Abstract
Purpose
Lysosomal protein transmembrane 4 beta-35 (LAPTM4B-35) is a tetra-transmembrane glycoprotein that is abundantly localized on membrane-bound organelles including endosomes and lysosomes, and promotes cell proliferation and tumorigenesis through regulation of cell cycle and signaling pathways. The aim of the present study is to determine the potential clinical implications of LAPTM4B-35 expression in hepatocellular carcinoma (HCC).
Methods
Immunohistochemistry assay was used to determine the expression of LAPTM4B-35 protein in normal and HCC tissues from 71 patients. The correlations of LAPTM4B-35 expression with clinicopathological parameters, including gender, age, background liver, viral status, tumor size, portal vein invasion, histopathological differentiation, serum AFP level, TNM staging and recurrence of HCC were assessed by Chi-squared test. Patient survival and their differences were determined by Kaplan–Meier method and log-rank test. Cox regression (Proportional hazard model) was adopted for multivariate analysis of prognostic factors.
Results
LAPTM4B-35 immunoreactivity was negative or low in normal liver tissues, but high in HCC tissues (51/71, 71.8%). The overexpression of LAPTM4B-35 was significantly associated with recurrence, TNM staging and portal vein invasion of HCC. Patients with high LAPTM4B-35 expression had significantly poorer overall survival (OS) and disease-free survival (DFS) (both P < 0.001) when compared with patients with the low expression of LAPTM4B-35. On multivariate analysis, LAPTM4B-35 expression was found to be an independent prognostic factor for OS and DFS (P = 0.018 and P = 0.001, respectively).
Conclusion
LAPTM4B-35 expression showed a strong association with the potencies of recurrence and metastasis and progression of HCC, and that may be applied as a novel marker for the prediction of recurrence and metastasis potency of HCC, and helpful for improving the diagnosis, prognosis and treatment of HCC.
Keywords: Lysosomal protein transmembrane 4 beta, Immunohistochemistry, HCC, Recurrence, Survival
Introduction
Hepatocellular carcinoma (HCC) is one of the five most common cancers worldwide and is the third leading cause of death from cancer. Although HCC occurs mostly in Southeast Asia and sub-Sahara Africa, the incidence and mortality rate of HCC are increasing in the United States and Western Europe over the past 25 years (Bosch et al. 2005; Llovet et al. 2003). The exact molecular mechanisms underlying hepatocarcinogenesis have not yet been elucidated, despite there are many evidences suggesting that risk factors, such as aflatoxin, alcohol, hepatitis B virus, hepatitis C virus and cirrhosis, etc. contribute to malignant transformation of normal liver cells (Farazi and Depinho 2006; Suriawinata and Xu 2004). Moreover, the development of HCC is a multistep process associated with complicated changes in host gene-expression patterns including tumor-suppressor genes and oncogenes and some cell-cycle regulators during the process of initiation and progression of HCC. HCC is so complex in etiology and pathogenesis, and different risk factors lead to different molecular pathways making pathogenesis of HCC more complex to interpret. Furthermore, the high frequency of tumor recurrence is a major cause for the high mortality of HCC after surgical resection and/or other therapeutic treatments (Kaibori et al. 2009; Kamiyama et al. 2009; Tanaka et al. 2009). To solve this big knotty problem, more prognostic markers that could predict the progress of tumor and/or be used as a target in individualization of adjuvant therapy must be explored to aid in the prediction of recurrence and prognosis of HCC so that appropriate therapeutic strategies could be applied in advance.
Lysosomal protein transmembrane-4 beta (LAPTM4B), a novel gene upregulated in HCC, was cloned using fluorescence differential display, RACE, and RT-PCR in our laboratory (Shao et al. 2003). It is mapped to chromosome 8q22.1 with seven exons and encodes a 35-kDa protein, LAPTM4B-35, which is a type-III transmembrane protein with four putative transmembrane regions. Northern blot analyses show that the levels of LAPTM4B mRNA are fairly low in adult normal liver, but markedly elevated in 87.3% (48/55) of the HCC tissues and correlated significantly with pathological grades of the disease (Liu et al. 2003, 2004; Shao et al. 2003). We also found that LAPTM4B-35 overexpressing cells greatly activated PI3K-AKT signaling pathway and displayed a higher survival rate of anti-apoptosis, faster proliferation rate, an increased efficiency of colony formation in soft agar and serum-independence growth, promoted migration and invasion, as well as tumorigenicity (unpublished data), suggesting that upregulated LAPTM4B-35 might act as an oncogene and involves in tumorigenesis and metastasis of HCC. But so far, there has not been any evidence concerning tumor recurrence, metastasis and clinicopathological relevance with LAPTM4B-35 upregulation in HCC tissues.
The present work aimed to investigate the expression of LAPTM4B-35 in HCC tissues by immunohistochemical and its association with clinicopathological features and post-resectional survival. Our results suggested that LAPTM4B-35 staining score can be used as an independent marker for tumor recurrence and prognosis of patients with HCC.
Materials and methods
Patients
Tissue samples were obtained from 71 patients with HCC who underwent surgical resection in Union Hospital Affiliated to Tongji Medical College from 2000 to 2002. The staging for each HCC was evaluated according to TNM staging guidelines. Among them, 59 were males and 12 were females, with the median age of 49 (range 33–74) years. The clinicopathological features of patients, including gender, age, background liver, viral status, tumor size, portal vein invasion, histopathological differentiation, serum AFP level, TNM staging and recurrence of HCC are summarized in Table 1. The Institutional Ethics Committee approval for the project was achieved before the study was started.
Table 1.
Relationship between LAPTM4B-35 expression and clinicopathological features of HCC
| Variables | Patients | LAPTM4B-35 expression | P a | |
|---|---|---|---|---|
| Low | High | |||
| All cases | 71 | |||
| Gender | ||||
| Male | 59 | 17 | 42 | 0.789 |
| Female | 12 | 3 | 9 | |
| Age (years) | ||||
| <60 | 47 | 12 | 35 | 0.489 |
| ≥60 | 24 | 8 | 16 | |
| Background liver | ||||
| Normal liver | 3 | 1 | 2 | 0.054 |
| Chronic liver | 21 | 10 | 11 | |
| Cirrhosis | 47 | 9 | 38 | |
| Viral status | ||||
| Hepatitis virus B | 51 | 14 | 37 | 0.518 |
| Hepatitis virus C | 12 | 5 | 7 | |
| Both B and C | 2 | 0 | 2 | |
| Non-B and non-C | 6 | 1 | 5 | |
| Tumor size | ||||
| <5 cm | 38 | 10 | 28 | 0.710 |
| ≥5 cm | 33 | 10 | 23 | |
| Portal vein invasion | ||||
| No | 52 | 20 | 32 | 0.001 |
| Yes | 19 | 0 | 19 | |
| Histopathological differentiation | ||||
| WD | 21 | 8 | 13 | 0.107 |
| MD | 26 | 9 | 17 | |
| PD | 24 | 3 | 21 | |
| Serum AFP level | ||||
| <25 ng/ml | 35 | 13 | 22 | 0.097 |
| ≥25 ng/ml | 36 | 7 | 29 | |
| TNM stage | ||||
| I–II | 25 | 14 | 11 | <0.001 |
| III–IV | 46 | 6 | 40 | |
| Recurrence | ||||
| No | 17 | 13 | 4 | <0.001 |
| Yes | 54 | 7 | 47 | |
LAPTM4B Lysosomal protein transmembrane 4 beta, HCC hepatocellular carcinoma
aChi-square test
Immunohistochemical staining
Lysosomal protein transmembrane 4 beta-35 expression was detected immunohistochemically for paraffin-embedded specimens from 71 patients with HCC. Surgical specimens were fixed in 10% formalin, embedded in paraffin, and were sectioned at a thickness of 4 μm. For heat-induced epitope retrieval, deparaffinized sections were soaked in 10 mM citrate buffer (pH 6.0) and treated at 95°C for 30 min in a microwave oven. IHC staining was performed using the avidin–biotin immunoperoxidase technique. Endogenous peroxidase activity was first blocked by incubating with 0.3% H2O2 in methanol for 15 min, and non-specific immunoglobulin binding was then blocked by incubating with 10% normal goat serum for 10 min. Sections were incubated at room temperature for 2 h with the purified anti-LAPTM4B-35 polyclonal antibody(LAPTM4B-N10-pAb) at 1:50 dilution. This antibody was raised in our laboratory, as previously reported (Yang et al. 2008). The sections were rinsed and incubated for 30 min with biotinylated second antibody. After washing, the sections were incubated for 30 min with horseradish peroxidase-conjugated streptavidin, and finally treated with 3, 3′-diaminobenzidine tetrahydrochloride in 0.01% H2O2 for 10 min. The slides were counterstained with Meyer’s hematoxylin. As a negative control, the primary antibody was replaced with normal rabbit IgG at an appropriate dilution.
Staining assessment and scoring
Lysosomal protein transmembrane 4 beta-35 expression levels were classified semiquantitatively based on the total combined scores of the percent positive staining tumor cells together with the staining intensity. A tumor was scored as ‘0’ if <5% of tumor cells stained positive, ‘1’ if 5–50% were positive, and ‘2’ if >50% of cells were positive stained. The staining intensity was scored as ‘0’ if no cells stained or there was only weak staining, ‘1’ if there was moderate staining, and ‘2’ in cases of strong staining. The final score of LAPTM4B-35 expression was defined as ‘Low LAPTM4B-35 expression’ if the sum of the positivity score and the staining intensity score was 0–2, and ‘High LAPTM4B-35 expression’ if the sum was 3–4. In each case, at least three different areas of tumor were valuated, and the mean of the results was taken as the final expression score. The scoring procedure was carried out twice by two independent pathologists without any knowledge of the clinical data and corresponding H&E slides. The concordance rate between the two primary pathologists was greater than 95%. In cases of significant disagreement, the slides in question were re-reviewed simultaneously by the original two pathologists, together with a third pathologist at a multiheaded microscope, in order to resolve the divergence of opinion.
Statistical analysis
The Chi-square test was used to show differences of categorical variables. Patient survival and their differences were determined by Kaplan–Meier method and log-rank test. Cox regression (Proportional hazard model) was adopted for multivariate analysis of prognostic factors. Statistical software package SPSS11.5 (SPSS Inc., Chicago, IL, USA) was employed for all analyses. Statistically significant P value was defined as <0.05.
Results
Clinical profiles of the patients
Of the 71 patients, 21 (29.6%) were histopathologically well differentiated, 26 (36.6%) were moderately differentiated and 24 (33.8%) were poorly differentiated. Portal vein invasion of tumor cells existed in 19 (26.8%) cases of total 71 patients and did not exist in other 52 (73.2%) patients (Table 1). Up to August 31, 2008 (the census date), 22 (31%) patients were alive, 49 (69%) patients had died of disease. Follow-up ranged from 3 to 93 months (median, 36 months).
Expression of LAPTM4B-35 and its relationship with clinicopathological features of hepatocellular carcinoma
Lysosomal protein transmembrane 4 beta-35 high expression was detected in HCC tissues from 51 patients (71.8%) in accordance with afore-mentioned criteria. Normal liver tissue as a control exhibited negative staining (Fig. 1a), 20 and 51 HCC samples exhibited staining score 0 and 1, respectively (Fig. 1b–d). The staining scores were significantly associated with recurrence, TNM staging and portal vein invasion of HCC (Table 1; P < 0.05), but not with age, gender, background liver, histopathological differentiation, viral status, tumor size and serum AFP level of tumor antigen marker (Table 1; P > 0.05).
Fig. 1.
Representative staining pattern of LAPTM4B-35 expression detected by IHC (positivity + intensity). a Non-cancer liver tissue, b–d HCC tissues. b Score of 0 + 0, c score of 1 + 1, d score of 2 + 2 (original magnification ×200)
The impact of LAPTM4B-35 expression on overall and disease-free survival in hepatocellular carcinomas
Using Kaplan–Meier method and log-rank test, HCC tissues with higher staining score of LAPTM4B-35 were correlated to shorter overall or disease-free survival (DFS) of patients (Fig. 2a, b; Table 2; log-rank value 21.40 and 26.36; P < 0.001 and 0.001, respectively). Besides, the survival benefits were also found in those with earlier TNM staging, higher histopathological differentiation grade, absence of portal vein invasion and better background liver for overall or DFS (Table 2; P < 0.05), whereas others were not of predictive values (Table 2; P > 0.05). Multivariate Cox regression analysis enrolling above-mentioned significant parameters revealed that LAPTM4B-35 staining score (RR 3.123, 95%CI, 1.221–7.992, P = 0.018), portal vein invasion (RR 3.906, 95%CI, 1.928–7.911, P < 0.001) and TNM stage (RR 2.699, 95%CI, 1.041–6.999, P = 0.041) were independent prognostic markers for overall survival (OS) of patients with HCC (Table 3; P < 0.05). Meanwhile, LAPTM4B-35 staining score (RR 4.418, 95%CI, 1.856–10.518, P = 0.001) and portal vein invasion (RR 2.972, 95%CI, 1.481–5.961, P = 0.002) were independent prognostic markers for disease-free survival DFS of patients with HCC (Table 3; P < .05).
Fig. 2.
Comparison of survival of patients with HCC after resection based on staining scores of LAPTM4B-35. Score 0, black solid line. Score 1, black dashed line. a Overall survival after resection. b Disease-free survival after resection
Table 2.
Univariate survival analysis of OS and DFS in 71 patients with HCC
| Variables | No. of cases | OS | DFS | ||||
|---|---|---|---|---|---|---|---|
| Mean ± SE (month) | 95% CI | P a | Mean ± SE (month) | 95% CI | P a | ||
| Gender | |||||||
| Male | 59 | 40 ± 5 | 31–49 | 0.323 | 34 ± 5 | 25–43 | 0.549 |
| Female | 12 | 44 ± 8 | 28–59 | 37 ± 8 | 22–51 | ||
| Age (years) | |||||||
| <60 | 47 | 41 ± 5 | 31–51 | 0.597 | 34 ± 5 | 24 –44 | 0.650 |
| ≥60 | 24 | 38 ± 6 | 27–50 | 32 ± 6 | 20–44 | ||
| Background liver | |||||||
| Normal liver | 3 | 69 ± 4 | 62–77 | 0.004 | 65 ± 4 | 56–74 | 0.002 |
| Chronic liver | 21 | 62 ± 8 | 46–78 | 57 ± 9 | 40–74 | ||
| Cirrhosis | 47 | 30 ± 4 | 22–38 | 23 ± 4 | 15–31 | ||
| Viral status | |||||||
| Hepatitis virus B | 51 | 39 ± 5 | 30–49 | 0.230 | 33 ± 5 | 24–43 | 0.510 |
| Hepatitis virus C | 12 | 41 ± 10 | 20–61 | 36 ± 11 | 15–58 | ||
| Both B and C | 2 | 19 ± 10 | 0–37 | 9 ± 4 | 2–15 | ||
| Non-B and non-C | 6 | 61 ± 9 | 43–79 | 51 ± 8 | 34–67 | ||
| Tumor size | |||||||
| <5 cm | 38 | 41 ± 6 | 31–52 | 0.967 | 34 ± 5 | 23–44 | 0.770 |
| ≥5 cm | 33 | 42 ± 6 | 30–54 | 36 ± 6 | 23–48 | ||
| Portal vein invasion | |||||||
| No | 52 | 53 ± 5 | 43–63 | <0.001 | 45 ± 5 | 36–55 | <0.001 |
| Yes | 19 | 12 ± 1 | 10–15 | 7 ± 1 | 5–10 | ||
| Histopathological differentiation | |||||||
| WD | 21 | 60 ± 8 | 44–75 | 0.005 | 49 ± 8 | 34–65 | 0.013 |
| MD | 26 | 41 ± 6 | 29–53 | 36 ± 6 | 23–48 | ||
| PD | 24 | 25 ± 5 | 15–36 | 19 ± 6 | 8–30 | ||
| Serum AFP level | |||||||
| <25 ng/ml | 35 | 45 ± 6 | 33–57 | 0.422 | 38 ± 6 | 27–50 | 0.413 |
| ≥25 ng/ml | 36 | 37 ± 5 | 27–47 | 30 ± 5 | 20–41 | ||
| TNM stage | |||||||
| I–II | 25 | 73 ± 6 | 61–85 | <0.001 | 64 ± 6 | 52–76 | <0.001 |
| III–IV | 46 | 25 ± 4 | 18–32 | 19 ± 4 | 11–26 | ||
| LAPTM4B-35 expression | |||||||
| Low | 20 | 76 ± 6 | 64–88 | <0.001 | 73 ± 7 | 60–85 | <0.001 |
| High | 51 | 26 ± 3 | 20–32 | 19 ± 3 | 13–25 | ||
LAPTM4B Lysosomal protein transmembrane 4 beta, HCC hepatocellular carcinoma, OS overall survival, DFS disease-free survival
aLog-rank test
Table 3.
Multivariate survival analysis of OS and DFS in 71 patients with HCC
| Variables | OS | DFS | ||||
|---|---|---|---|---|---|---|
| RR | 95%CI | P a | RR | 95%CI | P a | |
| TNM stage | 2.699 | 1.041–6.999 | 0.041 | 1.900 | 0.812–4.444 | 0.139 |
| Background liver | 1.386 | 0.682–2.816 | 0.367 | 1.664 | 0.881–3.145 | 0.117 |
| Portal vein invasion | 3.906 | 1.928–7.911 | <0.001 | 2.972 | 1.481–5.961 | 0.002 |
| Histopathological differentiation | 1.412 | 0.926–2.153 | 0.109 | 1.344 | 0.891–2.029 | 0.159 |
| LAPTM4B-35 expression | 3.123 | 1.221–7.992 | 0.018 | 4.418 | 1.856–10.518 | 0.001 |
LAPTM4B Lysosomal protein transmembrane 4 beta, HCC hepatocellular carcinoma, OS overall survival, DFS disease-free survival, RR relative risk, CI confidence interval
aCox regression test
Discussion
In this study, we analyzed the expression of LAPTM4B-35 in 71 HCC surgical specimens by IHC. We found that the high expression of LAPTM4B-35 was associated with high tumor recurrence, metastasis and poor survival, suggesting that LAPTM4B-35 could be an independent prognostic factor. This is the first study to demonstrate an association of clinicopathological features and prognostic impact of LAPTM4B-35 expression in HCC using clinical samples in detail.
We analyzed the correlation of LAPTM4B-35 expression with clinicopathological parameters in HCC. High LAPTM4B-35 expression was significantly correlated with recurrence, TNM staging and portal vein invasion of HCC, but not with age, gender, background liver, histopathological differentiation, viral status, tumor size and serum AFP level of tumor antigen marker in HCC. Moreover, patients with high LAPTM4B-35 expression had significantly poorer OS and DFS when compared with patients with the low expression of LAPTM4B-35. Multivariate analysis demonstrated that among the factors analyzed, with the exception of portal vein invasion, LAPTM4B-35 expression was an independent prognostic factor for OS and DFS in patients with HCC. These results clearly demonstrated that high LAPTM4B-35 expression is associated with the disease progression of HCC, and the patients with high LAPTM4B-35 expression had an unfavorable clinical outcome. This raises the possibility that LAPTM4B-35 may be a prognostic parameter for HCC which is as or more reliable than the clinicopathological factors currently in use, and suggests the possibility to use LAPTM4B-35 as a target in individualization of adjuvant therapy. It should be noted that several other molecular markers, such as LAMA4, CCT and TGF-alpha have been identified which show association with prognosis in HCC (Huang et al. 2008; Isokawa et al. 1999; Yeh et al. 2007). However, it is not yet clear whether such markers are effective for clinical application as replacements for, or in addition to, the prognostic factors currently in use. As such, further investigation is called for to determine whether combined detection of LAPTM4B-35 together with some of these molecules would be valuable in enhancing prognostic effectiveness.
The close relationship between LAPTM4B-35 expression and clinicopathological features predicted that LAPTM4B-35 might play an important role in carcinogenesis and tumor progression. Currently, there have been some clues that are able to help to explain its mechanisms. As described by some authors, the over-expression of LAPTM4B-35 could activate some proto-oncogenes, such as c-myc, c-jun and c-fos, and promote malignant transformation in some cell lines (He et al. 2003; Liu et al. 2003). Hence, the LAPTM4B gene may function as a proto-oncogene via its translation product, LAPTM4B-35 protein. Furthermore, coimmunoprecipitation assay indicated that LAPTM4B-35 interacted with integrin-α6ß1 in BEL7402 cells, which were enhanced by LN-1, and may play an important role in the integrin-α6 mediated signal transduction pathways (Liu et al. 2003). Moreover, a high homology (46%) to LAPTM4A suggested that LAPTM4B might have a similar role in multidrug resistance (Cabrita et al. 1999; Hogue et al. 1999). Therefore, targeted inhibition of LAPTM4B and/or LAPTM4B-35 protein levels might be a new idea for therapy of HCC. Certainly, further strong supports from basic investigations are needed.
Moreover, our results also suggested that LAPTM4B-35 staining score can be used as a novel marker for predicating tumor recurrence of patients with HCC. Mechanisms about this remain to be clarified, but the activation of PI3K-AKT signaling pathway may be one important reason to interpret this phenomenon. As reported by published paper, p-Akt was recognized as a risk factor for recurrence and poor prognosis of HCC (Nakanishi et al. 2005). The serine/threonine kinase Akt was identified originally as an oncogene in mouse thymoma and activated fully by phosphorylation after activation of PI3K, regulating multiple processes, such as apoptosis, cell proliferation and glucose utilization (Bellacosa et al. 1991; Datta et al. 1999; Kandel and Hay 1999). And based on the recent finding in our laboratory that LAPTM4B-35 can interact with p85α subunit of PI3K to activate PI3K-AKT pathway (unpublished data), we may infer that LAPTM4B-35 promoted the recurrence and metastasis of tumor through phosphorylating AKT and its staining score could be a novel marker for the predication of tumor recurrence in patients with HCC.
In conclusion, we demonstrated, here, that LAPTM4B-35 is overexpressed in a large proportion of patients with HCC and high LAPTM4B-35 expression correlated with the disease progression and poor clinical outcome in HCC. Furthermore, LAPTM4B-35 proved to be a risk factor for tumor recurrence and independent molecular marker of prognosis in HCC and may become a novel molecular target in the strategies for the prediction of tumor recurrence and prognosis or treatment of this disease.
Footnotes
Hua Yang and Fu Xia Xiong have contributed equally to this work
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