Detection of E6/E7 human papilloma virus (HPV) transcripts in lymph nodes of patients with cervical cancer

Persistent high risk Human Papillomavirus (HPV) infection is necessary for the development of cervical cancer (CC).HPV carcinogenesis is based on viral E6 and E7 proteins' capacity to interfere in cell proliferation control.The metastasis status of pelvic lymph nodes (PLN) is a critical parameter in post-operative decisions on adjuvant therapy, given its strong correlation with recurrence in CC.In order to complement the histopathological evaluation of subclinical node metastases, we evaluated the application of a commercial HPV E6 / E7 mRNA kit, to detect viral messenger RNAs in lymph nodes and tumors of patients with CC.Forty five cervical primary tumors and 152 PLN ( 3-4 from each patient) were included.HPVs were typed in tumors by PCR (polymerase chain reaction) using generic primers PGMY and reverse line blot hybridization (RLB) with type-specific oligo probes corresponding to 37 HPV types that infect the anogenital tract.PNL were collected in RNAlater (Invitrogen) and mRNA was extracted using the MiniMag (Biomerieux) system.Detection of E6 and E7 mRNAs corresponding to HPVs types 16, 18, 31, 33 and 45 was carried out using isothermal real time PCR (NucliSENS EasyQ HPV, Biomerieux).HPV was detected in the tumors of 42 patients; the viral types identified were HPV16 (n = 32), HPV18 (n = 5), HPV31 (n = 3), HPV45 (n = 2), HPV59 (n = 1) and HPV73 (n = 1).These last 2 cases were not considered in the study because the mRNA detection system does not include these viral types.Also excluded were 2 cases whose tumors were HPV negative.There was a high correlation between the histological and virological results.Seventy-two percent of the histologically positive PLN were positive for E6-E7 mRNA; while 93% (125/134) of the negative PLN were also negative for E6-E7 mRNA assay.There was, however 8% (9/134) of negative PLN in which viral messengers were detected.Patients' follow up was limited (4 years) and except for two patients who died (both positive for HPV 45) in the period under review, no recurrences were recorded in any of the patients included in the study.The presence of HPV in PLN may indicate a metastasis, as since the virus is not able of producing viremia or invading tissues it can only be "transported" by the cancer cell.The presence of HPV mRNA indicates viral genome transcription, a process which occurs in a lymph node only in CC metastatic cells, as these viruses can only replicate in epithelial-origin cells.Therefore, the findings of these mRNAs in the negative pelvic lymph nodes point out a very early metastasis, detectable at molecular level, but unobservable on histological diagnosis.The commercial kits for HPV E6/E7 mRNA detection, usually applied for the cervical disease management, may be used also to evaluate PLN biopsies, in hospitals without high complexity laboratories.The test might be a tool to complement morphological observation, particularly when PLN do not exhibit characteristics compatible with invasion, thus optimizing monitoring and decision making.