Introduction

Sub-Saharan African countries suffer from a high burden of cervical cancer (CC) and HIV infection. Effective cervical screening and treatment is not yet widely available and there is lack of colposcopy, histopathology and follow-up installed capacity. We present preliminary results of the initial implementation of the same-day screen-and-treat (with and without triage) approach for women living with HIV (WLWH) participating in the CESTA study.

Methods

In CESTA (Fig.1), WLWH aged 25-54 years are screened for HPV with a clinician sample tested directly on site in one hour using GeneXpert®. HPV positive WLWH are randomised in a 4:1 ratio into VIA triage followed by thermal ablation (TA) treatment (VIA-triage arm) or direct TA (no-triage arm). Ineligible women for TA are referred to colposcopy. Proportions of women completing the screen-and -treat or screen, triage, treatment approach are estimated.

Results

Except for one woman with repeated invalid sample for HPV testing, all women followed the same-day HPV-based screen-and-treat approach. 193 of 281 women were HPV positive (prevalence 69%, 95%CI: 63-74). In the VIA-triage arm (n=154), 137 WLWH were evaluable for VIA (89%, 95%CI: 83-95, Fig. 2); VIA positivity among them was 120/137 (88%, 95%CI: 81-93). Overall, 146/193 WLWH (74%, 95%CI: 69-82) received TA the same day. Colposcopy referral was mainly due to the squamous-columnar junction not fully visible (mostly WLWH older than 44 y/o, p<0.05). 163 of 193 randomised women completed the one visit screen-and-treat algorithm approach (84%, 95%CI: 79-89).

Conclusions

Same-day screen-and-treat approach is feasible, should reduce the lost to follow-up and provide ablative treatment on the same day for eligible women. However, it should be noted that a proportion of women will always be referred to colposcopy (i.e., older women, advanced disease). Finally, the high positivity of HPV DNA testing as well as visual triage on WLWH deserve further evaluation.

Introduction

The majority of cervical cancers occur at the cervical transformation zone (TZ), an epithelial site which is thought to be sustained by a specialized type of epithelial stem cell known as the ‘reserve cell’. Reserve cells, which resemble basal epithelial cells, are found beneath the columnar epithelium of TZ -and during metaplasia, can drive the development of a stratified epithelium like that of the ectocervix.

Methods

We have used immunofluorescence and RNAscope analysis to characterize cervical reserve cells in relation to their local niche within the TZ, their response to HPV infection, immune surveillance, and their role in cervical metaplasia using hysterectomy and LLETZ biopsies from uninfected women, women with HPV infection and HPV-HIV coinfections, treated for cervical precancer.

Results

Reserve cells were prominent at the entrance of cervical crypts. They resemble basal cells in expressing p63, a biomarker of stratification potential, along with the keratins K5 and K14 and more variably K17. At crypts entrances, HPV infection leads to an expansion of the infected K5/P63 reserve cell population, which showed high levels of HPV E6/E7 (CIN3) expressions, along with extensive P16 and MCM expression. By contrast, the epithelium between the cervical crypts, typically showed more modest levels of E6/E7 (CIN2). This contrasts with the very low E6/E7 levels seen in the p63-positive epithelial basal layer during productive papillomavirus infection. In the epithelia where immune infiltrates were apparent, E6/E7 expressing cells at crypts entrances were spared. The immune cells had a distinctive, non-uniform distribution and were typically most prominent in follicles adjacent to infected crypt entrances.

Conclusions

We suggest that E6/E7 may have dual roles in proliferation and immune evasion. Our work identifies an epithelial site at the TZ associated with ‘deregulated HPV gene expression’ and neoplasia, and suggests that HPV gene expansion modulates reserve cell expansion, immune evasion, and disrupts metaplastic processes.

Introduction

The International HPV Reference Center supports quality and order in HPV research and diagnostics. Notably, the center assigns HPV type numbers to novel HPV types, maintains a reference clone repository, and issues international proficiency panels for HPV genotyping and screening.

In 2022, we issued two different proficiency panels: The HPV DNA genotyping panel assesses the proficiency of the different HPV typing assays as used in different laboratories. The HPV DNA screening panel assesses the sensitivity and specificity of the various HPV screening assays, as used in different laboratories.

Methods

Participating laboratories were asked to perform HPV testing using one or more of their usual assays on coded samples composed of purified whole genomic plasmids of sixteen HPV types (HPV 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68a and 68b) in a background of human cellular DNA. Proficient genotyping requires detection in both single and multiple infections of 50 International Units of HPV 16 and HPV 18 DNA/ 5μl and 500 genome equivalents in 5 μl for the other types, with no false positive results. The screening study has the same requirements for HPV 16 and HPV 18. HPV 31, 33, 45, 52 and 58 are also include as single infections, whereas HPV types rarely found in cancers are included only as pools.

Results

The 2022 genotyping proficiency study was subscribed to by 73 different laboratories worldwide. The screening study had 92 panels distributed, particularly to laboratories from Latin America, Europe, and Asia. Both public health laboratories, research laboratories and diagnostic test manufacturers are participating.

Conclusions

A continuing global proficiency program will promote reliable laboratory services both for genotyping in HPV vaccine research and monitoring as well as for HPV-based cervical screening.

Introduction

Human papillomavirus (HPV) is a major etiological agent in cancer worldwide, and is associated with the development of cervical and oropharyngeal cancers (OPC), amongst others. Liquid biopsy is an emerging method for non-invasively monitoring diseases using bodily fluids such as blood, urine, and saliva. Circulating tumour (ct)DNA isolated from liquid biopsy has been shown to have clinical utility as a biomarker in cancer, especially for monitoring disease progression and recurrence.

Methods

In this study, we aimed to detect circulating HPV DNA across different analytes (plasma, saliva, urine, and vaginal swab) in patients with HPV-related cancer to determine the link between HPV-DNA in liquid biopsy and disease status. Samples were collected from 45 patients with prior to treatment for p16+ primary or recurrent cervical cancer or OPC at the McGill University Health Centre. Samples were analyzed using droplet digital PCR (ddPCR) with primers and probes for HPV16/18/33. ctDNA levels were correlated with disease course.

Results

HPV-ctDNA was detectable in 42/47 (89%) patients prior to treatment. In our cohort, 28 patients had matched pre- and post-treatment samples. All patients sampled longitudinally showed significant reductions in ctDNA post treatment compared to pre-treatment (range: 86-100%), with 25 patients having a complete loss of detectable ctDNA. Post-treatment patients with no detectable ctDNA had no signs of recurrence/residual disease on follow-up imaging. In contrast, patients who tested positive for ctDNA post-treatment showed signs of recurrence on follow-up imaging.

Conclusions

HPV16, 18, and 33 ctDNA was successfully detected in saliva and blood of patients with HPV-positive OPC, and vaginal swab, urine, and blood of patients with cervical cancer with 89% sensitivity. The inclusion of multiple analytes increased the sensitivity of the assay, especially in patients with low plasma cfDNA. The presence of ctDNA correlated with treatment response, indicating the potential of HPV ctDNA for monitoring tumor progression in patients.

Introduction

p16/Ki-67 dual-stain cytology has been proposed to triage HPV-positive women with good reproducibility and accuracy. However, its implementation could be challenging, particularly, in low-resource settings. We aimed to evaluate the reproducibility of the dual-stain cytology within ESTAMPA among local and external pathologists.

Methods

In 12 study centres across Latin-America, 42,502 women have been screened with cytology and HPV testing; those screened positive were referred to colposcopy with biopsy/treatment as needed. Residual material from HPV-positive women were centralized in Costa Rica for dual-stain processing. Dual-stain was performed using Ventana Benchmark Automated Stainer after preparation of the ThinPrep liquid-based cytology. Dual-stained slides were interpreted blindly to cytology, histology, and all clinical data by a local pathologist. Ten percent of interpreted slides are being randomly selected for a second reading by an external pathologist. So far, 92 dual-stained slides of 933 available by May 2022 have been reinterpreted. Reproducibility was measured using the percentage agreement and the Cohen's kappa coefficient (κ).

Results

4,228 HPV-positive women are being tested by dual-stain, including 493 CIN3+. Of 92 dual-stained slides reinterpreted, six and 11 were unsatisfactory due to low cellularity for the local and external pathologist, respectively. Pathologists agreed on 80 slides (54 negative, 21 positive and 5 unsatisfactory), leading to an overall agreement of 87% and concordance κ =0.74 (95%CI 0.61-0.87). The external pathologist reported five slides positive of which four were negative and one unsatisfactory for the local pathologist, one slide negative that was positive for the local, and six slides unsatisfactory that were reported as negative for the local pathologist (Figure1).

Conclusions

Preliminary results suggest that dual-stain exhibits good reproducibility. However, local pathologist seems to be less rigid to determine low cellularity. Further external review of more slides and discussion between both pathologists on discordant results will bring some new evidence to understand reasons behind discordance.

Introduction

The persistent infection of high-risk HPV in the cervical transformation zone is the cause of cervical cancer. The majority of sexually active people become infected with high-risk HPV at some point in their life, however, only a small proportion of infected females develop cancer. In general, after the initial infection, HPV develops a temporal productive lesion and then the infection is controlled by the host immune system, leading to the latent persistent infection. In later life, the infection may be re-activated, develop into a pre-cancerous lesion, and in rare cases, progress to malignant cancer. The aim of cervical screening is to identify HPV-associated lesions that may progress to cancer in order to allow treatment.

Methods

Infected lesions can be categorised from productive low-grade lesions to non-productive high-grade lesions. These lesions are characterised based on the regulation/expression of viral oncogenes, and their morphological phenotype which provides insight into the different probabilities of cancer progression. The phenotypes (the biological markers) include the surrogate molecular markers of viral oncogene expression (MCM, Ki67, and p16) and the viral productive life cycle (E4) as well as cellular morphologies.

Results

We showed a new method to lift the surface cells of the cervix (Cervical cell lift, CCL) and generate a spatial map of the biological markers there. We have successfully located and characterised the infected lesion on the CCL. Compared to the normal cytology, the major advantage of the CCL is the preservation of native cell topology, and by preserving this spatial information, the lesions can be visualised in their entirety. The CLL detected CIN2+ lesion with 80% of sensitivity.

Conclusions

The CCL potentially can replace the current triage test (cytology) of cervical screening by providing the location and the grade (CIN1-CIN3) of the infected lesion without biopsy and histopathological assessment.

Introduction

Although our knowledge of HPV-independent squamous cell cancers (SCC) of the cervix is growing, the current 2020 WHO classification does not describe HPV independent cervical precancers. The main reason for this was that these exceedingly rare cervix HPV-independent precancerous lesions were not described at time of publication.

Methods

This review will focus on recent aspects of HPV-independent cervical carcinogenesis.

Results

In 2020 we reported for the first time a preinvasive cervical lesion negative with 3 different HPV tests in a series of 474 cone specimens (Reich O. Gynecol Oncol 2020). In 2022 we demonstrated detailed characteristics of HPV-negative cervical intraepithelial precursors (Regauer S. Am J Surg Path 2022). HPV-negativity was defined as lack of both, DNA of 32 HPV subtypes and E6/E7 mRNA of 14 HPV subtypes, and additionally by the absence of HPV sequences in ~5 Mio´s WGS reads. The morphological hallmark of this cervical lesion was the presence of atypical keratinocytes confined to the basal and parabasal layers in squamous epithelium with hyper- and parakeratosis with elongated rete ridges. The subepithelial stroma had a dense inflammation with plasma cells and eosinophilic granulocytes. Finding an appropriate terminology for these differentiated intraepithelial precursor lesions, however, proves difficult. In analogy to terminology of vulvar carcinogenesis, differentiated cervical intraepithelial neoplasia (d-CIN) may be appropriate.

Conclusions

The existence of primarily HPV-negative squamous cervical precancers (d-CIN type and basaloid type) needs to be recognized (Regauer S. Int J Gynecol Cancer 2022). In a future classification squamous intraepithelial cervical precancers should be grouped into two categories: HPV-associated and HPV-independent.

Introduction

Colposcopy is an important part of cervical screening/management programs. Colposcopic appearance is often classified, for teaching and telemedicine, based on static images that do not reveal the dynamics of acetowhitening. We compared the accuracy and reproducibility of colposcopic impression based on a single image at one minute after application of acetic acid versus a time-series of 17 sequential images over two minutes.

Methods

Approximately 5,000 colposcopic examinations conducted with the DYSIS colposcopic system were divided into 10 random sets, each assigned to a separate expert colposcopist. Colposcopists first classified single two-dimensional images at one minute and then a time-series of 17 sequential images as ‘normal,’ ‘indeterminate,’ ‘high grade,’ or ‘cancer’. Ratings were compared to histologic diagnoses. Additionally, 5 colposcopists reviewed a subset of 200 single images and 200 time series to estimate intra- and inter-rater reliability.

Results

Of 4,640 patients with adequate images, only 24.4% were correctly categorized by single image visual assessment (11% of 64 cancers; 31% of 605 CIN3; 22.4% of 558 CIN2; 23.9% of 3412 <CIN2). Individual colposcopist accuracy was low; Youden indices (sensitivity plus specificity minus one) ranged from 0.07 to 0.24. Use of the time-series increased the proportion of images classified as normal, regardless of histology. Intra-rater reliability was substantial (weighted kappa=0.64); inter-rater reliability was slight (Fleiss’ unweighted kappa=0.17).

Conclusions

Substantial variation exists in visual assessment of colposcopic images, even when a 17-image time series showing the two-minute process of acetowhitening is presented. We are currently evaluating whether deep-learning image evaluation can assist classification.

Introduction

Human papillomavirus (HPV) has been detected in lung tumors, but its role as an etiologic agent for lung cancer remains controversial. HPV infection may foster a pro-inflammatory environment that increases lung cancer risk. We sought to examine differences in lymphocyte and cytokine/chemokine profiles between lung cancer cases and HPV-vaccinated controls, as well as whether cytokine/chemokine levels were associated with mortality among cases.

Methods

Peripheral blood mononuclear cells (PBMCs) and plasma were isolated from 137 newly diagnosed lung cancer cases and 11 HPV-vaccinated controls. Cells from controls and 45 randomly selected cases were exposed to media or to 3 doses of Gardasil as an HPV challenge. Interleukin (IL-) 4, IFN-g, and IL-17A secreting cells were quantified using an ELISpot assay. Individuals were categorized as HPV-responsive if the HPV challenge resulted in an increase of at least 2 types of secreting cells, compared to media-exposed cells. The Milliplex human cytokine assay was used to assess circulating levels of 38 cytokines in plasma of all cases and controls. Multivariable Cox proportional hazards models were used to estimate hazard ratios by high versus low cytokine levels.

Results

All 11 HPV-vaccinated controls demonstrated an increase in cells secreting IFN-g and IL-4. Of the 45 challenge cases, 20 (44.4%) were classified as HPV-responsive. Levels of IL-17A were significantly higher among HPV-responsive (89.1 cells/10⁶) compared to non-responsive cases (9.8 cells/10⁶) (p=0.012). There were no differences in circulating cytokines at baseline by HPV-responsiveness. High levels of circulating IL-10 (HR: 2.02, 95% CI: 1.18-3.46), IL-7 (1.94, 1.11-3.39), IL-9 (1.85, 1.08-3.17), and GRO (2.10, 1.08-4.09) were associated with mortality compared to cases with low levels. There were no differences in survival by HPV-responsiveness.

Conclusions

High cytokine levels at diagnosis were associated with survival which may indicate that immunoregulatory factors may be of importance in lung cancer prognosis.