The diagnosis of hepatocellular carcinoma (HCC) remains obscure in some complicated cases. Thyroid transcription factor-1 (TTF-1) was presumed to be helpful in the diagnosis of HCC. This paper aims to study the utility of TTF-1 in the diagnosis and differential diagnosis of HCC and to assign clinicopathological correlations. Immunohistochemical detection of TTF-1 is performed with tissue microarray which contains a total of 781 cases including 299 hepatocellular carcinomas (HCCs), 31 intrahepaticcholangiocarcinomas (ICCs), 86 metastatic adenocarcinomas (MACs), 5 metastatic hepatocellular carcinomas (mHCCs), 9 hepatoblastomas (HBs), 40 renal clear cell carcinomas (RCCs), 215 adjacent nonneoplastic hepatic tissue, and 96 normal hepatic tissue. The result shows that TTF-1 is positive in 55.2% (165/299) HCCs, 6.4% (2/31) ICCs, 3.5% (3/86) MACs, 60% (3/5) mHCCs, 33.3% (3/9) HBs, 0% (0/40) RCCs, 99.0% (213/215) adjacent nonneoplastic hepatic tissue, and 95.8% (93/96) normal hepatic tissue. Expression of TTF-1 is correlated with differentiation of HCC, but not with patients' age, gender, serum AFP, tumor size, number of tumor nodules, and tumor thrombus in vein or bile duct. These data suggest that TTF-1 antibody is sensitive and specific in HCC and might serve as a candidate for the diagnosis and differential diagnosis of HCC.
Hepatocellular carcinoma (HCC) is the most common malignancies in liver. Generally accurate pathological diagnoses are not difficult to be reached. However, confirmatory methods are still required in certain complicated cases. Although some immunohistochemical markers, such as
A total of 781 specimens included 299 primary hepatocellular carcinomas (HCCs), 31 intrahepatic-cholangiocarcinomas (ICCs), 86 metastatic adenocarcinomas (MACs) (50 are hepatic metastases from colorectal carcinomas, 36 are from stomach), 5 metastatic HCCs (mHCCs) (4 were vertebra metastasis, 1 was lymph node metastasis), 9 hepatoblastomas (HBs), 40 renal clear cell carcinomas (RCCs), 215 adjacent non-neoplastic hepatic tissue (hepatic tissue adjacent to HCC), and 96 normal hepatic tissue (hepatic tissue adjacent to hemangioma). 299 HCC cases were classified as well differentiated (grade I + II, 234 cases) and poorly differentiated (grade III + IV, 65 cases) according to Edmondson-Sterner grade criteria [
The tissue microarray block was constructed using a tissue microarray (Beecher Instruments, Silver Spring, MD) as described previously. The tissue samples consist of 299 HCCs, 31 CCs, 86 MACs, 40 RCCs, 215 adjacent non-tumor livers (liver tissue surrounding HCC), and 96 normal livers. Two cores were punched in each case; each of tissue cores is 1.0 mm in diameter; consecutive 4
The sections were deparaffinized in xylene and rehydrated in graded series of ethanol. For antigen retrieval, the sections were immersed in EDTA solution (pH 9.0) and processed in a heated steamer at 100°C for 5 minutes. After cooling at room temperature for 30 minutes, slides were washed in phosphate-buffered saline solution (pH 7.4) and treated with 3% hydrogen peroxide for 15 minutes. The sections were incubated for 1 hour with the monoclonal mouse antibody specific for TTF-1 (clone 8G7G3/1, Santa Cruz
Nuclear and/or cytoplasmic expression of TTF-1 was considered positive immunostaining. Expression within tumor stroma was not specifically recorded. For all, both extent (on the basis of the percentage of positive cells) and intensity of immunostaining were evaluated by a semiquantitative system. Extent was scored as follows: 0, ≤5%; 1 + (1 point), 6% to 25%; 2 + (2 points), 26% to 50%; 3 + (3 points), >51%. Intensity was arbitrarily scored as weak (0 point), moderate (1 point), or strong (2 points). Intensity was designated as weak when immunostaining was present but only barely detectable. To correlate extent and intensity of immunostaining, these values in positive cases were converted into composite immunohistochemical scores by multiplying the individual scores of extent by intensity (possible range of values from 0 to 6). For example, a moderate 3 + extent (3 points) and moderate intensity of immunostaining (2 points) would have an immunohistochemical composite score of
Chi-square analysis was used for statistical analysis. A
The clinicopathological features of the 299 patients with HCC in the study group are summarized in Table
Clinical and pathologic information.
Factor | Cases load |
---|---|
Age | |
≤35 y | 40 (13.4%) |
>35 y | 259 (86.6%) |
Sex | |
Male | 276 (92.3%) |
Female | 23 (7.7%) |
Serum AFP | |
≤400 ug/L | 157 (52.5%) |
>400 ug/L | 142 (47.5%) |
Tumor size | |
Single and <3 cm | 16 (5.7%) |
≥3 cm | 283 (94.6%) |
No. of nodules | |
1 | 178 (59.5%) |
≥2 | 121 (40.5%) |
Vein or bile duct tumor thrombus | |
Present | 93 (31.1%) |
Absent | 206 (68.9%) |
Edmondson-Sterner grade | |
I + II | 234 (78.3%) |
III + IV | 65 (21.7%) |
Histological pattern | |
Trabecular | 274 (91.6%) |
Pseudoglandular | 17 (5.7%) |
Solid | 8 (2.7%) |
TTF-1 was positive in 165 (55.2%) of 299 HCCs, within which 132 of 165 (80%) were scored as strong positivity (Table
TTF-1 expression in HCC.
Score | No. (% of all cases) | |
---|---|---|
Negative | 0 | 124 (41.4%) |
1 | 10 (3.3%) | |
Moderate positive | 2 | 23 (7.7%) |
3 | 10 (3.3%) | |
Strong positive | 4 | 21 (7%) |
6 | 111 (37.1%) |
TTF-1 expression in tissue microarray.
Case group | % of positive staining |
---|---|
HCC | 55.2% (165/299) |
ICC | 6.4% (2/31) |
MAC | 3.5% (3/86) |
mHCC | 60% (3/5) |
RCC | 0% (0/40) |
HB | 33.3% (3/9) |
Adjacent non-neoplastic hepatic tissue | 99.0% (213/215) |
Normal hepatic tissue | 95.8% (93/96) |
TTF-1 expression in different histopathological patterns of HCC.
Histopathological pattern | No. reactive (No. evaluated) | % of expression |
---|---|---|
Trabecular | 154 (274) | 56.2% |
Pseudoglandular | 9 (17) | 52.9% |
Solid | 2 (8) | 25.0% |
(a) (b) TTF-1 expressed in HCC, inset: high magnification of the cytoplasmic staining; (c) (d) TTF-1 staining in adjacent non-neoplastic hepatic tissue. Bile duct epitheliums, endothelial cells, were negative for TTF-1. Inset: high magnification of the cytoplasm staining (a, c: IHC
(a) HCC with clear cell (HE
Expression of TTF-1 correlated significantly with the grade of HCC, which was stronger in well-differentiated carcinoma (grade I + II) (64.5%; 151/234) than in poorly differentiated ones (grade III + IV) (21.5%; 14/65). The difference between high-grade and low-grade carcinoma was significant (
Clinicopathologic correlations between biologic factors and patients with HCC.
Factor | No. reactive (No. evaluated) | Rate of expression | |
---|---|---|---|
Sex | |||
Female | 13 (23) | 56.5% | |
Male | 152 (276) | 55.1% | |
Age | |||
≤35 y | 22 (40) | 55.0% | |
>35 y | 143 (259) | 55.2% | |
Serum AFP | |||
≤400 ug/l | 81 (157) | 51.9% | |
>400 ug/l | 84 (142) | 59.1% | |
Tumor size | |||
Single and <3 cm | 11 (16) | 68.7% | |
≥3 cm | 154 (283) | 54.4% | |
No. of nodules | |||
1 | 98 (178) | 53.9% | |
≥2 | 67 (121) | 53.7% | |
Vein or bile duct tumor thrombus | |||
Present | 54 (93) | 58.0% | |
Absent | 111 (206) | 53.8% | |
Edmondson-Sterner grade | |||
I + II | 151 (234) | 64.5% | |
III + IV | 14 (65) | 21.5% |
*
TTF-1 is a 38-kDa nuclear protein initially identified as a mediator of thyroid-specific gene transcription, the code gene located on chromosome 14q13. It is a member of the NKx2 family of homeodomain transcription factors and is expressed in thyroid and lung tissues [
Expression of TTF-1 was characteristically seen in the nuclei of tumor cells origin from thyroid and lung. Cytoplasmic staining was reported occasionally and presumed as a nonspecific staining pattern previously [
Nevertheless, the expression rate of TTF-1 in HCC in our study (55.2%) was lower compared with previous studies which may attribute to difference in number of cases in the studies, and different antibody agent applied as well. Furthermore, we investigated the correlations between the expression of TTF-1 in HCC and their clinicopathological features, which show that tumor grades are parallel with the level of TTF-1, with the expression rates being lower in high-grade cases (poorly differentiated) compared with low-grade cases (well differentiated), 21.5% versus 64.5%, respectively. However, there is no correlation between TTF-1 expression and sex, age, level of serum AFP, tumor size, number of nodules, and vein or bile duct tumor thrombus. In addition to this, an interesting phenomenon is found in HB. TTF-1 only expresses in fetal cells, but not embryonal cells, which are less differentiated in morphology than fetal cells in HBs [
In summary, our study focuses on the utility of TTF-1 in the diagnosis and differential diagnosis of HCC with a large number of cases using tissue microarray. Cytoplasmic TTF-1 staining is sensitive and specific to HCC. The clinicopathological correlations are analyzed in our study, which may help to understand what role cytoplasmic staining of TTF-1 plays in HCC exactly. In all, TTF-1 antibody may serve as a candidate for making diagnosis and differential diagnosis of HCC.