Introduction
The urachus is an extraperitoneal fibromuscular band that connects the bladder dome and the umbilicus. During fetal development, the urachus ensures the communication between the forming bladder and the allantois. After the fourth month of embryonic life, the urachus usually transforms into a fibromuscular strand (i.e. median umbilical ligament). In up to one third of adults, this obliteration can be incomplete resulting in microscopic urachal residues. Incomplete regression of the urachal structure may lead to various diseases including urachal cancer (UrC) [
1,
2].
UrC is an extremely rare disease with an estimated annual incidence of one in one million adults. The majority of UrC present as adenocarcinomas (~90%) with mucinous, intestinal, signet ring, not otherwise specified (NOS) or mixed histology. Because of its hidden anatomical location, UrC is mostly detected in progressed stages when patients present with hematuria resulting from its invasion into the urinary bladder. At this advanced stage, the overall 5-year survival is only about 50% [
3‐
5].
Although urachal and colorectal adenocarcinomas (CRC) are different types of cancer, they share remarkable histopathological and clinical similarities. Also, their immunophenotype shows overlapping staining characteristics with only a few exceptions [
4]. In contrast to CRC, the molecular background of UrC is only poorly understood. Data on UrC’s mutational pattern further highlighted its similarity to CRC with overlapping mutational patterns such as in
TP53,
KRAS,
SMAD4 and
NRAS [
6‐
17]. On the other hand, later studies also identified some significant molecular differences between UrC and CRC [
6‐
12,
14‐
18].
In CRC, the adenomatous polyposis coli (
APC) tumor suppressor gene has a critical role in the initiation of tumorigenesis.
APC mutations lead to abnormal ß-catenin accumulation in the nucleus. This nuclear ß-catenin gets in contact with a member of the TCF/LEF family and acts as a transcriptional regulator on specific proliferation-associated target genes. APC mutations occur in more than 80% of CRC [
17]. As a consequence, nuclear ß-catenin expression can be observed in the generality of CRC making ß-catenin a well established diagnostic and prognostic biomarker in CRC [
19,
20]. The occurrence and role of
APC-alterations and ß-catenin-expression are less well established in UrC.
Phosphatase and tensin homolog (PTEN) is a leading negative regulator of the PI3K signaling pathway and therefore known as a tumorsupressor gene. Its inactivating genetic mutations occur in up to 10% of CRCs [
17]. In addition to genetic loss, PTEN is frequently downregulated by epigenetic silencing, leading to loss of PTEN expression [
21]. Loss of PTEN protein expression was shown to be associated with resistance to anti-epidermal growth factor receptor (EGFR) therapy [
22], while this therapy demonstrated effectiveness in UrC [
14].
Therefore, we aimed to analyze relevant genetic alterations including the APC and PTEN genes as well as to assess the tissue protein expressions of β-catenin and PTEN in UrC.
Discussion
It is well known, that mutations of the
APC tumor suppressor gene have a critical role already in early stages of CRC development. The most important function of
APC is to establish an interaction with the ß-catenin protein, thus accelerating its degradation and regulation of the cadherin-mediated cell–cell adhesion system. Functional loss of
APC leads to abnormal ß-catenin accumulation and translocation from the plasma membrane to the nucleus [
20].
Singh et al. used a whole exome sequencing approach and found
APC mutations in 43% (3/7) of UrC samples. They detected one sample with a nonsense mutation, one with a frameshift mutation and one with a deletion. The frameshift mutation and deletion caused dysfunctional APC proteins [
16]. Collazo-Lorduy et al. described truncated
APC mutations (R1450*, R554*) in 22% (2/9) of UrC samples using a targeted exome sequencing approach [
14]. In addition, Lee et al. reported
APC mutations in 18% (3/17) of UrC cases including a frameshift deletion (K1444fs) and a stop-gain single nucleotide variant (SNV) (E1093*) [
10]. In the latest study, Kardos et al. performed targeted exon sequencing of 12 urachal adenocarcinoma cases and found 3 of 12 samples (25%) with
APC mutations [
9].
In our study, analyzing 34 UrC cases, deleterious alterations of APC tumor suppressor gene were present in 2 UrC samples. Neither mutation has been previously reported in UrC: p.Y1075* (c.3225 T > G), p.K1199* (c.3595A > T). Both of these nonsense mutations are predicted to cause a truncated, dysfunctional APC protein. Three additional alterations were considered benign. Presence of potentially pathogenic APC mutations tended to be associated with shorter OS, however, because of the low number of cases with APC mutation, this correlation has to be interpreted cautiously.
Summarizing our results with all currently available data on
APC status in UrC, an overall number of 14 of 141 (10%) UrC samples exhibited
APC alterations [
6‐
16,
18,
24‐
26] which is in clear contrast to the high
APC mutational rate (80%) found in CRC [
17]. This finding represents a further characteristic difference in the molecular taxonomy between CRC and UrC. Therefore, our results demonstrate that the Wnt pathway is probably less frequently involved in the pathogenesis of UrC compared to CRC.
ß-catenin is a well-established diagnostic and prognostic biomarker in CRC. Physiologically, ß-catenin staining is restricted to the membrane/cytoplasm and is involved in cadherin-mediated cell-cell adhesion and gene transcription regulation. Nuclear ß-catenin expression can be observed in CRC [
19]. Wong et al. demonstrated positive nuclear staining for ß-catenin expression in the vast majority of colorectal adenocarcinomas which reflects the high
APC mutation rate found in CRC [
27].
In a recent review of the UrC literature including own data, we found a low incidence of nuclear ß-catenin staining in UrC (14%, 9/63) [
4]. In the present study, we detected positive nuclear ß-catenin immunostaining in 29% (11/38) of UrC samples. In addition, ß-catenin nuclear expression was not associated with adverse OS (
p = 0.606). Interestingly, we observed a nuclear ß-catenin localization only in one of the two samples with truncating APC mutation. As a potential genetic mechanism for ß-catenin nuclear accumulation, Alomar et al. identified an activating mutation in exon 3 of
CTNNB1 (ß-catenin) gene which resulted in an amino acid change at phosphorylation sites of glycogen synthase kinase-3 (GSK-3β). Failing of phosphorylation was found to decrease sequestration of β-catenin by
APC [
28]. This effect might explain our finding with low APC mutational frequency but at the same time surprising high rate (29%) of nuclear ß-catenin positivity. It has also to be kept in mind that we used a 1% threshold for calling a case positive in case of nuclear β-catenin staining. In some cases, it is difficult to discriminate between a real nuclear staining event in the background of strong membranous/cytoplasmic staining especially in smaller tumor cells (i.e. signet ring cells).
The tumor suppressor PTEN is a negative regulator of the PI3K signaling pathway.
PTEN mutations occur in 4–10% of CRC and were suggested as potential markers of response to EGFR and mitogen-activated protein kinase (MAPK) inhibitor-based targeted therapies [
17]. Perrone et al. showed that inactivation of PTEN protein by mutation (P103S, E99*) or deletion (hemizygous) was responsible of anti-EGFR resistance [
29]. PTEN protein loss was detected in approximately 40% of all CRC patients, which is clearly higher compared to the rate of genomic loss. This suggests that PTEN is more frequently downregulated by epigenetic silencing. In accordance,
PTEN methylation was found to be significantly correlated with PTEN expression [
21]. Similar to the findings at the genomic level, Frattini et al. showed that loss of PTEN protein expression was associated with non-responsiveness to cetuximab [
22].
In the literature, we found three whole exome sequencing studies with an overall number of 19 UrC samples. None of them reported any
PTEN mutations [
10,
14‐
16]. In addition, none of the targeted sequencing studies reported any
PTEN mutations in UrC. Here, we identified a pathogenic frameshift
PTEN mutation (p.C211fs) in one of the 34 UrC cancer samples (3%), suggesting a low incidence for
PTEN mutation in UrC. Therefore, the
PTEN mutational frequency in UrC seems to be similar to that of reported in CRC (4–10%) [
17].
To the best of our knowledge, no published data is available on PTEN protein expression in UrC. In the present study, performing PTEN IHC analysis, we observed PTEN protein loss in 20% of UrC cases (6/30), which is somewhat lower than that of 30–40% described in CRC. The low rate of
PTEN loss at the DNA level, but relative high rate at the protein level suggests that
PTEN is predominantly silenced by epigenetic downregulation in UrC [
21]. The high rate of PTEN protein loss in UrC suggests that its immunohistochemical analysis may be important in order to predict potential inresponsiveness to anti-EGFR therapy. In contrast, the rare occurrence of
PTEN inactivating mutations suggests that these alterations are less important to analyze when considering the administration of an anti-EGFR drug, while activating
KRAS mutations are more common in UrC (28%) and are clinically important negative predictors of anti-EGFR therapy [
6‐
16,
18,
24‐
26].
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