Aminopeptidase N (CD13, APN) is a zinc dependent, cell membrane metallopeptidase, which has been shown to participate in the postsecretory processing of neuropeptides and peptide hormones. It is widely distributed and has been found in various cell types of organs and tissues, including benign and malignant tumours. The expression and putative pathophysiological role of CD13 has been studied in a variety of malignant tumours.26,27
The expression of CD13 has been linked to tumour cell proliferation, degradation of extracellular matrix, and metastatic behaviour.27–32
Almost all of these biological effects were attributed to the ectopeptidase activity.
Recently, it was shown that CD13 is also expressed by HCCs.23
Interestingly, CD13 mRNA showed no significant differences between non-tumorous liver and HCC, whereas CD13 protein values were slightly increased.23
CD13 may have a pathophysiological effect on hepatocarcinogenesis by cleaving regulatory peptides and peptide hormones. However, CD13 may have another role in HCC, which is unique to the liver—that is, the formation of bile canaliculi and production and secretion of bile acids. Previously, we found CD13 positive bile canaliculi in the fetal liver, focal nodular hyperplasia, non-tumorous liver, and HCC.23
This reflects its ubiquitous expression and its close association with the formation and function of bile canaliculi.33
CD13 positive bile canaliculi have been detected as early as 16 to 18 weeks of gestation,23
underscoring the role of this molecule in morphogenesis. Expression of CD13 is maintained at a constant level during liver regeneration.34
Thus, expression of CD13 seems to be required in various proliferation and differentiation states of the liver, which makes it an attractive diagnostic marker for surgical pathology.
“CD13 may have a role in hepatocellular carcinoma that is unique to the liver—that is, the formation of bile canaliculi and production and secretion of bile acids”
Because CD13 shows a specific canalicular staining pattern,23
similar to that seen for p-CEAcan
, our present study aimed to investigate the putative use of CD13can
in differentiating HCCs from non-HCCs.
In our current series, both HCCs and non-HCCs expressed CD13 in the cytoplasm, at the cell membrane, or both, and the detection of CD13 itself was of no use in differentiating HCCs from non-HCCs. However, only non-neoplastic liver tissue and HCC showed a characteristic canalicular staining pattern, similar to that seen for p-CEA and CD10; this pattern was considered to be specific for HCCs and yielded a sensitivity of 96.2% and a specificity of 97.0% in our series. To evaluate further the diagnostic use of CD13can
in differentiating HCC from non-HCC, we compared the sensitivity and specificity of CD13can
, HepPar1, and AFP—markers that have proved their diagnostic usefulness in surgical pathology.1–3,6,7,9,19–21
In our series, the sensitivity of CD13can
was greater than that of p-CEAcan
, HepPar1, and AFP. Previous reports have described a canalicular staining pattern for p-CEA in 24–90% of cases1–4,6,7
and for CD10 in 28–86% of cases1–4,6,7
; our figures were 81.1% and 62.3%, respectively. Thus, our values for canalicular immunostaining of anti-p-CEA and anti-CD10 are within the range of previous observations. Table 3 summarises the results of more recent studies investigating the diagnostic use of CD10can
. Table 3 shows that the sensitivity of CD10can
is much lower than that of p-CEAcan
. However, as shown in our present study, the difference between p-CEAcan
is less pronounced, although unlike p-CEA and CD10, CD13 stained the cytoplasm of tumour cells less often, facilitating the recognition of even low numbers of bile canaliculi, which we consider to be an advantage of CD13, particularly in poorly differentiated HCCs.
Table 3 Summary of recent studies investigating the sensitivity and specificity of canalicular immunostaining for CD10 and p-CEA
CD13 was also expressed in bile ducts and in a cholangiocarcinoma, here resembling canalicular immunostaining. However, cholangiocarcinomas have an abundant desmoplastic stroma that aids in differentiating them from HCC in most cases.
HepPar1 is a monoclonal antibody that recognises a mitochondrial antigen of hepatocytes.6,37
In the past few years, several studies have investigated the sensitivity and specificity of HepPar1,10–16,38
and these were shown to range from 75% to 100% and from 66% to 100%, respectively. In our series, we found HepPar1 staining in only 29 of the 53 (54.7%) HCCs. This rather low sensitivity may be a sampling error, because most of our specimens were biopsies: HepPar1 staining is not homogeneous and only eight specimens showed HepPar1 staining in more than 50% of the tumour cells, and in most of our cases HepPar1 stained less than 50% of the tumour cells. Furthermore, in contrast to CD13, p-CEA, and CD10, immunostaining with HepPar1 does not show a hepatocyte specific staining pattern. Between 44% and 47% of gastric cancers react with HepPar1,16,38
and differentiating poorly differentiated gastric cancer from HCC using HepPar1 immunostaining only can be difficult at times. HepPar1 also occasionally stains cholangiocarcinomas (our present study)11,15,38
, and pancreatic, colon, lung, adrenal, neuroendocrine, ovarian, and endocervical cancers.11,15,38
Thus, although HepPar1 frequently reacts with HCCs, it should be used cautiously and in conjunction with a panel of other antibodies, as recently stated by Fan et al
Take home messages
- Canalicular staining for CD13 (CD13can) is a highly specific marker of hepatocyte differentiation, with a sensitivity greater than that of p-CEAcan, CD10can, HepPar1, and α fetoprotein
- Although CD13can does not differentiate between benign and malignant lesions, it is clearly useful for differentiating hepatocellular carcinoma (HCC) from non-HCC lesions
- Further studies are needed to determine whether CD13 could replace p-CEA and CD10 in the diagnostic hepatopathology of HCCs and liver metastases
In our series, the sensitivity of CD13can
, and HepPar1 was superior to that of AFP; only 16 of the 53 HCCs expressed AFP. Previous studies have shown that between 17% and 62% of HCCs show immunostaining for AFP,1–3,6,7
and AFP immunostaining in our HCC specimens was within this range.
Sensitivity and specificity are influenced by many variables, with sampling being the most important. Biopsy specimens often provide only a small fraction of the tumour, so that a lack of immunostaining may simply be the result of inadequate sampling (see above). All five markers tested here are subject to sampling errors. Thus, to reduce sampling errors, using a battery of different markers has become common practice in cases where the histological diagnosis is not readily apparent from routine histochemical stains (for example, haematoxylin and eosin, periodic acid Schiff, and reticulin stain). By comparing the staining patterns of CD13, p-CEA, CD10can, HepPar1, and AFP, we were able to show that only one specimen was negative for all five markers. A combination of CD13can (as the most sensitive marker for the presence of bile canaliculi), AFP (as a sensitive marker for poorly differentiated HCCs), and HepPar1 staining was diagnostic in 98.1% of our HCCs, whereas CD13can and AFP together were diagnostic in 96.2% of cases. Future studies are needed to determine whether CD13 has the potential to replace p-CEA and CD10 in the diagnostic hepatopathology of HCCs and liver metastases.
In summary, canalicular staining for CD13 is a highly specific marker of hepatocyte differentiation, with a sensitivity greater than that of p-CEAcan and CD10can. Although CD13can does not differentiate between benign and malignant lesions, it is clearly of use in differentiating HCC from non-HCC.