ASPM Is a Novel Marker for Vascular Invasion, Early Recurrence, and Poor Prognosis of Hepatocellular Carcinoma

Abnormal spindle-like microcephaly associated (ASPM) gene is the human orthologue of the Drosophila abnormal spindle (asp) and the most commonly mutated gene of autosomal recessive primary microcephaly (1–5). The homozygous semilethal mutation of Drosophila asp causes abnormal spindles, frequent polyploid cells, and cytokinesis failure (6), leading to arrest of neuroblasts in metaphase (7, 8) and larval-pupal lethality (6, 9). In human, the defective neurogenesis caused by homozygous mutation of ASPM leads to microcephaly and mental retardation (4, 10). The ASPM gene encodes a large protein of 3,477 amino acids with a NH₂-terminal microtubule-binding domain, two calponin homology domains, 74 repeated calmodulin-binding isoleucine-glutamine domains, and a COOH-terminal region (4, 11, 12). The NH₂-terminal microtubule-binding domain is highly conserved in eukaryotes, suggesting that ASPM may possess the fundamental function in cytokinesis. However, the multidomain feature suggests that ASPM have more diverse function than the Drosophila counterpart.

Drosophila Asp protein associates with centrosomes and is involved in organizing microtubules at the spindle poles in mitosis and meiosis (6, 8, 9, 11, 13–16). Centrosome defects cause chromosome misaggregation and aneuploidy, leading to genetic instability, a major driving force for malignant transformation and tumor progression (17–22). Recently, ASPM is shown to express in nearly all transformed human cell lines and in multiple fetal tissues (23, 24). These findings suggest ASPM play an important role in cell cycle progression and cell proliferation in embryonic development and tumorigenesis.

Using differential display analysis of gene expression profile of hepatocelllular carcinoma (HCC), we found frequent overexpression of ASPM, which is located at chromosome 1q31, a region with frequent gain in HCC (25, 26). In this study, we showed that ASPM was often overexpressed in human HCC and associated with tumor progression, early tumor recurrence (ETR), and poor prognosis.

Differential display, reverse transcription-PCR, and definition of ASPM overexpression. Differential display was done using anchor primer HT11C (5′-AAGCTTTTTTTTTTTC-3′) and arbitrary primer HAP7 (5′-AAGCTTAACGAGG-3′) from the RNAimage kit (GenHunter) as described (27–31).

Reverse transcription-PCR assays in the linear range were used for the measurements of ASPM mRNA levels using ribosomal protein S26 (RPS26; 32) or porphobilinogen deaminase (PBGD) mRNA for internal controls (33) as described (29–31, 34). Primers for ASPM spanning intron 20 to avoid DNA contamination are ASPM-forward (5′-TAAAAAGACATCGAGCTGCTT-3′) and ASPM-reverse (5′-CCTCTCCATAATGCCTGAATT-3′). The primers for RPS26 are RPS26-forward (5′-CCGTGCCTCCAAGATGACAAAG-3′) and RPS26-reverse (5′-TGTCTGGTAACGGCAATGCGGCT-3′) and the primers for PBGD are PBGD-5′ (5′-TGTCTGGTAACGGCAATGCGGCT-3′) and PBGD-3′ (5′-GGTCCACTTCATTCTTCTCCAG-3′). Relative ASPM mRNA level was determined as the ratio of ASPM to PBGD as described (29–31, 34, 35) and scored as high (ratio ≥ 1.0; 128 cases), medium (0.5 < ratio < 1.0; 18 cases), low (ratio < 0.5; 39 cases), trace (ratio < 0.2; 44 cases), and negative (24 cases). In 227 nontumorous livers examined, none had a ratio exceeding 0.5. Hence, a ratio ≥ 0.5 was regarded as ASPM overexpression.

Patients, histologic study, ETR, follow-up observation, and p53 mutation. Between 1983 and 1997, 247 surgically resected, unifocal, primary HCC pathologically assessed at the National Taiwan University Hospital, as described (36–39), formed the basis of this study. This study was executed according to the regulations of the Ethical Committee of the National Taiwan University Hospital. These patients, ages 14 to 88 years (mean, 55.5 years) and with adequate liver function reserve, had survived for 2 months after hepatectomy, and none received transhepatic arterial embolization or chemotherapy before surgery. The tumor was ≤3 cm in 56 cases and >5 cm in 137 cases. The tumor grade was classified into grade I to IV. The tumor staging was classified into five groups, which closely correlated with survival in 781 unifocal surgical HCC patients, as described (35). Stage I and II HCC had no vascular invasion. In contrast, stage IIIA to IV HCC had vascular invasion, with various extents of microscopic intrahepatic spread in stage IIIB and IV HCC.

During the follow-up observation up to 236 months until January 2008, 245 (99%) cases had been followed for >5 years (90 cases) or until death (155 cases). Intrahepatic tumor recurrence and/or distant metastasis detected within 12 months after tumor resection were defined as ETR, which was detected in 107 (47%) of 226 eligible cases. The diagnosis of recurrence and/or metastasis was based on serum α-fetoprotein (AFP) elevation, imaging findings (ultrasonography, computer tomography, angiography, bone scan, and magnetic resonance imaging), and histology as described (30, 34, 35). The details of category in patients, histologic study, and ETR were described in Supplementary Material. In cases with ETR, patients with only early intrahepatic recurrence had much better survival.

Mutations of p53 tumor suppressor gene, spanning exons 2 to 11, were detected by DNA sequencing as described (38, 40, 41).

To validate the clinicopathologic significance of ASPM expression, patients were randomly assigned to two groups, the learning set (124 cases) and test set (123 cases).

Statistical analysis. The statistical significance of differences of selected clinicopathologic features between those with and without ASPM mRNA overexpression was assessed by χ² tests. For 5-year survival analysis, person-months were calculated from the date of resection to the date of death or 60 months for those who survived for >5 years. P values < 0.05 or a 95% confidence interval (95% CI), not including 1, were considered statistically significant. Multivariate analyses of grade and stage were conducted by fitting multiple logistic regression models (42) and then times to ETR and death were analyzed by fitting multiple Cox's proportional hazards models (43). Basic model-fitting techniques for (a) variable selection, (b) goodness-of-fit assessment, and (c) regression diagnostics (including residual analysis, influence analysis, and check of multicollinearity) were used in our regression analyses to assure the quality of analysis results (42, 43). Two-tailed P < 0.05 was considered statistically significant.

ASPM mRNA expression in fetal and adult tissues and various solid types of human cancer. By the differential display analysis, we identified a 240-bp cDNA, which was often expressed in HCC and had sequence identical to human ASPM (Genbank NM_018136; Fig. 1A). Using reverse transcription-PCR at the linear range, ASPM mRNA was expressed in relative abundance in most fetal tissues, including the liver (Fig. 1B), and all the cell lines examined (Fig. 1C), but the level was very low in most adult tissues (Fig. 1B).

In clinical tissue samples, ASPM mRNA overexpression was found in 162 (66%) of 247 surgically removed, unifocal, primary HCC (Fig. 2) but in none of hepatocellular adenomas (4 cases), focal nodular hyperplasia (10 cases), and nontumorous livers (227 cases).

Correlation of ASPM overexpression with HCC progression. To better understand the significance of ASPM in HCC, we correlated its mRNA expression with the major clinicopathologic features. As shown in Table 1, ASPM overexpression was strongly associated with high serum AFP level (>200 ng/mL; P = 1 × 10^-8). Importantly, ASPM overexpression closely correlated with high-grade HCC [grade II-IV; odds ratio (OR), 5.1; 95% CI, 2.6-10; P = 1 × 10^-7] and high-stage HCC (stages IIIA-IV versus II; OR, 30.6; 95% CI, 13.1-73.6; P = 1 × 10^-8). ASPM overexpression also occurred more often in bigger tumors (>5 cm) but less distinct (P = 0.014).

ASPM overexpression as an important predictive marker for ETR and poor prognosis. ETR is the most critical, early clinical factor predictive of poor prognosis of HCC after hepatectomy (30, 35). ETR occurred two times higher in HCC with ASPM overexpression than in HCC without the overexpression (P = 1 × 10^-8; Table 2). Early metastasis was also found more often in HCC with ASPM overexpression [28% (30 of 107) versus 11% (8 of 73); P = 0.006]. With the close correlation with high tumor stage and ETR, the two most crucial histopathologic and clinical unfavorable prognostic factors, HCC with ASPM overexpression had lower 5-year survival than those without the overexpression (P = 0.000001; Fig. 3). We then examined the postoperative treatment modalities and metastasis, which could affect significantly the patient's outcome, and found no significant difference in both groups of patients. Surgical tumor resection and/or transarterial chemoembolization of the intrahepatic recurrent and distant metastatic lesions were done in 50 (56%) of 90 HCC with ETR and ASPM overexpression and in 13 (76%) of 17 HCC with ETR but without ASPM overexpression (P = 0.108). Surgical resection was done for early metastatic tumor in 14% (4 of 29) of HCC with ASPM overexpression and in 25% (2 of 8) without ASPM overexpression (P = 0.591).

In addition to the close association with vascular invasion (stage IIIA-IV) and ETR, ASPM overexpression was also associated with higher ETR in stage II HCC, which had no evidence of vascular invasion [28% (12 of 31) versus 11% (10 of 68); P = 0.008].

Interaction of ASPM overexpression with p53 mutation and high AFP in relation to ETR and prognosis. The p53 gene is the most commonly mutated gene in HCC and associated with more aggressive tumor (38, 40). High serum AFP is the most widely used diagnostic marker and associated with more aggressive HCC, ETR, and poor prognosis (35). In this study, we showed that ASPM overexpression correlated with high AFP (P = 1 × 10^-8) and p53 mutation (P = 0.000026; Table 1). We then analyzed their roles and the major clinicopathologic factors in relation to ETR. As shown in Table 2, tumor size, tumor grade, and tumor stage were important histopathologic risk factors for ETR, particularly high tumor stage (P = 1 × 10^-8), whereas ASPM overexpression, high AFP, and p53 mutation were the molecular risk factors, particularly ASPM overexpression (P = 1 × 10^-8). By multivariate regression analysis, ASPM overexpression directly correlated with ETR after considering tumor size, tumor stage, and p53 mutation (Table 3). The multivariate regression analysis also showed that ASPM overexpression directly correlated with high tumor stage after considering tumor size (Table 3). Regardless of p53 mutation, ASPM overexpression was associated with vascular invasion (P values = 1 × 10^-8), ETR (P values < 0.001) (Supplementary Table S1), and hence lower 5-year survival rate in both non-p53-mutated and p53-mutated HCC (P = 0.0027 and 0.00008, respectively; Fig. 3).

ASPM plays an important role in the neuronogenesis and its mutation is the major cause of primary microencephaly (1–5, 44). Recent studies have shown that ASPM is located to the centrosomes, spindle poles, and midbody (23, 45) and plays an important role in cytokinesis and cell proliferation in the transformed human cell lines, fetal tissues, and human cancer cells (23). Despite these elegant observations, the significance of ASPM in human cancer has not been fully investigated. In this study, we showed ASPM mRNA overexpression in 66% of 247 surgical removed, unifocal primary HCC and in 54% of 167 other types of human cancer, including hepatoblastoma, cholangiocarcinoma, and carcinomas of other anatomic sites (data not shown), consistent with the observations reported by other investigators (23). Besides, ASPM mRNA was expressed abundantly in most fetal tissues, including the liver. In contrast, ASPM mRNA level was very low or undetectable in many adult tissues, including liver, and benign hepatic tumor conditions, such as hepatocellular adenoma and focal nodular hyperplasia. These findings suggest that ASPM is actively involved in cell proliferation in embryonic development and the tumorigenesis of HCC and various other types of human cancer, whereas ASPM down-regulation is associated with tissue maturation and cell differentiation.

To elucidate the role of ASPM in HCC, we correlated its mRNA expression with the major clinical and histopathologic variables associated with tumor progression. We showed that ASPM overexpression correlated with high serum AFP level (>200 ng/mL; P = 1 × 10^-8) and poorly differentiated (grade II-IV) tumor (P = 1 × 10^-7) but less significantly with bigger tumor (>5 cm; P = 0.014). These findings suggest that ASPM overexpression in HCC facilitates tumor cell proliferation, leading to bigger tumor and less differentiation, which are associated with high AFP level (35). Importantly, ASPM overexpression was associated with high-stage (stage IIIA, IIIB, and IV) HCC that had vascular invasion and various extents of intrahepatic metastasis (OR, 30.6; 95% CI, 13.1-73.6; P = 1 × 10^-8). These findings suggest that HCC with ASPM overexpression harbor enhanced invasion/metastasis potential.

HCC is a dreadful disease difficult to treat; surgical resection provides an opportunity for cure. Despite the significant improvement of earlier diagnosis and better management, the outcome of HCC after tumor resection remains unsatisfactory because of the high tumor recurrence rate (30, 46–48). ETR is the most crucial unfavorable, clinical prognostic factor for surgical HCC patients, and <20% of the patients with ETR could survive >5 years after tumor resection, whereas >50% of patients without ETR did (30). Hence, ETR can be regarded as a crucial clinical event before death, and markers for the prediction of ETR are needed. Although the list of molecular factors associated with ETR expands rapidly (30, 34, 35, 49–51), the number of markers useful as predictors of ETR remains limited and their interaction has not been well investigated. Here, we showed that HCC with ASPM overexpression had ETR ∼2-fold higher than HCC without the overexpression (OR, 6.49; 95% CI, 3.31-12.9; P = 1 × 10^-8). In addition to the close association with vascular invasion (stage IIIA-IV) and ETR, ASPM overexpression was also associated with higher ETR in stage II HCC (low stage), which had no microscopic evidence of vascular invasion (P = 0.008). These findings suggest that ASPM overexpression serves as a useful predictive marker to identify surgical HCC at high risk of ETR regardless of p53 mutation status and tumor stage.

Using univariate analysis, tumor size, tumor grade, and tumor stage were important histopathologic risk factors for ETR, particularly high tumor stage (P = 1 × 10^-8), whereas ASPM overexpression, high AFP, and p53 mutation were the molecular risk factors, particularly ASPM overexpression (P = 1 × 10^-8). The multivariate regression analysis showed that ASPM overexpression directly correlated with high tumor stage after considering tumor size and ASPM overexpression directly correlated with ETR after considering tumor size, tumor stage, and p53 mutation. These findings indicate that ASPM overexpression is an independent risk factor of high-stage HCC, and conditioning on high stage and tumor size, ASPM overexpression is also an independent risk factor of ETR.

With the close association with high tumor stage and ETR, the two most crucial histopathologic and clinical factors associated with poor prognosis, HCC with ASPM overexpression had a significantly lower 5-year survival than HCC without the overexpression (P = 0.000001). Together, our findings indicate that ASPM overexpression contribute to higher tumor stage and frequent ETR, leading to poor prognosis, and a combined analysis of multiple molecular factors provides a more accurate assessment of risk for ETR and prognosis of HCC.

In addition to the association with centrosomes and involvement in cell division (23, 45), we showed that ASPM was cell cycle regulated (data not shown). The p53 plays important roles in cell cycle progression, and p53 mutation is the most commonly mutated gene in HCC in association with more aggressive tumor (38, 40). We then analyzed the interaction between ASPM overexpression and p53 mutation, the latter would be accompanied by the loss of G₁ (52) and G₂-M checkpoints (53), in HCC progression. In both HCC with and without p53 mutation, ASPM overexpression was associated with higher vascular invasion (P values = 1 × 10^-8), ETR (P values < 0.001 and P = 1 × 10^-8, respectively), and lower 5-year survival rate (P = 0.0027 and 0.0008, respectively). These findings indicate that ASPM overexpression is an important factor associated with HCC progression. However, it was also noted that HCC with p53 mutation and ASPM overexpression had the highest frequencies of vascular invasion and ETR and the lowest 5-year survival. These findings suggest that the loss of p53-mediated checkpoint contributes cooperatively with the ASPM overexpression toward more advanced disease, with higher ETR and poorer prognosis.

No potential conflicts of interest were disclosed.

We thank Chia-Yen Su for excellent technical assistance.