© 2014 APMIS. Published by John Wiley & Sons Ltd. DOI 10.1111/apm.12245
APMIS 122: 1001–1006
Clinicopathological characteristics of PIK3CA and HBx mutations in Korean patients with hepatocellular carcinomas DONG CHOON KIM,1 WOO JIN CHUNG,1 JAE-HO LEE,2 BYUNG KUK JANG,1 JAE SEOK HWANG,1 KOO JEONG KANG3 and SUN YOUNG KWON4 Department of Internal Medicine, Keimyung University Dongsan Medical Center, Daegu; 2Department of Anatomy, Keimyung University School of Medicine, Daegu; 3Division of Surgery, Department of Surgery, Keimyung University Dongsan Medical Center, Daegu; and 4Department of Pathology, Keimyung University Dongsan Medical Center, Daegu, Korea 1
Kim DC, Chung WJ, Lee J-H, Jang BK, Hwang JS, Kang KJ, Kwon SY. Clinicopathological characteristics of PIK3CA and HBx mutations in Korean patients with hepatocellular carcinomas. APMIS 2014; 122: 1001–1006. Hepatocellular carcinoma (HCC) is the fourth most common form of cancer in the Korean population, caused primarily by infection with either the Hepatitis B or C virus. Progression of this disease is frequently associated with mutations in either phosphoinositide-3-kinase, catalytic, alpha (PIK3CA) or hepatitis B virus X (HBx) gene. Previous studies have examined the frequency of PIK3CA mutations in HCC, although the clinical significance of these mutations has not been studied in a Korean population. In addition, HBx appears to play a key role in modulating a wide range of cellular functions, leading to HCC. In this study, we examined microdissected tumor samples from 50 HCC patients who underwent hepatectomy at Keimyung University Dongsan Medical Center. These patients were screened for mutations in PIK3CA and HBx to identify the clinical outcomes associated with these mutations. Exons 9 and 20 of PIK3CA and the entirety of HBx were screened for mutations by polymerase chain reaction and direct DNA sequencing. PIK3CA mutations were detected in 7 of 50 patients (14%). Among the 42 patients who were seropositive for hepatitis B, 17 (40.5%) had HBx mutations and 4 (9.52%) had mutations in PIK3CA. PIK3CA mutations were strongly correlated with tumor size. Patients harboring HBx mutations exhibited a longer time to recurrence; this difference was statistically significant not only in comparison with the PIK3CA mutation but also compared with those without any mutations. This result suggests a role for PIK3CA and HBx mutations as prognostic markers in HCC. Key words: PIK3CA; HBx; hepatocellular carcinoma; mutation. Woo Jin Chung, Department of Internal Medicine, Keimyung University Dongsan Medical Center, 56 Dalsung-ro, Jung-gu, Daegu 700-712, Korea. e-mail: [email protected]
Hepatocellular carcinoma (HCC) represents one of the most important health problems in South Korea, serving as the fourth most common cancer, and the second leading cause of cancer mortality (1). However, the exact mechanisms driving hepatocarcinogenesis are not completely understood, leading to an increased focus on genetic and epigenetic changes in HCC. The phosphatidylinositol-3kinase-Akt (PI3K/Akt) pathway regulates multiple biological processes including cell proliferation, differentiation, apoptosis, and mortality (2). The PI3Ks are heterodimers consisting of a catalytic Received 2 January 2013. Accepted 3 December 2013
subunit (p110a, PIK3CA) and an adaptor/regulatory subunit (p85). These subunits are activated by tyrosine kinase growth factor receptors such as epidermal growth factor receptor (EGFR) and insulin-like growth factor-1 receptor (IGF-1R), cell adhesion molecules such as integrins, G-proteincoupled receptors (GPCRs), and oncogenes such as Ras (3). Alterations in the PI3K-Akt signaling pathway are frequently seen in the hepatobiliary system, including PI3K-Akt-related HCC (2, 4). The true rate of PIK3CA mutations in HCC remains controversial, with one study finding mutations in 35.6% of HCCs in a Korean population (5) compared to only 2% in another study (6). 1001
KIM et al.
The hepatitis B virus X (HBx) gene is the smallest of seven hepatitis B virus (HBV) genes and encodes a 154-amino-acid multifunctional protein, with an N-terminal negative regulatory/antiapoptotic domain and a C-terminal transactivation/proapoptotic domain (7, 8); this gene has also been shown to modulate a wide range of cellular functions (9). Previous studies have shown that mutations in the basic core promoter of HBx lead to development of HCC (10–12). HBx transactivates the hepatocyte a-fetoprotein (AFP) gene via the CAAT/enhancer binding protein a site. CAAT/enhancer binding protein a growth control may also be subverted by PI3K/Akt signaling, a process commonly seen in liver regeneration. Because the CAAT/enhancer binding protein a-blocking action of HBx is thought to have evolved via selection for HBVinfected hepatocyte survival, a tumor-promoting function of AFP is implied (13, 14). Despite clear associations between signaling pathways and HCC, few studies have sought to identify associations between specific mutations and clinical outcomes. Here, we examine the role of PIK3CA and HBx mutations in 50 Korean patients with HCC. The clinicopathological characteristics of these patients were analyzed according to their genetic status to identify potential biomarkers of HCC progression. MATERIALS AND METHODS
reverse primer sequences used to amplify exon 20 were 5′TTGATGACATTGCATACATTCG-3′ and 5′-AATTG TGTGGAAGATCCAATCC-3′, respectively. PCR was performed using AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA, USA). PCR conditions were as follows: 1 cycle of 95 °C for 11 min, 40 cycles of 95 °C for 30 s, 55 °C for 40 s, and 72 °C for 1 min, followed by 1 cycle of 72 °C for 10 min. PCR products were electrophoresed on a 1.5% agarose gel and stained with ethidium bromide to confirm the size of the bands. Nested PCR was used to amplify the HBx gene as described previously (16). The primers used for HBx gene amplification were as follows: forward (5′-CTCTGCC AAGTGTTTGCTGA-3′) and reverse (5′-CAAGGCAC AGCTTGGAGGCT-3′) for the first round of PCR amplification, and forward (5′-TTGCTCGCAGCCGGTCTGG A-3′) and reverse (5′-TGAACAGTAGGCATGAACA-3′) for the second round of amplification. The following conditions were used for both rounds: 94 °C, 2 min for initial denaturation, followed by 30 cycles of 94 °C for 30 s, 54 °C for 40 s, and 72 °C for 1 min; a final extension step was then performed at 72 °C for 10 min. PCR products were analyzed by electrophoresis on a 1.5% agarose gel to confirm product size, followed by direct DNA sequencing for PIK3CA and HBx mutations using the ABI 3730 DNA sequencer (Bionics Inc., Seoul, Korea).
Statistical analysis SPSS 18.0 was used for all statistical analyses (SPSS, Inc., Chicago, IL, USA). The Chi-squared or Fisher’s exact test was used for categorical variables, and the Mann–Whitney U-test or Kruskal–Wallis analysis of variance for continuous variables. p ≤ 0.05 were considered to indicate significance.
Patients and DNA extraction This study comprised of 50 patients with HCC who underwent liver resection surgery between April 2008 and March 2011. All tumor samples were provided by the Keimyung Human Bio-Resource Bank at Dongsan Medical Center (Daegu, South Korea). This study was approved by the Institutional Review Board of Keimyung University Dongsan Medical Center, and informed consent was obtained from all individuals involved in the study. All tumor and paired normal tissue samples were microdissected under a light microscope using a 30-gauge syringe to confirm the somatic nature of tumor mutations. Genomic DNA was extracted using the Absolute DNA extraction kit (BioSewoom, Seoul, Korea) according to the manufacturer’s instructions.
PIK3CA and HBx mutation analysis Two hot spot regions (exons 9 and 20) of PIK3CA were amplified by polymerase chain reaction (PCR) to identify mutations in this gene; these locations were chosen as more than 75% of PIK3CA missense mutations cluster within these regions (3). All PCR was performed as described previously (15). The forward and reverse primer sequences used to amplify PIK3CA exon 9 were 5′-C CAGAGGGGAAAAATATGACAA-3′ and 5′-ACCTG TGACTCCATAGAAA-3′, respectively. Forward and
1002
RESULTS Characteristics of hepatocellular carcinoma patients
Fifty HCC patients (57.9 11.11 years of age) were included in this study. All cases were confirmed histologically by two pathologists after surgery, and were classified by Edmondson–Steiner grade (Table 1). The causes of HCC were HBV (86%), alcohol ingestion (10%), and hepatitis C virus (4%). The liver function scores of all patients were classified as Child-Pugh class A. Median tumor size was 6.39 4.22 cm. All HCC masses were fully resected during the operation, as determined by the operating physician. PIK3CA mutations analysis
Mutations in PIK3CA exon 20 were detected in the 7 of 50 HCC patients (14.0%), with no mutations found in exon 9. Two types of point mutations were found, H1047R, a mutation frequently detected in a variety of cancers (5, 17–20), and K1041G. Clinicopathological characteristics associated with
© 2014 APMIS. Published by John Wiley & Sons Ltd
PIK3CA AND HBX MUTATIONS IN HEPATOCELLULAR CARCINOMAS
Table 1. Characteristics of the patients at baseline No. (% or range) Age (years, mean SD) 57.9 11.11 (32–77) Sex (M/F) 39 (78.0%)/11 (22.0) Background disease HBs-Ag 43 (86.0) HCV-Ab 2 (4.0) Alcohol 5 (10.0) Tumor size (cm, 6.39 4.22 (1.08–18) mean SD) Single/multiple tumor 46 (92.0)/4 (8.0) Edmondson–Steiner grade Grade II 8 (16.0) Grade III 33 (66.0) Grade IV 9 (18.0) AJCC 7th HCC stage Stage I 40 (80.0) Stage II 5 (10.0) Stage IIIA 1 (2.0) Stage IIIB 4 (8.0) TW < 5.0 mm 20 (40.0) AFP (ng/mL, mean SD) 5897.21 28 939.99 Patient demographics 26 (52) regarding recurrence Time to recurrence (days, 216.00 160.27 mean SD) AFP (ng/mL, mean SD) 302.96 603.60 Recurrent tumor size (cm, 1.96 1.92 mean SD) SD, standard deviation; AJCC, American Joint Committee on Cancer; Tw, microscopic resected stump with a margin from the edge of the tumor; AFP, a-fetoprotein.
PIK3CA mutations are presented in Table 2. Median tumor sizes were larger in HCC patients with PIK3CA mutations (9.50 4.60 cm) than in patients without PIK3CA mutations (5.88 3.99), this difference being statistically significant (p = 0.049). Margin involvement and surgical margin
APMIS 122: 1001–1006
Clinicopathological characteristics of PIK3CA and HBx mutations in Korean patients with hepatocellular carcinomas DONG CHOON KIM,1 WOO JIN CHUNG,1 JAE-HO LEE,2 BYUNG KUK JANG,1 JAE SEOK HWANG,1 KOO JEONG KANG3 and SUN YOUNG KWON4 Department of Internal Medicine, Keimyung University Dongsan Medical Center, Daegu; 2Department of Anatomy, Keimyung University School of Medicine, Daegu; 3Division of Surgery, Department of Surgery, Keimyung University Dongsan Medical Center, Daegu; and 4Department of Pathology, Keimyung University Dongsan Medical Center, Daegu, Korea 1
Kim DC, Chung WJ, Lee J-H, Jang BK, Hwang JS, Kang KJ, Kwon SY. Clinicopathological characteristics of PIK3CA and HBx mutations in Korean patients with hepatocellular carcinomas. APMIS 2014; 122: 1001–1006. Hepatocellular carcinoma (HCC) is the fourth most common form of cancer in the Korean population, caused primarily by infection with either the Hepatitis B or C virus. Progression of this disease is frequently associated with mutations in either phosphoinositide-3-kinase, catalytic, alpha (PIK3CA) or hepatitis B virus X (HBx) gene. Previous studies have examined the frequency of PIK3CA mutations in HCC, although the clinical significance of these mutations has not been studied in a Korean population. In addition, HBx appears to play a key role in modulating a wide range of cellular functions, leading to HCC. In this study, we examined microdissected tumor samples from 50 HCC patients who underwent hepatectomy at Keimyung University Dongsan Medical Center. These patients were screened for mutations in PIK3CA and HBx to identify the clinical outcomes associated with these mutations. Exons 9 and 20 of PIK3CA and the entirety of HBx were screened for mutations by polymerase chain reaction and direct DNA sequencing. PIK3CA mutations were detected in 7 of 50 patients (14%). Among the 42 patients who were seropositive for hepatitis B, 17 (40.5%) had HBx mutations and 4 (9.52%) had mutations in PIK3CA. PIK3CA mutations were strongly correlated with tumor size. Patients harboring HBx mutations exhibited a longer time to recurrence; this difference was statistically significant not only in comparison with the PIK3CA mutation but also compared with those without any mutations. This result suggests a role for PIK3CA and HBx mutations as prognostic markers in HCC. Key words: PIK3CA; HBx; hepatocellular carcinoma; mutation. Woo Jin Chung, Department of Internal Medicine, Keimyung University Dongsan Medical Center, 56 Dalsung-ro, Jung-gu, Daegu 700-712, Korea. e-mail: [email protected]
Hepatocellular carcinoma (HCC) represents one of the most important health problems in South Korea, serving as the fourth most common cancer, and the second leading cause of cancer mortality (1). However, the exact mechanisms driving hepatocarcinogenesis are not completely understood, leading to an increased focus on genetic and epigenetic changes in HCC. The phosphatidylinositol-3kinase-Akt (PI3K/Akt) pathway regulates multiple biological processes including cell proliferation, differentiation, apoptosis, and mortality (2). The PI3Ks are heterodimers consisting of a catalytic Received 2 January 2013. Accepted 3 December 2013
subunit (p110a, PIK3CA) and an adaptor/regulatory subunit (p85). These subunits are activated by tyrosine kinase growth factor receptors such as epidermal growth factor receptor (EGFR) and insulin-like growth factor-1 receptor (IGF-1R), cell adhesion molecules such as integrins, G-proteincoupled receptors (GPCRs), and oncogenes such as Ras (3). Alterations in the PI3K-Akt signaling pathway are frequently seen in the hepatobiliary system, including PI3K-Akt-related HCC (2, 4). The true rate of PIK3CA mutations in HCC remains controversial, with one study finding mutations in 35.6% of HCCs in a Korean population (5) compared to only 2% in another study (6). 1001
KIM et al.
The hepatitis B virus X (HBx) gene is the smallest of seven hepatitis B virus (HBV) genes and encodes a 154-amino-acid multifunctional protein, with an N-terminal negative regulatory/antiapoptotic domain and a C-terminal transactivation/proapoptotic domain (7, 8); this gene has also been shown to modulate a wide range of cellular functions (9). Previous studies have shown that mutations in the basic core promoter of HBx lead to development of HCC (10–12). HBx transactivates the hepatocyte a-fetoprotein (AFP) gene via the CAAT/enhancer binding protein a site. CAAT/enhancer binding protein a growth control may also be subverted by PI3K/Akt signaling, a process commonly seen in liver regeneration. Because the CAAT/enhancer binding protein a-blocking action of HBx is thought to have evolved via selection for HBVinfected hepatocyte survival, a tumor-promoting function of AFP is implied (13, 14). Despite clear associations between signaling pathways and HCC, few studies have sought to identify associations between specific mutations and clinical outcomes. Here, we examine the role of PIK3CA and HBx mutations in 50 Korean patients with HCC. The clinicopathological characteristics of these patients were analyzed according to their genetic status to identify potential biomarkers of HCC progression. MATERIALS AND METHODS
reverse primer sequences used to amplify exon 20 were 5′TTGATGACATTGCATACATTCG-3′ and 5′-AATTG TGTGGAAGATCCAATCC-3′, respectively. PCR was performed using AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA, USA). PCR conditions were as follows: 1 cycle of 95 °C for 11 min, 40 cycles of 95 °C for 30 s, 55 °C for 40 s, and 72 °C for 1 min, followed by 1 cycle of 72 °C for 10 min. PCR products were electrophoresed on a 1.5% agarose gel and stained with ethidium bromide to confirm the size of the bands. Nested PCR was used to amplify the HBx gene as described previously (16). The primers used for HBx gene amplification were as follows: forward (5′-CTCTGCC AAGTGTTTGCTGA-3′) and reverse (5′-CAAGGCAC AGCTTGGAGGCT-3′) for the first round of PCR amplification, and forward (5′-TTGCTCGCAGCCGGTCTGG A-3′) and reverse (5′-TGAACAGTAGGCATGAACA-3′) for the second round of amplification. The following conditions were used for both rounds: 94 °C, 2 min for initial denaturation, followed by 30 cycles of 94 °C for 30 s, 54 °C for 40 s, and 72 °C for 1 min; a final extension step was then performed at 72 °C for 10 min. PCR products were analyzed by electrophoresis on a 1.5% agarose gel to confirm product size, followed by direct DNA sequencing for PIK3CA and HBx mutations using the ABI 3730 DNA sequencer (Bionics Inc., Seoul, Korea).
Statistical analysis SPSS 18.0 was used for all statistical analyses (SPSS, Inc., Chicago, IL, USA). The Chi-squared or Fisher’s exact test was used for categorical variables, and the Mann–Whitney U-test or Kruskal–Wallis analysis of variance for continuous variables. p ≤ 0.05 were considered to indicate significance.
Patients and DNA extraction This study comprised of 50 patients with HCC who underwent liver resection surgery between April 2008 and March 2011. All tumor samples were provided by the Keimyung Human Bio-Resource Bank at Dongsan Medical Center (Daegu, South Korea). This study was approved by the Institutional Review Board of Keimyung University Dongsan Medical Center, and informed consent was obtained from all individuals involved in the study. All tumor and paired normal tissue samples were microdissected under a light microscope using a 30-gauge syringe to confirm the somatic nature of tumor mutations. Genomic DNA was extracted using the Absolute DNA extraction kit (BioSewoom, Seoul, Korea) according to the manufacturer’s instructions.
PIK3CA and HBx mutation analysis Two hot spot regions (exons 9 and 20) of PIK3CA were amplified by polymerase chain reaction (PCR) to identify mutations in this gene; these locations were chosen as more than 75% of PIK3CA missense mutations cluster within these regions (3). All PCR was performed as described previously (15). The forward and reverse primer sequences used to amplify PIK3CA exon 9 were 5′-C CAGAGGGGAAAAATATGACAA-3′ and 5′-ACCTG TGACTCCATAGAAA-3′, respectively. Forward and
1002
RESULTS Characteristics of hepatocellular carcinoma patients
Fifty HCC patients (57.9 11.11 years of age) were included in this study. All cases were confirmed histologically by two pathologists after surgery, and were classified by Edmondson–Steiner grade (Table 1). The causes of HCC were HBV (86%), alcohol ingestion (10%), and hepatitis C virus (4%). The liver function scores of all patients were classified as Child-Pugh class A. Median tumor size was 6.39 4.22 cm. All HCC masses were fully resected during the operation, as determined by the operating physician. PIK3CA mutations analysis
Mutations in PIK3CA exon 20 were detected in the 7 of 50 HCC patients (14.0%), with no mutations found in exon 9. Two types of point mutations were found, H1047R, a mutation frequently detected in a variety of cancers (5, 17–20), and K1041G. Clinicopathological characteristics associated with
© 2014 APMIS. Published by John Wiley & Sons Ltd
PIK3CA AND HBX MUTATIONS IN HEPATOCELLULAR CARCINOMAS
Table 1. Characteristics of the patients at baseline No. (% or range) Age (years, mean SD) 57.9 11.11 (32–77) Sex (M/F) 39 (78.0%)/11 (22.0) Background disease HBs-Ag 43 (86.0) HCV-Ab 2 (4.0) Alcohol 5 (10.0) Tumor size (cm, 6.39 4.22 (1.08–18) mean SD) Single/multiple tumor 46 (92.0)/4 (8.0) Edmondson–Steiner grade Grade II 8 (16.0) Grade III 33 (66.0) Grade IV 9 (18.0) AJCC 7th HCC stage Stage I 40 (80.0) Stage II 5 (10.0) Stage IIIA 1 (2.0) Stage IIIB 4 (8.0) TW < 5.0 mm 20 (40.0) AFP (ng/mL, mean SD) 5897.21 28 939.99 Patient demographics 26 (52) regarding recurrence Time to recurrence (days, 216.00 160.27 mean SD) AFP (ng/mL, mean SD) 302.96 603.60 Recurrent tumor size (cm, 1.96 1.92 mean SD) SD, standard deviation; AJCC, American Joint Committee on Cancer; Tw, microscopic resected stump with a margin from the edge of the tumor; AFP, a-fetoprotein.
PIK3CA mutations are presented in Table 2. Median tumor sizes were larger in HCC patients with PIK3CA mutations (9.50 4.60 cm) than in patients without PIK3CA mutations (5.88 3.99), this difference being statistically significant (p = 0.049). Margin involvement and surgical margin
