Cervical Cancer Screening: Impact of Human Papillomavirus mRNA Testing on Detecting High-Grade Lesions in Women with Normal Cytology

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors present the results of a cohort study of the impact of HPV mRNA testing on quality assurance of 61,635 women with normal pap smears obtained between 2016-2019.

They conclude: "HPV mRNA testing significantly augments traditional cytological screening, improving the early detection and management of cervical cancer. This supports the need for its broader integration into screening strategies."

Overall, the study was well-designed, analyzed and reported.  My comments are relatively minor:

1) I found the term "rescreening" confusing as used.  One hand, "rescreening" implies reassessing an existing pap smear from a QA perspective; i.e., a pap smear is re-read by a different cytopathologist.  One the other hand, as I believe is the case in this study, "rescreening" can indicate that a person was screened again at a later date.  I suggest the authors choose a different term than "rescreening" (preferably) and/or explicitly clarify their meaning of the term.

2) Similarly, there are no to minimal data regarding the timing of the subsequent follow-up ("rescreening"?) pap smears, colposcopic evaluations and biopsies.

3) Was HPV co-testing (DNA, mRNA) done at the time of rescreening?  If so, it would be helpful to include those results as existing data clearly demonstrates fluctuance in HPV expression.

4) Similar to my comment about "rescreening", the term "revised cytologic diagnoses" is unclear.  In general, that term implies the original diagnosis for a given test (pap smear in this case) has been revised upon further assessment. I believe the authors are using the term to indicate that the patient's diagnosis has been revised upon follow-up; e.g., from "normal" to "ASCUS".  The authors should clarify their meaning as it is confusing to readers.

5) Tables 3 and 4 should be combined for clarity and space limitations.

6) Section 3.3 is unclear. "In the cohort of 752 women with normal Pap smears but positive HPV mRNA tests, 5 cases of cancer (0.7%) were identified during the follow-up period. Notably, four of these cases had initially been rescreened and identified as ASC-US+, indicating abnormal cytology. This subset of ASC-US+ women, numbering 220 in total, accounted for the majority of the diagnosed cancer cases in the study (data not shown)." Please clarify.

7) Lines 253-257: "In 2022, of the 257 Norwegian women aged 25 to 69 diagnosed with cervical cancer, 45 had previously received a normal Pap smear result within 3.5 years prior to their diagnosis [27]. This stark finding underscores the potential for severe dysplasia (CIN3+) to develop into invasive cancer over 10-20 years and the critical need for more effective early detection methods." The italicized line, while true, is not supported by the preceding statement with regard to the time interval (3.5 years vs. 10-20 years.)

Author Response

Reviewer 1 

The authors present the results of a cohort study of the impact of HPV mRNA testing on quality assurance of 61,635 women with normal pap smears obtained between 2016-2019. 

They conclude: "HPV mRNA testing significantly augments traditional cytological screening, improving the early detection and management of cervical cancer. This supports the need for its broader integration into screening strategies." 

Overall, the study was well-designed, analyzed and reported.  My comments are relatively minor: 

Comment 1: I found the term "rescreening" confusing as used.  One hand, "rescreening" implies reassessing an existing pap smear from a QA perspective; i.e., a pap smear is re-read by a different cytopathologist.  One the other hand, as I believe is the case in this study, "rescreening" can indicate that a person was screened again at a later date.  I suggest the authors choose a different term than "rescreening" (preferably) and/or explicitly clarify their meaning of the term. 
 
Response 1: Thank you for your comment regarding the use of the term 'rescreening.' In our study, 'rescreening' specifically refers to the re-evaluation of the same cytological sample by a different cytotechnologist following a positive HPV mRNA test result, particularly when the initial screening categorized the sample as 'normal.' According to national guidelines, if rescreening results in findings of ASC-US or LSIL and the HPV mRNA test is positive for HPV types 16 or 18, the patient is recommended for colposcopy and biopsy. Alternatively, if the findings are ASC-US or LSIL and the HPV type is 45, the recommendation is for a follow-up with repeat cytology and HPV testing after twelve months. Should HPV persistence be observed after 12 months, a colposcopy and biopsy are then recommended. 

To avoid further confusion, we will revise our manuscript to replace 'rescreening' with 're-evaluation' of the same cytology sample to describe this process more accurately. 

 

Comment 2: Similarly, there are no to minimal data regarding the timing of the subsequent follow-up ("rescreening"?) pap smears, colposcopic evaluations and biopsies. 
 
Response 2: Thank you for your comment regarding the timing of follow-up procedures. To clarify: 

• Initial HPV mRNA Testing: The residual material from the initial liquid-based cytology (LBC), which initially showed normal results, was tested for HPV mRNA within 2-3 weeks. 

• Re-evaluation Process: If HPV mRNA results were positive for types 16, 18, or 45, the original cytology sample was re-evaluated by a different cytologist and double-checked by a pathologist. 

• Subsequent Procedures: 

• Colposcopy and Biopsy: If there was an indication for colposcopy and biopsy, this was recommended to be performed within 1-3 months. 

• Follow-Up Testing: If the biopsy was normal or showed low-grade lesions (CIN1), or if there was an initial indication for follow-up without immediate biopsy, a new cytology and HPV test was recommended after 12 months. 
• Long-Term Follow-Up: All women who had their initial cervical cytology between 2016 and 2019 were followed up through 2022. This corresponds to at least three years of follow-up, aligning with Norwegian national guidelines which recommend screening with cervical cytology every three years for women aged 25-69. 

We have updated the manuscript to include detailed information about the timing of these follow-up procedures to ensure clarity for our readers. 

 

Comment 3. Was HPV co-testing (DNA, mRNA) done at the time of rescreening?  If so, it would be helpful to include those results as existing data clearly demonstrates fluctuance in HPV expression. 

Response 3: Thank you for your query regarding HPV co-testing. In our follow-up process, all women with either abnormal cytology results or a positive HPV test undergo co-testing using both cytology and HPV DNA tests. These data are maintained in the SymPathy database at the Department of Clinical Pathology, University Hospital of North Norway (UNN). 

In this study, we focused on: 

• The baseline results from the initial re-evaluation of cytology following a positive HPV mRNA test. 

• Subsequent biopsy results through 2022. 

We did not include detailed sequential data on subsequent cytology or HPV test results unless they directly pertained to the follow-up biopsies. For instance, if the cytology and HPV tests at the 12-month follow-up were both normal, no biopsy was indicated. This decision was primarily based on subsequent negative HPV DNA test results, particularly for women who had a positive HPV mRNA test at baseline but did not undergo an initial biopsy. 

We have updated the Methods section of the manuscript to clarify these procedures and have also acknowledged this as a limitation in the relevant section of the manuscript. 

 

Comment 4: Similar to my comment about "rescreening", the term "revised cytologic diagnoses" is unclear.  In general, that term implies the original diagnosis for a given test (pap smear in this case) has been revised upon further assessment. I believe the authors are using the term to indicate that the patient's diagnosis has been revised upon follow-up; e.g., from "normal" to "ASCUS".  The authors should clarify their meaning as it is confusing to readers. 

Response 4: Thank you for highlighting the confusion surrounding the term "revised cytologic diagnoses." In our study, this term specifically refers to instances where the initial cytology results, originally deemed 'Normal,' were re-evaluated due to a positive HPV mRNA test at baseline. If changes were observed during this re-evaluation, the new findings were recorded as revised diagnoses, which could range from 'Normal' to 'ASC-US', 'LSIL', or higher. 

To eliminate any ambiguity, we have clarified this in the manuscript by revising the terminology and providing a detailed explanation of our diagnostic re-evaluation process. We now specifically define 'revised cytologic diagnoses' as the updated findings from the re-evaluation of the initial cytology due to discordant results with the baseline HPV mRNA test. This re-evaluation process is distinct from any follow-up cytology performed after 12 months, which does not trigger a new assessment of the baseline cytology results. 

We believe these amendments will enhance the clarity of the manuscript and ensure that the methodology is accurately understood by readers. 

 

Comment 5. Tables 3 and 4 should be combined for clarity and space limitations. 

Response 5: Thank you for the suggestion to combine Tables 3 and 4. We understand the intent to streamline the presentation and address space limitations. However, after careful consideration, we have decided to retain both tables separately for several key reasons: 

• Clinical Relevance of Separate Endpoints: Table 3 correlates cytology results with CIN2+ histology, which is the current threshold for clinical intervention in cervical lesions. Table 4, on the other hand, correlates cytology results with CIN3+ histology, a more severe endpoint and one that provides robust data for assessing the risk of significant precancerous lesions. The distinction between CIN2+ and CIN3+ is clinically significant, as it influences patient management decisions. 

• Scientific Accuracy: Many studies in HPV testing and cervical screening emphasize CIN3+ because it represents a more definitive precursor to invasive cancer, thus serving as a critical marker for evaluating the efficacy of screening methods. Presenting these data separately allows for a more precise comparison and clearer interpretation of the effectiveness of cytology re-evaluation in detecting higher-grade lesions. 

• Reader Clarity: While combining the tables could conserve space, it could also potentially obscure important nuances between the outcomes of CIN2+ and CIN3+. Separate tables ensure that the data is accessible and comprehensible, allowing readers to easily grasp the differential impact of cytology re-evaluation on detecting varying degrees of cervical pathology. 

Given these considerations, we believe that maintaining both tables separately best serves the study's purpose and the interests of clarity and detailed reporting for the scientific community. We have, however, taken steps to ensure that the presentation is as concise as possible without sacrificing these important distinctions. 

We appreciate your feedback and hope this explanation clarifies our rationale for retaining both tables in their current form. 

 

Comment 6: Section 3.3 is unclear. "In the cohort of 752 women with normal Pap smears but positive HPV mRNA tests, 5 cases of cancer (0.7%) were identified during the follow-up period. Notably, four of these cases had initially been rescreened and identified as ASC-US+, indicating abnormal cytology. This subset of ASC-US+ women, numbering 220 in total, accounted for the majority of the diagnosed cancer cases in the study (data not shown)." Please clarify. 

Response 6: Thank you for your feedback regarding the clarity of Section 3.3. We apologize for any confusion and appreciate the opportunity to clarify this section. 

In our study, 752 women had initially normal cytology results but tested positive for HPV mRNA. During the follow-up period, cervical cancer was detected in 5 of these women, which corresponds to a prevalence rate of 0.7%. Notably, four of these five cancer cases had their initial 'normal' cytology results revised to 'ASC-US+' upon re-evaluation prompted by the positive HPV mRNA test result. This re-evaluation was part of our quality assurance process using the 3-type HPV mRNA test, which specifically targets the most oncogenic HPV types and is sensitive to changes in cellular activity that may not be captured in primary cytology screenings. 

This subset of women with 'ASC-US+' revised diagnoses accounted for the majority of the diagnosed cancer cases in the study, underscoring the importance of HPV mRNA testing as a supplementary diagnostic tool. It highlights that without the HPV mRNA test, these cases might have progressed undetected until the next scheduled screening, potentially allowing the cancer to develop further. 

We have revised Section 3.3 to more clearly present these findings and the pivotal role of HPV mRNA testing in identifying cases that may require earlier intervention than what would be indicated by cytology alone. 

 
 
Comment 7: Lines 253-257: "In 2022, of the 257 Norwegian women aged 25 to 69 diagnosed with cervical cancer, 45 had previously received a normal Pap smear result within 3.5 years prior to their diagnosis [27]. This stark finding underscores the potential for severe dysplasia (CIN3+) to develop into invasive cancer over 10-20 years and the critical need for more effective early detection methods." The italicized line, while true, is not supported by the preceding statement with regard to the time interval (3.5 years vs. 10-20 years.) 

Response 7: Thank you for pointing out the discrepancy between the time intervals mentioned in our discussion. You are correct in noting that the transition from citing a 3.5-year interval to discussing a development period of 10-20 years for cervical cancer could confuse readers. 

To clarify, while we noted that 45 of the 257 Norwegian women diagnosed with cervical cancer in 2022 had received a normal Pap smear within the past 3.5 years, our intention was to highlight the longer, typical progression time of cervical cancer from severe dysplasia (CIN3+) to invasive cancer, which can span 10-20 years. This progression underscores the importance of detecting lesions as early as possible, which can sometimes be missed in the triennial screening cycle. 

To address this, we have revised the manuscript to better articulate the connection between the typical progression timeline of cervical cancer and the observed failures within the 3.5-year screening interval. This revision will clarify that the earlier data point serves to illustrate the limitations of current screening intervals, not to directly correlate with the longer development timeline of cervical cancer. 

Reviewer 2 Report

Comments and Suggestions for Authors

Longitudinally, the Nordic countries have been renowned for their effective cervical screening programs, underpinned by well functioning registries, sufficient resource allocation and rigorous audit. Norway perhaps excels in cost-effectiveness of primary and secondary cervical cancer prevention strategies.

The optimal cervical screening method tailored for individuals 25-30yrs has been debated over time; until recently most guidelines discouraged the use of molecular HPV DNA approaches because of increased positivity harvest and low specificity concerns (as the authors correctly point, lines 68-69). Lately, the incorporation of vaccinated cohorts in cervical screening, has mandated a global shift towards HPV-based approaches in these younger ages entering screening.

This is a well prepared submission assessing the potential of a validated mRNA HPV assay implementation conducted in a University Hospital in Norway, as a quality assurance retrospective and follow-up study, augmented by robust registry data.

Abstract: Descriptive and concise. The potential of cervical biopsies to reliably identify precancerous cervical lesions in older individuals with endocervical transformation zones (type 3 TZ’s) is questionable.

The introduction is helpfully organized in subsections. In Subsection 1.3: The phrase “For quality assurance, this study utilized a specific 3-type HPV mRNA test (PreTect SEE) to rescreen cytologically normal samples that tested positive for HPV types 16, 18 and 45” should be definitely rephrased for clarity. Readers might get the wrong impression this study is a secondary triage intervention [samples with known (DNA?) HPV positivity were re-tested with an HPV mRNA assay], whereas seemingly this is a primary triage intervention (samples were only previously tested with cytology once and judged as NILM).

Introduction, Subsection 1.5. Research Question: “Our analysis…follow-up” This should be a bold statement and not underrated in the context of an extensive Introduction section.

Materials and Methods: Well-appointed.

Results: Table 1: A 20.5% ASC-US percentage is marginally high for any cytology laboratory.

Table 2: In almost one fourth of the patients biopsies were not performed; please comment on the colposcopic impression in these cases. Also, as stated before, ectocervical punch biopsied have serious limitations in Type 3 transformation zones (i.e. will not exclude HG disease).

All Figures and Tables are carefully prepared and helpful.

Discussion: The author’s claim “our data supports direct colposcopy and biopsy for all women testing positive for HPV mRNA, irrespective of their rescreening cytology results” is fully justified. With research on methylation still evolving, validated mRNA assays currently represent is a pragmatic feasible option.

The authors might consider some additional remarks or comments, comparing between mRNA & DNA HPV assays in this particular context (screening/triage in various age groups) as sufficient literature has accrued over time.

Finally, despite outside the scopes of this study, of note is that mRNA HPV assays still require further optimization to be considered in cervical screening self sampling approaches (Arbyn et al, Lancet Oncol 2022).

Author Response

Reviewer 2 
 
Comment 1: Longitudinally, the Nordic countries have been renowned for their effective cervical screening programs, underpinned by well functioning registries, sufficient resource allocation and rigorous audit. Norway perhaps excels in cost-effectiveness of primary and secondary cervical cancer prevention strategies. 

Response 1: Thank you for recognizing the strengths of the cervical screening programs in the Nordic countries, particularly in Norway. Indeed, the effectiveness of these programs is greatly enhanced by comprehensive national registries and a robust social security number system, which facilitate meticulous tracking of individuals over time. This tracking system allows for precise follow-up and contributes significantly to the overall success of our primary and secondary cervical cancer prevention strategies. 

Additionally, the integration of sufficient resource allocation and rigorous audit processes ensures that our programs not only meet but often exceed the required standards for cost-effectiveness and efficacy in cancer prevention. We appreciate your acknowledgment of these factors and are committed to continuously enhancing these strengths through our ongoing research and implementation efforts. To reflect this commitment, we have updated the introduction of our manuscript. 

 

Comment 2: The optimal cervical screening method tailored for individuals 25-30yrs has been debated over time; until recently most guidelines discouraged the use of molecular HPV DNA approaches because of increased positivity harvest and low specificity concerns (as the authors correctly point, lines 68-69). Lately, the incorporation of vaccinated cohorts in cervical screening, has mandated a global shift towards HPV-based approaches in these younger ages entering screening. 

Response 2: Thank you for highlighting the ongoing debate around the optimal cervical screening methods for individuals aged 25-30 years. As you noted, this age group historically posed a challenge due to high HPV positivity rates, which were not always indicative of high cervical cancer risk, particularly in vaccinated cohorts. 

Indeed, there is always a trade-off between sensitivity and specificity when designing screening algorithms. Young women, even those vaccinated against HPV types 16 and 18, can still test positive for other high-risk HPV types included in broader spectrum tests. Recent studies, including a population-based HPV prevalence study in the Nordic region by Nygård et al., have suggested that restricting HPV screening tests to the types included in the nonavalent vaccine might optimize the balance of harms and benefits in the post-vaccination era (Nygard 2021). This study highlights that screening for all 14 HPV types might result in suboptimal outcomes. 

Furthermore, a Swedish study supports this selective approach, showing that a significant majority (85.3%) of screen-detected cervical cancers were associated with HPV 16, 18, 31, 33, 45, or 52, and that including the additional eight types covered by most tests only marginally increased detection rates by 1.5% [22]. Similarly, a Norwegian study found that these additional types were detected in only 1.4% of screen-detected cancers, with 93.0% (66/71) attributable to the HPV types 16, 18, 31, 33, and 45 (Sorbye 2023). 

In Norway, another study found that no cases of cervical cancer were detected during a 5-7 year follow-up among women under 40 with normal cervical cytology and negative tests for HPV types 16, 18, and 45 using the PreTect SEE HPV mRNA test (Al-Shibli 2022). This evidence suggests that co-testing with cytology and a 3-type HPV mRNA test might perform better than primary screening with a 14-type HPV DNA test in women aged 25-30 due to its higher specificity. However, as outlined in recent guidelines (Portnoy 2022), most countries might continue to employ broad-spectrum HPV DNA testing with triage of positive cases until all women younger than 30 have been vaccinated, reducing the need for extensive screening in this age group. 

We have updated the manuscript to clearly reflect these nuanced considerations in our discussion of screening strategies for younger women. 

 
Sundström K, Dillner J. How Many Human Papillomavirus Types Do We Need to Screen For? J Infect Dis. 2021 May 20;223(9):1510-1511. doi: 10.1093/infdis/jiaa587. PMID: 32941611. 
 
Nygård, M.; Hansen, B.T.; Kjaer, S.K.; Hortlund, M.; Tryggvadóttir, L.; Munk, C.; Lagheden, C.; Sigurdardottir, L.G.; Campbell, S.; Liaw, K.L.; et al. Human papillomavirus genotype-specific risks for cervical intraepithelial lesions. Hum. Vaccin. Immunother. 2021, 17, 972–981. 

Sørbye, S.W.; Falang, B.M.; Antonsen, M. Distribution of HPV Types in Tumor Tissue from Non-Vaccinated Women with Cervical Cancer in Norway. J. Mol. Pathol. 2023, 4, 166-177. https://doi.org/10.3390/jmp4030015 
 
Portnoy A, Pedersen K, Nygård M, Trogstad L, Kim JJ, Burger EA. Identifying a Single Optimal Integrated Cervical Cancer Prevention Policy in Norway: A Cost-Effectiveness Analysis. Med Decis Making. 2022 Aug;42(6):795-807. doi: 10.1177/0272989X221082683. Epub 2022 Mar 8. PMID: 35255741. 

Comment 3: This is a well prepared submission assessing the potential of a validated mRNA HPV assay implementation conducted in a University Hospital in Norway, as a quality assurance retrospective and follow-up study, augmented by robust registry data. 

Response 3: Thank you for your positive assessment of our submission. We are grateful for your recognition of the robustness of our study, which leverages comprehensive registry data to retrospectively evaluate the impact of the HPV mRNA assay as a quality assurance measure in cervical cancer screening. Indeed, to our knowledge, this is one of the largest studies of its kind that investigates the use of an HPV mRNA test to minimize the risk of cervical cancer following normal cytology results. This approach not only underscores the potential for improved screening protocols but also highlights the importance of integrating advanced molecular diagnostics in routine clinical practice. We believe our findings contribute significantly to the ongoing discussion about optimizing cervical cancer prevention strategies, particularly in settings with existing strong screening infrastructure. 

Comment 4: Abstract: Descriptive and concise. The potential of cervical biopsies to reliably identify precancerous cervical lesions in older individuals with endocervical transformation zones (type 3 TZ’s) is questionable. 

Response 4: Thank you for your observation regarding the reliability of cervical biopsies in identifying precancerous lesions in older individuals, especially those with type 3 endocervical transformation zones (TZ). We acknowledge the limitations in the sensitivity of colposcopically guided biopsies in cases where the transformation zone is not visible, a scenario common in older women. 

In two previous publications we have indeed highlighted these challenges. In Sorbye 2010, we suggested a "test-and-treat" approach for women over 40 years with HSIL cytology, and in Sorbye 2011, we extended this recommendation to cases following a positive HPV mRNA test 12 months after a negative cervical biopsy. These studies underscore the need for tailored strategies in managing older populations where traditional biopsy methods may fall short. 

However, the current study primarily focuses on quality assurance for women with normal cervical cytology. This context emphasizes the potential of HPV mRNA testing as an adjunct tool in identifying women at risk even when traditional cytological assessments show normal results. While the issue of biopsy reliability in older women with type 3 TZ is indeed crucial, it forms part of a broader conversation on optimizing cervical cancer prevention strategies across different subpopulations and diagnostic scenarios. 

 
Sørbye SW, Fismen S, Gutteberg T, Mortensen ES (2010) Triage of Women with Minor Cervical Lesions: Data Suggesting a “Test and Treat” Approach for HPV E6/E7 mRNA Testing. PLoS ONE 5(9): e12724. https://doi.org/10.1371/journal.pone.0012724 
 
Sørbye SW, Arbyn M, Fismen S, Gutteberg TJ, Mortensen ES (2011) HPV E6/E7 mRNA Testing Is More Specific than Cytology in Post-Colposcopy Follow-Up of Women with Negative Cervical Biopsy. PLoS ONE 6(10): e26022. https://doi.org/10.1371/journal.pone.0026022 

 

Comment 5: The introduction is helpfully organized in subsections. In Subsection 1.3: The phrase “For quality assurance, this study utilized a specific 3-type HPV mRNA test (PreTect SEE) to rescreen cytologically normal samples that tested positive for HPV types 16, 18 and 45” should be definitely rephrased for clarity. Readers might get the wrong impression this study is a secondary triage intervention [samples with known (DNA?) HPV positivity were re-tested with an HPV mRNA assay], whereas seemingly this is a primary triage intervention (samples were only previously tested with cytology once and judged as NILM). 

Response 5: Thank you for your critical observation on the description of the HPV mRNA test usage in our study. Your comment highlights a vital aspect of clarity that we need to address to prevent any misunderstanding of our methods. 

In our study, the 3-type HPV mRNA test (PreTect SEE) was indeed utilized as an additional quality assurance measure rather than a conventional triage tool. This test was applied retrospectively to the residual material from cytologically normal samples (judged as NILM—Negative for Intraepithelial Lesion or Malignancy). Importantly, these samples were not previously tested for HPV DNA; the mRNA test was employed to enhance detection sensitivity and potentially catch high-risk cases that the initial cytology might have missed. 

This approach was adopted to leverage the high specificity of mRNA testing for HPV types 16, 18, and 45, aimed at reducing the risk of cervical cancer development within the screening interval. This was not a secondary triage of known HPV-positive samples but rather a proactive measure to assess the risk in samples initially deemed normal by cytology alone. 

To better communicate this, we have revised Subsection 1.3 to clearly state that the 3-type HPV mRNA test was used as a supplementary quality assurance tool to assess samples initially tested only with cytology and not as a response to prior HPV DNA test results. This clarification should eliminate any confusion regarding the role of the HPV mRNA test in our study framework. 

We hope this amendment will clarify the purpose and implementation of the HPV mRNA test in our study and appreciate your help in improving the manuscript’s accuracy and readability. 

 

Comment 6: Introduction, Subsection 1.5. Research Question: “Our analysis…follow-up” This should be a bold statement and not underrated in the context of an extensive Introduction section. 

Materials and Methods: Well-appointed. 

Response 6: Thank you for your valuable feedback on the presentation of our research question in Subsection 1.5. We appreciate your suggestion to make this section more prominent and impactful within the extensive introduction. 

In response, we have revised Subsection 1.5 to foreground the importance and novel contribution of our study more clearly. We now begin the section with a bold statement that highlights the innovative aspect of employing a 3-type HPV mRNA test as a quality assurance measure alongside Pap smears. This approach not only challenges traditional screening methods but also aims to significantly enhance the detection of high-grade lesions in women who initially presented with normal Pap results. 

Here is the revised version of Subsection 1.5: 

"In a groundbreaking quality assurance initiative, this study at the Department of Clinical Pathology at the University Hospital of North Norway (UNN) evaluates the integration of a 3-type HPV mRNA test with traditional cervical cytology. Conducted from 2016 to 2019, the project targeted women who received normal cytology results but tested positive for HPV, focusing on the re-evaluation of their initial cytology samples. Our analysis meticulously examines the impact of this integration, particularly assessing the proportion of women with positive HPV mRNA tests, adjustments in cytological diagnoses, and the detection of high-grade lesions (CIN2+) during follow-up. The primary goal is to determine how effectively this strategy minimizes misinterpretations of cervical cytology and improves the early identification of women who require further intervention, adhering to national guidelines through December 2022. " 
 
We believe that this reformulation not only makes the research question stand out but also clarifies the scope and intent of our study, enhancing the reader's understanding of its significance within the field of cervical cancer screening. 

 
 
Comment 7: Results: Table 1: A 20.5% ASC-US percentage is marginally high for any cytology laboratory. 

Response 7: Thank you for your observation regarding the ASC-US percentage reported in our results. It is indeed critical to contextualize this figure appropriately. The 20.5% ASC-US rate does not apply to the total of 98,648 cervical samples analyzed as part of our primary screening. Instead, this figure represents the ASC-US outcomes specifically within the subset of 752 women who initially had normal cytology but tested positive for HPV via the 3-type HPV mRNA test and whose samples were subsequently re-evaluated. 

This re-evaluation led to the identification of ASC-US in 20.5% of these high-risk cases. While this percentage may seem high, it is actually reflective of a targeted re-assessment within a small, select group identified by the HPV mRNA test as being at potentially higher risk. When this 20.5% is factored into the total cohort of 98,648 cervical samples, it corresponds to an incremental increase of only 0.25% in the overall ASC-US rate. This slight increase has minimal impact on the overall number of follow-ups required but significantly enhances the detection of potential high-risk cases that might otherwise be missed, thereby improving our ability to prevent cervical cancer following initially normal cytology results. 

We have clarified this explanation in the manuscript to ensure the context and implications of these figures are well understood. 

 

Comment 8: Table 2: In almost one fourth of the patients biopsies were not performed; please comment on the colposcopic impression in these cases. Also, as stated before, ectocervical punch biopsied have serious limitations in Type 3 transformation zones (i.e. will not exclude HG disease). 

All Figures and Tables are carefully prepared and helpful. 

Response 8: Thank you for your important comments concerning the management of cases where biopsies were not performed, as well as addressing the limitations associated with Type 3 transformation zones. 

Colposcopic Impressions Where Biopsies Were Not Performed: In our study, the absence of biopsies was not due to colposcopic findings being considered insufficiently abnormal but rather scenarios where colposcopy was not conducted according to the guidelines. According to Norwegian national guidelines, a biopsy should always be performed if colposcopy is indicated. If colposcopy does not reveal visible lesions but is conducted due to persistent high-risk HPV or ASC-US/LSIL findings, blind biopsies from all four quadrants of the transformation zone and an endocervical curettage (ECC) are recommended. The cases where biopsies were not performed involved situations where a follow-up with repeat cytology and HPV testing was chosen instead, based on specific clinical judgments and patient follow-up protocols, aligning with a conservative management strategy particularly for women with transient infections. 

Management of Type 3 Transformation Zones: We recognize the inherent challenges in diagnosing high-grade disease in women with Type 3 transformation zones, where lesions may not be visible. For women with negative biopsy results and persistent high-risk HPV or escalating cytological abnormalities, follow-up protocols involve repeat cytology and HPV testing at 12-month intervals. If abnormalities persist, or if consecutive screenings raise increasing concern without clear biopsy confirmation, a diagnostic excisional procedure like conization (LEEP) is recommended, especially for women over 40 years of age. This ensures that potential high-grade lesions hidden within Type 3 TZs are not missed. 

We have clarified these aspects in the manuscript, ensuring that our study's protocols and adherence to national guidelines are transparent and understandable. This clarification will help readers appreciate the rigor and safety inherent in our clinical decision-making processes. 

Comment 9: Discussion: The author’s claim “our data supports direct colposcopy and biopsy for all women testing positive for HPV mRNA, irrespective of their rescreening cytology results” is fully justified. With research on methylation still evolving, validated mRNA assays currently represent is a pragmatic feasible option. 

Response 9: Thank you for acknowledging the justification of our approach in recommending direct colposcopy and biopsy for all women testing positive for HPV mRNA, irrespective of their cytology results. This strategy is based on the substantial risk associated with positive HPV mRNA test results, reflecting active viral transcription that may lead to high-grade lesions or cancer. 

While the research on methylation markers is indeed promising, these are not yet broadly integrated into national guidelines for HPV-positive triage. Our facility at the University Hospital of North Norway has incorporated a 7-type HPV mRNA test in the triage of HPV DNA-positive women, following its inclusion in the national guidelines for primary screening of women aged 34-69 in 2019 and later extended to include all women aged 25-69 in 2023. This approach is informed by our long-term experience and studies, which have shown the practical effectiveness and feasibility of HPV mRNA testing as a reliable triage method in a real-world clinical setting (Sorbye 2023). 

We believe that while alternative markers like dual staining (p16/Ki67) and extended genotyping are being used in some regions, the robustness and immediate applicability of HPV mRNA tests make them a particularly valuable tool in managing HPV-positive women effectively until such time as methylation markers may be more fully validated and recommended in guidelines. 
 
Sørbye, S.W.; Falang, B.M.; Antonsen, M. Performance of a 7-Type HPV mRNA Test in Triage of HPV DNA Primary Screen Positive Women Compared to Liquid-Based Cytology. J. Mol. Pathol. 2023, 4, 69-80. https://doi.org/10.3390/jmp4020008 

 

Comment 10: The authors might consider some additional remarks or comments, comparing between mRNA & DNA HPV assays in this particular context (screening/triage in various age groups) as sufficient literature has accrued over time. 

Response 10: Thank you for the suggestion to enhance our discussion on the comparison between HPV mRNA and DNA assays, especially in the context of screening and triage across various age groups. 

HPV mRNA assays are generally recognized for their higher specificity compared to HPV DNA assays. This specificity is particularly valuable in younger women, who are more likely to experience transient HPV infections. The higher specificity of HPV mRNA tests, especially those targeting a limited number of HPV genotypes, helps to reduce the number of false-positive results leading to unnecessary follow-up procedures in these age groups (Sorbye 2016). 

Regarding longitudinal studies, while there are extensive follow-ups for HPV DNA tests, with some studies extending up to 30 years (Riibe 2021), HPV mRNA tests have been followed up to 12 years in recent research (Rad 2023). Most studies involving the Norwegian HPV mRNA tests have primarily focused on the original 5-type assay (PreTect HPV-Proofer). However, recent studies have also incorporated the more limited 3-type assay (PreTect SEE) alongside traditional cytology, and the extended 7-type assay (PreTect HPV-Proofer'7) for broader screening contexts. 

In the Norwegian setting, while many studies examine HPV mRNA testing in referral populations with low-grade cytological abnormalities like ASC-US or LSIL, our study uniquely investigates the use of HPV mRNA testing in a quality assurance framework alongside primary screening. This approach underscores the potential utility of HPV mRNA testing not only in triage but also as a primary screening tool, particularly when aiming to enhance early detection and intervention strategies. 

We will expand our manuscript to include these comparative insights, providing readers with a broader understanding of how mRNA and DNA HPV assays perform across different settings and age groups. 

 
Sørbye SW, Fismen S, Gutteberg TJ, et alPrimary cervical cancer screening with an HPV mRNA test: a prospective cohort studyBMJ Open 2016;6:e011981. doi: 10.1136/bmjopen-2016-011981 
 
Riibe MØ, Sørbye SW, Simonsen GS, Sundsfjord A, Ekgren J, Maltau JM. Risk of cervical intraepithelial neoplasia grade 3 or higher (CIN3+) among women with HPV-test in 1990-1992, a 30-year follow-up study. Infect Agent Cancer. 2021 Jun 22;16(1):46. doi: 10.1186/s13027-021-00386-z. PMID: 34158090; PMCID: PMC8220730. 
 
Rad A, Sørbye SW, Tiwari S, Løchen ML, Skjeldestad FE. Risk of Intraepithelial Neoplasia Grade 3 or Worse (CIN3+) among Women Examined by a 5-Type HPV mRNA Test during 2003 and 2004, Followed through 2015. Cancers (Basel). 2023 Jun 8;15(12):3106. doi: 10.3390/cancers15123106. PMID: 37370716; PMCID: PMC10296297. 

See also the publication list at PreTect AS, the manufacturer of the Norwegian HPV mRNA tests PreTect SEE and PreTect HPV-Proofer, for more information: 

https://www.pretect.no/publications/list 

 
 

Comment 11: Finally, despite outside the scopes of this study, of note is that mRNA HPV assays still require further optimization to be considered in cervical screening self sampling approaches (Arbyn et al, Lancet Oncol 2022). 

Response 11: Thank you for pointing out the ongoing developments in HPV mRNA assay optimization for self-sampling, as highlighted by Arbyn et al. in 2022. Recent studies postdating the review by Arbyn have shown promising results regarding the use of HPV mRNA tests in self-sampling contexts, specifically the XytoTest device. 

Two notable studies by Flores in 2021 and 2024 have compared HPV DNA and HPV mRNA assays in both physician-collected and self-collected samples. These studies found that the positivity rates for HPV DNA and HPV mRNA were comparable across both sample types. Interestingly, the positivity rate for the 7-type HPV mRNA test was only a third of that observed with the 14-type HPV DNA test. Furthermore, both tests showed similar sensitivities for detecting CIN3+, but the 7-type HPV mRNA test demonstrated higher specificity and a higher positive predictive value (PPV) for CIN3+, which effectively reduced the number of unnecessary colposcopies. 

At the University Hospital of North Norway, we utilize the XytoTest self-sampling device with the 7-type HPV mRNA test for several groups: women who are underscreened, those uncomfortable with gynecological examinations, and women with abnormal screening results lacking adequate follow-up. Utilizing the HPV mRNA test in self-sampling circumvents the need for additional triage with cervical cytology before referring for colposcopy and biopsy—a common requirement when a HPV DNA test yields a positive result. This streamlined approach not only reduces patient visits but also minimizes the risk of loss-to-follow-up by eliminating repeated call/recall cycles before final diagnostic decisions. 

These practical applications of the 7-type HPV mRNA test in our hospital support its effectiveness and feasibility in self-sampling scenarios, reinforcing the findings from recent literature and demonstrating significant advancements in this area since the review by Arbyn et al. 

 
Aranda Flores CE, Gomez Gutierrez G, Ortiz Leon JM, Cruz Rodriguez D, Sørbye SW. Self-collected versus clinician-collected cervical samples for the detection of HPV infections by 14-type DNA and 7-type mRNA tests. BMC Infect Dis. 2021 May 31;21(1):504. doi: 10.1186/s12879-021-06189-2. PMID: 34058992; PMCID: PMC8165795. 

Flores, C.E.A.; Falang, B.M.; Gómez-Laguna, L.; Gutiérrez, G.G.; León, J.M.O.; Uribe, M.; Cruz, O.; Sørbye, S.W. Enhancing Cervical Cancer Screening with 7-Type HPV mRNA E6/E7 Testing on Self-Collected Samples: Multicentric Insights from Mexico. Cancers 2024, 16, 2485. https://doi.org/10.3390/cancers16132485 

Reviewer 3 Report

Comments and Suggestions for Authors

I would like to congratulate the authors on the work they put into preparing the manuscript. It is written legibly, clearly and intrigues the reader. The presented molecular methods may be of great importance in the future in detecting pre-cancerous lesions with a correct Liquid-based cytology result, and at the same time they are a minimally invasive method.

1.       The methodology does not specify whether the cytology was collected on a liquid medium (LBC) or on a slide - conventional cytology

2.       Please use LATS terminology currently preferred in publications.

3.       A huge advantage of the work is gathering a large research group. I congratulate the authors on assessing the original 76,138 samples

4.       The work indicates that new molecular methods, such as extracting HPV mRNA, can be a very helpful and non-invasive diagnostic test for detecting pre-cancerous conditions.

5.       The authors rightly point out that Pap smears, interpreted subjectively, carry risks of both false negatives and overlooked high-grade lesions. It is characterized by 50-70% sensitivity for detecting severe dysplasia (CIN3),

6.       The authors write that the number of cervical cancers has tripled since 1950, please refer to the vaccination situation in the country. Are they common and used in adults? Was the study group asked/checked in the database about the status of vaccinated patients? How many patients were diagnosed with cc?

7.       Please specify what the indications for the biopsy were

8.       In Table 1, in the second part, please correct "biopsy" instead of "biopsi"

9.       Please explain the abbreviations each time under the tables

10.    The quality of Figure 1 requires improvement; the text is illegible and the arrows are missing

11.    Table 3 - please correct the border, it is not uniform; what does "CIN 1-" mean?

12.    I encourage you to cite other works presenting the sensitivity and specificity of molecular tests detecting E6/E7 mRNA proteins; also showing high sensitivity for detecting CIN 2+ lesions also in the population of increasingly older patients, in the group up to 50 years of age.

Pruski D, Millert-Kalinska S, Lewek A, Kedzia W. Sensitivity and specificity of HR HPV E6/E7 mRNA test in detecting cervical squamous intraepithelial lesion and cervical cancer. Ginekol Pol. 2019;90(2):66-71. doi: 10.5603/GP.2019.0011. PMID: 30860271.

 Jin X, Liu F, Zhang Y, Ma Y, Yang L, Wang Y, Liu Y. Diagnostic value of high-risk HPV E6/E7 mRNA in patients with ASCUS. BMC Womens Health. 2023 Sep 14;23(1):489. doi: 10.1186/s12905-023-02599-3. PMID: 37710244; PMCID: PMC10503067.

Author Response

Reviewer 3 

I would like to congratulate the authors on the work they put into preparing the manuscript. It is written legibly, clearly and intrigues the reader. The presented molecular methods may be of great importance in the future in detecting pre-cancerous lesions with a correct Liquid-based cytology result, and at the same time they are a minimally invasive method. 

Comment 1: The methodology does not specify whether the cytology was collected on a liquid medium (LBC) or on a slide - conventional cytology 

Response 1: Thank you for highlighting this detail. In our study, all cervical cytology samples were collected using liquid-based cytology (LBC) on ThinPrep medium. We initially used the term 'Pap smear' generically to refer to cervical cytology, which might have caused some confusion regarding the specific technique employed. To clarify, we have updated the manuscript to replace all instances of 'Pap smear' with 'liquid-based cytology (LBC)' throughout the document to prevent any misunderstanding. 

Comment 2: Please use LATS terminology currently preferred in publications. 

Response 2: Thank you for your suggestion regarding the use of LAST terminology, which categorizes lesions as LSIL and HSIL, typically distinguished using p16 immunostaining. While LAST is widely recognized, it does not distinguish between CIN2 and CIN3 as directly as traditional terminology, which is crucial for clinical management under Norwegian guidelines. Notably, the management of CIN2, especially in women under 30, involves careful monitoring rather than immediate treatment, reflecting its potential for spontaneous regression (Castle 2009). Studies, including a systematic review by Tainio et al., indicate that about half of CIN2 lesions regress within two years, and fewer than 20% progress to more severe stages, with even higher regression rates in younger women (Tainio 2018). Furthermore, the US Preventive Services Task Force recommends focusing on immediate risks of CIN3+ rather than CIN2+ due to the high regression likelihood of the latter, aiming to balance the detection and treatment of significant pre-cancer stages against the harms of unnecessary interventions (Wentzensen 2024). Given this context, our study employs traditional CIN terminology, which better supports our clinical decision-making framework and aligns with the guidelines that advocate for nuanced treatment approaches based on precise histological evaluation. 

Castle, P.E.; Schiffman, M.; Wheeler, C.M.; Solomon, D. Evidence for frequent regression of cervical intraepithelial neoplasia-grade 2. Obstet. Gynecol. 2009, 113, 18–25. 
 
Tainio, K.; Athanasiou, A.; Tikkinen, K.A.; Aaltonen, R.; Cárdenas, J.; Glazer-Livson, S.; Jakobsson, M.; Joronen, K.; Kiviharju, M.; Louvanto, K.; et al. Clinical course of untreated cervical intraepithelial neoplasia grade 2 under active surveillance: Systematic review and meta-analysis. BMJ 2018, 360, k499. 
 
Wentzensen, N.; Garcia, F.; Clarke, M.A.; Massad, L.S.; Cheung, L.C.; Egemen, D.; Guido, R.; Huh, W.; Saslow, D.; Smith, R.A.; et al. Enduring Consensus Guidelines for Cervical Cancer Screening and Management: Introduction to the Scope and Process. J. Low Genit. Tract Dis. 2024, 28, 117–123. 

Comment 3: A huge advantage of the work is gathering a large research group. I congratulate the authors on assessing the original 76,138 samples 

Response 3: Thank you for recognizing the scope of our work. This study represents one of the largest applications of a 3-type HPV mRNA test for quality assurance in women with normal cytology. It encompassed all participants in the Norwegian cervical cancer screening program within Troms and Finnmark counties. The study’s scale was made possible by integrating routine cervical cytology screening conducted at the Department of Clinical Pathology at the University Hospital of North Norway with additional HPV mRNA testing performed by PreTect AS, who expanded their team specifically to manage the volume from our study. Annually, this involved the re-evaluation of about 3-4 samples per week over four years, which was manageable within our workflow. Importantly, each of the 752 women with a positive HPV mRNA test received follow-up consistent with national guidelines through December 2022, regardless of the revised diagnosis after the initial cytology re-evaluation. This comprehensive approach underscores the potential of integrating HPV mRNA testing into routine screening to enhance the detection and follow-up of high-risk cases. 

Comment 4: The work indicates that new molecular methods, such as extracting HPV mRNA, can be a very helpful and non-invasive diagnostic test for detecting pre-cancerous conditions. 

Response 4: Absolutely, and thank you for highlighting this crucial aspect of our research. One of the key takeaways from our study is that a positive HPV mRNA test indicates a high risk of CIN2+ lesions even in cases where cervical cytology results are normal. This molecular method allows us to identify a relatively small group of high-risk women (1.2% of those screened) who would benefit from more immediate and targeted follow-up. This targeted approach ensures that these women do not have to wait the standard three years until the next screening round, thereby enhancing early detection and timely intervention for those at greatest risk. 

Comment 5: The authors rightly point out that Pap smears, interpreted subjectively, carry risks of both false negatives and overlooked high-grade lesions. It is characterized by 50-70% sensitivity for detecting severe dysplasia (CIN3). 

Response 5: Indeed, the limited sensitivity of cytology-based screening, often cited as 50-70% for detecting severe dysplasia (CIN3), is a well-recognized limitation. This has prompted a shift in many countries from traditional cytology-based methods to HPV-based screening to enhance detection rates. When our study commenced in 2016, cytology was still the primary screening method in Norway, despite the known risk of missing significant cervical lesions. The findings from our study have been well-documented and widely disseminated through the Norwegian cancer registry, national cervical cancer screening program, and mass media. These efforts have significantly contributed to the updated national guidelines in 2023, which now recommend HPV-based screening for all women aged 25 to 69 years. This change is expected to reduce the incidence of cervical cancer by improving the early detection of high-grade lesions. 

Comment 6: The authors write that the number of cervical cancers has tripled since 1950, please refer to the vaccination situation in the country. Are they common and used in adults? Was the study group asked/checked in the database about the status of vaccinated patients? How many patients were diagnosed with cc? 

Response 6: In Norway, the HPV vaccination was introduced into the childhood immunization program in 2009 for 12-year-old girls and extended to include boys in 2018. Currently, vaccination coverage among 12-year-olds stands at approximately 90%. The first cohort of vaccinated girls reached the age of 25 in 2022, the starting age for participation in the national cervical cancer screening program, which covers women aged 25 to 69. According to data from the Norwegian Cancer Registry, only one woman who was vaccinated in 2009 or 2010 has been diagnosed with cervical cancer in 2022 or 2023. Adult vaccination is almost nonexistent in Norway. In our study, among the five cases of cervical cancer detected in women with initially normal cytology and positive HPV mRNA tests, four were identified following the re-evaluation of the initial normal cytology results, and one during the follow-up of women with positive HPV mRNA tests but without cytological abnormalities. None of the women diagnosed with cervical cancer in our study had received the HPV vaccine. This context underscores the significance of HPV vaccination in reducing cervical cancer incidence and highlights the impact of our findings in a largely unvaccinated adult population. 

Comment 7: Please specify what the indications for the biopsy were 

Response 7: According to national guidelines, colposcopy and biopsy were indicated under the following circumstances: 1) High-grade cytology findings (ASC-H/HSIL) regardless of HPV status; 2) Low-grade cytology findings (ASC-US/LSIL) coupled with a positive HPV test for HPV types 16 or 18; and 3) Persistent HPV positivity, demonstrated by consecutive positive HPV tests 12 months apart. These criteria ensure timely and targeted interventions for those most at risk of developing cervical cancer. 

Comment 8: In Table 1, in the second part, please correct "biopsy" instead of "biopsi" 

Response 8: Thank you for pointing out the typo. It has been corrected from 'biopsi' to 'biopsy' in Table 1. 

Comment 9: Please explain the abbreviations each time under the tables 

Response 9: We have updated the manuscript to include explanations for all abbreviations used, under each table where they appear, to ensure clarity for all readers. 

Comment 10: The quality of Figure 1 requires improvement; the text is illegible and the arrows are missing 

Response 10: We appreciate your feedback on the quality of Figure 1. We have completely revised this figure to improve clarity and legibility. All text has been made more readable, and we have ensured that all necessary arrows are included and clearly visible. The updated figure now meets the high standards required for publication. 

 
Comment 11: Table 3 - please correct the border, it is not uniform; what does "CIN 1-" mean? 

Response 11: Thank you for pointing out the issue with the border in Table 3; we will ensure that this is corrected in the final layout by the production team at MDPI Women before publication. Regarding the term 'CIN 1-', it is used to denote histological findings of CIN1 or less, encompassing normal histology and low-grade lesions. 'CIN2+' refers to high-grade histological findings, including CIN2, CIN3, adenocarcinoma in situ (ACIS), and invasive cervical cancer. We have now clarified these definitions within the manuscript to prevent any confusion. 

Comment 12: I encourage you to cite other works presenting the sensitivity and specificity of molecular tests detecting E6/E7 mRNA proteins; also showing high sensitivity for detecting CIN 2+ lesions also in the population of increasingly older patients, in the group up to 50 years of age. 

Response 12: Thank you for your suggestion to enhance our discussion with additional references regarding the sensitivity and specificity of E6/E7 mRNA tests. We have included relevant studies to underscore the high sensitivity of these tests for detecting CIN 2+ lesions, particularly in older patient groups up to 50 years of age. For example, a study from Poland involving 125 women tested with a 5-type HPV mRNA assay (NucliSENS EasyQ® HPV v1.1 by bioMérieux) reported a sensitivity for CIN2+ of 86.1% (31/36) (Pruski 2019). Additionally, a Chinese study of 1,387 patients with ASC-US cytology who tested positive for HPV E6/E7 mRNA using the Hologic APTIMA assay demonstrated that 62.2% (219/352) of women with histologically confirmed HSIL+ had HPV types 16, 18, or 45, and this included 67.2% (43/64) of the women diagnosed with cervical cancer (Jin 2023). These references illustrate the robust performance of HPV mRNA testing across different populations and age groups. 

Pruski D, Millert-Kalinska S, Lewek A, Kedzia W. Sensitivity and specificity of HR HPV E6/E7 mRNA test in detecting cervical squamous intraepithelial lesion and cervical cancer. Ginekol Pol. 2019;90(2):66-71. doi: 10.5603/GP.2019.0011. PMID: 30860271. 

 Jin X, Liu F, Zhang Y, Ma Y, Yang L, Wang Y, Liu Y. Diagnostic value of high-risk HPV E6/E7 mRNA in patients with ASCUS. BMC Womens Health. 2023 Sep 14;23(1):489. doi: 10.1186/s12905-023-02599-3. PMID: 37710244; PMCID: PMC10503067. 

Reviewer 4 Report

Comments and Suggestions for Authors

Dear Authors, I think that your research is very interesting and valuable. I encourage you to continue to work with this very important topic in gynecology and women's health in general.
In future investigations you can include men as well because that would be interesting to see since we do not know very much about men and HPV infections.

 

Author Response

Reviewer 4 

Comment 1: Dear Authors, I think that your research is very interesting and valuable. I encourage you to continue to work with this very important topic in gynecology and women's health in general. 
In future investigations you can include men as well because that would be interesting to see since we do not know very much about men and HPV infections. 

Response 1: Thank you for recognizing the value of our research and for your insightful suggestion regarding the inclusion of male participants in future studies. Indeed, understanding HPV's impact on men is equally crucial, as HPV is associated with various cancers beyond the cervix, including penile, anal, and oropharyngeal cancers. While our current national guidelines in Norway do not include HPV screening for men, they are included in the HPV vaccination program as of 2018. We acknowledge the gap in research concerning HPV infections in men and the potential outcomes of HPV mRNA testing in this population. Exploring this could provide valuable insights into broader preventative strategies and the efficacy of vaccination. Future studies could certainly benefit from incorporating a more inclusive approach to better understand HPV's implications across genders. 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have addressed the issues raised in the original review.

I still believe that the tables should be combined, but I will defer that decision to the editors.

Otherwise, acceptable for publication.

Reviewer 2 Report

Comments and Suggestions for Authors

In this revised version, all comments have been addressed.

The Introduction and Materials & Methods section have been essentially re-written in a more reader-friendly manner. The Discussion section, albeit lengthy, has been also improved.

Overall, a very well prepared manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

Thank You for improving manuscript. I recommend to publish it in the current form.