Nd:YAG/Er:YAG dual laser compared with topical steroid to treat vulvar lichen sclerosus: A randomised controlled trial.

OBJECTIVE: To evaluate the efficacy and safety of a novel non-ablative Nd:YAG/Er:YAG dual laser treatment for vulvar lichen sclerosus (LS) in comparison with the recommended first-line therapy with topical steroid. DESIGN: A randomised investigator-initiated active-controlled trial. SETTING: Single tertiary referral centre. POPULATION: Women with vulvar LS. METHODS: Randomisation (2:1) to Nd:YAG/Er:YAG laser therapy or topical clobetasol proprionate therapy. Four laser treatments at 0, 1, 2 and 4 months or decreasing doses of steroid for 6 months. MAIN OUTCOME MEASURES: The primary outcome was the change in objective validated clinical LS score in the laser arm between baseline and 6 months. Secondary outcomes were laser tolerability/safety, symptom scores and patient satisfaction. RESULTS: Sixty-six women were included, 44 in the laser group and 22 in the steroid group. The total LS score decreased by -2.34 ± 1.20 (95% CI -2.71 to -1.98) in women treated with laser compared with a decrease of -0.95 ± 0.90 (95% CI -1.35 to -0.56) in those receiving steroid applications (p < 0.001). Laser treatment was safe and well tolerated. Subjective severity scores (on visual analogue scale) and vulvovaginal symptoms questionnaire scores improved similarly for the laser and steroid arms without significant differences between the two treatments. Patient satisfaction was higher in the laser arm than in the steroid arm (p = 0.035). CONCLUSIONS: Non-ablative dual Nd:YAG/Er:YAG laser therapy was safe and significantly improved clinical outcome and subjective symptoms at the 6-month follow up. This suggests that laser may be a promising alternative to corticosteroid therapy. However, the authors caution regular follow ups because of the premalignant nature of the disease.

The Spectrum of HPV-independent Penile Intraepithelial Neoplasia: A Proposal for Subclassification.

Compared with vulva, precursor lesions of human papillomavirus (HPV)-independent invasive squamous cell carcinoma (SCC) of the penis are insufficiently characterized. We analyzed the histologic and immunohistochemical characteristics of 70 peritumoral precursor lesions and correlated them with the histology and mutational profile of the adjacent HPV-negative invasive penile SCC. Atypical basal keratinocyte proliferation with variously elongated epithelial rete with premature squamatiziation, but regular superficial cornification, termed differentiated penile intraepithelial neoplasia (d-PeIN), were identified adjacent to 42/70 (60%) SCC (36/42 keratinizing ( P <0.001); 3 papillary, and 1 each verrucous, clear cell, sarcomatoid SCC). d-PeIN were associated with chronic inflammatory dermatoses (32/42; P <0.001), p53 overexpression (26/42; P <0.001), and hotspot mutations in TP53 (32/42; P <0.001), CDKN2A (26/42; P <0.001) or both (21/42; P =0.003) in the adjacent SCC. Cytoplasmic p16 ink4a overexpression in 5/42 d-PeIN correlated with CDKN2A missense mutations in the adjacent SCC. In all, 21/70 (30%) cornified verrucous or glycogenated verruciform precursors with minimal atypia and wild-type p53 (18/21; P <0.001) occurred adjacent to verrucous or papillary SCC (17/21; P <0.001) and keratinizing (4/21) SCC, which harbored mutations in HRAS and/or PIK3CA (12/21; P <0.004). Undifferentiated p16 ink4a -negative full-thickness precursors were identified in 7/70 (10%) SCC. Four histologically different HPV-independent penile precursor lesions can be assigned to 2 major genetic/biological pathways with characteristic highly differentiated precursors requiring different clinical management decisions. These include d-PeIN in chronic inflammatory dermatoses, with p53 overexpression and TP53/CDKN2A mutations, and the p53 wild-type verrucous and verruciform precursors unassociated with dermatoses, but with mutations in oncogenes PIK3CA and HRAS .

The Histologic and Molecular Spectrum of Highly Differentiated HPV-independent Cervical Intraepithelial Neoplasia.

Recently, the World Health Organization (WHO) recognized the existence of human papillomavirus (HPV)-independent invasive cervical squamous cell carcinoma (SCC), but HPV-independent precursor lesions were not included due to the lack of description of this rare entity. We present the histologic spectrum of highly differentiated squamous HPV-negative and p16 ink4a -negative precursor lesions adjacent to and/or preceding invasive HPV-negative cervical SCC in 3 patients. The histologic features resembled those described for vulvar HPV-negative precursor lesions. One precursor featured a proliferation of atypical basal keratinocytes with mitotic activity, premature squamatization in elongated epithelial rete, and mostly regular superficial squamous differentiation with TP53 mutation and immunohistochemical p53 overexpression termed differentiated cervical intraepithelial neoplasia (d-CIN). The other 2 precursors included verruciform acanthosis with plump rete, minimal atypia, and an EGFR mutation that resembled vulvar acanthosis with altered differentiation, and an exophytic papillary proliferation with a PIK3CA mutation resembling the differentiated exophytic vulvar intraepithelial lesion. Two precursors that preceded the invasive SCC harbored an additional pathogenic SMARCB1 mutation. The cytologic smears of d-CIN revealed 3-dimensional branched basaloid tubular structures and eosinophilic squamous cell clusters mimicking the histologic features. In conclusion, highly differentiated cervical HPV-negative precursors are characteristic intraepithelial squamous lesions with somatic mutations that resemble those described in vulvar HPV-independent carcinogenesis. For optimal reproducibility, we propose a simplistic classification of these HPV-negative cervical precursors in TP53 -mutated d-CIN and p53 wild-type verruciform intraepithelial neoplasia.

Different Mutational Landscapes in Human Papillomavirus-Induced and Human Papillomavirus-Independent Invasive Penile Squamous Cell Cancers.

Penile squamous cell carcinomas (SCC) are rare cancers that arise after transforming human papillomavirus (HPV) infections or independent of HPV in the background of chronic dermatoses. Limited knowledge about genetic alterations driving penile carcinogenesis comes from studies of mainly small cohorts of typically mixed etiology. In this comparative genetic study of HPV-induced and HPV-independent invasive penile SCC of 156 patients from a single institution in a low-incidence country, hotspots of 50 cancer-relevant genes were analyzed with targeted next-generation sequencing. Seventy-nine of 156 SCC were classified as HPV induced, and 77 of 156 SCC arose independent of HPV. Only 28 (35%) of 79 HPV-induced penile SCC, but 69 (90%) of 77 HPV-independent SCC carried somatic gene mutations. PIK3CA, FGFR3, and FBXW7 mutations occurred in both groups in similar numbers as seen in other human cancers. In contrast, mutations in TP53 (44/77; 57%), CDKN2A (35/77; 45%), and HRAS (13/77; 17%) genes occurred with one exception of a HIV positive patient exclusively in HPV-independent SCC with a frequent co-occurrence of TP53 and CDKN2A mutations (28/77; 42%). Mutations in multiple genes occurred in 9 (11%) of 79 HPV-induced SCC versus 47 (62%) of 77 HPV-independent SCC (χ2; P < .001). More than one mutation per gene (multi hits) was characteristic for HPV-independent SCC in 14 (18%) of 77 compared with only 3 (4%) of 79 HPV-induced SCC (χ2; P < .001). The total number of mutations in HPV-induced penile SCC (47 mutations) was significantly lower than that in HPV-independent SCC (143 mutations; Welsh test; P < .001). The presence of somatic driver gene mutations did not correlate with the age of patients, histology, or tumor stage of the primary SCC in either etiologic group, suggesting that acquisition of driver gene mutations is an early event after invasion. This large cohort analysis identified characteristic differences in mutational landscapes for the 2 etiologies. While genetic mutations in tumor suppressor genes drive HPV-independent penile carcinogenesis, oncogenic action of E6 and E7 substitute for mutations in HPV-induced SCC. A subgroup of patients with advanced SCC may be candidates for targeted therapy and clinical trials, although the majority of advanced penile SCC remain a therapeutic challenge.

Nd:YAG/Er:YAG dual laser vs. topical steroid to treat vulvar lichen sclerosus: study protocol of a randomized controlled trial.

PURPOSE: Vulvar lichen sclerosus (LS) is a chronic debilitating inflammatory skin disease. Today, the gold standard is a life-long topical steroid treatment. Alternative options are highly desired. We present a study protocol of a prospective, randomized, active-controlled, investigator-initiated clinical trial comparing a novel non-invasive dual Nd:YAG/Er:YAG laser therapy with the gold standard for the management of LS. METHODS: We recruited 66 patients, 44 in the laser arm and 22 in the steroid arm. Patients with a physician-administered clinical LS score ≥ 4 were included. Participants received either four laser treatments 1-2 months apart, or 6 months of topical steroid application. Follow-ups were planned at 6, 12, and 24 months. The primary outcome looks at the efficacy of the laser treatment at the 6-month follow-up. Secondary outcomes look at comparisons between baseline and follow-ups within the laser or the steroid arm, and comparisons between laser vs. steroid arm. Objective (LS score, histopathology, photo documentation) and subjective (Vulvovaginal Symptoms Questionnaire, symptom VAS score, patient satisfaction) measurements, tolerability, and adverse events are evaluated. CONCLUSION: The findings of this trial have the potential to offer a novel treatment option for LS. The standardized Nd:YAG/Er:YAG laser settings and the treatment regime are presented in this paper. CLINICAL TRIAL IDENTIFICATION NUMBER: NCT03926299.

The European Society of Gynaecological Oncology (ESGO), the International Society for the Study of Vulvovaginal Disease (ISSVD), the European College for the Study of Vulval Disease (ECSVD), and the European Federation for Colposcopy (EFC) Consensus Statement on the Management of Vaginal Intraepithelial Neoplasia.

ABSTRACT: The European Society of Gynaecological Oncology (ESGO), the International Society for the Study of Vulvovaginal Disease (ISSVD), the European College for the Study of Vulval Disease (ECSVD), and the European Federation for Colposcopy (EFC) developed consensus statements on pre-invasive vulvar lesions in order to improve the quality of care for patients with vaginal intraepithelial neoplasia (VaIN). The management of VaIN varies according to the grade of the lesion: VaIN 1 (low grade vaginal squamous intraepithelial lesions (SIL)) can be subjected to follow-up, while VaIN 2-3 (high-grade vaginal SIL) should be treated. Treatment needs individualization according to the patient's characteristics, disease extension and previous therapeutic procedures. Surgical excision is the mainstay of treatment and should be performed if invasion cannot be excluded. Total vaginectomy is used only in highly selected cases of extensive and persistent disease. Carbon dioxide (CO2) laser may be used as both an ablation method and an excisional one. Reported cure rates after laser excision and laser ablation are similar. Topical agents are useful for persistent, multifocal lesions or for patients who cannot undergo surgical treatment. Imiquimod was associated with the lowest recurrence rate, highest human papillomavirus (HPV) clearance, and can be considered the best topical approach. Trichloroacetic acid and 5-fluorouracil are historical options and should be discouraged. For VaIN after hysterectomy for cervical intraepithelial neoplasia (CIN) 3, laser vaporization and topical agents are not the best options, since they cannot reach epithelium buried in the vaginal scar. In these cases surgical options are preferable. Brachytherapy has a high overall success rate but due to late side effects should be reserved for poor surgical candidates, having multifocal disease, and with failed prior treatments. VaIN tends to recur and ensuring patient adherence to close follow-up visits is of the utmost importance. The first evaluation should be performed at 6 months with cytology and an HPV test during 2 years and annually thereafter. The implementation of vaccination against HPV infection is expected to contribute to the prevention of VaIN and thus cancer of the vagina. The effects of treatment can have an impact on quality of life and result in psychological and psychosexual issues which should be addressed. Patients with VaIN need clear and up-to-date information on a range of treatment options including risks and benefits, as well as the need for follow-up and the risk of recurrence.

The European Society of Gynaecological Oncology (ESGO), the International Society for the Study of Vulvovaginal Disease (ISSVD), the European College for the Study of Vulval Disease (ECSVD), and the European Federation for Colposcopy (EFC) consensus statement on the management of vaginal intraepithelial neoplasia.

The European Society of Gynaecological Oncology (ESGO), the International Society for the Study of Vulvovaginal Disease (ISSVD), the European College for the Study of Vulval Disease (ECSVD), and the European Federation for Colposcopy (EFC) developed consensus statements on pre-invasive vulvar lesions in order to improve the quality of care for patients with vaginal intraepithelial neoplasia (VaIN). The management of VaIN varies according to the grade of the lesion: VaIN 1 (low grade vaginal squamous intraepithelial lesions (SIL)) can be subjected to follow-up, while VaIN 2-3 (high-grade vaginal SIL) should be treated. Treatment needs individualization according to the patient's characteristics, disease extension and previous therapeutic procedures. Surgical excision is the mainstay of treatment and should be performed if invasion cannot be excluded. Total vaginectomy is used only in highly selected cases of extensive and persistent disease. Carbon dioxide (CO2) laser may be used as both an ablation method and an excisional one. Reported cure rates after laser excision and laser ablation are similar. Topical agents are useful for persistent, multifocal lesions or for patients who cannot undergo surgical treatment. Imiquimod was associated with the lowest recurrence rate, highest human papillomavirus (HPV) clearance, and can be considered the best topical approach. Trichloroacetic acid and 5-fluorouracil are historical options and should be discouraged. For VaIN after hysterectomy for cervical intraepithelial neoplasia (CIN) 3, laser vaporization and topical agents are not the best options, since they cannot reach epithelium buried in the vaginal scar. In these cases surgical options are preferable. Brachytherapy has a high overall success rate but due to late side effects should be reserved for poor surgical candidates, having multifocal disease, and with failed prior treatments. VaIN tends to recur and ensuring patient adherence to close follow-up visits is of the utmost importance. The first evaluation should be performed at 6 months with cytology and an HPV test during 2 years and annually thereafter. The implementation of vaccination against HPV infection is expected to contribute to the prevention of VaIN and thus cancer of the vagina. The effects of treatment can have an impact on quality of life and result in psychological and psychosexual issues which should be addressed. Patients with VaIN need clear and up-to-date information on a range of treatment options including risks and benefits, as well as the need for follow-up and the risk of recurrence.

Acquisition of Genetic Aberrations During the Progression of High-Grade Intraepithelial Lesions/Micro-Invasive Squamous Cancers to Widely Invasive Cervical Squamous Cell Cancer.

CONTEXT.­: Acquisition of genetic aberrations during cervical carcinogenesis in individual patients is poorly documented. OBJECTIVE.­: To provide a comparative analysis of high-grade squamous intraepithelial lesions (n = 7) and pT1a squamous cancers (n = 1) and their recurrences, subsequent widely invasive cancers, and metastases developed during 1-24 years. DESIGN.­: Archival tissues of 8 patients were analyzed immunohistochemically for reserve-cell origin, human papillomavirus genotypes, mutations in 50 cancer genes, and chromosomal copy number variations. RESULTS.­: Intraepithelial lesions arose either from cytokeratin 17- or 7-expressing reserve cells. All preinvasive and invasive tumors carried human papillomavirus high-risk genotypes and lacked somatic mutations. Chromosomal copy number variations were identified in all intraepithelial lesions and invasive cancers. Four of 8 high-grade intraepithelial lesions progressed to invasive cancer after incomplete treatment, and 4 of 8 invasive cancers arose de novo after in sano resection. Four of 8 cancers carried mutations with high mutational frequency (PIK3CA E545K [n = 2]; PIK3CA and SMAD1 [n = 1]; HRAS, RB1, and EGFR [n = 1]), as did their corresponding regional metastases. One nonmetastasized cancer had a subclonal PIK3CA mutation, and an initially nonmutated, low-stage cancer developed ovarian metastases with PIK3CA amplification. One patient had neither mutations nor metastases. The patient with treated PIK3CA E545K-mutated pT1a cancer developed a subsequent nonmutated intraepithelial lesion that progressed to invasive cancer with a subclonal PIK3CA-H1047R mutation. Cancer-related deaths in 4 of 8 (50%) patients occurred independent of mutational status or metastatic disease. CONCLUSIONS.­: Recurrences arose after persistent or de novo human papillomavirus infection of residual reserve cells or squamous metaplasia. Activating driver mutations were identified in invasive cancers only. High mutational load correlated with metastases, which in turn represented clonal disease.

Elimination of reserve cells for prevention of HPV-associated cervical cancer.

Human papilloma viruses (HPV), that are causative for most squamous cell cervical cancers (SCC), have a simple structure with only a few genes (six early and two late genes). Two of the early HPV genes (E6 and E7) are capable of transforming normal squamous epithelium into cancer. In the last 10 years, a controversial discussion arose as to which cells are primarily involved in cervical carcinogenesis. Virologists traditionally use a research model of stratified squamous epithelium, a permissive environment for completion of a full HPV-life cycle. Basic insights on HPV tropism, HPV life cycle, HPV-uptake, HPV-replication, HPV-gene expression were gained from this model. Stratified squamous epithelium, however, is a low-risk area for SCC. Most SCC develop in an area of endocervical columnar epithelium that undergoes squamous metaplasia. SCC arise after infection of immature squamous metaplasia, proliferating reserve cells/reserve cell hyperplasia and reserve cells of the endocervical columnar epithelium. Study models investigating this pathway of carcinogenesis do not exist and therapeutic consequences deduced from this knowledge are lacking. This review describes in detail cervical carcinogenesis after HPV infection of subcolumnar reserve cells and discusses new intervention strategies for patients. The WHO-launched global strategy to eliminate HPV-associated cervical cancer builds primarily on prophylactic vaccination, screening and treatment. New insights in cervical pathogenesis, may assist in reaching this ambitious WHO goal.

Topical imiquimod versus surgery for vulvar intraepithelial neoplasia: a multicentre, randomised, phase 3, non-inferiority trial.

BACKGROUND: The optimal management of vulvar high-grade squamous intraepithelial lesions (vHSILs) is challenging. Surgery is the standard treatment, but recurrences are observed in half of patients. Medical treatment with imiquimod is an effective alternative, but the two modalities have not been compared in a randomised trial. The aim of this study was to compare the clinical effectiveness, histological response, human papillomavirus (HPV) clearance, acceptance, and psychosexual morbidity of primary imiquimod treatment versus surgical treatment in women with vHSIL. METHODS: This study was a multicentre, randomised, phase 3, non-inferiority clinical trial done by the Austrian Gynaecological Oncology group at six hospitals in Austria. We recruited female patients aged 18-90 years with histologically confirmed vHSIL with visible unifocal or multifocal lesions. Main exclusion criteria were clinical suspicion of invasion, a history of vulvar cancer or severe inflammatory dermatosis of the vulva, and any active treatment for vHSIL within the previous 3 months. Women with known immunodeficiency, who were pregnant, or who were lactating were excluded. Patients were randomly assigned (1:1) by block randomisation to imiquimod or surgery, and stratified by unifocal or multifocal disease. Treatment with imiquimod was self-administered in a slowly escalating dosage scheme up to three times per week for a period of 4-6 months. Surgery consisted of excision or ablation. Patients were assessed with vulvoscopy, vulvar biopsy, HPV tests, and patient-reported outcomes at baseline and after 6 months and 12 months. The primary endpoint was complete clinical response (CCR) at 6 months after local imiquimod treatment or one surgical intervention. Primary analysis was per protocol with a non-inferiority margin of 20%. This trial is registered at ClinicalTrials.gov, NCT01861535. FINDINGS: 110 patients with vHSIL (78% with unifocal vHSIL and 22% with multifocal vHSIL) were randomly assigned between June 7, 2013, and Jan 8, 2020. Clinical response to treatment could be assessed in 107 patients (54 in the imiquimod group and 53 in the surgery group), and 98 patients (46 in the imiquimod group and 52 in the surgery group) completed the study per protocol. 37 (80%) of 46 patients using imiquimod had CCR, compared with 41 (79%) of 52 patients after one surgical intervention, showing non-inferiority of the new treatment (difference in proportion -0·016, 95% CI -0·15 to -0·18; p=0·0056). Invasive disease was found in five patients at primary or secondary surgery, but not in patients with per-protocol imiquimod treatment. There was no significant difference in HPV clearance, adverse events, and treatment satisfaction between study groups. INTERPRETATION: Imiquimod is a safe, effective, and well accepted alternative to surgery for women with vHSIL and can be considered as first-line treatment. FUNDING: Austrian Science Fund and Austrian Gynaecological Oncology group.

Driver gene mutations in micro-invasive cervical squamous cancers have no prognostic significance.

OBJECTIVE: To evaluate the prevalence of somatic gene mutations in different stages of cervical carcinogenesis placing special emphasis on micro-invasive pT1a cervical squamous cell cancers (SCC). METHODS: Micro-dissected samples of 32 micro-invasive pT1a and 55 ≥ pT1b SCC were evaluated by next generation sequencing of 50 cancer genes (cancer hot spot panel). RESULTS: At primary diagnosis, 8/32 (25%) pT1a SCC, 10/28 (36%) pT1b SCC and 15/27 (56%) pT2/3 SCC carried somatic gene mutations. The most commonly affected gene was the PIK3CA gene in hot spot regions E545K and E453K in 5/8 (62%) pT1a SCC, 7/15 (70%) pT1b SCC and 10/15 (66%) pT2/3 SCC followed by FBXW7 (n = 4), KRAS and RB1 (n = 2 each). ERBB2, APC, ATM, MLP gene mutations occurred only once. Solitary activating oncogenic somatic mutations dominated over tumor suppressor mutation in 88% pT1a, 80% pT1b and 60% pT2/3 SCC. Concomitant mutations involved typically an activating oncogenic mutation and an inactivating tumor-suppressor gene mutation. All patients with pT1a SCC are alive without evidence of disease after surgical treatment, independent of mutational status or lympho-vascular space invasion. CONCLUSIONS: Activating oncogenic gene mutations, in particular in the PIK3CA gene, occur early in cervical carcinogenesis. Although driver gene mutations bestow tumor cells with a growth advantage, early detection and complete removal of all cancer cells - with or without somatic gene mutations - are essential for cure. In contrast to advanced inoperable SCC, where PIK3CA driver gene mutations carry an adverse prognosis, the mutational status in surgically treated micro-invasive SCC is prognostically irrelevant.

HPV-negative Squamous Cell Carcinomas of the Cervix With Special Focus on Intraepithelial Precursor Lesions.

Recently, the World Health Organization (WHO) recognized human papilloma virus (HPV)-independent invasive cervical squamous cell carcinoma (SCC) without recognizing the existence of precursor lesions. This is a detailed characterization of 3 preinvasive lesions and 6 invasive SCC negative for HPV-DNA (32 genotypes), HPV-mRNA (14 genotypes) and genomic HPV sequencing. We evaluated histologic features, expression of p16ink4a, p53, CK7, and CK17, aberrations in 50 cancer genes and chromosomal copy number variations. HPV-negative preinvasive lesions were extensive basaloid or highly differentiated keratinizing intraepithelial proliferations of 3 to 20 cell layers thickness, partly with prominent cervical gland involvement. Overall, 2/3 intraepithelial lesions and the in situ component of 1/6 SCC showed p16ink4a block staining, while 1/6 in situ component revealed heterogenous p16ink4a staining. All invasive components of keratinizing SCC were p16ink4a-negative. Preinvasive and invasive SCC showed inconsistent CK7 and CK17 staining. Nuclear p53 overexpression was restricted to the TP53 gene mutated SCC. The highly vascularized peritumoral stroma showed a dense inflammatory infiltrate including plasma cells and intratumoral and peritumoral eosinophilic granulocytes. Inconsistent somatic gene mutations (PIK3CA, STK11, TP53, SMARC2B, and GNAS) occurred predominantly in nonhotspot locations at low mutational frequency in 3/6 SCC. Consistent aberrations included the pathogenic (angiogenic) germline polymorphism Q472H in the KDR gene (7/9 patients), and chromosome 3q gains (4/9 patients). In conclusion, HPV-negative intraepithelial cervical precancerous lesions exist, either as highly differentiated keratinized intraepithelial proliferations reminiscent of differentiated vulvar intraepithelial neoplasia, or undifferentiated basaloid intraepithelial lesions with occasional p16ink4a block staining resembling high-grade squamous intraepithelial lesion. Gains of chromosome 3q, angiogenic germline variants the inflammatory infiltrate may contribute to progression of HPV-negative cervical carcinogenesis.

Cervical Precancers Originate From Infected Proliferating Reserve Cells: A Comparative Histologic and Genetic Study of Thin and Thick High-grade Squamous Intraepithelial Lesions.

Human papillomavirus (HPV)-induced invasive cervical squamous cell cancer (SCC) develop via high-grade squamous intraepithelial lesion (HSIL). In contrast to classic thick HSIL, thin HSIL (≤9 cell layers) are poorly documented. This study compares histology, HPV genotypes, and aberrations in 50 cancer genes of 45 thin HSIL to 45 thick HSIL, 20 pT1a SCC, and 40 ≥pT1b SCC. Thin HSIL arose from proliferating reserve cells within endocervical epithelium or immature metaplasia throughout the transformation zone after infection with high-risk HPV genotypes (36/45; 80%), and 20% non-high-risk HPV genotypes compared with 2.5% thick HSIL, pT1a SCC, and ≥pT1b SCC. Thin HSIL were multifocal proliferations with varying epithelial thickness between 1 and 2 to 9 cell layers, with occasional transitions to thick HSIL or concomitant lesions of thick HSIL. Overall, 40% thin HSIL were located distant to and most thick HSIL occurred near or at the squamocolumnar junction. Only 20% thick HSIL showed koilocytosis. All HSIL lacked somatic gene mutations, compared with 30% pT1a and 55%≥pT1b SCC. Overrepresented rare germline variants in the MET, JAK3, and FGFR3 genes occurred in all patient groups. In summary, thin and thick HSIL arose independently of somatic gene mutations. The maturation level of the squamous epithelium at the time of transforming infection determines if a thick HSIL develops directly from HPV-infected proliferating reserve cells via thin HSIL or in stratified glycogenated squamous epithelium via low-grade squamous intraepithelial lesion. These observations raise doubts about the biological relevance of separation into thin and thick HSIL. The oncogenic potential of HPV genotypes but also germline variants may influence the natural history.

The origin of Human Papillomavirus (HPV) - induced cervical squamous cancer.

Most research models of HPV-associated squamous cervical carcinogenesis focus on stratified glycogenated squamous epithelium, a permissive environment for HPV-life-cycle completion, while immature squamous metaplastic epithelium and reserve cells as targets of HPV-infection have received less attention. Subcolumnar reserve cells of urogenital sinus origin with a CK17/p63-phenotype serve as the primary stem cell for squamous metaplasia. The area of manifest or potential squamous metaplasia, referred to as transformation zone, is the site where most squamous cancers occur after a transforming HPV infection of proliferating reserve cells and/or metaplastic epithelium. Improper use of terminology, in particular confusion of transformation zone with transition zone (synonymous: squamous-columnar junction or SCJ), as well as poorly substantiated postulates of a stem cell niche at the squamous-columnar junction with 'embryonic stem cell markers' have complicated understanding of HPV-related squamous carcinogenesis. Reserve cells as target cells and reservoirs of HPV should move into future research focus.

The development of the human vaginal fornix and the portio cervicis.

INTRODUCTION: One of the transitional zones of the human body is situated in the cervix uteri. The developmental differentiation of epithelial and stromal characteristics in such a region is of high clinical interest. However, few studies have focused on the development of this region, and information in anatomical and clinical textbooks is limited. We therefore examined the development of the human vaginal fornix and the cervix uteri during prenatal development. MATERIALS AND METHODS: We examined 29 female embryos and fetuses between 20 and 34 weeks and two newborns using histology and immunohistochemistry. RESULTS: The characteristic shape of the portiocervicis and the vaginal fornix first became visible in mid-term fetuses because of the different muscular coats and of an uncategorized Müllerian-derived epithelium, which was rapidly replaced by a multilayered squamous epithelium. Only thereafter, in older fetuses, were there organogenetic differentiation of the epithelia and the underlying stroma of the cervical canal. UGS-derived p63/CK17-positive cells could be identified as precursor cells for the squamous epithelium, and Müllerian-derived CK7-positive cells for the columnar-type epithelium. Both cell types and different stromal zones were already present in a prenatal transformation zone. Initial functional differentiation could be observed in perinatal stages. CONCLUSIONS: Our results on prenatal human development strongly support the view that two different cell lineages meet at the transitional zone of the cervix uteri and that these lineages depend on alternative signals from the underlying stromal compartment.

Possibly carcinogenic HPV subtypes are a cause of HSIL and negative clinical HPV tests - A European prospective single center study.

OBJECTIVE: To correlate p16ink4a positive cervical precancers of 388 consecutive patients from a single European center with the preceding clinical HPV-DNA and HPV E6/E7 mRNA screening test. METHOD: 374/388 patients had a HSIL (CIN 2/3) and 14/388 AIS (6 pure and 8 combined AIS/HSIL). Lesional tissues of HSIL/AIS with negative Cobas and/or Aptima HPV tests underwent HPV genotyping with CHIPRON HPV 3.5 LCD-array. Selected cases were subjected to a cancer hot spot analysis. RESULTS: The Aptima test missed 10/388 (2.6%) and the Cobas test seven of 388 (1.8%) precancers associated HPV-HR. Both HPV tests were negative in 20/374 precancers (5.3%; 17 HSIL/CIN3, two HSIL/CIN2, one AIS). Due to insufficient DNA four of 20 double negative cases (three HSIL, one AIS) were not genotyped. In the remaining cases, two of 20 (10%) HSIL genotyping detected HR-HPV subtypes. 10/20 (50%) HSIL were associated with possibly carcinogenic and low risk HPV (four x HPV73, three x HPV 53, one x HPV 82, one x HPV 67 and one x HPV 6), all of which are not included in both HPV tests. Two of 20 (10%) HSIL were negative with all HPV tests; one of these HSIL had a somatic PIK3CA gene mutation and the other had a single nucleotide variant in the APC gene. Three of 20 HSIL (15%) were thin HSIL (≤9 cell layers thick). CONCLUSIONS: Possibly carcinogenic HPV subtypes not included in the clinical HPV tests may account for the small gap of missed HSIL in clinical HPV screening. True HPV negative HSIL are exceedingly rare. Expanding HPV testing to include more possibly carcinogenic HPV subtypes may further reduce cervical cancer.

Thin variant of high-grade squamous intraepithelial lesion - relationship with high-risk and possibly carcinogenic human papilloma virus subtypes and somatic cancer gene mutations.

AIM: To further characterise the thin variant of high-grade squamous intraepithelial lesions (HSILs) of the cervix defined by the World Health Organization as full-thickness HSILs with nine or fewer cell layers. METHODS AND RESULTS: We examined 31 excisional cervical specimens featuring exclusively p16INK4a -overexpressing thin HSILs with respect to size, location at the squamocolumnar junction or endocervical mucosa, human papilloma virus (HPV) subtypes (pretherapeutic clinical HPV tests and HPV genotyping on lesional tissue after excision), and somatic mutations in 50 cancer genes. Thin HSILs were typically solitary lesions, located at the squamocolumnar junction (20/31; 65%), in the endocervical columnar epithelium (6/31; 19%), and in both locations (5/31; 16%). The horizontal extension of thin HSILs ranged from 100 µm to 8 mm, with 30% being <1 mm. HPV data were available for 27 specimens. Twenty of 27 (74%) thin HSILs showed high-risk HPV subtypes: HPV16 (n = 8), HPV16 with coinfection (n = 2), HPV18 (n = 1), HPV31 (n = 1), HPV33 (n = 2), HPV52/58 (n = 2), and 'other' high-risk HPV genotypes (n = 4). Five of 27 (19%) thin HSILs showed possibly carcinogenic subtypes: HPV53 (n = 3), HPV73 (n = 1), and HPV82 (n = 1). One thin HSIL was induced by low-risk HPV6 and one by the unclassified subtype HPV44. Somatic gene mutations were not identified. CONCLUSION: Thin HSILs were typically small lesions without somatic gene mutations. Two-thirds of thin HSILs developed after a transforming infection with high-risk HPV subtypes, and one-third were induced by non-high-risk HPV subtypes. If cervical cancer screening relies solely on presently available clinical HPV DNA tests, a significant percentage of women with HSIL will be missed.