From 1996 to 2008, 642 patients newly diagnosed with HCC who underwent liver resection were entered into a computer database at the Tri-Service General Hospital, a tertiary referral medical center in Taipei, Taiwan. The diagnosis of HCC in all patients was established histologically from surgical specimens. HCC patients with an initial serum AFP level ≥ 200 ng/dL (n = 178) were selected and 82 had HCC recurrence during follow-up. Thirteen patients were excluded because of an American Joint Committee on Cancer (AJCC) stage IV diagnosis, lost to follow-up or missing data. Therefore, 69 patients were included in this study and grouped according to their serum AFP levels when HCC recurred (Figure 1).

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Figure 1 Flow chart for patient selection. HCC: Hepatocellular carcinoma; AFP: α-fetoprotein; AJCC: American Joint Committee on Cancer.

Sixteen patients with normal AFP levels (≤ 20 ng/dL) during HCC recurrence were assigned to group I; 24 patients with AFP levels between 20 ng/dL and 200 ng/dL were assigned to group II; and, 29 patients with AFP levels of 200 ng/dL or higher were assigned to group III. In our hospital, the normal AFP level is ≤ 20 ng/dL measured by radioimmunoassay using an anti-AFP antibody (ELSA 2-AFP kit, CIS Bio International, Cedex, France).

Preoperative clinical characteristics were collected from the database and compared. These characteristics included age, gender, tumor size, serum alanine aminotransferase (ALT) level, total serum bilirubin, serum albumin level, platelet count, vessel invasion, tumor number, clinical stage (AJCC, Child-Pugh classification), hepatitis B virus, hepatitis C virus, presence of cirrhosis, mean recurrence interval, mean recurrence-to-death time, mean survival time and location of recurrence. Salvage treatments for recurrent HCC included repeat liver resection, liver transplantation, transarterial chemo-embolization, radio-frequency ablation, and thalidomide therapy. The mean AFP levels at operation (AFP op) and at recurrence (AFP recur) were calculated for these three groups. Intra-hepatic metastasis was defined as recurrent HCC located at the adjacent segment, section and same lobe. In contrast to intra-hepatic metastasis, multicentric recurrence was defined as recurrent HCC located at the contralateral liver lobe. The pathological grade was defined as: grade I - well differentiated, grade II - moderate, and grade III - poorly differentiated, according to the report by the pathologist (Dr. Yu CP).

The mean recurrence interval was defined as the interval between the operation and HCC recurrence. The starting point of overall survival was the operation date, and recurrence-to-death was the date of recurrence diagnosis. The endpoint of accumulated recurrence rate was defined as the date of recurrence and the endpoint of recurrence-to-death survival was the date of death or the last follow-up date if the patients survived. The choice of salvage therapy was according to the willingness of the patient, the degree of tumor progression, and the criteria for treatment guidelines of the Cancer Committee at the Tri-Service General Hospital. The recurrence interval was defined as the day of the initial liver resection to the day of HCC recurrence diagnosis.

Three patients, one from each group, underwent a second liver resection for tumor recurrence. Surgical specimens from both operations in each of these three patients were reviewed by a pathologist (Dr. Yu CP). The specimens were examined by hematoxylin and eosin staining and immunohistochemistry for AFP levels and the differences were compared.

All patients were followed up regularly every month in the first year and every 3 mo thereafter. AFP level was monitored at each follow-up. Abdominal ultrasound imaging or computed tomography (CT) was performed every 3-6 mo in first two years, then annually, according to the NCCN practice guideline[15]. In some patients, magnetic resonance imaging, hepatic angiography, and percutaneous needle biopsy were considered if the CT images were not conclusive for a new hepatic lesion. All clinicopathological data were collected in the computer databases of the Cancer Board at the Tri-Service General Hospital, and the follow-up data were regularly updated. This study was approved by the Institutional Review Board of Tri-Service General Hospital. The follow-up period of this study ended on June 30, 2009.

Continuous clinical data were analyzed using the Student’s t test and ANOVA, and the categorical data were analyzed using Fisher’s exact test or the χ² test. Kaplan-Meier and Cox regression analyses were performed to determine the survival curves, and the survival relative ratio between the three groups of HCC patients. P < 0.05 was considered statistically significant. All statistical analyses were performed using statistical software (SPSS version 15.0 for Windows, Chicago, IL, USA).

The accumulated recurrence rates in the three groups were calculated using the Kaplan-Meier method (Figure 2A). Accumulated recurrence rates were calculated from the first liver resection date to the recurrence date. Recurrence took place in all patients and was compared among groups I, II, and III (P < 0.001). The relative ratios [95% confidence interval (CI)] of groups II and III compared with group I patients were 0.244 (0.119-0.498) and 0.591 (0.340-1.027), respectively, with group I as the reference.

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Figure 2 Accumulated recurrence rates and recurrence-to-death survival rates. A: Accumulated recurrence rates were calculated from the first liver resection date to recurrence date and compared between groups I, II, and III (P < 0.001); B: Recurrence-to-death survival rates were calculated from the recurrence date to last follow-up date and compared between groups II, I, and III (P = 0.016).

The risk factors for clinical outcome related to survival are shown in Table 2. The six factors affecting clinical outcome by Cox proportion hazard analysis were tumor size (> 5 cm), total bilirubin (> 1.2 mg/dL), vessel invasion (+), recurrence interval (< 12 mo), Child-Pugh classification (B), and group I and III. When Cox regression analysis was performed, vessel invasion, group III, and recurrence interval were identified as independent risk factors.

The recurrence-to-death survival rates were calculated from the recurrence date to the last follow-up date and compared among groups I , II, and III as shown in Figure 2B (P = 0.016). Group II had a significantly better survival rate than the other two groups. The relative ratios (95% CI) of groups II and III compared with group I were 0.550 (0.241-1.253) and 0.337 (0.153-0.743), respectively.

We compared the AFP immunohistochemistry results for a pair of specimens from each group. All groups revealed strong AFP staining in the primary HCC liver resection specimens. In the liver resection specimens from recurrent HCC, groups II and III showed strong AFP staining, but the specimen from group I showed reduced staining (Figure 3).

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Figure 3 Immunohistochemical staining of AFP in representative specimens from the primary (A) and recurrent (B) HCC resections from each group (I, II, III) × 400.

In our patients, some underwent resection twice for removal of HCC. The first and second specimens from liver resection did not show consistent histopathological findings in a patient from group I, but did show similar histopathological findings in patients from groups II and III. Although these paired pathological findings are not conclusive, they do hint at an inconsistent pathological presentation. In a previous study, the authors proposed that the chromosomal changes and clonality relationship between primary and recurrent HCC could be proved using a comparative genomic hybridization technique[16]. In that study, 35% of HCC patients were considered to have a second primary HCC. In our study, the mechanism of HCC recurrence in group I patients may be due to de novo HCC with low secretion of AFP. This mechanism in groups II and III may be due to intra-hepatic metastasis from the primary HCC. Many possible reasons for this phenomenon have been reported, including de novo HCC[16,17], a biologic tumor changed by dedifferentiation[18], or a multicentric origin[19].

In our study, we used Cox regression analysis of the risk factors for accumulated recurrence rate and no confounders were identified. Patients with inconsistent AFP levels had a longer accumulated recurrence rate than those in groups II and III. The second primary HCC might have needed a longer time to develop, resulting in a longer recurrence period. In groups II and III, the recurrent tumor was probably from an intra-hepatic metastasis or residual HCC resulting in a shorter recurrence interval than that suggested by other articles[20,21]. The recurrence-to-death survival rate of patients with inconsistent AFP levels was worse than that in group II where the AFP level was consistent.

Elevated serum AFP is useful for the diagnosis of HCC, and also implies a large tumor burden at presentation[6]. A low serum AFP level at recurrence in patients with an initially high AFP level at HCC diagnosis may be a good prognostic factor of survival[6]. However, this was not supported by our results. This poor outcome may be caused by a biological change in the malignancy or delayed diagnosis. Physicians need to diagnose recurrence as early as possible by regular surveillance to improve patient outcome. In the NCCN practice guidelines, version 1, 2009[15], surveillance for HCC recurrence in patients with initially elevated AFP levels includes checking AFP levels every 3 mo and performing imaging studies annually for 2 years after resection[15]. When physicians follow the NCCN guidelines, they should bear in mind that AFP level is not as sensitive as generally believed for tumor recurrence in this subgroup of HCC patients, similar to group I patients in our study. The results of our study showed that regular checks of AFP levels should be conducted every 1-3 mo, followed by imaging studies every 3 mo for patients with an initially elevated AFP level. Regular imaging studies should not be omitted even when the serum AFP level is normal. The results of our study are important for reminding practicing physicians that regular imaging is still the benchmark in the surveillance of HCC patients.

There were some limitations in this study. First, these preliminary data were from one medical center and the conclusion needs to be validated by a meta-analysis of multicentric data. Second, the diagnosis of HCC recurrence was not proven by pathologic findings. We diagnosed HCC recurrence by dynamic imaging studies, hepatic angiography, and clinical disease progression. In a further study, tumor biopsy should be performed before salvage therapy if possible. The results of histochemical staining in the recurrent tumor could explain the exact mechanism of this clinical presentation in patients with inconsistent AFP levels.

In conclusion, 23% of our selected patients demonstrated an inconsistency in serum AFP level between initial HCC diagnosis before resection and HCC recurrence diagnosis. To improve medical care and avoid missed or delayed diagnoses, regular checks of AFP level and imaging studies every three months are strongly recommended during follow-up, even for patients with normal serum AFP levels.

Serum α-fetoprotein (AFP) level is widely used as a tumor marker for the diagnosis and detection of both occurrence and recurrence of hepatocellular carcinoma (HCC). A high serum AFP level in HCC also indicates a poor outcome. However, inconsistent AFP levels have been found, where patients’ AFP levels were initially high when HCC was diagnosed, but were normal when HCC recurred. There is no research on the prognosis of this subgroup of HCC patients.

When clinical physicians follow the National Comprehensive Cancer Network guideline, they should bear in mind that AFP level is not as sensitive as generally believed for tumor recurrence in this subgroup of HCC patients. Regular checks of AFP level and imaging studies every three months are strongly recommended during follow-up, even for patients with normal serum AFP levels.

This is the first article to investigate the clinical outcomes of patients with inconsistent AFP levels. The clinical outcomes in this subgroup of HCC patients were not as good as those with normal AFP levels. To improve medical care and avoid missed or delayed diagnoses, clinical physicians should bear these findings in mind.

The authors modified the surveillance principle for this subgroup of HCC patients following the results of this study to improve the quality of medical care.

This is a retrospective analysis of a cohort of patients with HCC, it maybe interesting for the readers.