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. 2021 Feb 11;16(2):e0246902. doi: 10.1371/journal.pone.0246902

Histological outcomes in HPV-screened elderly women in Denmark

Gry St-Martin 1,*, Petra Hall Viborg 2, Ane Birgitte Telén Andersen 2, Berit Andersen 3,4, Jette Christensen 5, Dorthe Ejersbo 6, Hanne Nørgaard Heje 7, Kirsten Marie Jochumsen 8, Tonje Johansen 9, Lise Grupe Larsen 10, Elsebeth Lynge 1,11, Reza Rafiolsadat Serizawa 12, Marianne Waldstrøm 6,13
Editor: Clement A Adebamowo14
PMCID: PMC7877658  PMID: 33571319

Abstract

Introduction

Danish women exit cervical cancer screening at age 65 years, but 23% of cervical cancer cases occur beyond this age. In addition, due to gradual implementation of cervical cancer screening, older women are underscreened by today´s standards. A one-time screening with HPV test was therefore offered to Danish women born before 1948.

Methods

Register based study reporting histology diagnoses and conizations in women found HPV positive in the one-time screening. Number and proportion of women with severe or non-severe histology results were calculated for screened and HPV-positive women by age group or region of residence. Number of women with biopsy and/or conization per case of cervical intraepithelial neoplasia (CIN) grade 2 or worse (CIN2+) or CIN3+ were also calculated by age groups and region.

Results

4,479 (4.1% of screened women) had positive HPV test. 94% of these had one or more additional tests. 2,785 (62%) of HPV-positive women had histology results, and conization was performed in 1,076 (24% of HPV-positive and 1% of all screened women). HPV positivity and CIN3+ detection varied little between regions, but the proportions of HPV positive women undergoing histology varied between regions from 40% to 86% and the proportion with conization from 13% to 36%. Correspondingly, the number of histologies and conizations per CIN3+ detected varied from 5.9 to 11.2 and 1.8 to 4.7, respectively. In total, 514 CIN2+ (0.47% of screened women, 11% of HPV-positive) and 337 CIN3+ (0.31% of screened women, 7.5% of HPV-positive) were diagnosed, including 37 cervical cancer cases.

Discussion

HPV screening of insufficiently screened birth cohorts can potentially prevent morbidity and mortality from cervical cancer but longer follow-up is needed to see if cancer incidence declines in the screened women in the coming years. Management strategies differed among regions which influenced the proportions undergoing biopsy/conization.

Introduction

Cervical cancer screening has substantially reduced the incidence of cervical cancer [1]. Most countries in Europe recommend cervical screening of women up until the age of 60–65 years [2]. In many high-income countries, however, the cervical cancer incidence in women aged above 65 years is relatively high. This has led to considerations regarding optimal age to stop screening [3,4].

Denmark is no exception: women exit the screening program at age 60–64 years, but 23% of cervical cancer cases in 2011–16 were diagnosed in women aged 65 years or older [5]. This attracted attention and gave rise to debate among clinicians, politicians, and non-governmental organizations on a possible extension of the upper age limit for screening. The Danish cervical screening program was implemented gradually, until all women aged 23 to 59 years were covered in 2006. In 2007, the upper age limit was extended to 64 years, and since 2012, the national screening guidelines have recommended testing for high risk (hr) human papillomavirus (HPV) as an exit-test between age 60 and 64. Women aged below 60 are screened with cytology [6,7].

Realizing that the history of the Danish cervical cancer screening program had left women born before 1948 incompletely screened or unscreened [8], the government decided to offer a one-time HPV screening test to women born before 1948 [9,10]. This initiative was implemented in 2017–18, and 30% of invited women participated of whom 4.1% tested positive for hr-HPV [9]. In this article, we report on histological findings among these HPV-positive women with the aim of contributing to the evidence regarding cervical cancer screening beyond the currently recommended upper age limit.

Material and methods

The one-time screening initiative for women born before 1948 has been described in detail previously [9]. Briefly, the initiative was part of a wider, national plan for cancer developed by the Ministry of Health, which provided for a single contact to women in the targeted birth cohorts. No ongoing screening or reminders were included in the cancer plan for these cohorts. The initiative was implemented by the five administrative regions of Denmark, following joint planning by the national health authorities and the regions. All women in the targeted birth cohorts received a letter during 2017 from their region of residence with information about the initiative and an invitation to contact their general practitioner (GP) for an HPV screening test or, if needed, for individualized advice about participation. Denmark has a tax-financed, public healthcare system and participation in the screening was voluntary and free of charge to the women. The GP collected a cervical sample which was analyzed for hr-HPV at the public hospital pathology departments. Of 359,763 invited women, 108,585 (30%) were screened, of whom 4,479 (4.1%) were positive for hr-HPV, in the following abbreviated to HPV-positive [9].

Results of the initial HPV tests as well as triage and follow-up tests were recorded in the Danish Pathology Register (DPR), which holds complete cytology and histology results from all pathology departments in Denmark [11]. Test results for the 4,479 women who initially tested HPV-positive were retrieved from 2017 to October 2019 using the civil registration number, which provides a unique identifier for each woman. Data for this study was provided from the Quality Assurance Program of the Danish Regions (RKKP) and the Danish Quality Database for Cervical Cancer Screening (DKLS) aggregated by region or by 5-year groups of birth-year.

Although the initiative was centrally planned, the five regions had some autonomy in designing their screening algorithms, further testing, and management. The Capital region recommended referral of all HPV-positive women for colposcopy. In the four other regions, HPV type 16 and 18 were always recommended referral for colposcopy, and for other hr-HPV-types, cytology was performed on the screening sample and colposcopy recommended if atypical squamous cell of undetermined significance or worse (ASCUS+) was present. Otherwise, repeated HPV testing after 12 months was recommended.

Histology diagnoses in the DPR are coded according to the cervical intraepithelial neoplasia (CIN) classification. Histology was divided into severe diagnoses (CIN2, CIN3, cancer, and unclassifiable CIN) and non-severe diagnosis without indication of treatment (CIN1 and normal histology). Histology diagnoses may stem from biopsies, conizations, or hysterectomies. If more than one histology diagnosis was registered, the most severe was used for the analyses.

The following outcomes are reported by birth-year groups and by region:

  • Proportion testing HPV-positive

  • Proportion returning for further testing, i.e. followed up

  • Proportion having histological examination performed

  • Proportion with conization

  • Proportion with severe histology (CIN2+ and CIN3+)

  • Number of histologies and conizations per detected severe diagnosis (CIN2+ or CIN3+)

95% confidence intervals (CI) for proportions were calculated as Clopper-Pearson exact method. Differences between proportions was tested with two-sample test. Analyses were performed with STATA IC 15.1. This study was approved by the Danish Data Protection Agency via the Central Denmark Region which serves as ethical clearance according to Danish legislation.

Results

Of 108,585 screened women, 4,479 (4.1%) were HPV-positive, ranging from 4.3% in the youngest screened cohorts to 3.2% in the oldest. Of the 4,479 HPV-positive women, 4,222 (94%) had one or more additional tests performed during the period covered by this study; 2,785 (62% of HPV-positive, 2.5% of all screened women) had one or more histology result registered, 514 (11% of HPV positive, 0.47% of screened) had CIN2+, and 1,076 women (24% of HPV-positive, 1% of all screened women) had conization performed, Table 1.

Table 1. Danish women born before 1948 and HPV-screened in 2017–2018.

Screening outcome Birth year Total
1943–1947 1938–1942 1933–1937 1932 or earlier
Screened women 60,921 31,990 12,057 3,617 108,585
HPV-positive 2,621 1,322 420 116 4,479
 % of screened (95% CI) 4.3% (4.1–4.5) 4.1% (3.9–4.4) 3.5% (3.2–3.8) 3.2% (2.7–3.8) 4.1% (4.0–4.2)
# with follow-up registered 2,513 1,246 376 87 4,222
 % of HPV-positive (95% CI) 96% (95–97) 94% (93–95) 90% (86–92) 75% (66–83) 94% (94–95)
Histology performed1 1,661 817 251 56 2,785
 % of screened (95% CI) 2.7% (2.6–2.9) 2.6% (2.4–2.7) 2.1% (1.8–2.4) 1.5% (1.2–2.0) 2.6% (2.5–2.7)
 % of HPV-positive (95% CI) 63% (61–65) 62% (59–64) 60% (55–64) 48% (39–58) 62% (61–64)
 % of women w/follow-up (95% CI) 66% (64–68) 66% (63–68) 67% (62–72) 64% (53–74) 66% (65–67)
Follow-up but no histology2 852 429 125 31 1,437
 % of screened (95% CI) 1.4% (1.3–1.5) 1.3% (1.2–1.5) 1.0% (0.86–1.2) 0.85% (0.58–1.2) 1.3% (1.3–1.4)
 % of HPV-positive (95% CI) 33% (31–34) 32% (30–35) 30% (25–34) 27% (19–36) 32% (31–33)
 % of women w/follow-up (95% CI) 34% (32–36) 34% (32–37) 33% (28–38) 36% (26–47) 34% (33–35)
Conization performed 627 344 85 20 1,076
 % of screened (95% CI) 1.0% (0.9–1.1) 1.1% (0.9–1.2) 0.7% (0.6–0.9) 0.6% (0.3–0.9) 1.0% (0.9–1.1)
 % of HPV-positive (95% CI) 24% (22–26) 26% (24–28) 20% (16–24) 17% (11–25) 24% (23–25)
 % of women w/follow-up (95% CI) 25% (23–27) 28% (25–30) 23% (18–27) 23% (15–33) 25% (24–27)
 % of women w/histology (95% CI) 38% (35–40) 42% (39–46) 34% (28–40) 36 (23–50) 39% (37–40)

Numbers and proportions of women screened, HPV-positive, with follow-up, with histology, and with conization by birth-year group.

1: From biopsy or conization.

2: Follow-up with cytology and/or HPV-test.

Across all cohorts, about two-thirds of women for whom follow-up tests were registered had a cervical histology diagnosis. Due to the lower proportion of HPV-positive women and less follow-up in the older cohorts, the proportion with histology diagnosis varied from 2.7% in the youngest to 1.5% in the oldest cohorts of screened women, and from 63% to 48% of HPV-positive women. A similar pattern was seen for conization. In total, 1.0% of screened women underwent conization varying from 1.0% in the youngest to 0.6% in the oldest cohort, but in each cohort constituting about one-fourth of women with follow-up, Table 1.

Overall, CIN3 was diagnosed in 300 women and cervical cancer in 37. These cases constituted 0.31% of screened women and 7.5% of HPV-positive women. All cancer cases occurred in women born after 1937, Table 2. For CIN2+, the proportions were 0.47% of screened women and 11% of HPV-positive women. In total, 3.2 women underwent conization per detected CIN3+ case, and these numbers varied little across cohorts, apart from the oldest group where the numbers were small. For all histology results, including from biopsies, 8.3 women had histology performed for each CIN3+ case diagnosed, Table 2. Fig 1 shows the variation in follow-up screening outcomes by birth year group. Older birth year groups had less follow-up examinations but less variation in CIN3+ detection.

Table 2. Histological outcome and proportions with <CIN2, CIN2+ and CIN3+ of screened women, of HPV-positive and of women with follow-up.

Histology outcome Birth year Total2
1943–1947 1938–1942 1933–1937 1932 or earlier
Women with histology1 1661 817 251 53 2782
 No malignancy or dysplasia 1181 552 174 37 1944
 CIN 1 163 84 29 5 281
 CIN2 60 40 5 # 105
 CIN 3/AIS 173 87 29 11 300
 CIN unclassified 42 23 5 # 70
 Cancer 21 16 0 0 37
 Inconclusive or unknown 21 15 9 # 45
<CIN2 1344 636 203 42 2225
 % of screened (95% CI) 2.2% (2.1–2.3) 2.0% (1.8–2.1) 1.7% (1.5–1.9) 1.2% (0.8–1.6) 2.0% (2.0–2.1)
 % of HPV-pos. (95% CI) 51% (49–53) 48% (45–51) 48% (43–53) 36% (27–45) 50% (48–51)
 % of women w/histology 81% (79–83) 78% (75–81) 81% (75–86) 75% (62–86) 80% (78–81)
CIN2+ 296 166 39 13 514
 % of screened (95% CI) 0.49% (0.43–0.54) 0.52% (0.44–0.6) 0.32% (0.23–0.44) 0.36% (0.19–0.61) 0.47% (0.43–0.51)
 % of HPV-pos. (95% CI) 11% (10–13) 13% (11–14) 9.3% (6.7–12) 11% (6.1–18) 11% (11–12)
 % of women w/histology 18% (16–20) 20% (18–23) 16% (11–21) 23% (13–36) 18% (17–20)
CIN3+ 194 103 29 11 337
 % of screened (95% CI) 0.32% (0.28–0.37) 0.32% (0.26–0.39) 0.24% (0.16–0.35) 0.30% (0.15–0.54) 0.31% (0.28–0.35)
 % of HPV-pos.(95% CI) 7.4% (6.4–8.5) 7.8% (6.4–9.4) 6.9% (4.7–9.8) 9.5% (4.8–16) 7.5% (6.8–8.3)
 % of women w/histology 12% (10–13) 13% (10–15) 12% (7.9–16) 20% (10–32) 12% (11–13)
Numbers of procedures per diagnosis
 Conizations per CIN2+ case (95% CI) 2.1 (1.8–2.4) 2.1 (1.8–2.5) 2.2 (1.5–3.2) 1.5 (0.8–3.1) 2.1 (1.9–2.3)
 Conizations per CIN3+ case (95% CI) 3.2 (2.8–3.8) 3.3 (2.7–4.2) 2.9 (1.9–4.5) 1.8 (0.9–3.8) 3.2 (2.8–3.6)
 Histology performed per CIN2+ case 5.6 (5.0–6.3) 4.9 (4.2–5.8) 6.4 (4.6–9.0) 4.3 (2.4–7.9) 5.4 (4.9–6.0)
 Histology performed per CIN3+ case 8.6 (7.4–9.9) 7.9 (6.5–9.7) 8.7 (5.9–12.7) 5.1 (2.7–9.7) 8.3 (7.4–9.3)

Conizations per CIN2+ or CIN3+ detected case by birth-year group. For women with more than one histology diagnosis, the most severe is used.

1: From biopsy or conization.

2: Cells with fewer than 3 women are excluded for reasons of privacy (marked #). Totals may thus differ between tables.

Fig 1. Proportions of women who were HPV positive, had histology performed, had CIN3+, or underwent conization, by birth year, compared to national average.

Fig 1

The proportion of HPV-positive women varied from 3.4% of screened women in Southern Denmark to 4.8% in Central Denmark (p < 0.0001), with the other regions in between, Table 3. The follow-up proportions were high in all regions, but type of follow-up varied considerably. Follow-up with histology varied from 1.8% of screened women in Southern Denmark to 3.5% in the Capital Region (p < 0.0001). In the Capital Region, 90% of women with follow-up had histology performed, 42% in Central Denmark Region, with the other regions in between. Conizations varied from 0.5% of screened women in Southern Denmark to 1.9% in Northern Denmark (p < 0.0001). In total, 0.31% of screened women had CIN3+ detected, Table 4.

Table 3. Numbers and proportions of women screened, HPV positive, with follow-up, with histology, and with conization by region.

Screening outcome Region1 Total
Capital Zealand South Central North
Screened women 30,498 17,807 25,212 23,654 11,414 108,585
HPV-positive 1,246 736 855 1,130 512 4,479
 % of screened (95% CI) 4.1% (3.9–4.3) 4.1% (3.8–4.4) 3.4% (3.2–3.6 4.8% (4.5–5.0) 4.5% (4.1–4.9) 4.1% (4.0–4.2)
# with follow-up test(s) 1,193 688 789 1,078 474 4,222
 % of HPV positive (95% CI) 96% (94–97) 93% (91–95) 92% (90–94) 95% (94–97) 93% (90–95) 94% (94–95)
Histology performed2 1,073 488 451 453 320 2,785
 % of screened (95% CI) 3.5% (3.3–3.7) 2.7% (2.5–3.0) 1.8% (1.6–2.0) 1.9% (1.7–2.1) 2.8% (2.5–3.1) 2.6% (2.5–2.7)
 % of HPV positive (95% CI) 86% (84–88) 66% (63–70) 53% (49–56) 40% (37–43) 63% (58–67) 62% (61–64)
 % of women w/follow-up (95% CI) 90% (88–92) 71% (67–74) 57% (54–61) 42% (39–45) 68% (63–72) 66% (65–67)
Follow-up but no histology3 120 200 338 625 154 1,437
 % of screened (95% CI) 0.39% (0.33–0.47) 1.1% (0.97–1.3) 1.3% (1.2–1.5) 2.6% (2.4–2.9) 1.3% (1.1–1.6) 1.3% (1.3–1.4)
 % of HPV-positive (95% CI) 9.6% (8.0–11) 27% (24–31) 40% (36–43) 55% (52–58) 30% (26–34) 32% (31–33)
 % of women w/follow-up (95% CI) 10% (8.4–12) 29% (26–33) 43% (39–46) 58% (55–61) 32% (28–37) 34% (33–35)
Conization performed 449 130 136 144 217 1,076
 % of screened (95% CI) 1.5% (13–16) 0.73% (0.61–0.87) 0.54% (0.45–0.64) 0.6% (0.51–0.71) 1.9% (1.7–2.2) 1.0% (0.9–1.1)
 % of HPV-positive (95% CI) 36% (33–39) 18% (15–21) 16% (14–19) 13% (11–15) 42% (38–47) 24% (23–25)
 % of women with follow-up (95% CI) 38% (35–40) 19% (16–22) 17% (15–20) 13% (11–16) 46% (41–50) 25% (24–27)
 % of women with histology (95% CI) 42% (39–45) 27% (23–31) 30% (26–35) 32% (28–36) 68% (62–73) 39% (37–40)

1: The full names of the Danish regions are Capital Region, Region Zealand, Region of Southern Denmark, Central Denmark Region, North Denmark Region.

2: From biopsy or conization.

3: Follow-up with cytology and/or HPV-test.

Table 4. Histological outcome and proportions with <CIN2, CIN2+ and CIN3+ of screened women, of HPV-positive and of women with follow-up.

Conizations per CIN2+ or CIN3+ detected case by region.

Histology outcome Region1 Total3
Capital Zealand South Central North
Women with histology2 1,073 488 451 453 319 2,784
 Normal histology 789 376 318 273 188 1,944
 CIN 1 90 26 32 77 56 281
 CIN2 39 24 12 16 15 106
 CIN 3/AIS 87 43 63 62 45 300
 CIN unclassified 45 6 7 12 # 70
 Cancer 9 6 13 6 3 37
 Histology inconclusive or unknown 14 7 6 7 12 46
<CIN2 879 402 350 350 244 2225
 % <CIN2 of screened women (95% CI) 2.9% (2.7–3.1) 2.3% (2.0–2.5) 1.4% (1.2–1.5) 1.5% (1.3–1.6) 2.1% (1.9–2.4) 2.0% (2.0–2.1)
 % <CIN2 of HPV positive (95% CI) 71% (68–73) 55% (51–58) 41% (38–44) 31% (28–34) 48% (43–52) 50% (48–51)
 % <CIN2 of women with histology (95% CI) 82% (79–84) 82% (79–86) 78% (73–81) 77% (73–81) 76% (71–81) 80% (78–81)
CIN2+ 180 79 95 96 64 514
 % CIN2+ of screened women (95% CI) 0.59% (0.51–0.68) 0.44% (0.35–0.55) 0.38% (0.30–0.46) 0.41% (0.33–0.50) 0.56% (0.43–0.72) 0.47% (0.43–0.51)
 % CIN2+ of HPV positive (95% CI) 14% (13–17) 11% (9–13) 11% (9–13) 8% (7–10) 13% (10–16) 11% (11–12)
 % CIN2+ of women with histology (95% CI) 17% (15–19) 16% (13–20) 21% (17–25) 21% (17–25) 20% (16–25) 18% (17–20)
CIN3+ 96 49 76 68 48 337
 % CIN3+ of screened women (95% CI) 0.31% (0.26–0.38) 0.28% (0.20–0.36) 0.30% (0.24–0.38) 0.29% (0.22–0.36) 0.42% (0.31–0.58) 0.31% (0.28–0.35)
 % CIN3+ of HPV positive (95% CI) 7.7% (6.3–9.3) 6.7% (5.0–8.7) 8.9% (7.1–11) 6.0% (4.7–7.6) 9.4% (7.0–12) 7.5% (6.8–8.3)
 % CIN3+ of women with histology (95% CI) 8.9% (7.3–11) 10% (7.5–13) 17% (14–21) 15% (12–19) 15% (11–19) 12% (11–13)
Numbers of procedures per diagnosis
 Conizations per CIN2+ case (95% CI) 2.5 (2.1–3.0) 1.6 (1.2–2.2) 1.4 (1.1–1.9) 1.5 (1.2–1.9) 3.4 (2.6–4.5) 2.1 (1.9–2.3)
 Conizations per CIN3+ case (95% CI) 4.7 (3.8–5.8) 2.7 (1.9–3.7) 1.8 (1.4–2.4) 2.1 (1.6–2.9) 4.5 (3.3–6.2) 3.2 (2.8–3.6)
 Histology performed per CIN2+ case (95% CI) 6.0 (5.1–7.0) 6.2 (4.9–7.8) 4.7 (3.8–5.9) 4.7 (3.8–5.9) 5.0 (3.8–6.5) 5.4 (4.9–6.0)
 Histology performed per CIN3+ case (95% CI) 11.2 (9.1–13.8) 10.0 (7.4–13.3) 5.9 (4.7–7.6) 6.7 (5.2–8.6) 6.7 (4.9–9.0) 8.3 (7.4–9.3)

1: The full names of the Danish regions are Capital Region, Region Zealand, Region of Southern Denmark, Central Denmark Region, North Denmark Region.

2: From biopsy or conization.

3: Cells with fewer than 3 women are excluded for reasons of privacy (marked #). Totals may thus differ between tables.

As shown in Fig 2 and Table 3, quite different proportions of screened women underwent biopsy and/or conization in the five regions, in spite of smaller variations in HPV positivity rate and CIN3+ detection. Accordingly, the number of conizations per CIN3+ differed substantially between regions from 1.8 in Southern Denmark to 4.7 in the Capital Region, Table 4. The number of women with histology to CIN3+ cases diagnosed varied between regions, with Capital and Zealand regions above the national average. Capital Region had the highest numbers of both histology and conizations per CIN3+ case, whereas North Denmark Region had a high number of conization per CIN3+ case, while the number of histology per CIN3+ case was below the national average.

Fig 2. Regional proportions of women who were HPV positive, had histology performed, had CIN3+, or underwent conization, compared to national average.

Fig 2

Discussion

Main findings

In 2017, 108,585 Danish women aged 69 years or older had cervical samples tested for HPV, and 4.1% were HPV-positive. By far the majority of the HPV-positive women had one or more follow-up tests taken, and about two-thirds had at least one histology result. Conization was performed in 1% of screened women, corresponding to 24% of HPV-positive women. Cervical cancer was detected in 37 women, and CIN3 in 300 women, which was a CIN3+ detection rate of 0.31% in screened women and of 7.5% in HPV-positive women. More than three women underwent conization per detected CIN3+ case. Proportions of screened women testing HPV-positive, undergoing histology or conization varied little below age 79 but were lower in women above this age. CIN3+ detection rates were similar across age.

While the CIN3+ detection rates varied little across the five Danish regions, there was a considerable variation in proportions of women undergoing histology and/or conization. As a consequence, there was approximately a two-fold variation in the numbers of biopsies/conizations per severe histology across regions.

Other studies

A Swedish study including 1,051 women aged 60–89 years found that 4.1% were HPV- positive [12]. For HPV-positive women, testing was repeated after 3 months, and if negative again at 12 months. Overall, 30 (3%) women had two positive HPV-tests within one year, and 30% of those positive on first test were negative on both repeat-tests. In another Swedish study, where women initially HPV-negative were retested after a mean interval of 3.2 years, 2.6% were HPV-positive, of whom 52% were positive again on repeat-test after an average of 2.5 months [13]. In both studies, women with two positive HPV-tests had a high prevalence of CIN on histology, although mainly CIN1, but few had detectable cytological changes. Comparable results were obtained in another study where HPV-test was performed on self-collected samples [14]. When retesting HPV-positive or HPV-negative women, these studies thus demonstrated that test results in elderly women vary over even short time spans, although it is unknown to which extent this is due to cleared infections, reinfection, reactivation of latent HPV-infection or difficulty in obtaining representative samples from elderly women. In these Swedish studies, fewer severe histology lesions were found than in ours. This may be related to sample size, selection of studied women, to screening histories of studied women, or to the historically lower cervical cancer incidence in Sweden than in Denmark [15].

A Danish GRADE-based evidence review on management of elderly, HPV-positive women concluded that there is insufficient evidence on how to ensure reduction in cervical cancer risk while minimizing overtreatment [16]. Surveillance over time is complicated by the difficulty in obtaining representative samples from the cervix due to the age-related changes, and the Danish guidelines therefore recommend considering conization in women where the transformation zone cannot be visualized and/or biopsies have been unrepresentative.

The Horizon study compared screening by cytology to different HPV assays in Denmark [17]. For women aged 30–65 years screened with Cobas HPV test (which was also used in most regions for the one-time HPV screening of older women), the study found 129 biopsies and 37 CIN3+ cases among 465 women positive on Cobas, i.e. 8% of HPV positive women had CIN3+, and 3.5 biopsies were performed per CIN3+ case diagnosed.

A woman’s risk of cervical cancer in old age is related to her screening history [18,19], and at the population level, the cervical cancer incidence in a specific age-group depends on the screening history of the birth cohorts making up that age-group at that point in time [8]. The Danish data showed a steady decrease in HPV-prevalence after the age of 35 years [9]. 82% of cervical cancer cases diagnosed in women aged above 60 years occurred in never or insufficiently screened women, and the average age of non- or insufficiently screened cancer patients was 76 years, the same age as cancer incidence peaks in old age [9,20]. However, the Danish Pathology Register is only complete after 1998, and the screening history of older women is therefore underreported in the register. Consequently, studies show an increase in proportion screened over time, reflecting not only increased screening but also increased completeness of the pathology register [21].

A cohort study of more than 500,000 Swedish women found that screening between age 61 and 65 years was beneficial for women with an insufficient screening history, while there was no significant reduction in cervical cancer risk up to age 80 years in women adequately screened with normal test results at age 51–60 [18].

Strengths and limitations

The availability of the national pathology register and unique identifiers ensured complete follow-up. However, data on deaths and emigrations were not available, and we were therefore unable to censor these women in the analysis. At the population level, we know that the birth cohorts targeted by the one-time HPV-screening were insufficiently screened, but individual data on the screening history, previous histology findings or therapeutic procedures were not available, as the pathology register data are incomplete before 1998.

For some women, conization was performed after a biopsy had revealed a severe lesion, but for other women the procedure was diagnostic, when a representative biopsy could not be obtained. For some women, a conization may have been performed even in case of a non-severe/no lesion to avoid lengthy follow-up with repeated cervical sampling. Some women may still be in follow-up with HPV test and/or biopsy and may still be conizised after the period covered in this study. Conization rates could thus be slightly higher than reported here, as a decision on end of follow-up is not coded.

Women whose HPV screening test was negative were not retested, so intermittent HPV-positivity, as in the Swedish studies referenced above, would not have been detected, and the prevalence of histological lesions in women testing HPV negative was unknown. This limits the ability of the study to fully estimate the cancer preventive effect of a single HPV test in these age groups.

Part of the variation between regions could theoretically stem from differences in age distribution. The aggregate RKKP data did not allow for age adjustment, but there is little difference across regions in the age distribution of women in the studied age-range [22]. We consider it unlikely that age specific participation in screening should vary across regions, and consequently we consider it unlikely that the observed regional differences in histology and conization rate derived from difference in age distributions.

Clinical implications

The European guidelines for cervical cancer screening recommend that women exit cervical cancer screening at age 60–65 years provided they have had a recent negative test [23]. The US preventive services task force recommends against screening of women older than 65 years if they have had adequate prior screening and are not at high risk for cervical cancer [24].

A possible extension of existing screening programs beyond age 65 years has been subject of both scientific and political considerations in many countries. In Denmark, the decision was to target in incompletely screened birth cohorts with the one-time HPV screening described here. Our results suggest that, for these cohorts, HPV screening can potentially prevent some of the morbidity and mortality from cervical cancer, as severe lesions (CIN3+) were detected in 7.5% of HPV-positive women, including 37 women with cervical cancer. However, not all CIN3+ cases progress to cancer, and some of the 37 detected cervical cancer cases might not have become symptomatic in the women’s remaining lifetime. Whether this screening intervention translates into fewer cervical cancer cases and deaths will be visible only after a longer follow-up.

The results may not, however, be generalizable to birth cohorts after 1948 who have had a systematic screening offer throughout their adult life. It is also important to note that, although women older than 65 years constitute an increasing proportion of cervical cancer cases, the incidence in older women has declined over the past decades [15,25], reflecting the progressively lower risk in each birth cohort [8]. The age-specific mortality has also declined over time [15]. With primary HPV screening being implemented in many countries, the risk in elderly women may be even lower in the future.

An alternative to a general extension of screening age might be to consider more personalized approaches, factoring in each woman´s age, screening history, comorbidities, general health and expected remaining lifespan [26] but more evidence is needed to estimate effects of both general and personalized strategies. In Denmark, complete cervical cancer screening results are available from the Danish Pathology Register for birth cohorts aging in the future, thus enabling studies comparing cancer risk after screening age for women who did or did not meet exit criteria, including women whose exit test was an HPV test.

In all screening initiatives, it is important to consider the potential harms in addition to the benefits. Although conization is generally considered a minor procedure, the risk of bleeding and complications like perforations may be elevated in older women, and age-related anatomical changes may render the procedure more difficult. In addition, the therapeutic effect of conization is not well documented for women over 60 years [16]. On the other hand, obtaining representative biopsies in older women is not always possible and therefore conization may be necessary for providing a diagnosis and can be preferable to avoid lengthy follow-up with repeated cervical sampling [16]. As shown in this study, the choice of management strategy has an important impact on the number of biopsies/conizations per findings of CIN2+ or CIN3+, and conceivably also on the number of women recalled for repeated tests in the absence of conization. Although differences in study design do not allow for direct comparison with the Horizon study, the results do suggest that the yield of CIN3+ compared to the number having histology results is less favorable for older women, with 8.3 histologies per CIN3+ in our study vs. 3.5 in Horizon.

Conclusion

To address the concern about the relatively high proportion of cervical cancer cases diagnosed beyond screening age, in 2017, a one-time HPV-screening initiative was offered to Danish women born before 1948. These birth cohorts had not been offered sufficient systematic screening earlier in life. In total, 4.1% of participating women tested HPV-positive, and out of those 7.5% had CIN3+ detected. While these proportions varied little across the five Danish regions, there were considerable regional differences in proportions of women undergoing biopsy and conization, reflecting differences in management strategies.

Data Availability

All relevant data are within the manuscript tables. To comply with data privacy, numbers of patients are masked when below 3.

Funding Statement

This study was financially supported by Region Zealand, Southern Denmark Region, The Danish Cancer Society, and Fabrikant Einar Willumsens Mindelegat. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

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Decision Letter 0

Clement A Adebamowo

26 Nov 2020

PONE-D-20-34114

Histological outcomes in HPV-screened elderly women in Denmark

PLOS ONE

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Reviewer #1: This is an important work that provides an insight of how to manage cervical cancer screening in women that may have not had because of their age an adequate screening history. The authors provide a cohort and a regional analysis of HPV detection, precancer cases detected and invasive cervical cancer detected. The geographical variability is noted as well as the cohort effect where older women have lower burden of disease. The authors identify the strength and limitation of the study. They are caution in indicating that the impact of this additional effort will be seen in follow up data on incidence and mortality from cervical cancer.

I have one consideration is that the authors report that out of 359,763 invited women, only 108,585 (30%) were screened. Was there any attempt to re/contact these women?. Given the number of cases detected among those screened it seems reasonable to try to capture more women. I could not see a discussion on this issue.

Reviewer #2: This is an excellent manuscript reporting precancer/cancer outcomes in a 'catch-up' HPV screening population of women born before 1948 in Denmark. In general the results are well-described and sound, though several questions or clarifications remain to support the author's conclusion that these results do not justify a recommendation to increase the screening exit age (which may be appropriate but is not yet justified with the reported data, though the opportunity seems to exist):

1. As a comparator, the authors should have the ability to examine the rate of CIN3+ at younger ages among screened and HPV positive women (recognizing HPV testing has been more recently introduced into the national screening program, many large studies have been done in Denmark that could serve as a benchmark for comparison). Is the yield of CIN3+ per biopsy/cone similar, higher, lower compared to younger women? This seems to be a critical comparison in making an argument for screening women up to age 65 but not after given the high PPV of an HPV positive test observed here. Note that >95% of older women would just need to provide a cervicovaginal sample in the case of 1-2 additional screens in the lifetime for quite a considerable yield of precancer. Agree, however, that this must be balanced against harms, life expectancy, and women's preferences.

2. The authors state on page 16, lines 253-254 that screening history for individual women is not available - given that some of their references as well as many others from Denmark report screening history in women diagnosed from cancer, it is unclear why a similar methodology could not be used to assess this important information. This helps to understand residual risk at older ages in the context of different screening histories, which is critical for setting policy on exit ages.

3. While the authors are correct that in Denmark 80% of cervical cancer cases over age 60 years were insufficiently or never screened, it should be noted that insufficient screening in the elderly may reflect not having been screened recently because they exited screening ages. Doesn't this reflect the screening exit policy (a general response), and not a need to capture non-compliant screeners (a personalized, individual response)? This is again why it seems important to document how many women had a screening record to meet exiting criteria against the detected CIN3+ in this study (see Hammer et al. J Low Gen Tract Dis. 2018;22(1).). Linking screening history in this study would be broadly informative as this information is otherwise not available!

4. The authors conclude not to recommend extending screening exit ages due to the complications of diagnosis/treatment at older ages. Is there not an option to use the same registers to examine any adverse events that followed the procedures in these women and compare these to AEs in HPV-positive younger women? So far, these arguments are anecdotal and need to be substantiated with quantifiable data.

**********

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PLoS One. 2021 Feb 11;16(2):e0246902. doi: 10.1371/journal.pone.0246902.r002

Author response to Decision Letter 0


13 Jan 2021

To the editors,

We are grateful for the review of our manuscript Histological outcomes in HPV-screened elderly women in Denmark.

We have made the requested changes to style and formatting as per point 1 in the Decision letter. In accordance with point 2 in the decision letter, the updated competing interests statement is included in our cover letter.

We have read with great interest the reviewer comments and revised the manuscript to reflect their inputs. Below are our replies to their individual points, inserted in the text:

Reviewer #1: This is an important work that provides an insight of how to manage cervical cancer screening in women that may have not had because of their age an adequate screening history. The authors provide a cohort and a regional analysis of HPV detection, precancer cases detected and invasive cervical cancer detected. The geographical variability is noted as well as the cohort effect where older women have lower burden of disease. The authors identify the strength and limitation of the study. They are caution in indicating that the impact of this additional effort will be seen in follow up data on incidence and mortality from cervical cancer.

I have one consideration is that the authors report that out of 359,763 invited women, only 108,585 (30%) were screened. Was there any attempt to re/contact these women?. Given the number of cases detected among those screened it seems reasonable to try to capture more women. I could not see a discussion on this issue.

Reply: We thank this reviewer for the comments on our paper and for raising the question regarding renewed contact to non-participating women. The screening initiative was part of the MoH’s National plan for cancer from 2017 and only provided for contacting the women once. We have clarified this in the Materials and methods section of the revised manuscript.

Reviewer #2: This is an excellent manuscript reporting precancer/cancer outcomes in a 'catch-up' HPV screening population of women born before 1948 in Denmark. In general the results are well-described and sound, though several questions or clarifications remain to support the author's conclusion that these results do not justify a recommendation to increase the screening exit age (which may be appropriate but is not yet justified with the reported data, though the opportunity seems to exist):

Reply: we thank the reviewer for the comments. We did not intend to make firm recommendations for the current screening program but the reviewer comments helped us realize that our manuscript could give this impression. The birth cohorts born before 1948, targeted by the one-time screening initiative, were not systematically offered screening throughout their adult life and therefore have a higher cervical cancer risk than cohorts exiting the screening program now (cf. Lynge et al., Eur J Cancer, 2017). Our discussion aimed to stress this fact and we have made several revisions throughout the discussion to clarify.

1. As a comparator, the authors should have the ability to examine the rate of CIN3+ at younger ages among screened and HPV positive women (recognizing HPV testing has been more recently introduced into the national screening program, many large studies have been done in Denmark that could serve as a benchmark for comparison). Is the yield of CIN3+ per biopsy/cone similar, higher, lower compared to younger women? This seems to be a critical comparison in making an argument for screening women up to age 65 but not after given the high PPV of an HPV positive test observed here. Note that >95% of older women would just need to provide a cervicovaginal sample in the case of 1-2 additional screens in the lifetime for quite a considerable yield of precancer. Agree, however, that this must be balanced against harms, life expectancy, and women's preferences.

Reply: We thank the reviewer for suggesting this comparison which we had not considered. Data from the current screening program are not reported in a way that enables for direct comparison. We therefore cite in the revised manuscript the Danish Horizon study (Rebolj et al., PLoS ONE, 2016), which compared cytology to HPV primary screening among women aged 30-65. Although results are not directly comparable to ours due to different study designs, they do suggest that the yield of CIN3+ per histology performed is better for women under 65 than for our study cohorts.

2. The authors state on page 16, lines 253-254 that screening history for individual women is not available - given that some of their references as well as many others from Denmark report screening history in women diagnosed from cancer, it is unclear why a similar methodology could not be used to assess this important information. This helps to understand residual risk at older ages in the context of different screening histories, which is critical for setting policy on exit ages.

Reply: the Danish Pathology Register is only complete from 1998 onwards, and therefore we do not have complete screening histories for women in the age groups concerned by the study.

3. While the authors are correct that in Denmark 80% of cervical cancer cases over age 60 years were insufficiently or never screened, it should be noted that insufficient screening in the elderly may reflect not having been screened recently because they exited screening ages. Doesn't this reflect the screening exit policy (a general response), and not a need to capture non-compliant screeners (a personalized, individual response)? This is again why it seems important to document how many women had a screening record to meet exiting criteria against the detected CIN3+ in this study (see Hammer et al. J Low Gen Tract Dis. 2018;22(1).). Linking screening history in this study would be broadly informative as this information is otherwise not available!

Reply: We are grateful for suggesting this study by Hammer et al. to supplement the one already cited, and we now include both in the references. As discussed in the 2018 article by Hammer et al., the lack of screening history reported may be due to incompleteness of the register as well as to not being screened. Consequently the study shows an increase in proportion screened over time, reflecting not only increased screening but also increased completeness of the pathology register.

In all studies on cervical cancer screening and incidence, the effects of age, period and cohort are intertwined, rendering the discussion quite complicated. The reviewer comments helped us realize that some of our points may not have been sufficiently well explained and we have rephrased parts of the discussion.

4. The authors conclude not to recommend extending screening exit ages due to the complications of diagnosis/treatment at older ages. Is there not an option to use the same registers to examine any adverse events that followed the procedures in these women and compare these to AEs in HPV-positive younger women? So far, these arguments are anecdotal and need to be substantiated with quantifiable data.

Reply: The observations reported by clinicians who have the impression that complication rates are indeed increased merit attention because of potential impact on the harm vs. benefit of screening. We therefore strongly agree with the reviewer that there is a need for documentation and quantification. However, data on treatment and complications are not in the pathology register and studying these is beyond the scope of the present report on histology findings in the one-time screening.

We have found the reviewer comments most helpful in improving the manuscript and look forward to renewed decision from the editor.

On behalf of the authors,

Yours sincerely,

Gry St-Martin

Attachment

Submitted filename: Response to Reviewers.docx

Decision Letter 1

Clement A Adebamowo

28 Jan 2021

Histological outcomes in HPV-screened elderly women in Denmark

PONE-D-20-34114R1

Dear Dr. St-Martin,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Clement A. Adebamowo, BM, ChB Hons; FWACS, FACS, ScD, FASCO

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: (No Response)

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: (No Response)

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4. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #1: Yes

Reviewer #2: (No Response)

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #1: Yes

Reviewer #2: (No Response)

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6. Review Comments to the Author

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Reviewer #1: No additional comments. The authors have provided the review on the topics required. The flow of the paper is good and the data presented is very relevant for the management of older cohorts.

Reviewer #2: (No Response)

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Reviewer #1: No

Reviewer #2: No

Acceptance letter

Clement A Adebamowo

3 Feb 2021

PONE-D-20-34114R1

Histological outcomes in HPV-screened elderly women in Denmark

Dear Dr. St-Martin:

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Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Clement A. Adebamowo

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    Attachment

    Submitted filename: Response to Reviewers.docx

    Data Availability Statement

    All relevant data are within the manuscript tables. To comply with data privacy, numbers of patients are masked when below 3.


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