20131222 TASL HBV pre-S deletion mutants and HCC outcomes

B型肝炎病毒表面前區剔除性突變

與抗病毒藥物，對於肝細胞癌患者

接受切除手術後，腫瘤復發之影響

蘇建維1,2,3 邱昱維4 蔡宜璇1 鄧睿敦1

周嘉揚2,5 洪宏緒1,6 霍德義3,7 吳肇卿1,4

1陽明大學醫學院臨床醫學研究所2陽明大學醫學院醫學系

3台北榮民總醫院內科部胃腸科4台北榮民總醫院醫學研究部轉譯研究科

5台北榮民總醫院外科部一般外科6振興醫院內科部胃腸肝膽科

7陽明大學醫學院藥理學研究所

Nomogram for risk of HCC

Yang HI, et al. J Clin Oncol 2010;28:2437-44

HBV genotype, precore and BCP mutants and HCC

305.6

785.8

955.5

269.4358.7

1149.2

0

200

400

600

800

1000

1200

1400

Genotype B

Genotype C

Precore wild

Precore Mutant

BCP wild

BCP mutant

Incidence rate per 100,000 person-years

Yang HI, et al. JNCI 2008;100:1134-43

The life cycle of HBV

Chan HLY, J Hepatol 2011;55:1121-1131

Immune epitopes and functional domains within the HBV pre-S region

Su IJ, et al. J Gastroenterol Hepatol 2008Kao JH Liver Int 2012

Su IJ, et al. J Gastroenterol Hepatol 2008

Prevalence of pre-S deletion

Chen BF, Kao JH, et al. Gastroenterology 2006;130:1153-68

Chen CH, et al. Gastroenterology 2007;133:1466-74

Pre-S deletion and HCC

Combined risk of developing HCC associated with HBV mutants and HCV genotype

Hung CH, Chen CH, Lee CM, et al. Intervirology 2013;56:316-24

Schematic representation of factors that influence the progression of HBV-related liver disease

Host Factors

Age> 40 years

Gender: Male> Female

Immune status

Viral FactorsGenotype: C> B; D>A

Viral load:> 104

copies/mL

Genome Mutations: pre-S deletion, T1762/A1764

Environmental factors

Alcohol

Smoking

Aflatoxin exposure

Concurrent HCV, HDV, HIV infection

Liaw YF, Factors influencing liver disease progression in chronic hepatitis B. Liver Int 2006; 26: 23-29

The role of HBV pre-S deletion mutants in HCC patients after

curative therapies

GGH predicts prognosis of HCC patients after resection surgery

Tsai HW, Su IJ, et al. Cancer 2011; 117:2951-60

GGH predict prognosis of HCC patients after resection surgery


GGH predict prognosis of HCC patients after resection surgery


Small pre-S deletion predict poor prognosis after resection for HCC

Yeh CT, et al. Hepatology 2010;52:1922-33

178 patients from non-tumor part of liver specimens

Small pre-S deletion predict poor prognosis after resection surgery for HCC

Yeh CT, et al. Hepatology 2010;52:1922-33

Disease-free survival Overall Survival

Lack of Association Between HBVPre-S Mutations and Recurrence After Surgical

Resection in HCC

Heo NY, Lee HC, Park YK, et al. J Med Virol 2013; 85:589-96

All were genotype C19 (29.7%) of the 64 patients had pre-S deletion

Lack of association between HBVpre-S mutations and recurrence after resection in HCC




Recurrence-free survival Overall Survival




Pre-S deletion mutants are associated with HCC development.

However, the prognostic value of pre-S deletion mutants in HCC patients after

curative therapies is still unclear.

Hepatitis B Viral Load, Pre-S Deletion Mutations and Anti-viral Therapy Predict

Post-operative Recurrence of Hepatocellular Carcinoma

PLoS ONE 2013;8(6):e66457

Materials and Methods

• From 1989 to 2007, 333 patients with HBV-related HCC who underwent resection surgery at Taipei Veterans General Hospital were enrolled.

• A total of 62 patients received antiviral therapy for HBV infection after resection, while the remaining 271 patients did not take antiviral therapy during the follow-up period.

• All serum samples of enrolled patients were tested for HBV DNA levels and genotype; and clonal analysis of pre-S regions were performed in 216 patients.

• After a median follow-up of 45.9 months, 165 patients died and 208 patients had HCC recurrence after resection. The 5-year overall survival and recurrence-free survival rates were 55.4% and 35.3%, respectively.

Characterization of HBV related HCC patients between genotype B and genotype C

All patients

(n= 333)

B

(n=174)

C

(n=139)

P

Patient Demographics (continuous variables are expressed as median; 25 and 75 percentiles)

Age (years) 56; 47-67 56; 44-68.3 57; 48-64 0.692

Sex (M/F) (%) 271/42 (86.6%/13.4%) 153/21 (87.9%/12.1%) 118/21 (84.9%/15.1%) 0.537

HCC family history

(yes/no) (%)

71/235

(23.2%/76.8%)

32/126

(20.3%/79.7%)

36/93

(27.9%/72.1%)

0.168

Albumin (g/dL) 4.0; 3.8-4.3 4.1; 3.9-4.4 3.9; 3.7-4.2 <0.001

Total bilirubin (mg/dL) 0.9; 0.7-1.2 0.8; 0.7-1.1 0.9; 0.7-1.2 0.715

ALT (U/L) 42; 28-66 40; 26-61.5 46; 31.8-73.3 0.017

ICG-15R (%) 10.0; 6.0-16.0 10.0; 6.0-15.0 12; 7-17.5 0.024

Platelet (/mm3) 162000; 120500-

212500

167500; 132500-

215750

143000; 104000-

200000

0.023

PT INR 1.01; 0.95-1.07 1.00; 0.95-1.05 1.02; 0.96-1.13 0.011


All patients

(n= 333)

B

(n=174)

C

(n=139)

P

Tumor factors (continuous variables are expressed as median; 25 and 75 percentiles)

Tumor size (cm) 4.0; 2.5-6.8 4.1; 2.5-6.7 3.5; 2.5-6.3 0.975

Multinodularity (%) 141/191 (42.3%/57.7%) 74/100 (42.5%/57.5%) 60/79 (43.2%/56.8%) 1.000

Macroscopic venous invasion (%) 61/271 (18.4%/81.6%) 36/138(20.7%/79.3%) 20/118(14.5%/85.5%) 0.205

AFP (ng/ml) 45.5; 7.4-974.5 31.3; 7.0-996.5 51.1; 8.2-714.8 0.146

Histo-pathological findings

Cirrhosis (yes/no) (%) 143/180 (44.3%/55.7%) 58/108 (34.9%/65.1%) 76/61 (55.5%/44.5%) 0.001

Edmondson grading (I-II/III-IV)(%) 214/108 (66.5%/33.5%) 117/50 (70.1%/29.9%) 87/49 (64.0%/36.0%) 0.317

Microscopic venous invasion (%) 221/111 (66.6%/33.4%) 117/57 (67.2%/32.8%) 92/46 (66.7%/33.3%) 1.000


All patients

(n= 333)

B

(n=174)

C

(n=139)

P

Viral factors

HBVDNA(copies/mL) (median;

25 and 75 percentiles)

3.90 x 105;

1.73 x 104-7.64 x 106

3.21 x 105;

2.24 x 104-4.17 x 106

1.76 x 105;

2.45 x 104-1.39 x 106

0.390

HBsAg (IU/mL) (median; 25

and 75 percentiles)

830.0; 287.5-1830.0 935.0; 400.5-1781.3 897.0; 200.8-1959.4 0.241

Pre-core mutation (G1896A)

(Y/N) (%)

194/102 (65.5%/34.5%) 119/46 (72.1%/27.9%) 74/55 (57.4%/42.6%) 0.012

BCP (A1762T, G1764A)

mutation (Y/N) (%)

209/86 (70.8%/29.2%) 96/69 (58.2%/41.8%) 112/16 (87.5%/12.5%) <0.001

Pre-S deletion (yes/no) (%) 73/85 (46.2%/53.8%) 33/62 (34.7%/65.3%) 40/22 (64.5%/35.5%) <0.001

Antiviral therapy (Y/N) (%) 40/293 (12.0%/88.0%) 26/148 (14.9%/85.1%) 10/129 (7.2%/92.8%) 0.067

Overall survival

HCC tumorigenesis

Tumor factor

Liver functional

reserve

Hepatic fibrosis

Viral factors

Univariate analysis of factors associated with overall survival after resection for hepatocellular carcinoma.

(all patients)

Variable Number Median survival months (95% CI) Hazard ratio (95% CI) P

Age > 60 / 60 y/o 131/201 50.6(41.0-60.2)/115.1(78.9-151.3) 1.743 (1.283-2.367) <0.001

Sex Female/Male 45/287 128.3(25.7-230.9)/70.4(53.7-87.1) 0.690(0.422-1.126) 0.137

Albumin 4 / > 4 g/dL 165/163 46.1(36.0-56.2)/125.9(80.4-171.4) 1.843(1.345-2.526) <0.001

Bilirubin > 1.6 / 1.6 mg/dL 20/312 81.9(46.3-117.5)/71.2(48.0-94.4) 0.855(0.450-1.623) 0.631

ALT >40 / 40 U/L 178/154 70.4(52.0-88.8)/79.7(42.8-116.6) 1.087(0.799-1.479) 0.594

ALK-P >100 / 100 U/L 132/199 41.2(26.1-56.3)/100.9(73.1-128.7) 1.892(1.392-2.571) <0.001

GGT >60 / 60 U/L 131/197 46.7(35.1-58.3)/113.5(92.6-134.4) 1.843(1.356-2.506) <0.001

Platelet 105 / > 105 /mm3 51/262 60.3(34.9-85.7)/74.2(45.5-102.9) 1.149(0.776-1.702) 0.488

ICG-15R > 10% / 10% 160/170 52.3(34.2-70.4)/120.9(80.1-161.7) 1.707(1.250-2.330) 0.001

MELD ≧8/<8 136/190 57.5(37.4-77.6)/94.5(67.4-121.6) 1.405(1.032-1.914) 0.013


(all patients)

Variable Number Median survival months

(95% CI)

Hazard ratio

(95% CI)

P

HBV genotype C/B 138/174 72.8(52.2-93.4)/

69.9(23.6-116.2)

1.120

(0.821-1.529)

0.475

HBV DNA >106 / 106 cp/ml 136/184 75.1(55.8-94.4)/

70.4(29.6-111.2)

0.980

(0.715-1.344)

0.901

HBsAg > 1000 / 1000 IU/mL 121/157 79.7(45.1-114.3)/

100.0(64.0-136.0)

1.142

(0.803-1.624)

0.459

Precore mutation Y/N 193/102 94.5(61.6-127.4)/

54.5(33.7-75.3)

0.752

(0.544-1.040)

0.085

A1762T/G1764A mutation Y/N 209/85 71.2(56.8-85.6)/

70.9(0-165.9)

1.040

(0.722-1.499)

0.833

Antiviral therapy (N/Y) 62/271 54.5(40.0-69.0)/

NA

5.938

(2.915-12.094)

<0.001


(all patients)

Variable Number Median survival months (95% CI) Hazard ratio (95% CI) P

Tumor size > 5cm / 5cm 122/210 37.5(22.6-52.4)/100.0(68.0-132.0) 2.001 (1.472-2.721) <0.001

Multinodularity Y/N 141/191 45.7(33.6-57.8)/119.8(97.9-141.7) 2.182 (1.603-2.972) <0.001

Macroscopic venous invasion

Y/N

61/271 16.8(0-36.5)/94.5(67.3-121.7) 2.809 (1.976-3.984) <0.001

Cut margin 1cm/ >1cm 223/108 60.3(45.9-74.7)/113.5(89.3-137.7) 1.520 (1.079-2.141) 0.017

AFP >20 / 20 ng/ml 192/136 69.7(48.1-91.3)/81.9(48.4-115.4) 1.308 (0.951-1.799) 0.099

Microscopic venous invasion

Y/N

221/110 52.3(40.6-64.0)/119.8(103.7-135.9) 2.203 (1.527-3.185) <0.001

Cirrhosis on non-tumor part Y/N 143/179 54.2(40.9-67.5)/122.5(82.9-162.1) 1.706 (1.248-2.331) 0.001

Edmondson stage III or IV/ I or II 214/108 79.9(54.0-105.8)/64.0(38.3-89.7) 1.369 (0.997-1.879) 0.052

Overall survival: antiviral therapy

P < 0.001

The impact of anti-viral therapy on overall survival stratified by BCLC stage

Multivariate analysis of factors associated with overall survival after resection for HCC (all patients)

Variable Hazard ratio

(95% confidence interval)

Standard error P

ICG-15R > 10% 1.574(1.122-2.208) 0.173 0.009

ALK-P >100 U/L 1.762(1.267-2.450) 0.168 0.001

Macroscopic venous

invasion

2.326(1.580-3.425) 0.197 <0.001

Microscopic venous

invasion

1.848(1.225-2.786) 0.209 0.003

Without antiviral

therapy

8.275(2.631-26.023) 0.585 <0.001

Recurrence

Recurrence: antiviral therapy

The impact of anti-viral therapy on recurrence stratified by viral factors

HBV DNA levels

P< 0.001 P=0.038

The impact of anti-viral therapy on recurrence stratified by viral factors

Serum HBsAg levels

P= 0.001 P= 0.037

The effect of anti-viral therapy in decreasingrecurrence is irrespective of viral load

The impact of anti-viral therapy on recurrence stratified by BCLC stage

Impact of antiviral therapy on prognosis of HBV-related HCC after resection

Chan ACY, et al. Arch Surg 2011;146:675-81

AJCC stage I and II

AJCC stage III

Overall survival Disease-free survival

The effect of anti-viral therapy in improving overall survival is more apparent in early stage HCC

Multivariate analysis of factors associated with recurrence after resection for HCC (all patients)

Variable Hazard ratio

(95% confidence interval)

Standard error P

GGT >60 U/L 1.628 (1.210-2.190) 0.151 0.001

ICG-15 > 10% 1.386 (1.036-1.854) 0.148 0.028

Without antiviral therapy 2.296 (1.451-3.632) 0.234 <0.001

Macroscopic venous invasion 2.375(1.650-3.425) 0.186 <0.001

Microscopic venous invasion 1.499(1.080-2.083) 0.167 0.016

Strategy for Preventing HBV-HCC

Primary prevention

• HBV vaccination

Secondary prevention

• Adequate antiviral therapy

• Close surveillance (sonography and AFP)

Tertiary Prevention

• Adjuvant anti-tumor therapy (such as target therapy) for reducing early recurrence

• Antiviral therapy for improving liver functional reserve and reducing late recurrence

Summary

• Anti-viral therapy could improve the post-operativeprognosis both in terms of overall survival andrecurrence in patients with early stage HCC, but theeffect was less significant in those with BCLC stage C.

• As anti-viral therapy might confound the impact ofviral factors, to eliminate this bias, 62 patients whoreceived anti-viral therapy after resection surgerywere excluded and the remaining 271 patients werefurther analyzed for prognostic factors.

Multivariate analysis of factors associated with overall survival and recurrence after resection for HCC (without

antiviral therapy)

Variable HR (95% CI) Standard error P

Overall survivalICG-15 > 10% 1.618 (1.142-2.292) 0.178 0.007

ALK-P >100 U/L 1.441 (1.035-2.006) 0.169 0.031

Tumor size > 5 cm 1.602 (1.127-2.276) 0.179 0.009

Macroscopic venous invasion 2.155 (1.462-3.174) 0.198 <0.001

Microscopic venous invasion 1.842 (1.209-2.801) 0.214 0.004

Cirrhosis on non-tumor part 1.486 (1.043-2.116) 0.180 0.028

RecurrenceHBV DNA >106 copies/mL 1.428 (1.047-1.947) 0.158 0.024

GGT >60 U/L 1.700 (1.239-2.331) 0.194 0.001



Ongoing viral replication is a crucial predictor of post-operative HCC recurrence, anti-viral therapy can reduce recurrence and improve

prognosis.

Characterization of HBV related HCC patients with and without pre-S deletion (n=216)

Without Pre-S deletion(n=143)

With Pre-S deletion (n=73)

P

Patient Demographics

Age (years) 58; 46-69 54; 46-64 0.500

Sex (male/female) (%) 123/20 (86/0%/14.0%) 65/8 (89.0%/11.0%) 0.680

HCC family history (yes/no) (%)

28/100 (21.9%/78.1%) 20/47 (29.9%/70.1%) 0.292

Albumin (g/dL) 4.1;3.9-4.4 4.0; 3.7-4.2 0.018

Total bilirubin (mg/dL) 0.9; 0.7-1.2 0.9; 0.7-1.3 0.847

ALT (U/L) 42; 27.5-65.6 49; 39-74 0.084

ICG-15R (%) 11; 6-16 10; 7-17 0.735

Platelet (/mm3) 156000; 125000-199000 148000; 98000-209000 0.849

PT INR 1.01; 0.96-1.06 1.00; 0.94-1.12 0.571

MELD 7.7; 6.6-8.6 7.9; 6.9-9.3 0.796

15: pre-S143: pre-S215: pre-S1+ pre-S2




P

Tumor factors

Tumor size (cm) 3.4; 2.5-6.0 3.0; 2.1-6.0 0.191

Multi-nodularity/single tumor (%)

57/86 (39.9%/60.1%) 25/48 (34.2%/65.8%) 0.512

Macroscopic venous invasion (yes/no) (%)

21/121 (14.8%/85.2%) 12/61 (16.4%/83.6%) 0.906

AFP (ng/ml) 20.8; 6.7-752.3 50.6; 12.1-803.0 0.343

Cut margin ≤1/>1 cm (%) 101/42 (70.6%/29.4%) 45/27 (62.5%/37.5%) 0.294

Cirrhosis (yes/no) (%) 51/86 (37.2%/62.8%) 40/30 (57.1%/42.9%) 0.010

Edmondson grading (I-II/III-IV) (%)

47/91 (34.1%/65.9%) 18/53 (25.4%/74.6%) 0.258

Microscopic venous invasion (yes/no) (%)

93/49 (65.5%/34.5%) 47/26 (64.4%/35.6%) 0.992

BCLC (A/B/C) (%) 84/40/14(59.6%/28.4%/12.1%)

45/16/11(62.5%/22.2%/15.3%)

0.566




P

Viral factors

HBVDNA(copies/mL)* 4.85 x 105;3.67 x 104-1.08 x 107

1.61x 106; 9.95 x 104-1.51 x 107

0.055

HBsAg (IU/mL)* 896.3; 339.5-1732.4 1127.5; 405-2101 0.297

Genotype B/C (%) 98/44 (69.0%/31.0%) 33/40 (45.2%/54.8%) 0.001

Pre-core (G1896A) mutation (yes/no) (%)*

88/42 (67.7%/32.3%) 40/28 (58.8%/41.2%) 0.279

BCP (A1762T, G1764A) mutation (yes/no) (%)*

83/47 (63.8%/36.2%) 52/16 (76.5%/23.5%) 0.099

Multivariate analysis of factors associated with overall survival and recurrence after resection for HCC (without

antiviral therapy)

Variable HR (95% CI) Standard error P

Overall survivalAge >60 years 2.035 (1.288-3.215) 0.233 0.002

Alk-P >100 U/L 2.288 (1.451-3.606) 0.232 <0.001



G1896A mutation 0.598 (0.382-0.937) 0.229 0.025

RecurrenceMulti-nodularity 1.693 (1.115-2.571) 0.213 0.013

Macroscopic venous invasion 2.415 (1.418-4.098) 0.271 0.001

Cirrhosis 1.544 (1.047-2.279) 0.198 0.029

Edmondson stage III or IV 1.526 (1.029-2.263) 0.201 0.036

Pre-S deletion 1.564 (1.057-2.314) 0.200 0.025

Pre-S deletion and post-operative recurrence stratified by serum HBVDNA and HBsAg levels

P=0.028P=0.047

P=0.926 P=0.253

The impact of pre-S deletion mutants on recurrence for patients receiving anti-viral therapy

P=0.161

hepatocarcinogenesis and tumor recurrence induced by pre-S deletion mutants may be based on active HBV viral replication, the accumulation

of oxidative stress, and subsequent chromosomal instability

Conclusion

• On-going viral replication and the presence of pre-S deletion mutants are both crucial predictors of post-operative HCC recurrence.

• Hepatocarcinogenesis and tumor recurrence induced by pre-S deletion mutants may be based on active HBV viral replication, the accumulation of oxidative stress, and subsequent chromosomal instability

• Antiviral therapy may improve post-operative outcomes, especially in early stage HCC.

Thanks for Attendance

Health & Medicine

20131222 TASL HBV pre-S deletion mutants and HCC outcomes