Journal of Gastroenterology and Hepatology (1999) 14 (Suppl.) S32–S36

it was found to be associated with HCC. First, it wasdetected in the serum of mice with transplantable HCCby Abelev et al. in 19632 and then, in the following year, by Tatarinov in the serum of some patients withHCC.3 These early studies were based on variousimmunodiffusion methods and were essentially qualita-tive or, at best, semiquantitative. We now know that apositive test by the original immunodiffusion techniquerepresented a value of approximately 1 mg/mL.With thisapproach, the sensitivity of AFP for HCC was relativelylow, but detection of AFP was very specific for HCC.The source of AFP in the foetus was localized to theyolk sac in early foetal life and to the liver in later foetallife.4 It was thought that the protein was absent in theadult.

However, in the early 1970s, several groups devel-oped radioimmunoassays (RIA) which could detectAFP even in normal adults, albeit in concentrations of less than 10 ng/mL.5,6 Early case reports describedpreclinical detection of HCC using AFP as a screeningtest for high-risk patients and concluded that ‘. . . it isencouraging to note that a sensitive and specific screening method exists (for HCC) at a stage when surgical intervention and increased survival or cure may be possible’.7 However, on further study, it soonbecame apparent that with the increased sensitivity

INTRODUCTION

Thirty-five years after its discovery, serum alpha-fetoprotein (AFP) remains a model tumour marker.It is still widely used in the management of patients with hepatocellular carcinoma (HCC) and germ-celltumours. This brief review describes the current status of AFP as a marker for HCC and some method-ological advances that may secure its role well into the next century. First, we consider how the specificityof the AFP test for HCC can be increased and second, how AFP can be used to effectively monitorresponse to therapy, in non-surgical treatments whichare now being extensively developed. In the finalsection, brief mention is made of the use of AFP mRNAto detect circulating malignant cells in patients with HCC and how the relative specificity of AFP forHCC was used in early attempts at gene therapy forHCC.

HISTORICAL ASPECTS

Although AFP was recognized as a component of cordblood as early as 1956,1 it was not until the 1960s that

HEPATIC NEOPLASMS: ADVANCES IN TREATMENT

Role of alpha-fetoprotein in the diagnosis and management ofhepatocellular carcinoma

PHILIP J JOHNSON

Department of Clinical Oncology, Chinese University of Hong Kong, Shatin, Hong Kong

Abstract Thirty-five years after its first description, alpha-fetoprotein (AFP) remains the gold stan-dard by which other markers are judged. Serum levels above the reference range of 10 ng/mL occur inapproximately 75% of cases of hepatocellular carcinoma (HCC). In individual patients, the serum AFPlevel behaves as if it reflects tumour mass. However, the specificity of AFP is relatively low because mod-erately raised levels are also found in some patients with uncomplicated chronic liver disease. Recently,tumour-specific AFP assays have been developed. These are based on the carbohydrate side-chains onthe AFP molecule which exhibit characteristic differences in AFP of different origins. Monitoringresponse to treatment may often be more effectively carried out by serial estimation of AFP than byconventional imaging techniques. The relative specificity of AFP for HCC has also been employed todetect circulating HCC cells and to target gene therapy.

Key words: alpha-fetoprotein, hepatocellular carcinoma, tumour markers.

Correspondence: Philip Johnson, Department of Clinical Oncology, Chinese University of Hong Kong, Shatin, Hong Kong.

of RIA there was the inevitable knock-on effect forspecificity. Thus, although the AFP test was very sensi-tive, being detected above the reference range inapproximately 80% of HCC cases, it was a far less spe-cific test. Increased levels became detectable in patientswith other malignant diseases, particularly those of thegastrointestinal tract8,9 and also benign liver diseases,such as chronic hepatitis, fulminant hepatitis and cir-rhosis.10,11 The raised levels in patients with secondaryliver tumours was not of major concern because theywere uncommon and distinction of primary from sec-ondary liver tumours was seldom a major clinicaldilemma.

However, the loss of specificity due to raised levels inbenign liver disease did become a major drawback forthe test.This was because most patients with HCC havecoexisting chronic liver disease and one was often con-cerned to determine whether HCC had arisen in apatient with chronic liver disease. Although, as notedabove, some studies which applied AFP for HCCscreening came to positive conclusions, the number offalse positive results has generally led to disillusionmentwith the use of AFP for population screening.

The second role for AFP has been to monitorresponse to therapy.12 Until recently, this was confinedto surgical practice because all other forms of treatmentwere largely ineffective. It was clear that AFP was mostuseful in this respect. After successful HCC resection,AFP levels fell to within the normal range, with a half-life of approximately 6 days. Furthermore, if there wasa recurrence, levels would again rise, often before therewas any clinical evidence of disease. The rate of risecould be measured as a doubling time which rangedfrom 5 to 120 days.12 Also of importance was the obser-vation that, even if resection appeared complete, if theAFP did not reach the reference range, residual, clini-cally non-detectable tumour, was invariably present andthis presaged tumour recurrence. Thus, although thefactors influencing the absolute AFP levels were still notclear, it was apparent that the serum AFP level in theindividual patient, behaved as if it reflected tumourmass.

Role of AFP in management of HCC S33

TOWARDS ASSAYS FORHEPATOCELLULAR CARCINOMA-SPECIFIC ALPHA-FETOPROTEIN

The biochemical basis of disease-specific assaysdepends on the structure of AFP. Alpha-fetoprotein isa glycoprotein which consists of 591 amino acids andat least one oligosaccharide side-chain linked toasparagine 232.13 The oligosaccharide chain has thecommon pentasaccharide core but varies in the degreeof sialylation and fucosylation13–15 (Fig. 1). It appearsthat, while the protein structure of AFP from differentsources is constant, the carbohydrate side-chain is vari-able.The most effective methods to date mainly use thedegree of fucosylation as detected by AFP binding affinity to various lectins, in particular Lens culinarisagglutinin-A and erythro-agglutinating phytohaemag-glutinin-E4.16–19 Diagnostic kits based on lectin affinityelectrophoresis with detection by antibody-affinity blot-ting and immunoenzymatic amplification have beendeveloped. In general, the kits seem capable of differ-entiating between AFP of benign origin, in which thebinding is less than 15%, and that from HCC in whichthe binding is greater than 25%.16 Using a wider panelof lectins, four types of AFP can be distinguished: livertype, HCC type, gastrointestinal tumour type and yolksac tumour type.19

An alternative approach, which appears on the basisof our recent observations to be related to the degree ofsialylation, is based on isoelectric focusing (IEF). OnIEF, AFP is shown to exhibit three major bands, desig-nated +I, +II, and +III.20,21 Band +I is associated withchronic liver disease, band +II with HCC and band +IIIwith germ cell tumours. We have recently shown, in aprospective study, that this test will clearly distinguishbetween raised AFP of benign or malignant origin. Ofparticular interest, however, was the observation thatthe AFP band +II can be detected several months, oreven years, before the tumour becomes detectable by conventional imaging methods. Such tests forhepatoma-specific AFP may afford the opportunity ofvery early and specific diagnosis.22

Figure 1 Reported basicstructures of the oligosaccha-rides of alpha-fetoprotein. Thevariation between isoforms ofdifferent origins resides in thedifference in the terminal sialicacids (SA) and the proximalfucose (Fuc) molecule.

MONITORING RESPONSE TOTREATMENT

The second important role of AFP lies in detectingresponse to treatment. As noted above, until recentlythere was little in the way of effective treatment otherthan surgical resection. We have recently shown thatselective internal radiation with intra-arterial 90yttriummicrospheres is an effective way of palliating HCC.23 Ofparticular interest is the observation that such treatmentdoes not lead to complete remission and only a modestpercentage of partial remissions. Nonetheless, there wasa dramatic decrease in AFP levels in most cases. Theapparent dissociation between radiological and serolog-ical (AFP) responses was explained, in several cases,when initially inoperable cases became operable fol-lowing partial radiological response. On detailed patho-logical examination of the resected specimens, therewas a complete, or almost complete, histological remis-sion and, in such cases, the serum AFP had also fallento within the reference range. The implication is thatwhat is seen on conventional computed tomography orultrasound imaging may reflect necrotic or fibrotictissue whereas the serum AFP levels may better reflectviable malignant tissue.

We have had similar experience with a new systemiccombination therapy of PIAF (Platinum, interferon,adriamycin and 5-fluorouracil).24 Again, using conven-tional criteria, response rates were only modest but AFP levels showed consistent falls. In several cases(10–20%), initially inoperable tumours became opera-ble and again the resected specimens showed complete,or almost complete, pathological remission (Figs 2,3).This leads us to conclude that monitoring serum AFPmay be a better way of monitoring progress than con-ventional radiological techniques. The word ‘conven-tional’ is emphasized as there is no doubt that newer,more sophisticated techniques of imaging may be able

S34 PJ Johnson

to distinguish between viable and non-viable tumour.However, we might be led to wonder if HCC clinicaltrials, which relied on conventional radiological criteriato assess response, may have missed important activeagents.

DETECTION OF CIRCULATINGHEPATOCELLULAR CARCINOMACELLS

Several groups have proposed that it may be possible toinfer the presence of circulating HCC cells and, hence,the potential for metastasis formation, if mRNA ofhepatocyte-specific proteins can be detected. The twoproteins used have been albumin (alb) and alpha-feto-protein and their respective mRNA have been detectedin serum by reverse transcription-polymerase chainreaction (RT-PCR). Most studies have, indeed, shownthat detection of alb mRNA and AFP mRNA is strongly

Figure 2 Computed tomography scan changes in a patient with hepatocellular carcinoma (a) before and (b) after 6 monthsof systemic chemotherapy with an interferon and cytotoxic drug regimen. Note that the decrease in size of the tumour wouldonly qualify as a partial response. However, at resection the tumour was completely necrotic and no viable tumour cells couldbe detected.

Figure 3 Changes in alpha-fetoprotein (AFP) levels in thetumour shown in Figure 2. Note that the serum AFP level hadfallen to the reference range by the time complete pathologi-cal remission had been documented.

(a) (b)

associated with the presence of metastases.25–28 How-ever, the specificity of the RT-PCR assay appears not to be high enough to confidently differentiate HCCcells from other nucleated cells in the peripheral blood,as AFP mRNA can often be detected in patients withchronic hepatitis or cirrhosis. Also, we and others haveobserved that alb mRNA can be detected in normalsubjects, a phenomenon attributable to illegitimatetranscription of the alb gene.29 These difficulties haveprompted us to develop a semiquantitative estimationof the amounts of hepatocyte-specific mRNA in the circulation with reference to the equivalent number of hepatoblastoma cells (HepG2). This will help differentiate the presence of HCC from other normalnucleated cells in the peripheral blood. Our RT-PCRassay is sufficiently sensitive to detect one HCC cell in106 peripheral blood mononuclear cells.28 As well ashaving a possible prognostic role, such methodologyallows us to investigate interesting questions, such as‘does surgical resection of HCC lead to widespread dissemination of tumour cells into the peripheral circulation?’.

GENE THERAPY

As with many cancers, the possibility of gene therapy iscurrently being investigated in patients with HCC. Oneof the major problems is how to target the tumour cells:the relatively specific production of AFP by HCC canbe usefully applied here. The p53 tumour suppressorgene might possibly correct the genetic change respon-sible for the malignant phenotype in HCC. One idea isto use a replication-defective retrovirus containing wild-type p53 transcriptionally regulated by AFP transcrip-tional regulatory sequences.30 It has already been shownthat the new p53 gene is expressed specifically in AFP-positive HCC cells and that clonal growth of these cellsis inhibited.31 Such approaches are now undergoingclinical trials.

A second approach is to introduce enzymes by genetherapy, as per the suicide gene paradigm.32,33 Here, agene which codes for a specific enzyme, is introducedinto the relevant tumour cell.The new enzyme activatesa harmless ‘prodrug’ into a cytotoxic agent which cankill the cancer cell. In one example, the gene for thymi-dine kinase (TK, from herpes simplex type-1 virus) istransferred into hepatoma cells. The drug, gancyclovir,is then administered. Hopefully, the tumour cells which contain the functioning TK gene, will convert theharmless gancyclovir into its cytotoxic triphosphateform.Tumour cell specificity is again achieved by usinga replication-deficient adenovirus as the vector, inwhich expression of the TK gene is under the controlof the AFP promoter/enhancer. As in the previouslymentioned case, it has been shown that, in animalmodels, tumour cell growth can be inhibited and thatTK expression is confined to AFP-expressing tumours.

Taketa has called AFP the gold standard of tumourmarkers.17 In view of its continuing and expanding rolein the management and investigation of patients withHCC, it seems likely to maintain this status well intothe next century.

Role of AFP in management of HCC S35

ACKNOWLEDGEMENTS

The author gratefully acknowledges the support of theHong Kong Cancer Fund in this work and thanks WTThomas Leung for providing figures 2 and 3.

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