Hot Topic: Human Papillomavirus Molecular Detection with Genotyping
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Published: January 2014
Human papillomavirus (HPV) infection is a significant cause of morbidity and mortality worldwide and screening for this virus in the appropriate patients can have a dramatic impact on reducing death related to HPV-associated cervical cancer. The recommended cervical cancer screening guidelines outline what testing should be done and when this testing should be performed and suggest that early detection of HPV-16 and HPV-18 may prevent the progression of cervical cancer. Dr. Binnicker explains the appropriate use of new molecular testing for detection of HPV with reflex to genotyping for HPV-16 and 18.
Presenter: Matthew Binnicker, PhD
- Director of the Clinical Virology Laboratory in the Division of Clinical Microbiology at Mayo Clinic in Rochester, Minnesota.
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Welcome to Mayo Medical Laboratories Hot Topics. These presentations provide short discussion of current topics and may be helpful to you in your practice. Our speaker for this program is Matt Binnicker, PhD, Director of the Clinical Virology Laboratory in the Division of Clinical Microbiology at Mayo Clinic in Rochester, Minnesota. Dr. Binnicker describes new laboratory tests for the detection of high-risk human papillomavirus (HPV) with specific genotyping for high-risk HPV type16 and HPV type 18. Thanks for the introduction and thanks to each of you for joining me for this presentation on a very important topic that impacts thousands of women every year. Over the last several decades, we’ve come to recognize that human papillomavirus is a significant cause of morbidity and mortality worldwide, and screening for this virus in the appropriate patients can have a dramatic impact on reducing death related to HPV-associated cervical cancer.
Before we begin, I just want to mention that I don’t have any corporate or financial conflicts of interest to disclose.
Also I’d like to emphasize that we’re in a new era of health care and health care delivery in the United States, so as you view this presentation, I would encourage you to consider how the testing we’re going to discuss can best be used in your practice, when the tests should be used, and how the results will impact the patients that you are caring for? So let’s get started.
Human Papillomavirus (HPV) Backgroung and Transmission
Human papillomaviruses are members of the Papillomaviridae family, of which there are over 118 recognized genotypes. There is a worldwide distribution of HPV, and it is common and ubiquitous in all areas around the globe. Unlike some viruses, there is no seasonal distribution for HPV, and no known systemic or blood-borne phase of the infection. Because of this, transmission of HPV requires direct epithelium-to-epithelium contact, and is NOT believed to be spread through exposure to body fluids. It is well recognized that anogenital HPV is spread primarily by sexual contact.
As I mentioned on the previous slide, HPV infections are extremely common, with an estimated 20 million individuals infected worldwide and 6.2 million new infections each year. Among sexually active adults, approximately 80% are believed to have acquired at least 1 HPV infection. The incidence of HPV by the time patients reach college age is quite high, with nearly 40% of women being infected and over 60% of men being exposed to HPV. Importantly, these infections are often times transient in nature, with the average duration of infection in women being 6 to 12 months. This is due to the fact that the immune system can clear the infection over time in most cases. However, individuals can be re-infected, and they may be co-infected with multiple HPV types at the same time, including both high-risk and low-risk HPV genotypes. There are a number of clinical conditions that are associated with HPV infection, and these are listed on the next slide.
Clinical Conditions Associated With Certain HPV Types
You can see that a number of HPV genotypes can cause the same clinical condition, and some genotypes are more closely associated with lower or higher severity illnesses. For example, genotypes 6 and 11 are commonly associated with genital warts and low-risk genital lesions, while infection with genotypes 16 and 18 is now known to be associated with a higher risk of developing cervical cancer. It’s important to point out that infection with HPV does not always yield disease, and this is because HPV has a relatively complex natural history of infection, as is depicted on this slide.
Natural History of HPV Infection
You’ll see that following an initial infection with HPV, there are a number of possible outcomes. The first possibility is that, over the course of several months to a year, the immune system may clear the infection and no disease will occur. A second possible outcome of infection is that a low-grade lesion, called cervical intraepithelial neoplasia-1, or CIN-1, might develop. This can still be resolved by the activity of a proper immune response, or alternatively, the infection can persist and progress to cause more advanced lesions, which are termed CIN-2 or CIN-3 grade lesions. This process may take up to 5 years to advance from an initial infection to a CIN-2 or 3 lesion. At this point, it is still possible that the infection may be cleared by the immune system and the lesions resolved. However, in some patients, the infection may continue to progress over a period of years to decades, and ultimately, cause cervical cancer.
As we pointed out on the previous slide, one outcome of infection is that the immune system may clear the infection over a period of months to years. Because of this, the vast majority of infections are subclinical or asymptomatic. It is also possible that individuals may become infected with low-risk HPV, such as genotypes 6 and 11, which can cause benign warts that may be present on various anogenital sites. These same low-risk HPV types may also cause low-grade squamous intraepithelial lesions (or LSILs) that are observed on Pap smear. LSILs may show pathologic changes such as koilocytotic atypia, which is a characteristic finding of nuclear atypia and a clear perinuclear halo.
In contrast, high-risk HPV infection of the genital tract may result in more severe disease, including high-grade squamous intraepithelial lesions and invasive cancer. HSILs are a true cancer precursor, and most commonly found in the cervix. Importantly, it has been identified that approximately 60% to 70% of anogenital cancers are caused by 2 genotypes of HPV, types 16 and 18. Infection with high-risk HPV can also result in cervical intraepithelial neoplasia, or CIN, which is graded 1 to 3 based on the severity and extent of tissue involvement.
I should point out that although most of the screening that is performed for HPV is done so in women, high-risk HPV can cause anogenital infection in males, including cancer and benign warts. In addition, HPV is an increasingly recognized cause of oral infection, which is rarely symptomatic, but may cause head and neck cancer, especially if the infection is due to genotypes 16 and/or 18. Now that we have a better understanding of the outcomes of HPV infection, let’s turn our attention to laboratory testing for this virus and how we can heSlp prevent patients infected with HPV from progressing to cervical cancer.
Interestingly, HPV cannot be cultured, and so the diagnosis depends on a number of laboratory methods, including microscopic exam, such as Pap smear or cervical biopsy, antigen immunohistochemistry, for example targeting the p16 tumor suppressor protein in infected tissue, and finally, molecular detection of HPV DNA or mRNA using PCR. Let’s take a closer look at these laboratory methods.
For many years, the mainstay of lab testing for HPV has been the Pap smear, which is a common screening test for precancerous or cancerous cells in the endocervical canal. Pap smears can be collected by applying cervical specimens directly to a microscope slide, or by placing the Pap specimen in a bottle of preservative, such as ThinPrep or SurePath media prior to submitting the sample to the testing laboratory. One major advantage of placing the sample in a liquid-based cytology media is that this allows the sample to be tested by both Pap smear AND a HPV DNA test. In addition, other sexually transmitted diseases, such as chlamydia, gonorrhea, and trichomoniasis are now more frequently being offered on samples collected in ThinPrep media.
Pap smears are read to identify cytologic changes that may be consistent with, or suggestive of, cervical cancer, and results are reported according the Bethesda reporting system. The results of a Pap smear can be negative, in which no cytologic abnormalities are observed, ASC-US, in which atypical squamous cells of undetermined significance are seen, all the way to HSIL or full-blown squamous cell carcinoma. The results of Pap smear are not entirely specific, and therefore, they may prompt further testing, such as cervical biopsy, if clinically indicated.
Nucleic Acid Amplification Tests (NAATs)
To increase the sensitivity and specificity of HPV screening, several nucleic acid amplification tests, or NAATs, have been developed. These tests are most commonly performed on liquid-based cytology specimens, such as cervical samples collected in ThinPrep or SurePath media. At the bottom of this slide, I’ve listed several of the most common, commercially available HPV NAAT tests. In parentheses, I’ve also included the nucleic acid target, for instance DNA or RNA, and the type of technology that is used, such as PCR, transcription-mediated amplification, or signal amplification. Now that we’ve spent some time discussing the types of HPV screening tests, let’s turn our focus to how these tests should be used, and the recommended cervical cancer screening guidelines.
Currently, the American Cancer Society, the American Society for Colposcopy and Cervical Pathology and the American Society for Clinical Pathology recommend that routine screening for HPV should not start until a woman reaches the age of 21. This is because sexually active women under the age of 21 are frequently infected with HPV, but most commonly clear the infection over 6 to 12 months. Between the ages of 21 and 29, the current recommendations are that women be screened at 3-year intervals using cytology alone. At age 30, patients can continue to be screened by cytology alone every 3 years, OR alternatively, by co-testing using cytology AND a HPV NAAT test every 5 years. This extended interval is due to the higher sensitivity of the HPV NAAT tests, and the greater negative predictive value of these assays. There are a few important caveats to point out. First, co-testing by cytology and HPV NAAT tests is currently not recommended in women less than 30 years of age, and second, primary screening using only a HPV NAAT test is currently not recommended. A common outcome of cytology screening is a result of ASC-US, or atypical squamous cells of undetermined significance.
Management of Women with ASC-US
On this slide, we can see how women with ASC-US cytology results can be managed according to the latest recommendations. One option is to repeat cytology testing in 6 to 12 months. The other alternative is to test the same sample by a HPV nucleic acid amplification test. If the result of the HPV test is negative, the patient can be seen again in 3 years for repeat co-testing by cytology and HPV NAAT. However, if the HPV test is positive, the recommendation is to refer the patient to colposcopy.
Is HPV Genotyping Important?
As we discussed earlier in the presentation, certain genotypes of HPV are more closely associated with the development of cervical cancer. So over the past several years, a lot of research has focused on determining whether HPV genotyping is important, and can this information give health care providers a better idea of whether their patient is at greater absolute risk for the development of cervical cancer. A recent study published in the American Journal of Clinical Pathology evaluated the role of HPV-16 and -18 genotyping in women with a positive high-risk HPV NAAT result, but negative cytology result. The data from this study suggested that women with HPV-16 or HPV-18 had an absolute risk of 11.4% for the development of CIN-2 or greater lesions. This was in contrast to a 6.1% absolute risk in women with ‘other’ high-risk HPV that was determined not to be genotypes 16 or 18. Importantly, women that were negative for high-risk HPV altogether had an absolute risk of only 0.8%. So these data suggest that genotyping is important in triaging women with high-risk HPV, and this conclusion has been supported by other studies, including one published in 2013 that concluded that “Incorporating screening with HPV and triage of HPV-positive women by a combination of genotyping for HPV 16/18 and cytology provided a good balance between maximizing sensitivity and specificity by limiting the number of colposcopies.” So it seems like there is a very good case for incorporating genotyping in the testing algorithm for HPV screening, but how exactly should we use these tests?
HPV - Role of Genotyping
On this slide, you can see the recent recommendation from ASCCP on the use of HPV genotyping in women that are positive by a high-risk HPV test, but negative by cytology. In these women, if the HPV-16 and 18 genotype results are negative, the patient can be seen again in 12 months for repeat co-testing. If however, the HPV-16 and/or 18 genotype is determined to be positive, the recommendation is to refer those women directly to colposcopy. This is a significant change in the testing and triage of women that are screened for HPV, and this recommendation is based on data suggesting that early detection of genotypes 16 and/or 18 needs to be investigated sooner than later, and by doing so, we may be able to better prevent the progression of cervical cancer.
New HPV Tests
Due to these important updates in the screening guidelines for HPV, Mayo Medical Laboratories now offers testing for the detection of high-risk HPV, with genotyping for HPV-16 and 18. The validated sources for this test include, cervical/endocervical samples that are collected in ThinPrep/PreservCyt media (this is the FDA-approved source), and alternatively, cervical/endocervical samples collected in SurePath media, or vaginal samples collected in ThinPrep or SurePath media. It is important to point out that samples submitted in SurePath media should be tested within 14 days of collection, so please submit SurePath samples to MML as soon as possible following collection.
Advantages of the New Test
Offering this new test will provide several advantages, including 3-in-1 testing on every sample. The assay will be able to detect the 14 high-risk HPV types that are associated with cervical cancer, and if they are present, provide specific genotype results for HPV-16 and/or HPV-18. Again, this genotyping information can be used to better triage women with positive high-risk HPV, but negative cytology results. Women that are negative for genotypes 16 and 18 can have repeat co-testing in 12 months, while women with positive results for HPV-16 and/or 18 should be referred to colposcopy.
Thanks again for joining me today for this discussion on HPV. If you have questions about the testing that Mayo Medical Laboratories offers for HPV, please feel free to contact us.