Prognostic value of molecular classification in stage IV endometrial cancer.

OBJECTIVES: Multiple studies have proven the prognostic value of molecular classification for stage I-III endometrial cancer patients. However, studies on the relevance of molecular classification for stage IV endometrial cancer patients are lacking. Hypothetically, poor prognostic molecular subtypes are more common in higher stages of endometrial cancer. Considering the poor prognosis of stage IV endometrial cancer patients, it is questionable whether molecular classification has additional prognostic value. Therefore, we determined which molecular subclasses are found in stage IV endometrial cancer and if there is a correlation with progression-free and overall survival. METHODS: A retrospective multicenter cohort study was conducted using data from five Dutch hospitals. Patients with stage IV endometrial cancer at diagnosis who were treated with primary cytoreductive surgery or cytoreductive surgery after induction chemotherapy between January 2000 and December 2018 were included. Exclusion criteria were age <18 years or recurrent disease. The molecular classification was performed centrally on all tumor samples according to the World Health Organization 2020 classification (including POLE and estrogen receptor status). The Kaplan-Meier method was used to calculate progression free and overall survival in the molecular subclasses, for the different histological subtypes and for estrogen receptor positive versus estrogen receptor negative tumors. Groups were compared using the log-rank test. RESULTS: 164 stage IV endometrial cancer patients were molecularly classified. Median age of the patients was 67 years (range 33-86). Most patients presented with a non-endometrioid histological subtype (58%). Intra-abdominal complete cytoreductive surgery was achieved in 60.4% of the patients. 101 tumors (61.6%) were classified as p53 abnormal, 35 (21.3%) as no specific molecular profile, 21 (12.8%) as mismatch repair deficient, and 6 (3%) as POLE mutated. Molecular classification had no significant impact on progression free (p=0.056) or overall survival (p=0.12) after cytoreductive surgery. Overall survival was affected by histologic subtype (p<0.0001) and estrogen receptor status (p=0.013). CONCLUSION: The distribution of the molecular subclasses in stage IV endometrial cancer patients differed substantially from the distribution in stage I-III endometrial cancer patients, with the unfavorable subclasses being more frequently present. Although the molecular classification was not prognostic in stage IV endometrial cancer, it could guide adjuvant treatment decisions.

Identification of HPV genotypes causing cervical precancer using tissue-based genotyping.

Identification of high-risk human papillomavirus genotypes causing cervical precancer is crucial for informing HPV vaccine development and efficacy studies, and for determining which types to include in next-generation genotyping assays. Co-occurrence of hrHPV infections is common and complicates carcinogenicity assessment; accurate attribution requires tissue-based genotyping of precancers. We included all women with cervical intraepithelial neoplasia Grade 2 or worse (CIN2+) from the Biopsy Study, an observational study of 690 women enrolled between 2009 and 2012 at the University of Oklahoma. Tissue-based genotyping, including whole tissue sections (WTS) and laser-capture microdissection (LCM), was performed on all precancers with multiple hrHPV infections detected in cytology, totaling over 1,800 HPV genotyping assays. Genotype attribution was compared to hierarchical and proportional hrHPV-type attribution models. Of 276 women with CIN2+, 122 (44.2%) had multiple hrHPV genotypes in cytology. Of 114 women with genotyping data, 94 had one or more hrHPV detected in tissue. Seventy-one women (75.5%) had a single causal hrHPV genotype, while 23 women had multiple hrHPV genotypes causing CIN2+. Ten women had multiple causal infections in a single biopsy, contrary to the previous notion that each lesion is caused by a single type only. While HPV16 was the predominant causal hrHPV genotype using all approaches, the hierarchical model overattributed HPV16, whereas other causal hrHPV genotypes, particularly HPV18 and HPV35, were underattributed. Understanding true causal genotypes is important for the evaluation of vaccine efficacy, to estimate the extent of unmasking, and for type-specific risk assessment in screening and management.

CADM1 and MAL methylation status in cervical scrapes is representative of the most severe underlying lesion in women with multiple cervical biopsies.

Recent studies have shown that CADM1/MAL methylation levels in cervical scrapes increase with severity and duration of the underlying cervical intraepithelial neoplasia (CIN) lesion. Multiple lesions of different histological grades and duration are frequently present on the cervix. To gain more insight into the possible epigenetic heterogeneity and its consequences for the methylation status in cervical scrapes, we performed an exploratory study of CADM1/MAL methylation in different grades of CIN lesions present in women with multiple cervical biopsies. CADM1-M18 and MAL-M1 methylation was assessed using a standardised, multiplex, quantitative methylation specific PCR on 178 biopsies with various grades of CIN in 65 women, and in their corresponding cervical scrapes. CADM1/MAL methylation positivity increased with disease severity, from 5.5% in normal biopsies to 63.3% and 100% in biopsies with CIN3 and cervical cancer, respectively. In the majority (8/9) of women where besides a CIN2/3 lesion a biopsy from normal cervical tissue was present, the CIN2/3 biopsy was CADM1/MAL methylation positive and the normal biopsy was CADM1/MAL methylation negative. A good concordance (78%) was found between CADM1/MAL methylation results on the scrapes and the biopsy with the worst diagnosis, particularly between samples of women with CIN3 and cervical cancer (92% and 100% concordance, respectively). Thus, in women with multiple cervical biopsies, CADM1/MAL methylation increases with severity of the lesion and is lesion-specific. CADM1/MAL methylation status in cervical scrapes appears to be representative of the worst underlying lesion, particularly for CIN3 and cervical cancer.

Investigating Diagnostic Problems of CIN1 and CIN2 Associated With High-risk HPV by Combining the Novel Molecular Biomarker PanHPVE4 With P16INK4a.

Grading cervical intraepithelial neoplasia (CIN) determines clinical management of women after abnormal cytology with potential for overdiagnosis and overtreatment. We studied a novel biomarker of human papillomavirus (HPV) life-cycle completion (panHPVE4), in combination with the minichromosome maintenance (MCM) protein cell-cycle marker and the p16INK4a transformation marker, to improve CIN diagnosis and categorization. Scoring these biomarkers alongside CIN grading by 3 pathologists was performed on 114 cervical specimens with high-risk (HR) HPV. Interobserver agreement for histopathology was moderate (κ=0.43 for CIN1/negative, 0.54 for CIN2/≤CIN1, and 0.36 for CIN3). Agreement was good or excellent for biomarker scoring (E4: κ=0.896; 95% confidence interval [CI]: 0.763-0.969; p16INK4a : κ=0.798; 95% CI: 0.712-0.884; MCM: κ=0.894; 95% CI: NC (this quantity cannot be calculated). Biomarker expression was studied by immunofluorescence and immunohistochemistry and was correlated with 104 final CIN diagnoses after histologic review. All 25 histologically negative specimens were p16INK4a and panHPVE4 negative, although 9 were MCM-positive. There were variable extents of p16INK4a positivity in 11/11 CIN1 and extensive panHPVE4 staining in 9/11. Ten CIN2 lesions expressed panHPVE4 and p16INK4a, and 13 CIN2 expressed only p16INK4a. CIN3 showed extensive p16INK4a positivity with no/minimal panHPVE4 staining. PanHPVE4, unlike MCM, distinguished CIN1 from negative. PanHPVE4 with p16INK4a separated CIN2/3 showing only expression of p16INK4a, indicating transforming HR-HPV E7 expression, from CIN1/2 showing completion of HR-HPV life cycle by E4 expression and variable p16INK4a expression. PanHPVE4 and p16INK4a staining are complementary markers that could provide simple, reliable support for diagnosing CIN. Their value in distinguishing CIN1/2 that supports HR-HPV life-cycle completion (and which might ultimately regress) from purely transforming CIN2/3 needing treatment warrants further research.

The Value of Endocervical Curettage in Addition to Biopsies in Women Referred to Colposcopy.

OBJECTIVE: Performing endocervical curettage (ECC) at colposcopy may increase the yield of cervical intraepithelial neoplasia grade 2 (CIN2) or worse (CIN2+) compared to biopsies alone. The additional benefit of ECC in detecting CIN2+ was studied in women with lesion-targeted biopsies (low-grade or worse impression) and women with biopsies of normal-appearing cervix (less than low-grade impression). METHODS: In this subanalysis of a multicenter study, 126 women referred to colposcopy who had an ECC were included. Multiple directed biopsies were taken from lesions, and a nontargeted biopsy was added if fewer than 4 biopsies were collected. Risk strata of CIN2+ were evaluated based on cytology and colposcopic appearance to identify women for whom ECC would be most valuable. RESULTS: The CIN2+ yield of ECC in addition to biopsies was 15 (11.9%) of 126. In women with lesion-targeted biopsies and ECC, the CIN2+ yield of targeted biopsies was 34 (51.5%) of 66, the yield of additional nontargeted biopsies was 1 (1.5%) of 66, and the additional CIN2+ yield of ECC was 5 (7.6%) of 66. The yield in women with nontargeted biopsies only and ECC was 5 (8.3%) 60, and the additional yield for ECC was 10 (16.7%) of 60. Endocervical curettage did not find disease in women with atypical squamous cells of undetermined significance/low-grade squamous intraepithelial lesion. CONCLUSIONS: In women with less than low-grade impression and especially those with unsatisfactory colposcopy, the yield of CIN2+ was higher for ECC compared to nontargeted biopsies. The highest yield of CIN2+ from ECC was observed in women with high-grade squamous intraepithelial lesion and less than low-grade impression, suggesting that disease is higher up in the endocervix in this group.

Attributing oncogenic human papillomavirus genotypes to high-grade cervical neoplasia: which type causes the lesion?

Human papillomavirus (HPV) is found in most women with high-grade cervical intraepithelial neoplasia (CIN) 2/3 in cervical cytology and biopsies. Multiple high-risk HPV (hrHPV) genotypes are present in 15% to 50% of cytology samples. We have shown by laser-capture microscopy (LCM)-polymerase chain reaction (PCR) that each lesion is associated with a single hrHPV type. Attribution of hrHPV types to CIN2/3 is important to understand the oncogenic role of different types and the limitations of cytologic typing. We studied hrHPV genotypes in 257 women with histologic CIN2/3 referred on the basis of abnormal cytology. HPV typing was done on cytology and CIN2/3 biopsies. If the whole-tissue section of the biopsy was positive for multiple hrHPV types, LCM-PCR was performed. We found 181 (70%) single and 71 (28%) multiple hrHPV infections in cytology, with 5 (2%) cases HPV-positive only on whole-tissue section PCR. Of cases with multiple cytologic hrHPV infections, 47/71 (66%) showed a single type in CIN2/3 lesions. In total, in 232 of 257 (90%) women with CIN2/3, a single hrHPV type caused CIN2/3. One was nonattributable on the LCM level. The remaining 24 women had 2 or more contiguous or separated lesions, each associated with a single hrHPV infection. The probability of HPV16 being present in CIN2/3, if detected in cytology, was 0.96 (95% confidence interval=0.90-0.98). LCM-PCR confirms that only 9% of histologic CIN2/3 is associated with multiple hrHPV types, much less than cytology would indicate, and each lesion was associated with a single hrHPV infection.

Oncogenic human papillomavirus-infected immature metaplastic cells and cervical neoplasia.

Persistent cervical high-risk human papillomavirus (HR-HPV) infection results in high-grade cervical intraepithelial neoplasia (CIN2/3) and cervical carcinoma. The susceptibility of the cervix to HPV carcinogenesis and the importance of HPV18 in cervical carcinoma despite relative infrequency in CIN2/3 could be linked to HR-HPV infection of immature metaplasia (IM) at the squamocolumnar junction. Atypical IM (AIM) is an equivocal category used to describe changes in IM suggestive of high-grade neoplasia, which causes diagnostic and management problems. We used laser capture microscopy combined with polymerase chain reaction in 24 women with HPV18, HPV16, or other HPV infections on cytologic analysis and a cervical loop electrosurgical excision procedure to locate HR-HPV in cervical tissue. HPV18-positive AIM and CIN2/3 were present in 7/12 cases with HPV18 on cytologic analysis. In 2 cases with HPV18 and other HPV types, HPV18 was only present in AIM and not in CIN2/3. HPV16-positive AIM was present in 3/7 and HPV16-positive CIN2/3 in 5/7 cases with HPV16. No cases had HPV16 AIM without CIN2/3. Other HR-HPV-positive AIM and CIN2/3 cases were present, respectively, in 1/6 and 5/6 cases positive for HR-HPV types other than HPV16/18. In a subset, 94% HPV18 AIM regions showed CK17 and p16 positivity, and 41% were CK7 positive. CIN2/3 and AIM with other HR-HPVs showed similar patterns. AIM was a particular feature of HPV18 infection in women with CIN2/3. HR-HPV infection of CK7/17-positive AIM expressing p16 was particularly seen for HPV18 with and without classical CIN2/3 and should be regarded as a high-grade precancer.

Molecular mapping of high-grade cervical intraepithelial neoplasia shows etiological dominance of HPV16.

Women with high-grade cervical intraepithelial neoplasia (HGCIN) frequently present with multiple cervical lesions and multiple concomitant Human papillomavirus (HPV) genotype infections. To elucidate HPV genotype attribution in different regions on the cervix, we performed molecular mapping of cervical disease in women with HGCIN. Thirteen subjects referred to colposcopy for abnormal cervical cancer screening results were included. A cervical smear and biopsies from 4 different areas on the cervix were collected. HPV genotyping using Linear Array (for cytology) or SPF(10) LiPA(25) (for histology) were performed in 13 smears, 52 whole sections from biopsies and 138 tissue regions isolated with laser capture microdissection (LCM). Twelve subjects had a diagnosis of CIN3 and one subject had a diagnosis of CIN2 based on the worst histology found in 4 biopsies. Eight of the 13 smears (62%) showed multiple genotype infections. Four of 13 women (31%) had multiple HPV infections in their biopsies. After performing LCM-PCR, only one woman (8%) had two different carcinogenic HPV types in morphologically distinct, but colliding HGCIN lesions. HPV16 was identified as the causal type in all women with HPV16 in cytology. A large proportion of other HPV types found in cervical smears were not detected at the tissue level. Using tissue-based genotyping and LCM-PCR analysis, we were able to attribute an individual HPV type to each area of CIN lesions. We demonstrate that HPV16 is even more etiologically dominant than previously thought, based on various genotype attribution models.

Increased incidence of pregnancy complications in women who later develop scleroderma: a case control study.

INTRODUCTION: Studies have shown that fetal progenitor cells persist in maternal blood or bone marrow for more than 30 years after delivery. Increased trafficking of fetal cells occurs during pregnancy complications, such as hypertension, preeclampsia, miscarriage and intra-uterine growth restriction (IUGR). Women with these pregnancy complications are significantly more often HLA-class II compatible with their spouses. Women who later develop scleroderma also give birth to an HLA-class II child more often. From these prior studies we hypothesized that preeclampsia and other pregnancy complications could be associated with increased levels of fetal cell trafficking, and later be involved in the development of scleroderma. METHODS: This study was a retrospective multi-centre matched case-control study. One-hundred-and-three women with systemic sclerosis (SSc) and 103 women with no history of SSc or other autoimmune disease were given a questionnaire regarding complications during pregnancy, such as hypertension, intra-uterine growth restriction (IUGR) and miscarriage. Conditional logistic regression analysis was used to assess associations. RESULTS: We found a statistically significantly increased incidence of having had a pregnancy history of hypertension or a fetus with IUGR in women who subsequently developed SSc compared to healthy controls. We found an odds ratio of 2.6 (95% confidence interval (CI): 1.1 to 4.6) for hypertensive complications during pregnancy and an odds ratio of 3.9 (95% CI: 1.2 to 12.3) for intra-uterine growth restriction for women with SSc compared to healthy controls. CONCLUSIONS: This is the first study to show an association between hypertensive complications during pregnancy or IUGR and the development of SSc at a later age. We speculate that the pregnancy abnormalities may have resulted in increased fetomaternal trafficking, which may have played a role in the increased incidence of SSc. Further studies are indicated to examine this putative relationship.