Detection of HPV and Human Chromosome Sites by Dual-Color Fluorescence In Situ Hybridization Reveals Recurrent HPV Integration Sites and Heterogeneity in Cervical Cancer.

Jinfeng Xiong,Chun Gao,Xiaomin Li,Hui Shen,Hui Wang,Jing Cheng,Wencheng Ding,Ci Ren,Liming Wang,Tong Zhu,Da Zhu

doi:10.3389/fonc.2021.734758

Abstract

Human papillomavirus (HPV) integration in the human genome is suggested to be an important cause of cervical cancer. With the development of sequencing technologies, an increasing number of integration “hotspots” have been identified. However, this HPV integration information was derived from analysis of whole cervical cancer tissue, and we know very little about the integration in different cancer cell subgroups or individual cancer cells. This study optimized the preparation of probes and provided a dual-color fluorescence in situ hybridization (FISH) method to detect HPV integration sites in paraffin-embedded cervical cancer samples. We used both HPV probes and site-specific probes: 3p14 (FHIT), 8q24 (MYC), 13q22 (KLF5/KLF12), 3q28 (TP63), and 5p15 (TERT). We detected HPV signals in 75 of the 96 cases of cervical cancer; 62 cases showed punctate signals, and 13 cases showed diffuse punctate signals. We identified 3p14 as a high-frequency HPV integration site in 4 cervical cancer cases. HPV integration at 8p14 occurred in 2 cases of cervical cancer. In the same cervical cancer tissue of sample No.1321, two distinct subgroups of cells were observed based on the HPV probe but showed no difference in cell and nucleus morphology. Our study provides a new method to investigate the frequent HPV integration sites in cervical cancer and reports the heterogeneity within cervical cancer from the perspective of HPV integration.

Highlights

Cervical cancer remains the leading cause of gynecological tumor-related mortality worldwide and the second most common malignancy in women, with 570,000 women diagnosed with cervical cancer and 311,000 dying from the disease each year [1, 2].Most Human papillomavirus (HPV) infections are cleared by the immune system, but in some cases, the infection persists
Persistent infection with high-risk HPV leads to cervical intraepithelial neoplasia (CIN), which occasionally develops into cervical cancer [3]
HPV16/18 probes were hybridized in SiHa and CaSki cells, two smaller HPV signals were observed in SiHa cells with only two copies of HPV16 (Figure 1A), and 7 to 8 signals of different sizes were observed in dots and clumps in CaSki cells with 500 HPV16/18 copies (Figure 1D)

Summary

Introduction

Cervical cancer remains the leading cause of gynecological tumor-related mortality worldwide and the second most common malignancy in women, with 570,000 women diagnosed with cervical cancer and 311,000 dying from the disease each year [1, 2]. Most HPV infections are cleared by the immune system, but in some cases, the infection persists. Persistent infection with high-risk HPV leads to cervical intraepithelial neoplasia (CIN), which occasionally develops into cervical cancer [3]. High-risk HPV types include 16, 18, 31, 33, 58 and other subtypes. HPV16 and 18 infection is common and can be detected in approximately 70% of cervical cancer cases. The virus can remain in the episome or integrate into the human genome, and the two patterns may coexist [4]

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Results

Conclusion