A 51-year-old woman was referred to our Gynecology Unit in July 2012, with hypermenorrhea and dysmenorrhea. The patient reported regular menstrual periods, two previous pregnancies with spontaneous deliveries and negative previous pap smear. Patient’s body mass index (BMI) was 23. The anamnesis of the patient was negative for any comorbidities. Family history of cancer was negative, too.
She reported a previous diagnostic hysteroscopy with negative endometrial sampling followed by 5-months progestins treatment without benefits.
On clinical examination and pelvic ultrasound her uterus appeared enlarged (length, anteroposterior and transverse diameter were 107x75x83 mm respectively) due to two intramural and sub-serosal uterine myomas (6 and 5 cm, respectively), with regular endometrium (thickness 8 mm) and ovaries.
Due to symptoms and age, hysterectomy was recommended. The patient declined surgical treatments and was switched to medical therapy with Gonadotropin Releasing Hormone (GnRH) analogue (Leuproreline 3.75 mg monthly intramuscular injections) for 6 months. Six months later, the patient reported recurrent symptoms and bilateral inguinal lymph node enlargement.
Further clinical examination and pelvic ultrasound showed unchanged uterine myomas, regular endometrium (5 mm) and enlarged, fixed right inguinal lymph node of 2 cm. Because of failure of medical therapy, surgical intervention was again proposed. In February 2013 the patient was referred to open surgical intervention: the exploration of all peritoneal surfaces and all abdominal cavities did not uncover any sign of malignancy or endometriosis. The uterus was enlarged (longitudinal diameter 10 cm), its surface was smooth and regular, ovaries were small and regular. Surface of peritoneum and all abdominal organs were free from disease and no enlarged pelvic and para-aortic lymph node were found. Surgical resection included hysterectomy, bilateral salpingo-oophorectomy, pelvic and bilateral inguinal lymphadenectomy and peritoneal washing for cytology. Intra-operative frozen sections of right inguinal lymph node dissection were positive for EC, although frozen sections of the endometrium were negative for EC. Due to cancer diagnosis, omentectomy and multiple peritoneal biopsies were performed. No post-operative complications were observed and the patient was discharged from the hospital in good conditions 6 days after surgery.
Final histology identified a focus of well differentiated uterine intramucosal endometrioid adenocarcinoma, without any sign of myometrial invasion (Fig. 1a). Cervix, tubes and ovaries were disease-free. No metastases were found in the peritoneal biopsies or in the pelvic and retroperitoneal removed lymph nodes. However, histology confirmed bilateral inguinal lymph nodes (2 out of 6 overall) positive for endometrial adenocarcinoma (Fig. 1b), with immunohistochemistry positive for CK7, ER, VIM, and negative for CK20 (typical in EC). Moreover, cytology of the peritoneal fluid showed atypical cells.
Positron Emission Tomography (PET), performed 5 weeks after surgery, showed uptake in the right inguinal area referable to residual disease or inflammation, without other pathological areas (Fig. 2). The patient was submitted to pelvic external beam radiotherapy (4500 cGy in 25 fractions) with concomitant weekly cisplatin and subsequent boost on the inguinal region (2000 cGy in 10 fractions). Moreover, 4 additional cycles of Cisplatin (70 mg/mq) every 21 days were administered. To date, the patient is disease-free.
In order to confirm histological examination and to understand whether the metastatic inguinal lymph nodes were derived from the EC, we used mitochondrial genome sequencing [6]. Samples of tumor tissue and metastatic lymph nodes were formalin-fixed and paraffin-embedded. Haematoxylin and eosin sections were reviewed to identify paraffin blocks with tumor areas. A control (non tumor) DNA specimen was obtained from the patient’s saliva after collection of informed consent. Whole mtDNA sequencing was performed on EC, metastatic inguinal lymph node and saliva-derived total DNA as previously described [5,6,7]. We used a PCR-based resequencing system which enables identification of sequence variations in the entire human mitochondrial genome and its control region. Total DNA was used for PCR amplification of 46 overlapping fragments covering the entire mtDNA using a set of 46 primer pairs. Overlapping regions of the mitochondrial genome are amplified with specific primer pairs tailed with universal M13 sequences at their 5′ end to generate resequencing amplicons. Amplicons are then used as templates for quick sequencing using universal M13 primers. All primer pairs are ready to use and anneal at the same temperature. The purified PCR product was used for direct sequencing with BigDye kit version 1.1 (Thermo Fisher Scientific). Sequences were run in an ABI 3730 Genetic Analyzer automated sequencing machine. Electropherograms were analyzed with SeqScape version 2.5 software. Mitochondrial DNA mutations detected in this first phase were confirmed using a second PCR reaction. When the latter showed the same mitochondrial DNA variant of interest, the mutation was confirmed on a second extraction of DNA from the same sample to exclude DNA contamination or sample mix-up. The informative nature of mitochondrial mutations was ascertained by sequencing mitochondrial DNA from saliva.
Sequence analysis was performed with MToolBox [8] and the genomes were deposited in public human mitochondrial database HmtDB [9] with the following identifiers: endometrial tumor: PA_EU_IT_0271, metastasis: PA_EU_IT_0272, saliva: PA_EU_IT_0273. The analysis revealed the presence of several informative variants. The m.3170C > A in the MT-RNR2 gene was found heteroplasmic in the saliva and homoplasmic both in the tumor and metastatic nodes (Fig. 3a). By using denaturing high pressure liquid chromatography (DHPLC) (Fig. 3b-c), a method shown to be sensitive enough to detect heteroplasmy levels as low as 2% [10] we were able to confirm the heteroplasmy status of these mutations in the saliva. The m.15851A > G in MT-CYB and the m.15927G > A in MT-TT were found homoplasmic only in the metastasis specimen. The m.15924A > G in MT-TT was present in homoplasmy in the saliva, in heteroplasmy in the tumor tissue and was not present in the metastatic lymph nodes, indicating a reverse shift occurring stepwise during progression from primary tumor to nodal metastasis. Similarly, to the latter, the shift to homoplasmy of the m.3170C > A exclusively occurring in the EC and in the metastatic tissues may be considered as a marker of clonal origin of the two tissues.
