In Situ Hybridization in Diagnostic Pathology
Case 3 -
Ricardo V. Lloyd and Arie Perry
This 36-year-old man presented with an enlarged right upper quadrant mass.
The biopsy showed carcinoma composed of cells with abundant eosinophilic cytoplasm which
grew in sheets and nests. Other areas of the tumor showed glandular differentiation.
Immunohistochemical staining was diffusely positive for keratin (CAM 5.2). There was positive staining
for polyclonal CEA in a focal canalicular pattern. Hepatocyte paraffin 1 immunostaining was positive in
the hepatocellular carcinoma areas but not in the cholangiocarcinoma areas. In situ hybridization for
albumin mRNA was diffusely positive in both areas.
Combined hepatocellular-cholangiocarcinoma (HCC-CC) is a rare primary tumor of the liver
showing features of both hepatocellular and biliary epithelial differentiation
. In a review of 24
cases of these tumors, Goodman et al,  classified the tumors into three histologic types.
Type I or collision tumors (4 cases) had a coincidental occurrence of both tumors in the
Type II or transitional tumors (n = 12) had intermediate differentiation and an
identifiable transition between hepatocellular and cholangiocarcinoma.
Type III or fibrolamellar tumors (n = 8) resembled fibrolamellar variant of hepatocellular
carcinoma which also contained mucin-producing pseudoglands. Type III tumors occurred at a younger age
in the absence of cirrhosis and had a slightly larger survival.
In a series from Hong Kong (n = 21), most of the tumors (n = 18) were Type II. Two patients had Type
I tumors, and one had Type III or fibrolamellar variant . These combined HCC-CC had clinical and
pathological features more similar to regular HCC. Cirrhosis or chronic hepatitis was present in 77.8 %
of patients; 75% of patients had hepatitis B surface antigen positivity. Elevated serum AFP was present
in 61.5% of patients, while 55% of the tumors had immunoreactive AFP .
Molecular studies of these combined tumors had shown loss of heterozygosity on 4 q and 17 p most
commonly followed by 8 p and 16 q. Of the 11 cases analyzed, 8 showed identical allelic classes in both
In addition to alpha-fetoprotein and polyclonal CEA, newer immunohistochemical markers
that are useful in the diagnosis of HCC include hepatocyte paraffin 1 and CD10
Albumin protein is present in high concentrations in the serum, so immunohistochemical
staining for albumin normally results in strong non-specific background staining. However, albumin mRNA
is largely restricted to a few cells and tumors with hepatocytes and hepatocellular neoplasms being the
principal synthesizers of albumin. The albumin gene was cloned many years ago
, and most molecular
biology studies showed no difference between the normal liver and hepatocellular carcinomas in the use of
multiple sites for transcriptional initiation and polyadenylation of the albumin gene
et al. used a digoxigenin-labeled 51 bp oligonucleotide probe to study albumin expression
in the liver and reported that 9/12 (75 percent) hepatocellular carcinomas examined were positive
. Other investigators have utilized oligonucleotide and RNA probes to detect albumin mRNA in primary
and metastatic liver lesions
Table 1. Oligonucleotide Sequences of Albumin Probe Cocktail
|Probe ||Sequence ||Nucleotides|
|1 ||51 GAG AAA AAG GGA AAT AAA GGT TAC CCA CT ||42-70|
|2 ||51 GCA TCT CGA CGA AAC ACA CCC CTG GAA AT ||87-115|
|3 ||51 CAA CCT CTG GTC TCA CCA ATC GGG GGA GGT ||441-470|
|4 ||51 AGC AGG CAT CTC ATC ATT TTC CAC TTC GGC ||981-1010|
|5 ||51 CTC GGC AAA GCA GGT CTC CTT ATC GTC AGC ||1791-1820|
Because albumin synthesis is largely restricted to the
liver, detection of albumin mRNA in a poorly differentiated neoplasm, can be very useful in
distinguishing a primary, and a metastatic lesion in the liver or in diagnosing metastatic hepatocellular
carcinoma as in the current case.
Staining for albumin mRNA is highly specific (Table 2).
Table 2. Analysis of Albumin mRNA by In Situ Hybridization
| ||% Positive|
|Diagnosis ||Yamaguchi et al.  ||Papotti et al.  ||Salomao et al.  ||Krishna et al. |
|Hepatocellular Carcinoma ||50/53 (94%) ||26/30 (87%) ||27/33 (82%) ||22/23 (96%)|
|Adenoma ||1/1 (100%) ||-- ||-- ||7/7 (100%)|
|FNH ||1/1 (100%) ||-- ||-- ||7/7 (100%)|
|Cholangiocarcinoma ||0/8 (0%) ||-- ||0/7 (0%) ||0/9 (0%)|
|Metastatic Adenocarcinoma ||0/14 (0%) ||0/10 (0%) ||0/13 (0%) ||0/16 (0%)|
In the two studies done at
the Mayo Clinic
, the only other neoplasm that had a positive ISH signal was a malignant mixed
germ cell tumor which had hepatoid areas. Hepatoblastomas are also positive for albumin mRNA , so
this ISH test should assist in distinguishing hepatoblastomas from other childhood neoplasms in the
differential diagnosis. Albumin mRNA has also been useful in separating clear cell carcinoma of the
liver from other clear cell neoplasms in the abdomen .
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