Original article

Preoperative serum hyaluronic acid level as a prognostic factor in patients undergoing hepatic resection for hepatocellular carcinoma K. Mima1 , T. Beppu1,2 , T. Ishiko1 , A. Chikamoto1 , S. Nakagawa1 , H. Hayashi1 , M. Watanabe1 , K. Sakamaki3 and H. Baba1 1

Department of Gastroenterological Surgery, Graduate School of Medical Sciences, Kumamoto University, and 2 Department of Multidisciplinary Treatment for Gastroenterological Cancer, Kumamoto University Hospital, Kumamoto, and 3 Department of Biostatistics and Epidemiology, Graduate School of Medicine, Yokohama City University, Yokohama, Japan Correspondence to: Professor H. Baba, Department of Gastroenterological Surgery, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556, Japan (e-mail: [email protected])

Background: Hyaluronic acid (HA) probably plays a critical role in tumorigenesis. The clinical

significance of serum HA concentration in patients with hepatocellular carcinoma (HCC) remains to be elucidated. This study analysed the relationship between preoperative serum HA levels and prognosis after hepatic resection in patients with HCC. Methods: Consecutive patients who underwent hepatic resection for HCC between September 1999 and March 2012 were included in this retrospective study. Serum HA levels were measured within 4 weeks before surgery by an immunoturbidimetric automated latex assay. The cut-off level for preoperative serum HA was validated using a time-dependent receiver operating characteristic (ROC) curve analysis. The prognostic impact of preoperative serum HA levels was analysed using Cox proportional hazards models. Results: A total of 506 patients of median age 66 years (405 men, 80·0 per cent) were analysed. The median length of follow-up was 32 months. High serum HA levels (100 ng/ml or above) were associated with shorter recurrence-free survival (P < 0·001) (hazard ratio (HR) 1·50, 95 per cent confidence interval 1·17 to 1·93; P = 0·002) and overall survival (P = 0·001) (HR 1·46, 1·03 to 2·07; P = 0·033). In patients with HCC without severe liver fibrosis, serum HA level was correlated with multiple tumours (P = 0·039), early recurrence (P = 0·033), and poor recurrence-free (P < 0·001) and overall (P = 0·024) survival. Conclusion: High preoperative serum HA levels predict poor prognosis in patients with HCC after hepatic resection, and may serve as a future biomarker. Paper accepted 18 September 2013 Published online in Wiley Online Library (www.bjs.co.uk). DOI: 10.1002/bjs.9343

Introduction

Hepatocellular carcinoma (HCC) is the fifth most prevalent and third most deadly cancer1 . Hepatic resection is one option for the treatment of early-stage HCC. However, the prognosis of HCC is poor owing to tumour invasiveness, intrahepatic spread, extrahepatic metastasis and resistance to chemotherapy2,3 . The identification of new recurrence and survival markers is thus of great importance to help improve the outcome of patients with HCC. Hyaluronic acid (HA), also known as hyaluronan, is an unbranched glycosaminoglycan consisting of repeating disaccharides of glucuronic acid and N -acetylglucosamine.  2014 BJS Society Ltd Published by John Wiley & Sons Ltd

HA is a major component of the extracellular matrix of most mammalian tissues and plays an important role in tissue homeostasis and biomechanical integrity4 . Serum HA concentration is easy to measure with commercially available assays, and is used as a marker for liver fibrosis and cirrhosis in clinical practice5,6 . HA is known to play a critical role in tumorigenesis and tumour metastasis. It is overproduced by many types of tumour, and the interaction between HA and its receptor, CD44, promotes tumour progression and multidrug resistance4,7 . HA strongly promotes anchorageindependent tumour cell growth, survival and migration, thereby increasing metastatic spread8 . A reduction in HA production causes decreased tumour growth9 . CD44 BJS

K. Mima, T. Beppu, T. Ishiko, A. Chikamoto, S. Nakagawa, H. Hayashi et al.

increases tumour cell invasion, and high CD44 expression is associated with poor prognosis of HCC after hepatic resection10 . Although experimental evidence suggests that HA plays a critical role in tumorigenesis, the clinical significance of serum HA levels in patients with HCC remains largely unknown. The aim of the present study was to investigate the significance of serum HA levels in patients who had hepatic resection for HCC. Methods

Consecutive patients who underwent hepatic resection for HCC between September 1999 and March 2012 at Kumamoto University Hospital were included in this retrospective study. All patients had histologically confirmed HCC. This study was approved by the Human Ethics Review Committee of the Graduate School of Medicine, Kumamoto University, Kumamoto, Japan.

Serum hyaluronic acid assay Serum HA levels were measured within 4 weeks before surgery by an immunoturbidimetric automated latex assay using a commercially available kit (LPIA Ace HA; Mitsubishi Chemical Medience Corporation, Tokyo, Japan). Briefly, serum samples were aspirated and added to a reagent containing latex particles to enhance the HA–hyaluronic acid binding protein (HABP) reaction. This reaction was then quantitated by changes in the diffusion of a monochromatic light beam. The assay was linear for HA concentrations ranging from 10 to 800 ng/ml, and samples exceeding 800 ng/ml were diluted and retested. Normal serum HA levels are less than 50 ng/ml. The coefficient of variance is less than 15 per cent for serum HA levels using this assay.

Hyaluronic acid staining of tissue sections Sample processing and haematoxylin and eosin staining were performed as described previously10 . Sections were incubated in biotinylated HABP (Hokudo, Sapporo, Japan), 2 µg/ml, overnight at 4◦ C. The specificity of the staining for HA was confirmed by digesting sections with hyaluronidase (Santa Cruz Biotechnology, Dallas, Texas, USA) before incubating with biotinylated HABP.

Surgical treatment Indications for hepatic resection and the type of liver resection for HCC were based on the results of preoperative diagnostic imaging, intraoperative ultrasonography, and  2014 BJS Society Ltd Published by John Wiley & Sons Ltd

assessment of liver function. Briefly, operative decisions were based on an algorithm that included presence of ascites, total serum bilirubin level, indocyanine green tolerance test, and hepatic function as determined by technetium-99 m-diethylenetriaminepenta-acetic acid– galactosyl human serum albumin single-photon emission computed tomography (CT)/CT fusion images11 . Surgical procedures were as described previously12 . Major hepatic resection was considered as the excision of three or more liver segments13 .

Pathological assessments Pathological diagnoses and clinicopathological factors were established using the general guidelines for primary liver cancer of the Liver Cancer Study Group of Japan and the American Joint Committee on Cancer/International Union Against Cancer staging system14 – 16 . Severity of fibrosis and inflammation activity in the background liver were recorded using the Inuyama classification14,15 . Fibrosis severity (F score) was classified into four subgroups: F0 (no fibrosis); F1 (mild fibrosis), fibrous portal expansion; F2 (moderate fibrosis), bridging fibrosis; F3 (severe fibrosis), bridging fibrosis with distorted acinar architecture; and F4, cirrhosis. Liver inflammation activity (A score) was classified into four subgroups based on observation of piecemeal necrosis, spotty necrosis or bridging necrosis: A0, no activity; A1, mild activity; A2, moderate activity; and A3, severe activity. Vascular invasion was defined as portal vein (third branch, second branch, first branch or trunk) or hepatic vein (hepatic vein trunk or inferior vena cava) invasion by macroscopic examination of resected specimens.

Patient follow-up All patients were followed up with physical examination, determination of α-fetoprotein and des-γcarboxyprothrombin levels, and dynamic CT or magnetic resonance imaging (MRI) every 3 months for the first and second year after surgery, and every 6 months thereafter. Recurrences were defined as the appearance of a lesion with radiological features typical of HCC as confirmed on ultrasound imaging, CT and MRI. If recurrence was detected, the patient received further treatment with hepatic resection, radiofrequency ablation or transarterial chemoembolization. Cumulative recurrence-free survival (RFS) was defined as the interval between surgery and date of first recurrence, death, or last follow-up. Overall survival (OS) was calculated from surgery to death or last follow-up. Postoperative complications were graded as defined by the Clavien–Dindo classification17,18 . www.bjs.co.uk

BJS

Serum hyaluronic acid levels and hepatocellular carcinoma

Statistical analysis Categorical variables were compared using the χ2 test. Multiple logistic regression analyses were performed to confirm the relationship between variables and serum HA level. RFS and OS curves were determined with the Kaplan–Meier method and compared according to serum HA concentration with the log rank test. To investigate the prognostic impact of preoperative serum HA level, multivariable Cox proportional hazards regression analyses were performed. Only factors demonstrating an association with RFS and OS with P < 0·100 on univariable analysis were included in multivariable analysis. To validate the cut-off value for preoperative serum HA concentration, the C statistic for survival data was used with multivariable Cox regression, which is calculated by means of the timedependent receiver operating characteristic (ROC) curve19 . Table 1

To investigate the impact of serum HA on HCC progression unrelated to liver fibrosis, the relationship between clinicopathological factors and serum HA levels was analysed in a subgroup of 199 patients with HCC without severe liver fibrosis (defined as an F score of 0, 1 or 2). Statistical analyses were performed with Ekuseru-Toukei 2008 software (Social Survey Research Information Company, Tokyo, Japan) and R 2·15·3 (http://www.R-project.org). Differences were considered significant at P < 0·050. Results

Patient characteristics and preoperative serum hyaluronic acid level During the study interval, 605 consecutive patients underwent hepatic resection for HCC. Ninety-nine

Relationship of patient characteristics and preoperative serum hyaluronic acid levels in univariable and multivariable analysis Multivariable analysis† Low serum HA (n = 283)*

High serum HA (n = 223)*

95 (33·6) 188 (66·4)

96 (43·0) 127 (57·0)

236 (83·4) 47 (16·6)

169 (75·8) 54 (24·2)

94 (33·2) 189 (66·8)

38 (17·0) 185 (83·0)

125 (44·2) 158 (55·8)

141 (63·2) 82 (36·8)

95 (33·6) 188 (66·4)

100 (44·8) 123 (55·2)

35 (12·4) 248 (87·6)

54 (24·2) 169 (75·8)

152 (53·7) 131 (46·3)

172 (77·1) 51 (22·9)

131 (46·3) 152 (53·7)

163 (73·1) 60 (26·9)

78 (27·6) 205 (72·4)

109 (48·9) 114 (51·1)

35 (12·4) 248 (87·6)

69 (30·9) 154 (69·1)

5 (1·8) 278 (98·2)

11 (4·9) 212 (95·1)

Age (years) > 70 ≤ 70 Sex M F HBsAg status Positive Negative HCV-Ab status Positive Negative No. of tumours Multiple Solitary Serum albumin (g/dl) ≤ 3·5 > 3·5 ICGR15 (%) > 10 ≤ 10 Platelet count (× 104 /µl) ≤ 15 > 15 F score‡ F4 F0–3 A score‡ A3 A0–2 Surgical margin Positive Negative

Univariable P§

Odds ratio

0·029

P 0·309

1·25 (0·81, 1·91) 1·00 (reference) 0·034

0·154 0·69 (0·42, 1·15) 1·00 (reference)

< 0·001

0·014 0·49 (0·28, 0·86) 1·00 (reference)

< 0·001

0·867 1·04 (0·65, 1·67) 1·00 (reference)

0·001

0·138 1·36 (0·91, 2·05) 1·00 (reference)

< 0·001

0·079 1·60 (0·95, 2·70) 1·00 (reference)

< 0·001

< 0·001 2·11 (1·36, 3·26) 1·00 (reference)

< 0·001

< 0·001 2·25 (1·47, 3·43) 1·00 (reference)

< 0·001

0·032 1·60 (1·04, 2·46) 1·00 (reference)

< 0·001

0·003 2·15 (1·29, 3·57) 1·00 (reference)

0·043

0·073 3·05 (0·90, 10·30) 1·00 (reference)

Values in parentheses are *percentages and †95 per cent confidence intervals. ‡Fibrosis severity (F score) and inflammation activity (A score) in background liver were recorded according to the Inuyama classification. HA, hyaluronic acid; HBsAg, hepatitis B surface antigen; HCV-Ab, hepatitis C virus antibody; ICGR15, indocyanine green retention rate at 15 min. §χ2 test.

 2014 BJS Society Ltd Published by John Wiley & Sons Ltd

www.bjs.co.uk

BJS

K. Mima, T. Beppu, T. Ishiko, A. Chikamoto, S. Nakagawa, H. Hayashi et al.

100

100 Low serum HA High serum HA

80 Overall survival (%)

Recurrence-free survival (%)

80

60

40

60

40

20

20

0

1

2

0

3

a

183 124

111 65

2

3

Time after resection (years)

Time after resection (years) No. at risk Low serum HA 283 High serum HA 223

1

76 38

Recurrence-free survival

No. at risk Low serum HA High serum HA

b

283 223

247 182

172 144

128 98

Overall survival

Fig. 1 Kaplan–Meier survival curves for a recurrence-free and b overall survival after hepatic resection in 506 patients with hepatocellular carcinoma and low or high serum hyaluronic acid (HA) levels. a P < 0·001, b P = 0·001 (log rank test)

patients were excluded as their preoperative serum HA level had not been determined. The remaining 506 patients were analysed. Median age was 66 (range 30–86) years, and 405 (80·0 per cent) were men; 132 patients (26·1 per cent) were hepatitis B surface antigen (HBsAg)-positive and 266 (52·6 per cent) were hepatitis C virus antibody (HCVAb)-positive. The median serum HA level was 84 (i.q.r. 41–168) ng/ml. Serum HA levels increased with increasing severity of liver fibrosis (Fig. S1A, supporting information). C statistics for survival data were estimated to validate the cut-off value for preoperative serum HA; serum HA cutoff values of at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 and 150 ng/ml were tested (Fig. S1B, supporting information). Because scores for both RFS and OS were highest when high serum HA was defined as 100 ng/ml or above, this was assumed to be a valid cut-off value for serum HA concentration. Based on this cut-off, the 506 patients were divided into a high HA group (223 patients) and a low HA group (283). Comparison of the background characteristics of these two groups is shown in Table 1. In univariable analysis, age, sex, HBsAg positivity, HCV-Ab positivity, number of tumours, serum albumin, indocyanine green retention rate at 15 min (ICGR15), platelet count, liver fibrosis severity, liver inflammation activity and surgical margin positivity were significantly associated with serum HA level. There  2014 BJS Society Ltd Published by John Wiley & Sons Ltd

were no significant differences in surgical factors, including extent of surgery, operating time, blood loss, red blood cell transfusion and postoperative complications (Table S1, supporting information). In multivariable analysis HBsAg positivity (P = 0·014), high ICGR15 (P < 0·001), low platelet count (P < 0·001), liver fibrosis severity (P = 0·032) and liver inflammation activity (P = 0·003) remained significant independent risk factors for high serum HA concentration (Table 1).

Prognosis and preoperative serum hyaluronic acid level The median length of follow-up was 32 (i.q.r. 17–58) months. High serum HA concentration was associated with shorter RFS (P < 0·001) and OS (P = 0·001) (Fig. 1), and recurrence within the first 2 years after resection (defined as early recurrence): 55·2 per cent (123 of 223) versus 37·5 per cent (106 of 283) in the low HA group (P < 0·001).

Prognostic impact of preoperative serum hyaluronic acid level after hepatic resection Factors associated with RFS and OS with P < 0·100 in univariable analysis (Tables S3 and S4, supporting information) were included in the multivariable analysis (Table 2 and Table S2, supporting information). Multivariable analysis www.bjs.co.uk

BJS

Serum hyaluronic acid levels and hepatocellular carcinoma

Multivariable Cox regression analysis of recurrence-free and overall survival in 506 patients with hepatocellular carcinoma after hepatic resection

Table 2

Hazard ratio Recurrence-free survival Age > 70 years Male sex Multiple tumours Vascular invasion* Serum albumin ≤ 3·5 g/dl AFP > 20 ng/ml Red blood cell transfusion Postoperative complications† Serum HA level ≥ 100 ng/ml Overall survival Tumour size > 3 cm Multiple tumours Serum albumin ≤ 3·5 g/dl AFP > 20 ng/ml Red blood cell transfusion Serum HA level ≥ 100 ng/ml

P

1·51 (1·18, 1·94) 1·45 (1·07, 1·96) 1·91 (1·49, 2·44) 1·56 (0·99, 2·46) 1·54 (1·15, 2·05) 1·34 (1·05, 1·71) 1·39 (0·95, 2·03) 1·49 (1·12, 1·99) 1·50 (1·17, 1·93)

0·001 0·016 < 0·001 0·052 0·004 0·021 0·093 0·006 0·002

1·92 (1·26, 2·94) 1·92 (1·34, 2·74) 2·25 (1·51, 3·34) 1·48 (1·04, 2·11) 2·01 (1·25, 3·24) 1·46 (1·03, 2·07)

0·003 < 0·001 < 0·001 0·031 0·004 0·033

Preoperative serum hyaluronic acid level in patients without severe liver fibrosis or cirrhosis

Values in parentheses are 95 per cent confidence intervals. *Portal vein (third branch, second branch, first branch or trunk) or hepatic vein (trunk of hepatic vein or inferior vena cava) invasion defined via macroscopic examination of resected specimens. †Defined as Clavien–Dindo grade III, IV or V. All variables included in multivariable analyses for recurrence-free and overall survival are shown in Table S2 (supporting information). AFP, α-fetoprotein; HA, hyaluronic acid.

revealed that high preoperative serum HA levels independently predicted poor RFS (hazard ratio (HR) 1·50, 95 per cent confidence interval 1·17 to 1·93; P = 0·002) and OS (HR 1·46, 1·03 to 2·07; P = 0·033). Age above 70 years was significantly associated with poor RFS in the multivariable analysis. With regard to

The relationship between clinicopathological factors and serum HA concentration in a subgroup of 199 patients with HCC without severe liver fibrosis or cirrhosis (defined as F score 0, 1 or 2) was analysed (Table S6, supporting information). In these patients high serum HA levels were significantly associated with increased age (P < 0·001), multiple tumours (P = 0·039) and early recurrence (P = 0·033). High serum HA levels tended to be associated with large tumour size more than low serum HA levels, but this correlation was not statistically significant (74 versus 59·2 per cent respectively; P = 0·055). High serum HA levels were associated with poor RFS (P < 0·001) and OS (P = 0·024) in this group of patients (Fig. 2).

Localization of hyaluronic acid in hepatocellular carcinoma To investigate the localization of HA in HCC tissues, HA was stained using biotinylated HABP (Fig. 3). HA staining was increased in the tumour stroma. The

Low serum HA High serum HA

100

100

80

Overall survival (%)

Recurrence-free survival (%)

clinicopathological factors, a significant correlation was demonstrated between age greater than 70 years and sex (P = 0·003), HBsAg negativity (P < 0·001), HCV-Ab positivity (P < 0·001), large tumour size (P < 0·001), low serum albumin level (P = 0·012), high ICGR15 (P = 0·001) and high serum HA level (P = 0·029) (Table S5, supporting information).

60 40 20

0

1

2

3

80 60 40 20

0

Time after resection (years) No. at risk Low serum HA 142 High serum HA 57

a

Recurrence-free survival

100 30

63 19

1

2

3

Time after resection (years) 43 11

No. at risk Low serum HA 142 High serum HA 57

b

126 41

98 32

68 19

Overall survival

Fig. 2 Kaplan–Meier survival curves for a recurrence-free and b overall survival after hepatic resection in 199 patients with hepatocellular carcinoma without severe liver fibrosis or cirrhosis, and with low or high serum hyaluronic acid (HA) levels. a P < 0·001, b P = 0·024 (log rank test)

 2014 BJS Society Ltd Published by John Wiley & Sons Ltd

www.bjs.co.uk

BJS

K. Mima, T. Beppu, T. Ishiko, A. Chikamoto, S. Nakagawa, H. Hayashi et al.

a

Haematoxylin and eosin

b

Incubation with HABP

c

After digestion with hyaluronidase

Localization of hyaluronic acid in hepatocellular carcinoma. a Haematoxylin and eosin staining (scale bar, 100 µm). Specific histochemistry with hyaluronic acid binding protein (HABP) b before and c after treatment with hyaluronidase (scale bars, 100 µm)

Fig. 3

intensity of stromal HA staining varied within each tumour. In contrast, tumour cells were negative for HA staining. Discussion

This study has demonstrated that high preoperative serum HA levels (defined as 100 ng/ml or more) independently predict RFS and OS after hepatic resection for HCC. Furthermore, in patients with HCC without severe fibrosis or cirrhosis, high serum HA levels were associated with multiple tumours, early recurrence and poor prognosis following hepatic resection for HCC. Consistent with previous studies5,6 , serum HA levels were associated with liver fibrosis severity, liver inflammation activity, low platelet count and high ICGR15, which reflects hepatic dysfunction. These results indicate that determination of serum HA concentration, which is readily clinically available, may have prognostic value for patients undergoing hepatic resection for HCC. Tumour recurrences after hepatic resection for HCC have been divided into intrahepatic metastases (true metastasis of the primary tumour) and de novo tumours20 . Intrahepatic metastases generally arise within the first 2 years after resection (defined as early recurrence). The present study showed that high serum HA levels were significantly associated with early recurrence. These findings suggest that HA may be associated with the potential for intrahepatic metastasis. In the present study, multivariable analysis revealed that age above 70 years was associated with poor RFS. This suggests that these patients may require careful monitoring for HCC recurrence after resection; further studies are needed to investigate the mechanisms of increased age underlying HCC recurrence. Accumulating experimental evidence suggests that HA plays a critical role in tumour progression4 .  2014 BJS Society Ltd Published by John Wiley & Sons Ltd

The phosphatidylinositol 3-kinase (PI3K)–Akt signalling pathway is inappropriately activated in many cancers, leading to increased cellular growth and survival21 . HA activates the PI3K–Akt signalling pathway, which promotes cell survival22 . This effect is reversed when the constitutive HA–CD44 interactions are inhibited23 . HA is also thought to be important for tumour invasiveness. The matrix metalloproteinases (MMPs) play important roles in cancer cell invasion and metastasis, including extracellular matrix degradation and facilitation of tumour growth at the secondary site24 . The HA–CD44 interaction promotes the binding of MMP9 and CD44, leading to increased tumour cell invasiveness and angiogenesis25 . These observations support the findings in this study that high serum HA levels were associated with a more aggressive HCC subtype, early recurrence and poor prognosis after hepatic resection. In the present study, HA staining was not detected in tumour cells of resected liver specimens, but was found in the tumour stroma. This suggests that HA accumulates mainly in tumour stroma in HCC. It is thought that the production of HA in stromal cells is stimulated by interaction with tumour cells4 . However, little is known about the mechanisms underlying HA accumulation in HCC. Therefore, further studies are needed to investigate these mechanisms and the association between serum HA levels and HA accumulation in HCC. A limitation of the present study is that it was retrospective, although it included more than 500 patients. Further clinical validation of serum HA levels in a large prospective trial is needed. Acknowledgements

The authors thank K. Miyake and N. Yokoyama for their valuable technical assistance. Disclosure: The authors declare no conflict of interest. www.bjs.co.uk

BJS

Serum hyaluronic acid levels and hepatocellular carcinoma

References 1 El-Serag HB. Hepatocellular carcinoma. N Engl J Med 2011; 365: 1118–1127. 2 Lim KC, Chow PK, Allen JC, Siddiqui FJ, Chan ES, Tan SB. Systematic review of outcomes of liver resection for early hepatocellular carcinoma within the Milan criteria. Br J Surg 2012; 99: 1622–1629. 3 Thelen A, Benckert C, Tautenhahn HM, Hau HM, Bartels M, Linnemann J et al. Liver resection for hepatocellular carcinoma in patients without cirrhosis. Br J Surg 2013; 100: 130–137. 4 Toole BP. Hyaluronan: from extracellular glue to pericellular cue. Nat Rev Cancer 2004; 4: 528–539. 5 Frebourg T, Delpech B, Bercoff E, Senant J, Bertrand P, Deugnier Y et al. Serum hyaluronate in liver diseases: study by enzymoimmunological assay. Hepatology 1986; 6: 392–395. 6 Par´es A, Deulofeu R, Gim´enez A, Caballer´ıa L, Bruguera M, Caballer´ıa J et al. Serum hyaluronate reflects hepatic fibrogenesis in alcoholic liver disease and is useful as a marker of fibrosis. Hepatology 1996; 24: 1399–1403. 7 Toole BP, Slomiany MG. Hyaluronan: a constitutive regulator of chemoresistance and malignancy in cancer cells. Semin Cancer Biol 2008; 18: 244–250. 8 Kosaki R, Watanabe K, Yamaguchi Y. Overproduction of hyaluronan by expression of the hyaluronan synthase Has2 enhances anchorage-independent growth and tumorigenicity. Cancer Res 1999; 59: 1141–1145. 9 Simpson MA, Wilson CM, McCarthy JB. Inhibition of prostate tumor cell hyaluronan synthesis impairs subcutaneous growth and vascularization in immunocompromised mice. Am J Pathol 2002; 161: 849–857. 10 Mima K, Okabe H, Ishimoto T, Hayashi H, Nakagawa S, Kuroki H et al. CD44s regulates the TGF-β-mediated mesenchymal phenotype and is associated with poor prognosis in patients with hepatocellular carcinoma. Cancer Res 2012; 72: 3414–3423. 11 Yoshida M, Shiraishi S, Sakaguchi F, Utsunomiya D, Tashiro K, Tomiguchi S et al. Fused 99 m-Tc-GSA SPECT/CT imaging for the preoperative evaluation of postoperative liver function: can the liver uptake index predict postoperative hepatic functional reserve? Jpn J Radiol 2012; 30: 255–262. 12 Beppu T, Ishiko T, Chikamoto A, Komori H, Masuda T, Hayashi H et al. Liver hanging maneuver decreases blood loss and operative time in a right-side hepatectomy. Hepatogastroenterology 2012; 59: 542–545.

 2014 BJS Society Ltd Published by John Wiley & Sons Ltd

13 Strasberg SM. Nomenclature of hepatic anatomy and resections: a review of the Brisbane 2000 system. J Hepatobiliary Pancreat Surg 2005; 12: 351–355. 14 Liver Cancer Study Group of Japan. The General Rules for the Clinical and Pathological Study of Primary Liver Cancer (5th edn, revised version). Kanehara: Tokyo, 2009. 15 Minagawa M, Ikai I, Matsuyama Y, Yamaoka Y, Makuuchi M. Staging of hepatocellular carcinoma: assessment of the Japanese TNM and AJCC/UICC TNM systems in a cohort of 13 772 patients in Japan. Ann Surg 2007; 245: 909–922. 16 Vauthey JN, Lauwers GY, Esnaola NF, Do KA, Belghiti J, Mirza N et al. Simplified staging for hepatocellular carcinoma. J Clin Oncol 2002; 20: 1527–1536. 17 Dindo D, Demartines N, Clavien PA. Classification of surgical complications: a new proposal with evaluation in a cohort of 6336 patients and results of a survey. Ann Surg 2004; 240: 205–213. 18 Clavien PA, Barkun J, De Oliveira ML, Vauthey JN, Dindo D, Schulick RD et al. The Clavien–Dindo classification of surgical complications: five-year experience. Ann Surg 2009; 250: 187–196. 19 Heagerty PJ, Zheng Y. Survival model predictive accuracy and ROC curves. Biometrics 2005; 61: 92–105. 20 Llovet JM, Schwartz M, Mazzaferro V. Resection and liver transplantation for hepatocellular carcinoma. Semin Liver Dis 2005; 25: 181–200. 21 Engelman JA. Targeting PI3K signalling in cancer: opportunities, challenges and limitations. Nat Rev Cancer 2009; 9: 550–562. 22 Sohara Y, Ishiguro N, Machida K, Kurata H, Thant AA, Senga T et al. Hyaluronan activates cell motility of v-Src-transformed cells via Ras-mitogen-activated protein kinase and phosphoinositide 3-kinase–Akt in a tumor-specific manner. Mol Biol Cell 2001; 12: 1859–1868. 23 Ghatak S, Hascall VC, Markwald RR, Misra S. Stromal hyaluronan interaction with epithelial CD44 variants promotes prostate cancer invasiveness by augmenting expression and function of hepatocyte growth factor and androgen receptor. J Biol Chem 2010; 285: 19 821–19 832. 24 Kessenbrock K, Plaks V, Werb Z. Matrix metalloproteinases: regulators of the tumor microenvironment. Cell 2010; 141: 52–67. 25 Yu Q, Stamenkovic I. Cell surface-localized matrix metalloproteinase-9 proteolytically activates TGF-beta and promotes tumor invasion and angiogenesis. Genes Dev 2000; 14: 163–176.

www.bjs.co.uk

BJS

K. Mima, T. Beppu, T. Ishiko, A. Chikamoto, S. Nakagawa, H. Hayashi et al.

Supporting information

Additional supporting information may be found in the online version of this article: Fig. S1 Serum hyaluronic acid levels in 506 patients with hepatocellular carcinoma by severity of liver fibrosis, and estimated C statistic for recurrence-free and overall survival by cut-off value of high serum hyaluronic acid concentration (TIFF file) Table S1 Univariable analysis of the association between patient characteristics and preoperative serum hyaluronic acid levels (Word document) Table S2 Multivariable Cox regression analysis of recurrence-free and overall survival after hepatic resection in 506 patients with hepatocellular carcinoma (Word document) Table S3 Univariable analysis of recurrence-free survival after hepatic resection in 506 patients with hepatocellular carcinoma (Word document) Table S4 Univariable analyses for overall survival after hepatic resection in 506 patients with hepatocellular carcinoma (Word document) Table S5 Relationships between patient characteristics and patient age (Word document) Table S6 Relationships between patient characteristics and preoperative serum hyaluronic acid levels in 199 patients with hepatocellular carcinoma without severe liver fibrosis or cirrhosis (Word document)

If you wish to comment on this, or any other article published in the BJS, please visit the on-line correspondence section of the website (www.bjs.co.uk). Electronic communications will be reviewed by the Correspondence Editor and a selection will appear in the correspondence section of the Journal. Time taken to produce a thoughtful and well written letter will improve the chances of publication in the Journal.

 2014 BJS Society Ltd Published by John Wiley & Sons Ltd

www.bjs.co.uk

BJS

Preoperative serum hyaluronic acid level as a prognostic factor in patients undergoing hepatic resection for hepatocellular carcinoma.

Hyaluronic acid (HA) probably plays a critical role in tumorigenesis. The clinical significance of serum HA concentration in patients with hepatocellu...
212KB Sizes 0 Downloads 0 Views