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Quality Improvement to Demonstrate the Lack of Reliability of the Human Papillomavirus mRNA Assay to Identify Women With Latent Human Papillomavirus Infections

Cotton, Sarah, BS; Brown, Robert, E., MD; Nugent, Elizabeth, K., MD; Robazetti, Sonia, C., MD; Berens, Pamela, D., MD; Smith, Judith, A., PharmD

doi: 10.1097/AOG.0000000000002530
Contents: Clinical Practice and Quality
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OBJECTIVE: To assess the consistency between human papillomavirus (HPV) mRNA testing in women with a history of previous HPV infections diagnosed by HPV DNA assay and the potential effects on follow-up HPV screening.

METHODS: This was a quality improvement study that used data from a pathology laboratory software database reviewed from November 2014 to June 2016 to identify female patients aged 30 years or older with greater than one HPV-positive result, including one or more HPV mRNA assay results and one or more documented HPV DNA assay results for comparison. Previous correlative cytology and colposcopic histopathology were also documented. American College of Obstetricians and Gynecologists' cervical cancer screening guidelines were used to compare potential differences in follow-up recommendations.

RESULTS: Four hundred twenty-five charts for female patients 30 years of age or older were identified with one or more prior high-risk HPV infections by DNA assay. There was a 69.3% difference in HPV mRNA results compared with previous HPV DNA–positive results. There was a potential change in follow-up for 71.7% of patients with one prior high-risk-HPV-positive result and 60.0% of patients with two or more prior high-risk HPV-positive results. There were 231 colposcopy reports evaluated in this study. Of these, 62 (26.8%) were abnormal colposcopy reports, including 45 low-grade squamous intraepithelial lesions, 15 high-grade squamous intraepithelial lesions, and two cancers. Twenty-five (40.3%) abnormal colposcopy findings were in patients with a history of at least than two prior HPV DNA–positive results and a report of currently being HPV-negative with the mRNA assay.

CONCLUSION: The HPV mRNA assays are less sensitive for detection of latent HPV infections compared with HPV DNA assays. Based on these data and the potential change in follow-up care, the HPV mRNA assay should not be used for a primary screening tool for cervical cancer. Many pathology laboratories have shifted to using the HPV mRNA assay without clear discussion with gynecologists about the effects on patient follow-up. The type of HPV assay being used should be documented and any HPV mRNA result confirmed by HPV DNA assay.

Because human papillomavirus mRNA and DNA assays differ in sensitivity for detecting latent human papillomavirus infections, clinicians should be aware of the testing type used in the clinical laboratory.

Departments of Obstetrics, Gynecology, and Reproductive Sciences and Pathology and Laboratory Medicine, McGovern Medical School at UTHealth, UTHealth-Memorial Hermann Cancer Center-TMC, and the Department of Pharmacy, Memorial Hermann Hospital-TMC, Houston, Texas.

Corresponding author: Judith A. Smith, PharmD, Division of Gynecologic Oncology, Department of Obstetrics, Gynecology & Reproductive Sciences, UTHealth McGovern Medical School, 6431 Fannin Street, Room 3.152, Houston, TX 77030; email:

Financial Disclosure The authors did not report any potential conflicts of interest.

Each author has indicated that he or she has met the journal's requirements for authorship.

Human papillomavirus (HPV) is a double-stranded DNA virus that infects squamous and glandular epithelial cells in the ectocervix and endocervix and integrates its genome into the host DNA, causing either an active or a latent infection without detectable symptoms.1,2 It is the most common cause of invasive cervical cancer. In the majority of women, the host immune system will eliminate the HPV infection within 6–18 months. Approximately 10% of patients have a persistent infection that fluctuates between latent (inactive infection) and active infection. Active infection can result in cell damage with resultant dysplasia, which can lead to cancer.

The American College of Obstetricians and Gynecologists (ACOG) identifies two primary indications for testing for high-risk HPV infection: reflex testing, to determine the need for colposcopy in women with an atypical squamous cells of undetermined significance cytology result, and cotesting as an adjunct to cytology in women ages 30–65 years of age for cervical cancer screening.3 High-risk HPV strains 16 and 18 are responsible for 80% of all HPV-associated cancers, 66% of cervical cancer in the United States, and, as such, are the primary targets in HPV testing.4

The two most common methods of testing for HPV DNA are Hybrid Capture 2 and polymerase chain reaction.3,5,6 The DNA-based HPV assay detects the presence or absence of an HPV infection and cannot distinguish between an active or a latent infection. Typically active HPV infections will cause cellular damage and latent infections do not, but it is important to note that a latent infection may convert to an active infection at any time of physiologic stress or immunosuppression. A negative DNA test signifies that there is no HPV infection present (Table 1).

Table 1

Table 1

Follow-up screening guidelines for high-risk HPV-positive results are based on the patient's age and clinical history of abnormalities or dysplasia, as shown in Table 2 in ACOG Practice Bulletin No. 168, available at,7 To minimize unnecessary follow-up and interventions based on HPV-positive results alone, methods to detect active HPV infections and identify patients who may go on to have dysplasia or abnormalities have been introduced.

Table 2

Table 2

One of these is the E6/E7 mRNA-based HPV test. This test detects the presence of E6 and E7 mRNA within squamous and glandular epithelial cells in the cervix. The E6 and E7 oncoproteins are a necessity for malignant transformation of epithelial cells and are almost always detected in cancerous lesions.8 Both oncoproteins degrade tumor suppressor proteins controlling proliferation; E6 degrades p53 and E7 degrades retinoblastoma protein.1,8 Together, these two oncoproteins create a synergistic effect that ultimately leads to the transformation of HPV-infected epithelium into tumorigenic cells.8 Detection of the E6 and E7 mRNA indicates active HPV infection and is highly correlated with both histologic and cytologic abnormalities. The more severe the lesion, the larger number of E6 and E7 mRNA copies and likelihood of positive mRNA results.9–11

A negative E6 and E7 mRNA result is not equivalent to HPV DNA–negative because mRNA testing only detects an active HPV infection with active transcription of the viral genome. The presence of a latent HPV infection will not be detected with E6 and E7 mRNA testing.12,13 Compared with the HPV DNA assay, the HPV mRNA assay is more specific in predicting the presence of dysplasia but less sensitive in detecting the presence of HPV latent infection.14 For screening purposes, the HPV DNA test is preferred because it will detect latent infection.

During the initial enrollment phase for an HPV research study, an unusual observation was made that multiple patients with a 10-year history or greater of high-risk HPV positivity being screened for the study now had a negative HPV test. After exploration of this unusual trend, it was found that the collaborating pathology laboratory had recently just switched from an HPV DNA to an HPV mRNA assay. These samples were then retested with a HPV DNA test and four of five results returned high-risk HPV-positive. This quality improvement study was developed because the clinical consequences of using the HPV mRNA assay as a primary cervical cancer screening tool to detect latent infections are so unclear.15 The purpose of the study was to assess the frequency of discordant results between the two tests with any documented previous cytology and biopsies results within the past 5 years to evaluate the potential changes in follow-up and treatment after the pathology laboratory had switched primary HPV screening tools in the clinical setting.

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This retrospective study was reviewed and approved to proceed by the University of Texas Health Sciences Center at Houston institutional review board as a quality improvement research study. The HPV test results were obtained from the University of Texas Health Sciences Center Department of Pathology APeasy (anatomic pathology laboratory software) database, created by Small Business Computers of New England. The mRNA test utilized is the APTIMA HPV assay and the DNA test used is the Cervista HPV-HR assay. Both tests were created by Hologic and detect the same 14 HPV high-risk types: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68. The sensitivity and specificity for each respective assay are readily available from Hologic.

In this retrospective study, pathology charts were electronically searched to identify women older than age 30 years who had had at least one positive HPV DNA result reported in the laboratory system before November 2014 and also had results from at least one HPV mRNA assay reported between November 2014 and June 2016. The age of 30 years was selected intentionally because “older” age is associated with a greater likelihood of a persistent HPV infection. Pathology charts with missing data were excluded.

All HPV test results, cytology, and colposcopic-directed biopsy results were entered into a deidentified electronic database by the data extractor. An independent review of the data entry was done to confirm accuracy of data collection. Results of the HPV DNA and HPV mRNA were compared. Using the guidelines from ACOG from 2016 and the American Society for Colposcopy and Cervical Pathology 2012 guidelines16,17 as best practice, we then assessed how the use of the HPV mRNA test may have altered patient management. Actual patient chart and clinical follow-up were not evaluated in this study. Data were then stratified by age (30–39, 40–49, 50–59, 60+ years), cytology, and history of abnormal biopsy results from colposcopy examinations. Descriptive statistics were completed to describe the HPV assay results.

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There were 425 patient pathology charts who met the inclusion criteria. In 68.0% of patients (289/425), a negative HPV mRNA test followed the prior HPV DNA test. When the negative HPV mRNA assay results in women with a previous positive HPV DNA data were evaluated, there was consistently a potential change in follow-up as recommended by the 2016 ACOG cervical cancer screening guidelines. Of the 289 patients with discordant results, 201 (69.6%) likely would have had their recommended follow-up altered based on the negative HPV mRNA results. Among women with only one prior HPV DNA–positive result, 170 of 237 (71.7%) may have altered recommended clinical follow-up, and this was true for 31 of 52 (59.6%) of those with at least two prior HPV DNA results. When stratified by age, the 40- to 49-year-old group and those older than age 60 years both had greater discrepancy compared with other age groups. Figure 1 shows the summary of discrepancies between assay results across all age groups.

The deidentified data then were further stratified by those more likely to have had latent HPV infection, defined as having greater than two previous HPV DNA–positive results and analyzed for discrepancy. There were 89 (20.9%) patients with two or more prior HPV DNA–positive results. Of these, 52 (58.4%) had current HPV mRNA–negative results. These data were also stratified by age, which revealed a 55.0% discrepancy for patients aged 30–39 years, a 65.2% discrepancy for patients aged 40–49 years, a 52.9% discrepancy for patients aged 50–59 years, and a 66.7% discrepancy for those 60 years and older (Fig. 2).

The 289 patients with discrepancies of HPV results then were analyzed based on their history of abnormal cytology as shown in Table 2. Of these patients, 146 (50.5%) had no history of abnormal cytology. However, 143 (49.5%) patients did have a history of abnormal cytology, including 89 (30.8%) with a 1-year history and 54 (18.6%) with a 2-year or greater history of abnormal cytology. Table 3 summarizes the history of abnormal cytology for the overall population. Of the 289 patients who had discrepancies of HPV test results, 110 (38.1%) patients had a prior Pap cytology diagnosis of atypical squamous cells, cervical intraepithelial neoplasia 1, 2, or 3, or cancer within the previous 5 years (Table 4). Of patients with a prior cervical intraepithelial neoplasia 2 or 3 diagnosis, 12 were aged 30–39 years, four aged 40–49 years, five aged 50–59 years, and two patients aged 60 years and older. Two patients had a previous HPV-related cancer diagnosis.

Table 3

Table 3

Table 4

Table 4

Finally, the data from patients with discrepancies between HPV assay results then were analyzed by history of colposcopies. There were 231 colposcopy reports evaluated in this study. Of these, 62 (26.8%) were abnormal colposcopy histopathology reports, including 45 low-grade squamous intraepithelial lesion diagnoses, 15 high-grade squamous intraepithelial lesion diagnoses, and two cancer diagnoses. Specifically, 25 (40.3%) of these abnormal colposcopy findings were in patients with a history of at least two prior HPV DNA–positive results and a report of currently being HPV-negative with the mRNA assay. Table 5 summarizes the history of abnormal colposcopy findings for the overall population.

Table 5

Table 5

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Approximately 70% of women with one or more prior HPV DNA–positive results had a current negative HPV mRNA result. Clinical recommendations based on best practice for women with a negative follow-up HPV result likely would change for these 70% of women. Based on the prestudy analysis we did, an estimated 80% of women with a prior positive HPV DNA test with a negative HPV mRNA test would have a positive HPV DNA result indicating a latent infection and no change in follow-up would be recommended. Some may argue women with one prior HPV DNA test may have cleared the infection by the time the HPV mRNA testing was done because the majority of women clear HPV infection without any intervention in 6–18 months. Even if analysis is focused on just the 89 patients more likely to have had a persistent HPV infection defined in this study as greater than two previous HPV DNA results, 60% had a current negative HPV mRNA test result. Furthermore, in this group of women with presumably persistent HPV infection, review of pathology records revealed that 75% have had at least one abnormal Pap test result and 36.5% have had at least one abnormal colposcopy. This points to the strong possibility that these patients are still have infection and need closer screening to prevent development of cancer. There was a potential change in follow-up in an overwhelming majority of women who had pathology charts reviewed in this study, which is concerning because the HPV mRNA test is becoming more prevalent as a primary screening tool. As such, it is important for the clinician to know what type of test their clinical laboratory uses for HPV screening and to use a laboratory that uses HPV DNA testing for screening purposes.

The ACOG 2016 guidelines used to evaluate whether discrepancy between HPV assay results could potentially affect clinical interventions, follow-up, or both do not clearly distinguish which HPV assay should be used in primary screening; however, the referenced data are based on the HPV DNA assay.7 The current Centers for Disease Control and Prevention HPV screening guidelines do specify that the HPV DNA test should be used for preliminary screening with subsequent triage with the HPV mRNA assay to guide additional interventions and follow-up.17 Recently the American Society of Clinical Oncology published guidelines for secondary prevention of cervical cancer that does specify that the HPV DNA assay should be used for primary HPV screening.18

As a quality improvement study, the results suggest that the HPV DNA assay for primary screening should be reinstituted as a standard assay for HPV screening. We demonstrated the limitations and potential inaccuracy and unreliability if the HPV mRNA assay is used “off-label” as primary screening rather than its intended U.S. Food and Drug Administration-approved use to triage positive HPV DNA test results to more accurately distinguish which patients should undergo colposcopy and biopsy. The main limitation of this study is that the samples were not simultaneously tested using both the HPV mRNA and HPV DNA assays.

The HPV mRNA assay is automated, requires less manual sample preparation and processing, is low-cost, and has therefore been adopted into current pathology practice in many cases replacing the HPV DNA assay, perhaps without clear discussion or consultation with the clinicians caring for these patients. At minimum, the type of HPV assay being used should be documented and any HPV mRNA screening result that is negative should be confirmed by a HPV DNA assay. Preferably, HPV DNA testing will be the primary test used by clinical laboratories for HPV screening. Hopefully, the data presented here will start conversations between gynecologists and pathologists to be sure the HPV DNA test is used for primary screening to identify those at potential long-term risk who should have closer monitoring and follow-up.

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© 2018 by The American College of Obstetricians and Gynecologists. Published by Wolters Kluwer Health, Inc. All rights reserved.