Immunohistochemistry panel segregates molecular types of hepatocellular carcinoma in Brazilian autopsy cases

doi:10.3748/wjg.v22.i27.6246

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World Journal of Gastroenterology

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1007-9327

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Baishideng Publishing Group Inc, 7041 Koll Center Parkway, Suite 160, Pleasanton, CA 94566, USA

Case Control Study

World J Gastroenterol. Jul 21, 2016; 22(27): 6246-6256
Published online Jul 21, 2016. doi: 10.3748/wjg.v22.i27.6246

Table 1 Antibodies and immunohistochemistry parameters used in this study

Antibody	Manufacturer	Clone	Species	Dilution	Retrieval¹	Positive control	Staining pattern
EGFR PharmDx	Dako	2-18C9	Mouse	Pre-diluted	Proteinase K	Provided by manufacturer	Membrane
Ki67 Ag	Dako	MIB-1	Mouse	1:400	Standard	Tonsil	Nucleus
Caspase 3	DBS	3C SP03	Mouse	1:400	Standard	Tonsil/stomach	Cytoplasm
ERK1	DBS	Polyclonal	Rabbit	1:100	Standard	Breast carcinoma	Cytoplasm
ERK2	DBS	Polyclonal	Rabbit	1:400	Standard	Breast carcinoma	Cytoplasm
MET	Cell signaling	Polyclonal	Rabbit	1:50	Standard	Breast carcinoma	Cytoplasm
mTOR	Cell signaling	Polyclonal	Rabbit	1:50	Standard	Lymphoid tissue	Cytoplasm
K19	Novocastra	b170	Mouse	1:300	Standard	Cholangio-carcinoma	Cytoplasm
Vimentin	Dako	Vim3B4	Mouse	1:3000	Standard	Kidney	Cytoplasm
p53	Dako	DO7	Mouse	1:100	Standard	Breast carcinoma	Nucleus
Beta-catenin	BD	14Beta-catenin	Mouse	1:800	Standard	Tonsil	Membrane/nucleus
Cyclin D1	Dako	SP4	Rabbit	1:100	Steamer 10 mmol/L TRIS, 1 mmol/L EDTA, pH 9	Tonsil/intestine	Nucleus

¹Antigen retrieval was performed by steam heating for 40 min in 10 mmol/L citrate buffer, pH 6. BD: BD biosciences™; DBS: Diagnostic bioSystems™; IHC: Immunohistochemistry; K19: Keratin 19; mTOR; Mechanistic target of rapamycin.

Table 2 Clinicopathological features of 80 hepatocellular carcinoma autopsy cases

Feature	n (%)
Sex
Male	62 (77.5)
Female	18 (22.5)
Age, yr
Median	59.5
Range	28-82
With cirrhotic liver	72 (90.0)
With incidental small HCC, < 2.0 cm	4 (5.0)
Etiology
HCV only	27 (33.7)
HCV + alcohol	9 (11.2)
HCV + HBV	2 (2.5)
HCV + HBV + alcohol	1 (1.3)
Alcohol only	11 (13.8)
HBV only	9 (11.2)
HBV + alcohol	3 (3.8)
Hemochromatosis	2 (2.5)
Cryptogenic	10 (12.5)
Data not available	6 (7.5)
Larger tumor size, cm
Median	4
Range	0.8-18.0
Non-available/non-sizable cases	22 (27.5)
Number of tumor nodules
1	24 (33.3)
2	4 (5.6)
3	1 (1.4)
≥ 4	43 (53.8)
Non-available/uncountable cases	8 (10.0)
Gross pattern
Nodular	31 (38.8)
Massive with satellite lesions	25 (31.3)
Diffuse	8 (10.0)
Non-available/non-sizable cases	11 (13.8)
Histological grade, Edmondson-Steiner grade
1 + 2	22 (27.5)
3	47 (58.8)
4	11 (13.8)
Predominant histological pattern or variant
Trabecular	36 (45.0)
Acinar/pseudoacinar	9 (11.2)
Solid/macrotrabecular	24 (30.0)
Mixed	9 (11.2)
Clear cell	2 (2.5)

HBV: Hepatitis B virus; HCC: Hepatocellular carcinoma; HCV: Hepatitis C virus.

Table 3 Dissemination in 80 hepatocellular carcinoma autopsy cases

Metastasis feature	n (%)
Large vein invasion	12 (15.0)
Extrahepatic metastases	30 (37.5)
Extrahepatic metastases sites, cases by site
Lungs	21 (26.3)
Lymph node	6 (7.5)
Adrenal, bone, spleen, diaphragm, peritoneum	2 (2.5)
Small bowel, bladder, colon, pancreas, pituitaries, thyroid, pleura	1 (1.3)

Table 4 Overall expression and cut-off points for immunohistochemical markers in non-neoplastic liver, primary hepatocellular carcinoma and metastases n (%)

IHC marker	Cut-off	Non-neoplastic	Primary HCC	Metastases	P value
IHC marker	Cut-off	26 cases	75 cases	17 cases	P value
EGFR overexpression	≥ 200/300¹	17 (65)	29 (39)	6 (38)²	0.054
Ki67	≥ 0.1%	2 (8)^a	45 (63)⁴	13 (76)	< 0.001^a
K19	> 1%	0 (0)^a	12 (16)³	5 (29)	0.011^a
Vimentin	> 0/300	0 (0)	4 (6)³	3 (18)	0.064
Caspase 3 loss	≤ 10/300	2 (8)^a	26 (36)³	7 (41)	0.009^a
Cyclin D1	> 0.1%	4 (15)	16 (23)⁵	9 (53)^a	0.018^a
mTOR expression	≥ 100/300¹	8 (31)	14 (21)⁵	5 (31)²	0.533
MET expression	≥ 100/300¹	10 (38)	12 (17)⁴	5 (29)	0.068
ERK1 overexpression	≥ 200/300¹	0 (0)^a	10 (14)⁴	5 (33)³	0.006^a
ERK2 overexpression	≥ 200/300¹	0 (0)^a	10 (14)⁴	4 (24)	0.029^a
p53	≥ 10%	2 (8)^a	28 (42)⁵	8 (50)²	0.002^a
Beta-catenin, membrane	≥ 100/300¹	10 (38)	42 (61)⁵	11 (65)	0.113
Beta-catenin, nucleus	2 or 3+	0 (0)	9 (9)⁵	3 (18)	0.070

¹Score value;

²One case lost during technical processing (n-1);

³Two cases lost during technical processing (n-2);

⁴Three cases lost during technical processing (n-3);

⁵Four or more cases lost during technical processing.

^aP < 0.05 (outliers in pairwise test). EGFR: Epidermal growth factor receptor; ERK: Extracellular signaling-related kinase; HCC: Hepatocellular carcinoma; IHC: Immunohistochemistry; MET: MET Receptor tyrosine kinase; mTOR: Mechanistic target of rapamycin.

Table 5 Expression of immunohistochemical markers in categories of histological grade for primary hepatocellular carcinoma cases n (%)

IHC marker	Cut-off	Cases/valid samples			P value
IHC marker	Cut-off	Grades 1 + 2	Grade 3	Grade 4	P value
EGFR overexpression	≥ 200/300¹	14 (67)^a	12 (27)	3 (30)	0.008^a
Ki67	≥ 0.1%	6 (30)^a	31 (74)	8 (80)	0.002^a
K19	> 1%	0 (0)	7 (17)	5 (45)^a	0.005^a
Vimentin	> 0/300	0 (0)	3 (7)	1 (9)	0.465
Caspase 3 loss	≤ 10/300	3 (15)	16 (38)	8 (73)^a	0.006^a
Cyclin D1	> 0.1%	4 (20)	9 (21)	3 (30)	0.847
mTOR expression	≥ 100/300¹	7 (37)	17 (45)	1 (10)	0.134
MET expression	≥ 100/300¹	8 (42)^a	6 (14)	0 (0)	0.012^a
ERK1 overexpression	≥ 200/300¹	0 (0)	8 (20)	2 (18)	0.081
ERK2 overexpression	≥ 200/300¹	1 (5)	6 (14)	3 (30)	0.148
p53	≥ 10%	5 (28)	16 (42)	7 (64)	0.164
Beta-catenin, membrane	≥ 100/300¹	13 (65)	25 (64)	4 (40)	0.343
Beta-catenin, nucleus	2 or 3+	0 (0)	1 (3)	1 (10)	0.352

¹Score value.

^aP < 0.05 (outliers in pairwise test). EGFR: Epidermal growth factor receptor; ERK: Extracellular signaling-related kinase; HCC: Hepatocellular carcinoma; IHC: Immunohistochemistry; MET: MET receptor tyrosine kinase; mTOR: Mechanistic target of rapamycin.

Table 6 General correlation between expression scores for immunohistochemical markers and categories of histological grade for primary hepatocellular carcinoma

	Grade	Ki67	K19	p53	ERK1	BcatN	ERK2	Vim	CKD1	BcatM	mTOR	MET	EGFR	Group No.
Ki67	0.43^a													Group 1
K19	0.37^b	0.03
p53	0.36^c	0.29^d	0.08
ERK1	0.34^c	0.39^b	0.37^b	0.17
BcatN	0.23	0.44^a	0.13	0.24	0.18
ERK2	0.19	0.63^a	0.22	-0.10	0.36^c	0.24
Vim	0.14	0.11	0.51^a	0.02	0.31^c	0.08	0.14
CKD1	0.03	0.46^a	-0.32^c	-0.10	0.03	0.26^d	0.29^d	-0.19						Group 2
BcatM	-0.08	0.26^d	-0.01	-0.23	0.17	-0.07	0.42^a	0.01	0.25^d
mTOR	-0.24^d	-0.02	-0.20	-0.21	0.25^d	-0.03	0.13	-0.13	0.17	0.44^a
MET	-0.32^c	0.16	-0.20	-0.35^c	-0.01	0.22	0.39^b	-0.18	0.38^b	0.18	0.40^b
EGFR	-0.34^c	0.00	-0.14	-0.13	-0.04	-0.24	0.22	-0.07	0.13	0.20	0.11	0.23		Group 3
Casp3	-0.42^a	-0.18	-0.25^d	-0.46^a	-0.19	-0.06	0.06	-0.27^d	0.19	0.16	0.32^b	0.41^a	0.35^c	Group 3

^aP < 0.0005,

^bP≤ 0.001,

^cP < 0.01,

^dP < 0.05 by Spearman’s rho. Cases with positivity (group 1) and negativity (group 3) for immunohistochemical markers as associated with the histological grade and cell proliferation; Cases in group 2 showed intermediate features, between groups 1 and 3. BcatM: Membrane beta-catenin; BcatN: Nuclear beta-catenin; Casp3: Caspase 3; CKD1: Cyclin D1; EGFR: Epidermal growth factor receptor; HCC: Hepatocellular carcinoma; mTOR: Mechanistic target of rapamycin; Vim: Vimentin.

Citation: Felipe-Silva A, Wakamatsu A, dos Santos Cirqueira C, Alves VAF. Immunohistochemistry panel segregates molecular types of hepatocellular carcinoma in Brazilian autopsy cases. World J Gastroenterol 2016; 22(27): 6246-6256
URL: https://www.wjgnet.com/1007-9327/full/v22/i27/6246.htm
DOI: https://dx.doi.org/10.3748/wjg.v22.i27.6246