HPV E6/E7 mRNA In Situ Hybridization in Endocervical Adenocarcinoma: Implications for Prognosis and Diagnosis

Abstract

Background: Human papillomavirus (HPV) E6/E7 mRNA in situ hybridization (HPV E6/E7 RNAscope) appears to be a sensitive and specific technique in detecting transcriptionally active HPV. We aimed to examine the diagnostic utility of this technique in endocervical adenocarcinoma (ECA), to explore the prognostic factors for ECA patients and develop a clinically useful nomogram based on clinicopathological parameters to predict it.

Methods: We retrospectively analyzed 200 patients with ECA who had undergone surgery at Sun Yat-sen University Cancer Center from 2010 and 2014. The diagnostic performance of HPV E6/E7 RNAscope were evaluated by receiver operating characteristic (ROC) curve. A prognostic nomogram model including HPV E6/E7 RNAscope was generated based on multivariate Cox regression analysis, then we compared the predictive accuracy of the prognostic model with FIGO staging and treatment using concordance index (C-index), time-dependent ROC (tdROC), and decision curve analysis (DCA).

Results: The sensitivity and specificity of HPV E6/E7 RNAscope for distinguishing HPV-associated adenocarcinoma (HPVA) from non-HPV-associated adenocarcinoma (NHPVA) in the whole cohort were 75.8% and 80%, respectively. According the univariate analysis and multivariate logistic regression analysis, age, lymphovascular invasion (LVI), lymph node involvement (LNI), and HPV E6/E7 RNAscope were valuable predictive factors for OS. These parameters were incorporated into the nomogram model (nomogram A) compared with FIGO stage and treatment. The C-index of nomogram A for predicting OS was 0.825, which was significantly higher than FIGO stage (C-index = 0.653, p=0.002) and treatment (C-index = 0.578, p < 0.001).

Conclusions: HPV E6/E7 RNAscope is highly specific for ECA, and the 4-variable nomogram showed more accurate prognostic outcomes in patients with ECA. 

Introduction

Endocervical adenocarcinoma (ECA) accounts for 15–20% of all cervical carcinomas. Studies have shown that ECA is increasingly reported in young women[1], with a projected 5-year overall survival (OS) rate of only 20.3% (95% confidence interval [CI]: 14.2–27.1%) compared to the squamous type (31.3%, 95%CI: 29.2–33.3%)[2]. The International Federation of Gynecology and Obstetrics (FIGO) staging system is the gold standard for predicting outcomes in patients with ECA, and the mainstay treatment of advanced ECA is concurrent chemoradiotherapy. However, ECA patients with the same FIGO stage can have marked heterogeneities in their outcomes [3, 4]. The new FIGO 2018 amendments put forward by the gynecological oncology committee agree that staging is an ongoing process informed by data on prognosis and survival, and therefore, treatment should be individualized and not merely based on staging given the variability of the resource across regions. FIGO 2018 also recommends any imaging modality and/or pathological findings like lymphovascular invasion (LVI) for allocating staging[3].

A new classification—the International Endocervical Adenocarcinoma Criteria and Classification (IECC) has replaced the 2014 WHO classification and categorizes ECA based on the morphological features linked to the etiology (i.e., HPV infection), thus grouping them into HPV-associated (HPVA) and non-HPVA (NHPVA) types[4, 5]. Comparing HPVA with NHPVA types reveals essential differences in tumor biology and patient survival, with NHPVA-type tumors significantly associated with worse outcomes[6, 7]. It is apparent that a classification based on the pathogenesis and other clinicopathological factors may be more clinically useful and reproducible than the current FIGO scheme. The other shortcoming of FIGO is that it doesn't stratify patients based on histology and HPV status. Hence, novel prognostic determinants to complement the FIGO staging system is needed. The validity of this new classification is supported by HPV status but with limited clinical data[8, 9].

In the present studies, the following tests were used to detect HPV infection: HPV DNA, genotype, RNAscope, and immunochemistry (IHC) against p16 protein. Plasma HPV DNA levels can be determined using PCR and is a potential marker for cervical cancer[10]. The other 3 assays can be conducted on formalin-fixed, paraffin-embedded (FFPE) samples. Immunostaining for p16 is a cost-effective and highly sensitive marker but with a low specificity[11]. PCR for HPV genotyping is the gold-standard assay to diagnose active HPV infection[12]. Chromogenic in situ hybridization against RNA can identify HPV in situ via microscopic observation[13]. The technique is expensive but can be used on FFPE samples[14]. In situ detection of HPV E6/E7 mRNA using the RNAscope also appears to be a sensitive and a specific method; hence, we aimed to investigate the potential prognostic utility of this technique in ECAs.

Numerous controversies surround the FIGO staging system, wherefore ECAs are morphologically stratified into A, B, and C subgroups using the Silva based on their LVI or lymph node involvement (LNI) status[15]. Tumors characterized by well-demarcated glands with rounded contours are designated as pattern A; tumors with early or limited, localized destructive invasion or inflamed stroma adjacent to an intact gland are designated pattern B, while tumors with more aggressive features, including diffusely infiltrative glands, usually associated with extensive, diffuse desmoplastic response are designated pattern C. Therefore, clinicians could provide precision treatment to patients with pattern C tumors. However, stratifying patients based on a single biomarker, such as LVI or LNI alone, can lead to misclassification of tumors and poor patients' prognostication, hence the two parameters should be combined[16]. There is currently no such available nomogram model for patients with ECA that combines HPV, LVI, and LNI status, And other survival prediction models that follow the FIGO staging system have been insufficient.

In the present study, we aim to validate the utility of the novel RNA scope method for detecting HPV RNA in tissue samples for proper initial risk stratification of patients. We also built a nomogram that will compliment FIGO staging for prognostic classification of EAC. We hope the link between these two modalities will bring new insights into the clinical care of EAC patients.

Patients & Methods

Results

Discussion

Our study is the first of its kind to describe a prognostic nomogram model for invasive ECA in a large Chinese series by reporting HPV E6/E7 RNAscope using a new RNA ISH assay that recognizes 18 high-risk HPV types. The most important finding of our research are as follows: (1) the validity of the HPVA and NHPVA categories were supported by p16 immunophenotyping and HPV status; (2) HPV E6/E7 RNAscope is more sensitive and specific than p16 and HPV genotype in identifying HPVA; (3) we established a prognostic nomogram model based on ECA pathogenesis that adjusted for age, LVI, HPV E6/E7 RNAscope, and LNI, which had a greater predictive efficiency than the current conventional staging system; (4) in the future, we believe, based on our model, ECA patients could be divided into low and high-risk groups.

HPV E6/E7 RNAscope measurement is not routinely available in most hospitals; hence the methodology cannot be extrapolated to standard practice. About 94% of adenocarcinomas are associated with HPV, particularly high risk strains like 16 and 18[5], which is consistent with our results. In the present study, 87% (160/184) of HPVA samples overexpressed p16 or were HPV positive by RNAscope, validating the IECC criteria and previous studies[9, 20]. In terms of the IECC criteria, the HPV RNAscope is more specific for clinical practice, and it is more robust than p16 IHC, HPV DNA, and HPV genotype in identifying HPVA. Despite this, occasional p16 and/or HPV negative HPVAs were also identified. When outlying cases were excluded from the statistical analysis, 25 of the 184 HPVA cases were p16- and HPV-positive. All HPVA cases in the present study were systematically reviewed.. The specimens were more than 5 years old and may not have been optimal tissues for the performance of HPV RNAscope and p16. A recent study reported that the HPV associated ECA might represent unusual morphological variants of gastric-type carcinoma[21]. Furthermore, rare HPV genotypes not included in the RNA ISH probe set may be responsible for the negative HPV results. There are precedents in the literature for p16- and HPV-associated neoplasia. Notably, p16-positive invasive squamous carcinomas of the cervix have been reported[22] to be closely associated with methylation-induced inactivation of the p16 gene and allelic loss of p16[23, 24]. Other studies have reported that the HPV genome can be differentially expressed with varying heterogeneity between the primary and the metastatic sites suggesting that some HPV-related usual-type adenocarcinomas could have detectable HPV in multiple sections of primary carcinoma; hence all metastatic sites should be tested[20, 25, 26].

Clinicopathological variables carry a prognostic significance and usually affect clinical management[27, 28]. Depending on variables such as LVI and desire for fertility, histological-based risk evaluation should be considered during tissue sampling at initial diagnosis[15, 29]. The National Comprehensive Cancer Network guidelines recommend chemotherapy or radiotherapy as the standard treatment for patients with advanced ECA, hence, the performance of nomograms must be examined separately in patients treated using these therapies. The C-indices of nomograms A and B for predicting OS in treated patients were significantly higher than those of conventional classification, indicating that nomograms still have significant clinical value in patients with ECA. With the addition of the FIGO stage and treatment into nomogram B, the added values of these parameters over nomogram A were 0.836 and 0.825, respectively, across the entire population. There were no significant differences between nomograms A and B, probably due to the small sample size of patients with advanced-stage ECA.

The present study had several limitations. Firstly, there may have been selection bias in our patient cohort. Secondly, we lacked data on the impact of our nomogram on other prognostic endpoints, such as disease-free survival (DFS) prediction. Thirdly, in situ measurement of HPV RNA using RNAscope is not easily available and not globally standardized. Fourthly, our sample size was relatively small and from only one center. In order to validate our results, a study with a larger sample with multicenter data is required. Lastly, it remains unclear whether our nomogram can be applied to ECA patients with advanced-stage that is, stage III and IV.

Conclusions

We generated new nomograms that incorporate HPV E6/E7 mRNA in situ hybridization and patients’ clinical characteristics in order to prognosticate the OS rate in ECA patients. Our simple and explicit nomograms have a good clinical application value, with a good diagnostic discernment and calibration ability. They may be a useful tool for assessing the prognosis and the management of ECA patients.

Abbreviations

Declarations

Ethics approval and consent to participate

The Sun Yat-sen University Cancer Center Ethics Committee approved this study. 

Consent for publication

Not applicable.

Availability of data and materials

The authenticity of this article has been validated by uploading the key raw data onto the Research Data Deposit public platform (www.researchdata.org.cn) with the approval RDD number RDDA2020001638.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by the National Natural Science Foundation of China (No. 82072853). The Natural Science Foundation of Guangdong province (No. 2021A1515010688).

Authors’ contributions

Conception and design: Li-Li Liu. Performing experiments: Mei Li, Yue Li and Xia Yang. Drafting of the article: Rong-Zhen Luo and Shu-Lin Chen. Acquisition and interpretation of data, review, editing and approval of the manuscript: all authors.

Acknowledgments

The authors gratefully acknowledge the assistance from professor Jing-Ping Yun from Sun Yat-sen University Cancer Center.

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