Discussion
Early in embryological development, two mesonephric (Wolffian) ducts begin to form and connect the mesonephros to the cloaca around the fourth week of gestation. In the presence of testosterone, these ducts will give rise to the epididymis, seminal vesicles, vas deferens, and ejaculatory ducts in males. In females, the mesonephric ducts regress. However, vestiges of these structures may persist along the female genital tract in the form of epithelial inclusions – the so-called mesonephric remnants – that may be found adjacent to the ovarian hilum, in the thickness of the broad ligament, in the vagina and, more frequently, in the lateral walls of the cervix [
7]. The prevalence of mesonephric remnants varies from 1 to 22% in adults and up to 40% in children [
8,
9].
The epithelia of the mesonephric remnants may expand into benign or malignant lesions (See Table
2). There is a biphasic variant of mesonephric adenocarcinoma with a sarcomatoid component that can display homologous or heterologous differentiation, named malignant mixed mesonephric tumor (MMMT) [
9,
10]. Typically, a homologous component resembling either endometrial stroma or a non-specific spindle cell sarcoma is found in the setting of MMMT, although heterologous elements such as atypical cartilage, osteosarcoma, and rhabdomyosarcoma have been described [
11,
12].
Table 2
Mesonephric-derived entities: Benign and malignant lesions
Mesonephric remnants (MRs)
| Typically identified in asymptomatic women in reproductive and postmenopausal age groups. MRs can be seen in up to 22% of adults and 40% of newborns and children. The lateral wall of the cervix (3 and 9 o’clock) is the most frequent location. Not associated with increased risk of malignancy. | MRs are non-mass forming and thus are not clinically or grossly apparent. | Clusters or linear arrays of small tubules lined by bland cuboidal epithelia, lacking mucin. | PAX8, GATA3, and CD10 (+); calretinin 10% (+); ER, PR, p16, and p53 (−) | No studies have evaluated molecular alterations. | Mesonephric hyperplasia, endometrial adenocarcinoma with cervical stroma invasion. |
Gartner’s duct cyst (mesonephric cyst)
| Uncommon (< 1%); typically located in the lateral or anterior wall of the vagina. May be associated with renal and ureteral abnormalities. No increased risk of malignancy. | Presentation is similar to other vaginal cysts. | Bland, cuboidal to low columnar non-mucinous epithelia | CD10, GATA3, PAX8, and calretinin (+) | No studies have evaluated molecular alterations. | Müllerian cysts, Bartholin duct cysts (showing mucinous epithelia) |
Mesonephric hyperplasia (MH)
| Usually an incidental microscopic finding in reproductive and postmenopausal age groups. May be rarely associated with erosion, nodularity, or an abnormal Pap smear. | Usually not apparent on gross examination. Occasional thickening of the cervical wall. Formation of a discrete mass is rare. | Similar to mesonephric remnants, the proliferations are larger (> 6 mm) and more numerous, with more extensive involvement of the cervix. The most common type is a lobular variant. | PAX8, GATA3, and CD10 (+); calretinin 10% (+); ER, PR, p16, and p53 (−) | Activating KRAS and NRAS mutations are not found. | Mesonephric adenocarcinoma, endometrial adenocarcinoma with invasion of the cervical stroma, endocervical adenocarcinoma |
Mesonephric adenocarcinoma
| The vast majority of cases arise in the uterine cervix. Represents less than 1% of all carcinomas at this site. Patients commonly present with abnormal bleeding and/or an exophytic polypoid mass protruding into the cervical canal. | Firm mass in the lateral wall of the cervix. Diffusely thickened cervix may be an alternative presentation. | Often widely infiltrative. May display a variety of patterns: ductal, tubular, solid, papillary, retiform, and sex cord–like. Depending on the pattern, epithelial cells may be cuboidal or columnar. Rare cases are biphasic tumors, which disclose a sarcomatoid component. | CD10, CK7, PAX2, and PAX8 (+); GATA3 (+), but to a lesser extent compared with GATA 3 results in MRs and MH; TTF-1, calretinin, and inhibin are variably (+); CEAm, ER, and PR (−) | Canonical activating KRAS mutations, NRAS mutations, gain of 1q, no microsatellite instability. TP53 mutations are variably present. | Mesonephric hyperplasia, endometrioid adenocarcinoma, mesonephric-like adenocarcinoma, clear-cell carcinoma, serous carcinoma |
Mesonephric adenocarcinoma is a rare, non-mucinous cervical tumor. It accounts for less than 1% of all tumors at this site and is not related to human papillomavirus (HPV). It is usually located deep in the lateral cervical stroma, but rare cases of primary vaginal and uterine corpus MNAC have been reported [
13,
14]. The average age of presentation is 53 years, and there is no apparent peak, since its prevalence in age groups from the third through the sixth decade is similar, with 26% of patients being younger than 40 years. Diagnosis is usually made on biopsy specimens, endometrial curettings, or hysterectomy specimens. Clinically, this type of tumor usually presents as abnormal vaginal bleeding or as a cervical mass on pelvic examination. Tumors may be discovered incidentally in some cases; less commonly, they may involve the entire cervical circumference, presenting as a barrel-shaped cervix. On gross examination, mesonephric adenocarcinoma may be in the form of an exophytic, nodular, or friable polypoid mass [
3,
15,
16]. In our case, the patient presented with a polypoid mass protruding into the cervical canal with no abnormal bleeding.
Histologically, MNAC is usually widely infiltrative and may display numerous architectural growth patterns (See Table
2). The tubular pattern consists of back-to-back, small, round to oval glands that are closely packed together and lined by low columnar, cuboidal, or flattened cells, with some lumens containing dense eosinophilic secretions such as those seen in mesonephric remnants [
17,
18]. In the ductal pattern, the tumor exhibits large glandular spaces with occasional intraluminal infoldings or papillae, lined by one to several layers of tall columnar cells with hyperchromatic nuclei. The retiform pattern is characterized by elongated, slit-like branching tubules variably containing intraluminal papillae with hyalinized fibrous cores and zigzag shaped glandular spaces resembling the rete ovarii [
3,
19,
20]. A variation of the retiform pattern is a sieve-like pattern with cystic spaces lined by flattened cells. Cysts may be empty or contain colloid-like material. The sex cord–like pattern consists of cells growing in cords and trabeculae with scant cytoplasm [
18]. A pathological parameter regarding solid/spindled morphology portends a worse prognosis.
Cytologically, the tumors disclose relatively uniform columnar cells with scant eosinophilic cytoplasm. The nuclei are usually oval and often have irregular membranes and frequent grooving. Nuclear pseudoinclusions may be also found, as well as mild to moderate atypia [
7]. Marked nuclear atypia is not seen. Prominent nucleoli may occasionally be present. The mitotic index ranges from 1 to 50 mitoses per 10 HPFs (high-power fields) and may vary from case to case [
3,
21]. Mesonephric hyperplasia of the lobular or diffuse pattern may be present in the background of MNAC.
Immunohistochemically, mesonephric adenocarcinoma is usually diffusely and strongly positive for CD10 (apical and luminal), CK7, PAX8, EMA (epithelial membrane antigen), and vimentin. PAX2 is usually positive, but a strong and diffuse expression is more likely to be associated with benign mesonephric lesions [
22]. Other markers including calretinin, inhibin, and androgen receptors (AR) are variably positive. CEAm and CK20 are consistently negative markers. ER and PR are uniformly negative or only focally positive in MNAC [
23‐
25]. No immunoprofile is diagnostic, but positive immunostaining for CD10, CK7, and calretinin along with negative immunostaining for CEAm is suggestive of mesonephric adenocarcinoma [
20]. Although TTF-1 (thyroid transcription factor 1) is generally considered a biomarker for lung and thyroid carcinoma, it may be positive in MNAC. In our case, immunostaining of the metastatic specimen from the lingula showed positivity for TTF-1 and PAX8, the latter confirming the gynaecological origin of the metastasis (See Table
3). HNF1B (hepatocyte nuclear factor 1-beta), while considered a marker of clear cell carcinoma, may be expressed in a subset of mesonephric adenocarcinoma [
26]. MNAC usually shows negative or weak focal staining for p16, which does not correlate with the presence of HPV. This p16 staining pattern correlates with the one seen in our case.
Table 3
Immunohistochemical and in situ hybridization for both the primary and the metastatic tumors
Beta-catenin | NP | + (membrane stain) |
Calretinin | – | – |
CEAm | – | NP |
CD10 | + (luminal pattern) | + (luminal pattern) |
Cytokeratin AE1/AE3 | NP | + |
Cytokeratin 7 | + | NP |
Cytokeratin 20 | – | NP |
Estrogen Receptor | – | – |
Inhibin, alpha | + | – |
p16INK4A | + (Non-block) | + (Non-block) |
p53 | Negative (Wild type) | NP |
PAX-2 | + | + |
PAX-8 | NP | + |
Progesterone Receptor | – | NP |
TTF-1 | NP | + |
Vimentin | NP | + |
WT-1 | + | + |
1q | NAa | Gain |
KRAS/
NRAS mutations are the most common molecular alterations detected in mesonephric adenocarcinomas. MNAC is characterized by recurrent
KRAS mutations [
22].
KRAS mutations have also been documented in mesonephric-like adenocarcinomas of the female genital tract [
16]. Besides,
KRAS are more common than
NRAS mutations
, and the two are mutually exclusive. The chromatin remodeling genes
ARID1A/B are frequently mutated as well. Common genetic aberrations found in endometrial and other types of cervical adenocarcinoma, such as
PTEN and
PIK3CA, are not reported in MNAC.
TP53 is uncommonly mutated in mesonephric adenocarcinoma and other cervical adenocarcinomas, while more than 90% of endometrial serous carcinomas harbor
TP53 aberrations. Thus,
KRAS or
NRAS mutation in combination with the lack of
PIK3CA,
PTEN, and
TP53 mutations would support a diagnosis of mesonephric adenocarcinoma [
27,
28].
Different copy number variations (CNVs) have been reported in MNAC. A gain of 1q is the most common CNV associated with MNAC. In the series reported by Mirkovic et al. [
22] and Na et al. [
18], a 1q gain was detected in 12 out of 17 cases and 11 out of 12 cases, respectively. Interestingly, a 1q gain is also the most common copy number alteration among endometrial carcinomas [
29]. We identified a gain of 1q by CISH and calculated the ratio of hybridization signals for 1p36 and 1q25 on 100 tumoral nuclei (3.8 for 1q25 and 1.8 for 1p36). It is worthy of note that focal amplification of 1q may influence the oncogenic potential of tumor cells. Approximately 10% of all cancers show a focal amplification of chromosome 1q21.2, a region harboring the antiapoptotic gene MCL1 [
30]. In addition, in cases of multiple myeloma, many other genes located on the proximal region of chromosome 1q, such as
CKSB1 and
PDZ1, have proven to portend a worse prognosis and resistance to certain chemotherapy agents. Regarding MNAC, current data suggest that a 1q as well as a 10q gain may be indicators of aggressive behavior and may increase the risk of developing metastasis [
18,
22]. Other known arm-level chromosomal abnormalities included loss of chromosomes 1p and 9p and gain of chromosomes 10 and 19 [
18,
22]. Notably, no evidence of microsatellite instability or hypermutation has been identified in MNAC.
To the best of our knowledge, alterations of CTNNB1 in mesonephric adenocarcinoma have not been documented so far. In our case, next-generation sequencing technology detected KRAS G12C and CTNNB1 G34R mutations, absence of microsatellite instability and low tumor mutation burden (three mutations per megabase). This is the first case of MNAC reporting CTNNB1 gene mutation.
The
CTNNB1 gene is located on the short arm of chromosome 3 and encodes beta-catenin 1. This protein is part of a molecular complex related to the adherens junctions of epithelial cells and maintenance of cell adhesion [
31,
32]. Mutation and deletion of this gen were each reported in only 2% of cases of cervical squamous cell carcinoma and endocervical adenocarcinoma in the TCGA (The Cancer Genome Atlas) database.
Beta-catenin 1 is part of the Wnt cell signaling pathway. Specifically, Wnt4 is involved in inhibiting the differentiation of mesonephric duct–derived tissue, during gonadal development [
33]. Normally, activation of the Wnt pathway induces cytoplasmic accumulation of free beta-catenin 1 and the consequent expression of target genes. When not activated, the
CK1 (casein kinase 1) and
GSK3β (glycogen synthase kinase 3β) kinases phosphorylate specific amino acids in beta-catenin 1 and signal it for degradation in the proteasome. These amino acids are encoded by a region of the
CTNNB1 gene located in exon 3, which was indeed mutated in our case [
34,
35]. This mutation leads to a constitutive stabilization of beta-catenin 1, inducing cell proliferation and decreasing intercellular adhesion. Therefore, we speculate that the mutation found in this case could be related to the development of mesonephric adenocarcinoma because of a failure in the inhibition of mesonephric duct–derived tissue. Moreover, we think it is possible that there is a relationship between the mutation described and the presence of pulmonary metastasis in this case. It is interesting that a case of malignant mesonephric tumor with pulmonary metastasis as initial clinical presentation has been reported, but no molecular alterations were sought or informed [
36].
However, there is no saying whether the mutation of CTNNB1 is an early phenomenon in tumorigenesis contributing to the appearance and growth of the tumor or a later event caused by the accelerated cell division characteristic of neoplasms. Furthermore, it would be necessary to analyze the role of the CTNNB1 mutation in the set of molecular alterations presented in this case (KRAS gene mutation and 1q gain). To determine whether there is a relationship between oncogenesis and the development of metastasis with the mutation described, it would be necessary to expand the study of CTNNB1 gene mutation (especially activating mutations that affect exon 3) in other reported cases of mesonephric adenocarcinoma.
In this context, it is interesting to note that a FATWO (female adnexal tumor of probable Wolffian origin) case has been reported in which a missense mutation was identified in the
CTNNB1 gene. However, of the three tumors included in the study, it was the only one that showed this gene mutation [
37].
On the other hand, mutations of the
CTNNB1 have been associated with other malignant tumors, such as melanoma, renal cell carcinoma, hepatocarcinoma, medulloblastoma, colon cancer, lung cancer, and ovarian cancer, among others [
31,
38,
39].
MNAC has a broad differential diagnosis. On the benign spectrum, it should be differentiated from MH and MR (See Table
2). Ki-67 immunostaining may be helpful, since it has been reported to show positivity in only 1–2% of cells in MH versus 5–20% or more in carcinoma [
40,
41]. In our case, the percentage of Ki-67 positive cells in the primary tumor was 46%. On the malignant spectrum, the ductal variant of the tumor should be differentiated from endometrioid adenocarcinoma, which is usually positive for ER, PR, and vimentin. Tumors with papillary and slit-like arrangements can be confused with serous carcinoma, which is CEAm (+), ER (−), and PR (−). Primary cases are very rare, and most often represent metastasis from ovarian serous carcinoma. Foci of hobnail cells in MNAC can resemble clear-cell carcinoma, which is characteristically Bcl-2 (+). Medium-size or duct-like formations may also mimic endometrioid adenocarcinoma [
28]. In contrast to MNAC, these HPV-negative cervical tumors show high nuclear grade.
Mesonephric adenocarcinomas can be very aggressive, even when low stage. Yap et al. reviewed a total of 31 cases of MNAC in which most patients (82%) presented at International Federation of Gynecology and Obstetrics (FIGO) stage 1B; one third of FIGO stage 1 patients developed recurrence even after curative resection; and one fifth of the patients with stage 1B disease had a fatal course between 1 and 9 years after diagnosis [
4]. Local recurrence and distant metastases were common findings in this study, and median and mean times to recurrence were 2.1 and 3.6 years, respectively. In another series, Dierickx et al. reported a recurrence rate of 32% in patients with stage 1 MNAC [
1]. Most patients died within one year after recurrence, despite therapy. Nevertheless, MNAC may have a better prognosis than Müllerian counterparts [
2]. For most patients, treatment consists of hysterectomy, bilateral salpingo-oophorectomy, and pelvic lymphadenectomy, depending on the stage of disease at diagnosis.
A malignant clinical course has been reported in about 40% of MNAC cases. Distant metastases at initial diagnosis are detected in less than 5% of patients [
18]. In our case, the patient had no metastasis at the time of diagnosis. Mirkovic et al. reported that all patients with MNAC and confirmed metastasis (four out of sixteen patients) had a
KRAS mutation, as in our case.
TP53 mutations were not present in any of the tumors of metastatic patients. Notably, most of the metastatic tumors (75%) exhibited gains of chromosomes 10 and 12, and none of the non-metastatic cases had this finding. Metastases were more common when the tumor exhibited a sarcomatous component [
4]. Frequent sites of distant metastases included bones, lungs, pleura, and liver. In our case, metastasis to the left lung developed three years after diagnosis in absence of a spindle-cell component in the tumor.