HPV genotyping and site of viral integration in cervical cancers in Indian women

Abstract

Persistent HPV infection plays a major role in cervical cancer. This study was undertaken to identify HPV types in a cohort of Indian women with locally advanced cervical cancer as well as to determine the physical state and/or site of viral integration in the host genome. Pretreatment biopsies (n = 270) from patients were screened for HPV infection by a high throughput HPV genotyping assay based on luminex xMAP technology as well as MY09/11 PCR and SPF1/2 PCR. Overall HPV positivity was observed to be 95%, with HPV16 being most common (63%) followed by infection with HPV18. Integration status of the virus was identified using Amplification of Papillomavirus Oncogene Transcripts (APOT) assay in a subset of samples positive for HPV16 and/or HPV18 (n = 86) and with an adequate follow-up. The data was correlated with clinical outcome of the patients. Integration of the viral genome was observed in 79% of the cases and a preference for integration into the chromosomal loci 1p, 3q, 6q, 11q, 13q and 20q was seen. Clinical data revealed that the physical state of the virus (integrated or episomal) could be an important prognostic marker for cervical cancer.

Figure 1. Flowchart depicting summary of the study.

Genotyping was carried out on 270 advanced stage cervical cancer samples by high-throughput, GP5⁺/6⁺ primers based luminex array; consensus MY09/11 and SPF1/2 primers. HPV positivity was 95% (257/270). APOT assay was done on 86 HPV16⁺ and/or HPV18⁺ samples, with good clinical follow-up and good quality RNA. In 18 samples, only episomal form of HPV was identified, rest 68 hinted toward possible integration. Site of integration could be predicted with high score by BLAST and/or BLAT in 48 samples.

Figure 2. Frequency of 24 HPV types as detected by Luminex array.

The graph depicts frequency of 24 HPV types in 178 cervical cancer biopsy samples which were found to be positive GP5⁺/6⁺ primers. HPV16 infection predominated in the samples. Each bar represents different HPV types.

Figure 3. Kaplan-Meier analysis for episomal vs. integrated viral genome.

Kaplan-Meier survival curve for patients with episomal form of virus (n = 18) vs. integrated form (n = 68) is depicted. Most of the patients with episomal form (16 out of 18) had a disease free survival as compared to patients with integrated form, indicating a good clinical outcome, although with a borderline significance (p = 0.067).

Figure 4. Frequency of HPV integration into different chromosomal loci.

Site of integration as determined by APOT assay in 48 cases positive for HPV16, HPV18 or both and with high prediction score using BLAST/BLAT. Integration event was found to be more common in 1p and 3q chromosomal loci. Each bar represents different chromosomal locus.