Clinical Laboratory No. 1505 Molecular Diagnostics Katie Cavnar, MS, MLS(ASCP) Instructor, Clinical Laboratory Sciences Program University of West Florida Margaret Rhoden, MLS(ASCP), MB(ASCP)CM Medical Technologist, Molecular Pathology Pensacola Pathologists Peter Cavnar, PhD Assistant Professor Biology Department University of West Florida

Pensacola, Florida

L EARNING OBJECTIVES On completion of this exercise, the participant should be able to 

differentiate between a transient and persistent human papillomavirus (HPV) infection.



describe the role of E6 and E7 genes in high-risk HPV.



list the 2 genotypes of HPV responsible for most infections that cause cervical cancer.



briefly describe the specific targets detected by the 3 types of FDA-approved molecular testing methods discussed in this exercise.



explain how sensitivity and specificity relate to the HPV molecular tests discussed in this exercise.



describe why HPV molecular testing is not recommended for screening women younger than 30 years.

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I NCREASING HUMAN PAPILLOMAVIRUS TESTING SENSITIVITY WITH M OLECULAR DETECTION M ETHODS: RECOMMENDATIONS AND GUIDELINES Human papillomavirus (HPV) is a small, nonenveloped, double-stranded DNA virus with a genome approximately 8 kilobase pairs (kbp) long that infects epithelial cells of the skin and mucosal membranes.1 The HPV genome consists of the late genes, L1 and L2, which make up the icosahedral viral capsid, and the early genes, E1 through E8. Among the early genes, E6 and E7 are viral oncogenes. Malignant transformation of HPV depends on the expression of these oncogenes.1,2

According to the World Health Organization, cervical cancer is the second most common cancer in women worldwide, with an estimated 530,000 new cases and 270,000 deaths each year.3 In the United States, there are 12,360 estimated new cases and 4020 deaths annually.4 Since cervical cancer screening was implemented in the mid-20th century, there has been a significant decline in morbidity and mortality.5 More than 99% of cases of cervical cancer are caused by HPV.6 There are more than 100 types of HPV, of which more than 30 are known to infect cervical epithelium. Of these, less than half are considered high-risk HPV (hrHPV), which can integrate into epithelial cells and transform them into malignant cells.7 Human papillomavirus genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 are considered high risk for the development of severe dysplasia or carcinoma.8,9 Genotypes 16 and 18 are associated with 70% to 80% of all squamous cervical carcinoma worldwide, with HVP16 implicated in 60% of these infections.9,10 Human papillomavirus 16, 18, and 45 are implicated in approximately 94% of cervical adenocarcinomas.10 Women infected with HPV16 or 18 have a 10% to 20% higher risk for cervical cancer over a 10-year period compared with women with other hrHPV infections.11

Cytology The Bethesda system for cervical cytology classifies the stages of cervical neoplasia. A “normal” cytology report implies that no abnormal cells were present. Squamous cells with abnormalities that do not meet criteria for intraepithelial lesions are termed “atypical squamous cells of undetermined significance.” Low-grade

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squamous intraepithelial lesions include mild cervical intraepithelial neoplasms (CIN1) and other lesions associated with transient HPV infections.8 High-grade squamous intraepithelial lesions include moderate dysplasia (CIN2) and severe cervical intraepithelial dysplasia/carcinoma in situ (CIN3).8 The last stage is invasive carcinoma, either squamous cell carcinoma or glandular cell carcinoma (adenocarcinoma).8

Epidemiology Genital HPV infection is acquired through skin-to-skin contact. The infections can, but do not always, cause genital warts (intraepithelial lesions). Ninety percent of genital HPV infections are transient and do not transform into invasive cervical carcinoma.5 The infections are cleared by the immune system and usually become undetectable within 1 to 2 years. Persistent infections can lead to precancerous (dysplastic) lesions, especially from the more prevalent high-risk genotypes, such as HPV16. Left untreated, CIN3 has a 30% probability of progressing to invasive cancer over a 30-year period.5

Integration of HPV into the human genome is not fully understood; however, it is thought that HPV integration combined with host cell de-repression of viral gene expression contributes to the development from HPV infection into cervical cancer.1 In the normal HPV life cycle, the oncogenes E6 and E7 are strictly regulated, eliminating the risk for cervical carcinoma.1 Increased expression of E6 and E7 genes occurs with HPV integration and may increase the proliferation of cervical cancer. E6 and E7 proteins interact with cell proteins that normally regulate apoptosis, DNA repair, and tumor suppression.1 Smoking, long-term use of oral contraceptives, and infection with human immunodeficiency virus (HIV) are known co-risk factors for cervical cancer, and herpes simplex virus-2 (HSV-2) infection, diet and nutrition, Chlamydia trachomatis infection, and immunosuppression are probable cofactors.2

Diagnosis Cytology (Papanicolaou [“Pap”] test) screening is still a primary method for detecting hrHPV, although falsepositive results can occur.5 Because the hrHPV types cause 99.7% of cases of cervical cancer, there are several FDA-approved molecular tests that target either hrHPV DNA or RNA (Table I).6 Abnormal cytologic and molecular findings can lead to a referral for a colposcopy, a medical procedure to examine the cervix and perform biopsies if needed.

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The 2012 screening guidelines for the prevention and early detection of cervical cancer by the American Cancer Society (ACS), the American Society for Colposcopy and Cervical Pathology (ASCCP), and the American Society for Clinical Pathology (ASCP) recommend that molecular testing not be used alone to screen patients for HPV; this testing should be used in concurrence with cytology.5 However, in April 2014, the US Food and Drug Administration (FDA) approved the cobas HPV Test (Roche Molecular Systems) for use as a primary cervical screening test.

The first FDA-approved molecular test for HPV was the automated Hybrid Capture 2 (HC2) assay (QIAGEN, Digene).13 Currently, there is a second HC2 assay that detects high-risk HPV only. The HC2 detects viral DNA in a nucleic acid hybridization assay with chemiluminescent detection; this is a qualitative test for HPV, and it is unable to determine the specific genotype of HPV present.12 Testing should be done using only collection media that have been approved by the FDA for use with the assay. Collection vials with formaldehyde require complex sample preparation to reverse the cross-linking of proteins and nucleic acids caused by the formaldehyde; inadequate sample preparation can cause false-negative results.

The cobas HPV test is fully automated and uses real-time polymerase chain reaction (PCR) to detect HPV DNA.14 It detects 14 hrHPV strains and is able to detect the subtypes HPV16 and HPV18. A 2011 clinical trial, Addressing the Need for Advanced HPV Diagnostics (ATHENA) evaluated the performance of the cobas HPV test and demonstrated that HPV16 was identified in 12.8% of CIN1 cases, 29.7% of CIN2, and 51.2% of CIN3.15 A subanalysis of the study showed that HPV testing was more sensitive but less specific than cytology for the identification of CIN3 or more advanced stages.14

The Aptima HPV assay (Hologic) is an FDA-approved in vitro nucleic acid amplification test that detects HPV E6/E7 viral mRNA from hrHPV types in cervical samples collected in preservative medium. It is performed on fully automated systems.16 The assay involves isolation of the target HPV mRNA, amplification through targetmediated reverse transcriptase and t7 RNA polymerase enzymes, and detection with chemiluminescent labels.17 The Aptima HPV assay detects 14 hrHPV but it does not subtype. A second assay, the Aptima HPV 16 18/45 genotype assay (Hologic Gen-Probe), qualitatively detects E6/E7 viral mRNA from hrHPV types 16, 18 and 45, which are found in most invasive cervical cancers. It uses the same principles as the Aptima HPV assay.

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In a persistent infection, integration of hrHPV into the host genome leads to an increase in the expression of E6 and E7 genes; therefore, mRNA levels of E6 and E7 increase with lesion severity, demonstrating a higher risk for progression to carcinoma (Figure).18,19 hrHPV DNA molecular testing detects high-grade lesions, but it also detects transient infections, which are less important clinically because they are not likely to develop into highgrade lesions.1,19,20

Comparison of Testing Methods Sensitivity is the measure of true-positive results; it is presented as a percentage of all results that should have been positive if the assay is performed correctly. Specificity is the measure of true-negative results; it is presented as a percentage of all results that should have been negative.21 hrHPV DNA testing has been shown to have a higher sensitivity than cytology alone, but it has lower specificity because it cannot differentiate between transient and persistent infections.20,22 The cobas real-time PCR HPV test eliminates the need for retesting and has the further advantage of genotyping for HPV16 and 18, which the hybrid capture DNA test cannot.23 Several studies have shown that mRNA testing with the Aptima HPV assay has the same high sensitivity as both DNA tests (>90%) but is significantly more specific than either test, and distinctively more specific for CIN2+ or more advanced lesions than hybrid capture DNA testing.20,24-26 However, like hybrid capture DNA testing, the Aptima test may over-detect CIN2 lesions in younger women, which are likely to regress spontaneously.26 One laboratory reported that by switching from the DNA capture technique to mRNA detection of E6/E7, they were able to decrease the number of quantity not sufficient (QNS) samples by 90% and reduce colposcopy referrals by 21%.19

Screening Guidelines for Cervical Cancer The updated 2012 guidelines from the ACS, ASCCP, and ASCP make several recommendations for implementing screening (Table II).5 Women younger than 21 years should not be screened. Women aged 21 to 29 years should be screened every 3 years with cytology alone.5 There is a high prevalence of HPV infection in women younger than 30; however, most of those infections are transient and will be resolved by the woman’s immune system. Persistent infections that lead to precancerous lesions and cervical cancer are much more common in women 30 years or older.5 Based on the 2012 screening guidelines, HPV molecular testing should not be used in women 21 to 29 years of age, either as a cotest with cytology or as a sole screening test.5 If women in this age group have positive cytology results, management should be done in accord with recommendations by the ASCCP, which are available on their website (www.ascccp.org). The new FDA5

approved HPV test for primary cervical screening, the cobas HPV Test, expands the use of the test as either a cotest or a primary screening test in women 25 years and older.27 The FDA states, however, that this approval does not change current medical practice guidelines for cervical cancer screening.27

To date, 2 HPV vaccines have been licensed. These protect against infection with HPV16 and HPV18, the 2 types of cervical HPV that cause most cervical cancer. Because recommendations include vaccination of patients up to the age of 26 years, a woman might be vaccinated after HPV infection has occurred. Thus, the guidelines for screening do not change for women who have been vaccinated.5

Women in the 30- to 65-year age group should preferably be screened with both cytology and HPV molecular testing every 5 years or, alternatively, with cytology alone every 3 years.5 Adding HPV molecular testing to cytology screening increases the detection of CIN3+ or invasive cancer in this age group.5 Women who test positive for HPV with molecular testing but negative with cytology should either have cotesting repeated in 12 months or have immediate genotype testing for HPV16 or for HPV16/18 if possible. If the result of genotype testing is positive for HPV16 and/or 18, the patient should be referred for colposcopy.5 If the patient is negative for HPV on molecular testing but cytology shows atypical squamous cells of undetermined significance (ASCUS), she should continue routine screening.5 Screening women older than 65 years depends on history of positive cytologic results.

Case Conclusion Several studies have suggested that cotesting has little benefit over HPV molecular testing alone because cotesting only slightly increased sensitivity but decreased specificity.14,22,26 However, the ACS/ASCCP/ASCP guidelines do not support HPV molecular testing alone at this time. The guidelines state that, although randomized controlled trials have shown an increase in sensitivity with HPV molecular testing, they have not been as successful at demonstrating specificity.5 The authors recognize the potential for HPV testing alone but acknowledge that further evaluation is needed.

Summary The incidence of cervical cancer has been greatly decreased by screening. Molecular testing is an important adjunct to cytologic screening, especially in women 30 years and older. Cervical cancer is caused by a small number of high-risk HPV types, with HPV16 and HPV18 the most prevalent in high-grade and cancerous 6

lesions. Transient cervical HPV infections are common and usually resolve without leading to cancer. Persistent infections that integrate into the host human DNA are more likely to lead to higher-grade lesions and cancer. Cytology in conjunction with HPV molecular testing has been useful in determining hrHPV infection. It may be advantageous to use HPV molecular testing that can differentiate between transient and transforming HPV infections.20 Development of tests that detect biomarkers that indicate HPV integration, such as E6/E7 gene expression, with improved specificity will be beneficial. Although the incidence of cervical cancer has greatly decreased with screening, there is still a need to discern which methods of screening best serve the patient population and the clinical laboratory’s capabilities.

QUICK STOPS 

The “Pap smear” is named for pathologist George Papanicolaou, who introduced the Papanicolaoustained smear in 1949.8



The HPV E6/E7 oncogenes cooperate to induce abnormal mitotic spindle formation during cell division causing genomic instability.28



The HeLa cell line, originally isolated from Henrietta Lacks, contains extra DNA in its genome that originated from infection by HPV type 18.29

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Figure. Levels of E6/E7 mRNA increase with the progression of cervical cancer. Reprinted with permission from HOLOGIC®, Inc. and affiliates.

Table I. FDA-Approved Molecular HPV Tests and Methods Assay Manufacturer Method Hybrid Capture 2 QIAGEN, Digene Nucleic acid high-risk HPV hybridization assay DNA Test targets viral DNA

Genotyping No

cobas HPV Test

Roche Molecular Systems

Real-time PCR detects viral DNA

Yes, subtypes for HPV16 and 18

APTIMA HPV Assay

Hologic Gen-Probe

No

APTIMA HPV 16 18/45

Hologic Gen-Probe

Nucleic acid amplification test detects E6/E7 viral mRNA Nucleic acid amplification test detects HPV types 16, 18, and 45

Yes, detects HPV subtypes 16,18, and 45

Abbreviations: FDA, US Food and Drug Administration; HPV, human papillomavirus; and PCR, polymerase chain reaction.

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Table II. Overview of American Cancer Society Screening Guidelines for Cervical Cancer Age Group, y Recommendation Reasoning Younger than 21 Should not be screened Cervical cancer at this age is rare; most infections at this age are transient, and screening may lead to unnecessary evaluation1 21-29 Every 3 years with cytology alone Most HPV infections in this age are transient and will be resolved without developing into cervical cancer 30-65 1. Every 5 years with both cytology and HPV Adding HPV molecular testing molecular testing (preferredl) increases detection of CIN3+ or 2. Every 3 years with cytology alone cancer (alternative) Older than 65 Depends on history of positive results on cytology Vaccinated Follow age-specific guidelines Women may have been population, any age vaccinated after HPV infection has occurred

Abbreviation: HPV, human papillomavirus.

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REFERENCES 1.

Raybould R, Fiander A, Hibbits S. Human papillomavirus integration and its role in cervical malignant progression. Open Clin Cancer J. 2011;5:1-7.

2.

Lie A, Kristensen G. Human papillomavirus E6/E7 mRNA testing as a predictive marker for cervical carcinoma. Expert Rev Mol Diagn. 2008;8(4):405-415.

3.

World Health Organization. Human Papillomavirus (HPV) and cervical cancer. WHO website. http://www.who.int/mediacentre/factsheets/fs380/en/. Published September 2013. Accessed March 28, 2014.

4.

National Cancer Institute. Cervical Cancer. NIH website. http://www.cancer.gov/cancertopics/types/cervical. Published 2014. Accessed March 28, 2014.

5.

Saslow D, Solomon D, Lawson H, et al. American Cancer Society, American Society for Colposcopy and Cervical Pathology, and American Society for Clinical Pathology screening guidelines for the prevention and early detection of cervical cancer. Am J Clin Pathol. 2012;137:513-542.

6.

Wallbloomers J, Jacobs M, Manos M, et al. Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol. 1999;189:12-14.

7.

Doorbar J. Molecular biology of human papillomavirus infection and cervical cancer. Clin Sci. 2006;110:525-541.

8.

Burd E. Human papillomavirus and cervical cancer. Clin Microbiol Rev. 2003;16(1):1-17.

9.

Li N, Franceschi S, Howell-Jones R, et al. Human papillomavirus type distribution in 30,848 invasive cervical cancers worldwide: variation by geographical region, histological type and year of publication. Int J Cancer. 2011;128:927-935.

10.

de Sanjose S, Quint W, Alemany L, et al. Human papillomavirus genotype attribution in invasive cervical cancer: a retrospective cross-sectional worldwide study. Lancet Oncol. 2010;11:1048-1056.

11.

Khan M, Castle P, Lorinez A, et al. The elevated 10-year risk of cervical precancer and cancer in women with human papillomavirus (HPV) type 16 or 18 and the possible utility of type-specific HPV testing in clinical practice. J Natl Cancer Inst. 2005;97(14):1072-1079.

12.

Hybrid Capture High-Risk HPV DNA Test package insert. Gaithersburg, MD: Qiagen Digene; 2007.

13.

Naryshkin S, Austin R. Limitations of widely used high-risk human papillomavirus laboratorydeveloped testing in cervical cancer screening. Drug Healthc Patient Saf. 2012;4:167-172. 10

14.

Castle P, Stoler M, Wright T, et al. Performance of carcinogenic human papillomavirus (HPV) testing and HPV16 or HPV18 genotyping for cervical cancer screening of women aged 25 years and older: a subanalysis of the ATHENA study. Lancet Oncol. 2011;12:880-890.

15.

Wright T, Stoler M, Behrens C, et al. The ATHENA human papillomavirus study: design, methods, and baseline results. Am J Obstet Gynecol. 2012;206:46.e1-11.

16.

Heidman D, Hesselink A, van Kemenade F, et al. The Aptima HPV assay fulfills the cross-sectional clinical and reproducibility criteria of international guidelines for human papillomavirus test requirements for cervical screening. J Clin Microbiol. 2013;51(11):3653-3657.

17.

APTIMA HPV Assay package insert. San Diego, CA: Hologic Gen-Probe Inc; 2011.

18.

Castle P, Dockter J, Giachetti C, et al. A cross-sectional study of a prototype carcinogenic human papillomavirus E6/E7 messenger RNA assay for detection of cervical precancer and cancer. Clin Cancer Res. 2007;13(9):2599-2605.

19.

Sauter J, Mount S, St. John T, et al. Testing of integrated human papillomavirus mRNA decreases colposcopy referrals: could a change in human papillomavirus detection methodology lead to more costeffective patient care? Acta Cytol. 2014;58:162-166.

20.

Reuschenbach M, Clad A, von Knebel C, et al. Performance of p16INK4a –cytology, HPV mRNA, and HPV DNA testing to identify high grade cervical dysplasia in women with abnormal screening results. Gynecol Oncol. 2010;119:98-105.

21.

Ball, M. Clinical Laboratory Mathematics. Boston, MA: Pearson Education Inc; 2014.

22.

Mayrand M, Duarte-Franco E, Rodrigues I, et al. Human papillomavirus DNA versus Papanicolaou screening tests for cervical cancer. N Engl J Med. 2007;357(16):1579-1588.

23.

Rao A, Sandri M, Sideri M, et al. Comparison of hybrid capture 2 high-risk HP results in the low positive range with cobas HPV test results from the ATHENA study. J Clin Virol. 2013;58:161-167.

24.

Abryn M, Roelens J, Cuschieri K, et al. The APTIMA HPV assay versus the hybrid capture 2 test in triage of women with ASC-US or LSIL cervical cytology: a meta-analysis of the diagnostic accuracy. Int J Cancer. 2013;132:101-108.

25.

Szarewski A, Cadman L, Austin J, et al. Comparison of seven tests for high-grade cervical intraepithelial neoplasia in women with abnormal smears: the predictors 2 study. J Clin Microbiol. 2012;50(6):1867-1873.

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26.

Monsonego J, Hudgens M, Zerat L, et al. Evaluation of oncogenic human papillomavirus RNA and DNA tests with liquid-based cytology in primary cervical cancer screening: the FASE study. Int J Cancer. 2011;129:691-701.

27.

FDA approves first human papillomavirus test for primary cervical cancer [news release]. Silver Spring, MD: U.S. Food and Drug Administration; April 24, 2014. http://www.fda.gov/newsevents/newsroom/pressannouncements/ucm394773.htm Accessed August 30, 2014.

28.

Duensing S, Lee L, Duensing A, et al. The human papillomavirus type 16 E6 and E7 oncoproteins cooperate to induce mitotic defects and genomic instability by uncoupling centrosome duplication from the cell division cycle. Proc Natl Acad Sci U S A. 2000;97(18):10002-10007.

29.

Picken R, Yang H. The integration of HPV-18 into HeLa cells has involved duplication of part of the viral genome as well as human DNA flanking sequences. Nucleic Acids Res. 1987;15(23):10068.

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CMLE QUESTIONS 1.

Why do the 2012 American Cancer Society guidelines not recommend human papillomavirus (HPV) molecular testing for screening purposes for younger women? A.

Younger women are naturally immune to high-risk HPV infections.

B.

Most younger women are vaccinated against HPV, and therefore testing isn’t necessary.

C.

Most HPV infections in younger women are transient and cleared by the individual’s immune system.

D.

2.

3.

4.

Only older women are infected with high-risk HPV.

Malignant transformation of HPV infection depends on which 2 HPV genes? A.

L1 and L2

B.

E6 and E7

C.

E3 and E8

D.

L2 and E7

Vaccines are directed against the 2 HPV genotypes that cause most cervical cancer. Which 2 are they? A.

HPV16 and HPV33

B.

HPV31 and HPV45

C.

HPV18 and HPV31

D.

HPV16 and HPV18

The Aptima HPV assay (Hologic, Gen-Probe) tests for A.

viral mRNA of E6 and E7 genes.

B.

viral DNA.

C.

viral proteins.

D.

human DNA integration.

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5.

Which of the following statements regarding sensitivity of the HPV molecular methods discussed in this exercise is true? A.

The mRNA assay is more sensitive than either DNA method.

B.

The DNA hybrid capture assay is more sensitive than the APTIMA mRNA assay.

C.

All of the methods have poor sensitivity.

D.

All of the methods have similarly high sensitivity (>90%).

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