detect the HPV DNA or RNA from the samples. This category of methods is easily adapted for semi-automation or automation and do not need DNA or RNA extraction. Due to the nature of signal amplification, rather than target amplification, false-positive results can be drastically reduced.
Target Amplification-Based Methods
Target amplification-based methods amplify the target molecules (such as DNA) to achieve the detection goal. PCR is the most common method for this category.
Non-amplification methods detect the target using typical DNA hybridization methods. Most common methods include Southern blot, dot blot hybridization, and in situ hybridizations. Due to the low sensitivity and time consumption, this category of methods is not suitable for large-scale screening and not commonly used.
How Are the HPV Tests Used in Clinical Testing?
There are different purposes for using HPV testing:
Primary screen for high-grade dysplasia in HPV-positive 30+ year old women
Triage of ASC-US cytology results
• Monitoring women surgically treated for CIN2+ for recurrence
HPV E6/E7 mRNA Detection Powered by SuperbDNA™ Technology
We have developed a sandwich nucleic acid hybridization assay using SuperbDNA™ Technology to amplify the signals. The assay can simultaneously detect E6/E7 mRNAs of all 14 high risk HPV subtypes directly from Pap smear samples without RNA purification and RT-PCR. All HPV mRNA targets are captured through cooperative hybridization of multiple probes and the probe set design determines the specificity of each HPV subtype of E6/E7 mRNA.
Probe set oligonucleotides bind a contiguous region of the target E6/E7 mRNAs and selectively capture target RNAs to a solid surface during hybridization. Signal amplification is performed via sequential hybridization of the Pre-Amplifier, Amplifier and label probes.
Step 1: Lyse Samples
Step 2: Capture E6/E7
Step 3: Amplifier
Step 4: Label Probe - AP
Step 5: Substrates
Step 6: Read and Report
QuantiVirus™ HPV E6/E7 mRNA Test Analytical Performance
Analytical Performance - Limit of Detection (LOD)
The LDD was determined by examining the detection rate of 10 replicates of low concentration of ssDNA or HeLa cells and the 95% detection limit was calculated.
|HeLa* Cells||High Risk (14)||2.35 Cells|
|Lysate||HPV 18||1.9 Cells|
|HeLa cells Lysate||HPV 18||1,960 Copies|
|HPV 18 ssDNA||HPV 16||3,538 Copies|
Analytical Performance - No Cross-Reactivity
QuantiVirus™ HPV E6/E7 mRNA test has no cross-reactivity with low-risk HPV subtypes. QuantiVirus™ test showed a strong signal for pap smear samples with high-risk HPV subtypes, especially for CIN III samples. For pap smear samples with low-risk HPV subtypes, no significant increase in the signal was observed regardless of clinical stages.
Product Specifications for QuantiVirus™ HPV E6/E7 mRNA Test
Offerings - QuantiVirus™ HPV E6/E7 mRNA Test for Cervical Cancer
CE/IVD Catalog Number: DC-01-0001
Research-Use-Only (RUO) Catalog Number: DC-10-0001R
Offerings - QuantiVirus™ HPV E6/E7 mRNA Test for Head and Neck Cancer
CE/IVD Catalog Number: DC-01-0002
Research-Use-Only RUO) Catalog Number: DC-10-0002R
For in vitro diagnostic use (CE/IVD) or for research use
Sample types include cervical samples, saliva, FFPE and head-neck samples
Requirement: Capable of maintaining a constant temperature between 46–55 °C ±1 °C)
Validated Machine: VznHealth™ Thermal Shaker Incubator
Specification: Read 420nm wavelength, read time of 0.2 seconds/well, linear dynamic range ≥ 5-log (optional) and well-to-well uniformity < ± 5%)
Validated Machine: VznHealth™ Benchtop Luminometer and Molecular Device SpectraMax L Microplate Reader
Less than 8 hours
Stable for 12 Months at -25°C to -15°C
Title: Predicting Pre-Cancer in Cervical Screening: Can HPV mRNA Help?
Speaker: Mauro Scimia, M.D. (Strategic Consultant, DiaCarta, Inc.) and Jinwei Du,
- HPV mRNA: looking BEYOND the infection
- Introducing QuantiVirus® HPV E6/E7 mRNA Test
- Clinical data
- Running the QuantiVirus® HPV assay
What is the workflow for the HPV test?
The workflow will be doing the HPV 14 high-risk assay first. For the samples with the positive results, HPV 16 and 18 genotyping will be conducted to identify if the sample is HPV 16 or/and 18, or neither of them. One can select the 14-high risk test without the HPV 16 and 18 genotyping, or just do the HPV 16 and 18 genotyping without the 14 high-risk HPV test, depending on the needs.
What is the shortest time to complete the whole HPV test?
The shortest time from the beginning to finish is about 48 hours. The 14 high-risk HPV test will take 24 hours and the HPV 16 and 18 genotyping will take another 24 hours.
How many samples can I run on a 96-well plate if I just run a single test for each sample?
The test has three positive controls and three negative controls (blanks). Therefore, it will be for 90 samples if a single test is done per sample.
If I can’t use up the whole 96-well plate, what can I do with he rest of the wells?
We suggest you use a plastic plate sealer or aluminium foil to cover the rest of the wells so they are not contaminated.
Do I have to buy a luminometer to run the HPV test?
If you have a microtiter plate reader in the test labs for 420 nm wavelength measurement, you do not have to buy a luminometer. Some microtiter plates have the filter for 420 nm reading.