HPV DNA integration site as proof of the origin of ovarian metastasis from endocervical adenocarcinoma: three case reports

A 55-year-old nulliparous postmenopausal woman with no medical history was managed for FIGO (International Federation of Gynecology and Obstetrics classification) stage IIIB cervical cancer. Cervical biopsies showed HPV18-related moderately differentiated invasive adenocarcinoma. Magnetic resonance imaging (MRI) revealed a 4 cm anterior mass extending to the uterine isthmus, uterine corpus, left parametrium and superior third of the vagina. No other lesion was visualized on abdominal computed tomography (CT) and positron emission tomography (PET-CT). Concomitant external beam pelvic radiation (45 Gray (Gy) in 1.8 Gy daily fractions) and six cycles of chemotherapy (weekly cisplatin 40 mg/m²) were administered. The patient was reevaluated by MRI at the end of treatment, showing less than 50% size response with persistent parametrial involvement. Adjuvant brachytherapy (25 Gy) and external beam pelvic radiation (8 Gy in 3 daily fractions) was therefore decided. Two months later, the lesion had completely resolved on MRI. After 3 years of follow-up, MRI revealed a pelvic mass with no increased uptake on PET-CT. Bilateral salpingo-oophorectomy was performed based on a diagnosis of right ovarian mass without peritoneal carcinomatosis or other distant disease. Histological examination concluded on invasive mucinous adenocarcinoma.

To determine the origin of the ovarian lesion and in view of the synchronous HPV18-positive cervical carcinoma, molecular analyses were performed, showing that the ovarian tumor was HPV18-positive, strongly suggesting a primary cervical origin. As previous analyses of the cervical tumor identified the HPV integration site in chromosome 13, the ovarian tumor was screened to determine whether the ovarian metastasis presented the same HPV integration site. The same HPV integration site at locus 13q22.1 was demonstrated in ovarian tumor DNA, clearly confirming that the ovarian mass was a metastasis from the cervical adenocarcinoma (Fig. 1). Six cycles of chemotherapy (weekly paclitaxel and carboplatin) were therefore administered. Eighteen months later, the patient presented recurrence in the form of peritoneal carcinomatosis and a rectal nodule confirmed histologically at laparoscopy. Treatment of the recurrence consisted of six cycles of carboplatin and paclitaxel, which was early stopped because of neuropathy toxicity. After a therapeutic break requested by the patient, she was included in a phase 1 study for advanced gynecological cancers (clinicaltrials.​gov identifier: NCT 02978755). The patient is still on treatment.

A 45-year-old smoking primiparous woman with no medical history presented with ascites and a left ovarian mass. Histological examination of the mass revealed grade 1 ovarian endometrioid carcinoma. Staging surgery with total hysterectomy, bilateral salpingo-oophorectomy, omentectomy, and pelvic and para-aortic lymphadenectomies were therefore performed. No peritoneal carcinomatosis was observed.

HPV18-positive in situ endometrioid adenocarcinoma was found in the endocervix and HPV18-positive invasive endometrioid adenocarcinoma was found in the endometrium and both ovaries on final histological examination. The same HPV integration site in locus 2q22.3 was demonstrated in the ovarian tumor DNA, clearly confirming that the ovarian mass was a metastasis from the cervical adenocarcinoma (Fig. 2). The patient was then treated with radiotherapy and brachytherapy.

A 30-year-old nulliparous woman with no medical history, regularly screened by PAP-smear test presented with postcoital metrorrhagia. In situ carcinoma was diagnosed on cervical biopsies. MRI and abdominal CT imaging were normal. A large loop cervical excision and endometrial curettage were performed and confirmed the presence of a well differentiated HPV18-related in situ adenocarcinoma and high-grade cervical intraepithelial neoplasia (CIN III) with negative surgical margins. No obvious infiltration was observed on the examined slides. Nine years later, after several failures of in vitro fertilization, the patient experienced abdominal pain. Ultrasound imaging revealed a 9.5 cm complex left adnexal mass. Examination of the laparoscopic left salpingo-oophorectomy revealed mucinous cystadenoma of the intestinal type with borderline character traits and extensive foci of intraepithelial carcinoma. Seventeen months later, after being lost to follow-up, a 10 cm right ovarian mass was discovered. Total hysterectomy, right salpingo-oophorectomy, appendectomy, omentectomy were performed, revealing a 2.5 cm HPV18-related cervical cancer extending to the uterine isthmus with mucinous proliferation of intestinal type involving the cervix and right ovary. The HPV insertion site could not be determined due to insufficient tumor tissue. One month later, the patient was referred to our oncologic surgery department, where complementary laparoscopic pelvic and para-aortic lymphadenectomies were performed. Carcinomatous metastasis and intestinal involvement were discovered during laparoscopy. The patient was treated with FOLFOX chemotherapy for 3 months and then underwent complete cytoreductive surgery with peritonectomy, multiple bowel resections and intraperitoneal hyperthermic chemotherapy due to a partial response to chemotherapy. Examination of the resected tissues showed carcinomatous cells partly modified by chemotherapy and a high mitotic index (Ki 67: 70%). The patient is currently on follow-up.

Total DNA, isolated from formalin-fixed tissue blocks, was used for HPV typing. Real-time PCR using Sybr®Green PCR Master mix at a final concentration of 1X, specific primers for HPV16, HPV18 and HPV33 [2] at a final concentration of 660 nM each and 10 ng of tumor DNA was performed on a 7900HT Fast Real-Time PCR System in a final volume of 26 μL (Applied Biosystems, Foster City, CA). PCR reaction were run under the following program: 95 °C 15 min, 40 cycles of (95 °C 1 min, 55 °C 1 min, 72 °C 1 min), followed by a dissociation stage (95 °C 15 s, 60 °C 1 min, 95 °C 15 s).

For the identification of HPV integrations sites in patient 1 and patient 2, the DIPS-PCR method was performed on total DNA isolated from cryopreserved tumor tissue [7]. Briefly, after overnight ligation to an adapter, digested DNAs were submitted to two runs of PCR. For the analysis, 10 HPV18 primers matching the frequent viral breakpoint areas (E1, E2, L1, and L2 genes) were used. PCR products were run on an agarose gel and selected amplicons were analyzed by Sanger sequencing. Genomic chromosome-viral breakpoints were identified by alignment of the sequence with the HPV18 genome using the nucleotide BLAST tool from NCBI (https://​blast.​ncbi.​nlm.​nih.​gov/​Blast.​cgi) and human genome (hg19 reference) using the blat tool (https://​genome.​ucsc.​edu).

Presence of HPV of the same integration site in both cervical and ovarian tumors were confirmed by PCR using specific primers followed by Sanger sequencing, as shown by Figs. 1 and 2, parts B and C.

Immunohistochemistry was performed for p16 on formalin-fixed, paraffin-embedded tissue sections using the prediluted E6H4 antibody (Ventana Medical Systems, Inc.) on a Leica Bond automated instrument (Leica Biosystems, Inc.) with standard antigen retrieval and incubation times. Both nuclear and cytoplasmic staining was required for a cell to be considered “positive”.