These are some of the questions mtm is frequently asked about the CINtec® products. Simply click on the question to get the answer:
How do cervical cancer and its precursor lesions develop?
There are about 450,000 new cases of cervical cancer worldwide each year. Cervical cancer develops gradually over a long period of time through a sequence of precursor lesions. Persistent infections with high-risk human papillomaviruses (HR-HPV) have been demonstrated to be causatively involved in the pathogenesis of cervical cancer and its precursor lesions.
What is HPV?
Human papillomaviruses (HPV) are a group of closely related viruses which infect cutaneous and mucosal stratified squamous epithelia producing host cell proliferation leading for example to skin warts or mucosal condylomas. Over 100 HPV types, defined on the basis of DNA sequence homology, are presently known. At least 23 HPV types have been recognized to infect the cervical epithelium. The anogenital infections are usually sexually transmitted, and in women up to the age of 30 an infection with HPV can be detected in up to 30%. The vast majority of HPV infections heal unnoticed. However, in a small percentage of women the infection persists and the oncogenic potential of some of these viruses can take effect.
What are high-risk and low-risk HPVs?
In regard to their ability to transform cells, two risk groups of mucosal HPVs have been defined. The high-risk HPVs (e.g. 16, 18, 31, 35, 45, and others) are able to cause cervical dysplasias and carcinomas, whereas low-risk HPVs (e.g. 6, 11, 40, and 42) display transforming capacities in exceptional cases only. The gene products of the high-risk HPV E6 and E7 genes mediate the oncogenic potential of these viruses.
What is detected by a DNA-based HPV test?
A positive HPV test shows an infection with the virus. Since most infections get cleared by the immune system and thus do not lead to cervical cancer or its precursor lesions, a positive HPV test only gives an indication that the woman has a slightly higher absolute risk (in comparison to the total female population) to develop a preneoplastic lesion or cervical cancer.
What is detected by an HPV test, based on antibodies against viral capsid proteins?
Some HPV tests are based on the detection of viral capsid antigens. Usually viral replication occurs in differentiated keratinocytes. Under the influence of viral proteins, the host cell provides all the components needed for assembly of new infectious particles. So the viral capsid proteins are expressed in differentiating epithelial cells. In HPV-related carcinogenesis normal cellular processes such as differentiation are greatly disturbed, and as transformation proceeds, proper viral propagation is prevented. Therefore expression of viral capsid proteins in high-grade lesions and in invasive cervical carcinomas is rarely detected even though genetic material of HPV can be found. Detection of the viral capsid proteins is more a characteristic of an acute HPV infection, versus p16INK4a over-expression being a marker of a transforming HPV infection.
What is p16INK4a?
p16INK4a is a cyclin-dependent kinase inhibitor. This protein plays a crucial role in the retinoblastoma protein (pRB)-mediated control of the G1-S-phase transition of the cell cycle. Loss of p16INK4a expression or a functional inactivation of p16INK4a has been shown in many tumor entities. In contrast, for some cancer entities including cervical cancer, a strong over-expression of p16INK4a has been reported.
Are there differences in the staining pattern of p16 in dysplastic cells where the HPV genome is episomal in contrast to cells where the HPV-DNA is integrated into the chromosomal DNA of the host cells?
There is no evidence for differences in p16 staining patterns in relation to the episomal as opposed to the integrated forms of HPV-DNA up to now. In the cause of HPV-related carcinogenesis, disturbances of the spindle pole apparatus in proliferating cells are a well-known phenomenon. This is considered to be a strong promoter for chromosomal breakage and recombination leading to integration of episomal viral DNA into the host genome. p16 over-expression, however, should be detected regardless whether the HPV genome is integrated or episomal.
How does detecting p16 differ from detecting Ki-67?
p16 is a cellular protein that is significantly over-expressed during HR-HPV cellular oncogenic transformation and is thus linked to the development of disease. In contrast, Ki-67 is a proliferation associated antigen that is a marker for cellular proliferation (not differing between normal versus undesired cell proliferation). Differences between the two test methods used on biopsy tissues are obvious from the staining pattern as well as from the kind of cells stained, i.e. Ki67 also stains inflammatory cells.
Why use CINtec® Kits rather than a Single Antibody?
In deciding to use an antibody to detect p16, it is important to consider a number of factors first. CINtec® Kits are superior to laboratory derived antibodies and will enable a clinician to benefit from the following:
- The E6H4™ antibody clone has been specifically designed and selected to allow for the sensitive and specific detection of p16 over-expression in both formalin-fixed paraffin-embedded tissue sections and alcohol-fixed cervical cytology preparations. Specificity and performance of the optimized antibody clone E6H4™ has been demonstrated in a wealth of peer reviewed clinical publications.
- Simple, 2 step process that is validated to provide a reproducible result which is clinically relevant.
- GMP manufactured for reliability with every batch. The products are validated to have CE Marks in Europe. The CINtec® Histology Kit is available as Class 1 IVDs in the US.
- Cost of single antibodies, secondary reagents, additional work load, repeated validation and other ancillary reagents need to be considered as part of the pricing as well as other components.
- Roche mtm laboratories AG is undergoing large clinical trials to demonstrate significant clinical utility of the CINtec® products.
What are the appropriate storage conditions for CINtec® p16 kits?
CINtec® Kits should be refrigerated at 2-8 centigrade.
What detection system does the CINtec® p16 Histology Kit include?
The mtm CINtec® p16 Histology Kits provide a two step-protocol based on a polymer reagent conjugate with horse-radish peroxidase for signal generation.
What type of pre-treatment is included?
CINtec® Kits include a heat-induced epitope retrieval step using specific epitope retrieval solutions provided with the kits. The protocol that has been validated for the kit uses a 10 min incubation period at 95 degrees Celsius in the water bath.
What chromogen is included (DAB, AEC or other) in the CINtec® p16 Histology Kit?
DAB is provided as the chromogen; it is a two-component system that is used to prepare the final DAB chromogen working solution.
What kind of wash buffer is required to use for the washing steps, or is it included in the kits?
The wash buffer is a 10x concentrated, Tris/HCl-based buffer (pH 7.6) with detergent and an antimicrobial agent. The product is provided in units of one liter filled into an appropriate bottle. This wash buffer is provided as a separate product to be used with the kit; it is not packaged within the original CINtec® Kit box, instead, it is supplied and shipped in parallel to each CINtec® Kit ordered.
Additional supplies of the wash buffer may be ordered and shipped separate from the CINtec® Kit, if needed.
How many tests are provided per kit?
For the CINtec® Histology Kit, we have included reagents for 50 slides stained for p16 as well as for 50 slides stained with the Negative Reagent Control. The number of tests per kit is based on a calculation that 200 µL of the reagents are used per slide.
What platforms are CINtec® products compatible with?
CINtec® products are compatible with the following platforms:
- Manual immunostainining
- Autostainer (Labvision, Dako)
- Ventana Benchmark
Are the kit contents available in concentrated form or ready-to-use form?
The primary antibody reagents, which are the p16 specific E6H4™ monoclonal antibody and the isotype control antibody used as a Negative Reagent Control (CINtec® Histology Kit), as well as the secondary reagent, are ready-to-use solutions. The amount of antibody in the kit is based on 200µL per test. Wash buffer and epitope retrieval buffer are 10x concentrated solutions to be diluted with water.