Matthew M. Yeh, MD, PhD, Anne M. Larson, MD,w Jean S. Campbell, PhD,

Nelson Fausto, MD, Stephen J. Rulyak, MD,w and Paul E. Swanson, MD

Many risk factors for development of HCC are well known ,Ex: chronic hepatitis and cirrhosis.

Underlying molecular mechanisms leading to hepatocarcinogenesis

remain largely unclear.

Transforming growth factor-a (TGF-a) is a mitogen

1)synthesized as a transmembrane polypeptide

2)cleaved to a 50 amino acid diffusible form

TGF-a---EGFR

bind the EGFR (epidermal growth factor receptor) and activate the signaling cascade from this receptor.

TGF-a---transgenic mice

Overexpression of TGF-a in the liver of transgenic mice induces increased proliferation, dysplasia, adenoma, and carcinoma.

TGF-a---HCC and adjacent liver

Previous studies have also confirmed the presence of TGF-a in a significant portion of HCC and adjacent liver.

DNA topoisomerase II-a (Topo II-a) is a nuclear protein

targeted by several chemotherapeutic agents and has been shown to be overexpressed in HCC.

Ki-67 is a nuclear protein that is detected in proliferating cells (late G1, S, G2, and M phase), but absent in resting cells (G0 phase).

The expression of TGF-a or Topo II-a in dysplastic nodules has not been investigated.

most previous studies investigating the expression of TGF-a in HCC were performed in cirrhotic livers.

examined the patterns of Ki-67, TGF-a, and Topo II-a expression in liver cirrhosis, low-grade dysplastic nodules (LGDN), HGDN, and HCC, using an immunohistochemical approach

to define the possible relationships of these markers to tumor progression.

evaluated the difference in expression of TGF-a in HCC between cirrhotic and noncirrhotic livers.

Explanted liver specimens from patients with cirrhosis who underwent liver transplantation surgery at the University of Washington Medical Center from 1989 to 2002 were retrospectively reviewed.

Resected liver specimens in patients who underwent surgery for HCC at the University of Washington Medical Center from 1989 to 1992 were also retrospectively reviewed.

The morphologic diagnoses of the liver lesions were confirmed by the consensus opinion of 2 pathologists (M.M.Y. and P.E.S.)

using the criteria published by the International Working Party(IWP).

Monoclonal anti-TGF-a Monoclonal anti-Topo II-a monoclonal anti-Ki-67

Immunohistochemical analyses were performed on representative sections of HCC, HGDN, LGDN, cirrhosis, and noncirrhotic liver parenchyma.

The intensity of TGF-a immunostaining: graded by the 2 pathologists (M.M.Y.

and P.E.S.) from 0 to 4.

0-no staining; 1-focal weak staining in the

hepatocytic cytoplasm; 2-diffuse weak staining in the

hepatocytic cytoplasm; 3-diffuse weak and focal strong

staining in the hepatocyticcytoplasm; 4-diffuse strong staining in the

hepatocytic cytoplasm,

The percentage of nuclei positive for Topo II-a and Ki-67

was obtained by manually counting 500 hepatocytes in the most intensively stained regions.

in 52 cirrhotic livers from explanted or resected specimens: 46 HCC, 17 HGDN, and 12 low-grade dysplastic nodules were identified.

18 surgically resected cases of noncirrhoticliver with HCC were identified.

TGF-a-- HCC arising in the noncirrhotic and cirrhotic

The mean intensity score for TGF-a staining was 0.8 and 1.8 in HCC arising in the noncirrhotic and cirrhotic background, respectively (P=0.003).

Most previously reported genomic and molecular studies have focused on fully developed HCC.

postulated that the expression patterns of TGF-a,Ki-67, and Topo II-a should differ in the progression from cirrhosis through LGDN, HGDN to HCC.

Also compared the expression of TGF-a in HCC arising in cirrhotic livers with that of HCC arising in noncirrhotic livers.

Ki-67--- index of proliferation

Ki-67’s expression has been considered a reliable index of proliferation.

Increased proliferation--- stomach and esophagus

Increased proliferation has been observed in premalignant and malignant conditions of other organs, such as the stomach and esophagus.

Proliferation index---HCC

proliferation indices have been well-described in HCC.

Watanuki et al ---have reported a mean Topo II-a index

of 14.1% in HCC, with a mean Ki-67 index of 25.6% in the same lesion.

Proliferation index---HCC

In the current study, ---Topo II-a and Ki-67 proliferative indices

in HCC were similar to those of Watanuki and colleagues (18.9±10.4 and 26.1±13.6, respectively)

Topo II-a and Ki-67--- decreased survival and earlier cancer-related death

Increased Topo II-a and Ki-67 protein expression in HCC correlates with decreased recurrence-free survival and earlier cancer-related death.

Ki-67 and Topo II-a--- poor v.s. well or

moderately differentiated HCC

Previous studies have shown that poorly differentiated HCC has more immunoreactivity to Ki-67 and Topo II-a than well or moderately differentiated HCC.

the place of LGDN

In the current study, there was no significant difference in Ki-67 and Topo II-a staining between LGDN and the adjacent cirrhotic nodules.

the place of LGDN

further study with expanded numbers will be necessary to better define the place of LGDN within the concept of stepwise progression.

TGF-a--- mice, human HCC

Overexpression of TGF-a in the livers of transgenic mice leads to the development of HCC.

Previous studies have demonstrated that human HCC expresses TGF-a as well

TGF-a—cirrhotic v.s. nocirrhotic

The current study demonstrates that cirrhotic livers exhibit stronger expression of TGF-a when compared with noncirrhotic hepatic parenchyma.

TGF-a--- cirrhosis-associated HCC v.s. HCC in noncirrhotic livers

the intensity of TGF-a immunoreactivity in cirrhosis-associated HCC was stronger than that of HCC in noncirrhotic livers.

TGF-a--- decline expression

there was also a decline in TGF-a expression from cirrhosis, through LGDN and HGDN to HCC.

TGF-a---etiology-dependent

Kiss and colleagues: HBV-positive, higher proportion of HCC

overexpressing TGF-a was observed compared with the HBV-negative group (21% and 6%, respectively).

suggesting that TGF-a as a role in human hepatocarcinogenesis may be etiology-dependent.

the decrease in TGF-a expression from cirrhosis, through LGDN and HGDN to HCC supports the premise that TGF-a is more important to the early events in hepatocarcinogenesis.

Although the relatively lower expression of TGF-a in HGDN and HCC seems counterintuitive (because overexpression of TGF-a in transgenic mice results in the development of dysplasia and HCC)

we might plausibly argue that TGF-a is actively produced in cirrhotic liver and is gradually consumed in the process of hepatocarcinogenesis,

whereas other growth factors involved in the later stages of hepatocarcinogenesis may continue to be produced.

TGF-a – less v.s. well differentiated HCC

Morimitsu et al ---have observed decreased expression

of TGF-a in less differentiated HCC compared with well-differentiated HCC.

Proliferative activity increases from cirrhosis to HCC, with HGDN showing an intermediate proliferative index.

---This finding provides another compelling line of evidence that HGDN is an advanced premalignant lesion in hepatocarcinogenesis.

TGF-a plays an early role in cirrhosis associated hepatocarcinogenesis and that this process may be etiology-dependent.