The Role of Serum Alpha-Fetoprotein estimation in the Diagnosis and Management of Hepatocellular Carcinoma

LIVER TUMORS

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THE ROLE OF SERUM ALPHAFETOPROTEIN ESTIMATION IN THE DIAGNOSIS AND MANAGEMENT OF HEPATOCELLULAR CARCINOMA Philip J. Johnson, MD, FRCP

Alpha-fetoprotein (AFP), the major protein component of fetal serum, is synthesized by the visceral endoderm of the yolk sack during early fetal life and subsequently by the fetal liver. Immediately after birth levels fall rapidly to become virtually undetectable and rise again only when certain pathologic conditions develop. It has been known for almost 40 years that re expression of AFP consistently occurs in patients with hepatocellular carcinoma (HCC), and on this basis AFP became one of the first clinically useful tumor markers. For those involved in the management of patients with HCC, AFP plays a central role in day-to-day clinical practice. STRUCTURE OF SERUM ALPHA-FETOPROTEIN The AFP gene is part of a supergene family, the other members of which are genes for serum albumin, vitamin D binding protein, and alpha albumin, all situated on the long arm of chromosome 4 (4qll-q13). The proteins share a considerable sequence homology and are all synthesized in the liver and secreted into serum.9

Supported by grants from the University Grants Committee of Hong Kong (225/96M and 4261/97M)

From the Department of Clinical Oncology, Chinese University of Hong Kong Prince of Wales Hospital, Shatin, Hong Kong SAR, China

CLINICS IN LIVER DISEASE VOLUME 5 * NUMBER 1 * FEBRUARY 2001

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Alpha-fetoprotein is a glycoprotein consisting of 591 amino acids and 4% carbohydrate residues, giving a molecular mass of about 70 kd. The carbohydrate residue, purified from the ascitic fluid of a patient with HCC, was reported to consist of a single sugar chain of the biantennary complex-type, N-linked to asparagine 232 of the protein backbone (Fig. 1):2, This basic structure was confirmed by mass spectrometric analysis of AFP derived from the hepatoblastoma cell line Hep G2,’9, 57 and by paper electrophoretic and chromatographic analysis of pooled human-cord serum AFP at term pregnancy.63With isoelectric focusing and lectin electrophoretic techniques? 14, 21 human AFP exhibits microheterogeneity that results from differing degrees of terminal sialylation and fucosylation of the sugar side-chain(s)(Fig. 1 ) ? O Recent studies using more sensitive analytical techniques including fluorescence labeling, sequential exoglycosidase digestion high performance liquid chromatography (HPLC), and mass spectrometry have revealed that up to 11 major types of sugar side-chain (glycans) can be detected, of which 7 are Nlinked and 4 are O-linked to the protein backbone.z3The structure of the Nlinked glycans is, as suggested previously, of the biantennary complex-type with varying degrees of sialylation, fucosylation, and galactosylation. The 0-linked glycans, 3 of which were of the mucin 0-GalNAc -type with variable degrees of sialylation, were a novel finding (Fig. 1).The identification of 0-linked glycans was supported by the prediction of potential 0-GalNAc glycosylation sites on the protein backbone by analysis of the AFP structure by molecular modeling.23 These different isoforms (also sometimes known as glycofomzs) of AFP may be

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Figure 1. The structures of the main N- (upper) and 0- (lower) linked glycans in human alpha-fetoprotein (AFP). The charge heterogeneity is related to the number of sialic acid (SA) molecules at the sites underlined and, to a lesser extent, the fucose molecule, also underlined. The precise site of the attachment of the 0-linked glycan (s) to threonine in the protein backbone is not known. The isoforms associated with heptocellular carcinoma are predominantly the monosialylated species. (Adapfed from Johnson PJ, Poon TCW, Hjelm NM, et al: Glycan composition of serum alpha-fetoprotein in patients with hepatocellular carcinoma and non-seminomatous germ cell tumor. Br J Cancer 81:1188, 1999)

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disease-specific. They therefore form the basis of attempts to develop more specific assays for the detection of HCC, as discussed later. DETECTION AND QUANTITATIVE ASSESSMENT OF ALPHAFETOPROTEIN IN PLASMA AND SERUM

Alpha-fetoprotein was first recognized as a distinct band, next to albumin (hence alpha) by electrophoresis of human cord blood in 1956,'O but the association with HCC was not made until 1963 when Abelev detected AFP in the blood of mice with transplantable HCCs.' The following year Tatarinov reported finding AFP in the serum of patients with HCC,5s and these studies were confirmed by Stanislawski-Birenewajget a1 in chemically induced rat tumors52 and in humans. Early studies applying serum AFP estimation to the diagnosis of HCC used immunodiffusion techniques and were essentially qualitative or semiquantitative. It is now known that a positive AFP test using these techniques represents a value of about 1 to 2 pg/mL (i.e., 1000-2000 ng/mL) or more. With this approach, a collaborative study by OConnor et a1,44involving several different countries and large numbers of patients, found that 60% to 75% of HCC cases were positive using a modified Ouchterlony technique. Not surprisingly, the higher percentage was in patients with a histologic diagnosis, and the lower percentage was in patients with a purely clinical diagnosis. The occasional falsepositive results came from patients in the control group with cirrhosis, other primary tumors (stomach or pancreas), or testicular teratocarcinomas. Although the authors reported that their results were not affected by racial or geographical factors, most patients came from areas with high incidence of HCC in Africa. Alpert et al, quantifying AFP levels using counter-immunoelectrophoresis,reported similar results, except that levels of positivity were significantly lower in white patients with HCC, an observation that has withstood the test of time? In the early 1970s, several groups successfully purified AFP from human serum and developed sensitive radioimmunoassays that could detect AFP even in healthy adults, albeit at levels of less than 10 ng/mL.15,36,45 A range between 0.1 and 5.8 ng/mL (mean = 2.6 ? 1.6 ng/mL), as determined by radioimmunoassay, was reported by Masseyeff et aP6 in healthy subjects. Cut-off values for the upper limit of the reference range have since been set variously at 10 or 25 ng/mL, because routine assays are not highly accurate at these low levels. With this increased sensitivity, AFP could be detected in more than 80% of HCC cases, although there was the inevitable concomitant effect of decreased specificity.This loss of specificity fell into two broad categories: patients with other malignant diseases, particularly of the gastrointestinal 35, 39 and those with chronic liver diseases.l*, 24, 35 DISEASE ASSOCIATIONS

Two aspects of the association between serum AFP levels and different disease states deserve consideration. First, a percentage of patients with a particular disease have levels raised above the reference range. Second, for each pathologic condition there is a characteristic median and range of values. For example, in HCC the positivity rate is around 8O%, and the yunge of values is from 1 to lo7 ng/mL, with a median of around lo3 ng/mL. Nonseminomatous germ cell tumors are the only other adult condition that have a similar positivity rate

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and a similar range of levels.20, 29 As a general rule, for the patient with HCC, the positivity rate is not stage-dependent; if a tumor is AFP-negative at presentation, it remains so for the remainder of the clinical course. On the other hand, in the absence of treatment, levels do increase with time in patients with AFPpositive tumors: the doubling time is between 5 and 100 days. Other Malignant Diseases

The positivity rate is much lower in patients with other malignant tumors, as are the median and range. The major class of malignant tumors involved are those of the gastrointestinal tract, as first systematically studied by McIntire et al.39These workers confirmed several earlier isolated reports of AFP-positive gastrointestinal tumors28,40 and reported raised AFP levels in 15% of stomach cancer cases and 25% of cases of cancer of the biliary tract and pancreas. Levels were distinctly lower than those seen in patients with HCC: whereas the levels were raised above 1000 ng/mL in more than 40% of patients with HCC, that level was seen in only 1%of the other gastrointestinal tumors. Generally, these data have held up over the years and have been explained by the observation that developmentally the hepatic diverticulum, the pancreatic buds, and the stomach are all derived from the same endodermal lining of the foregut. Numerous subsequent reports have described isolated cases of other, nongastrointestinal cancers associated with AFP production. These observations show that serum AFP estimation is unlikely to find a major role in the differential diagnosis of malignant liver tumors, although the value of AFP determination may vary depending on the geographic areas in which the situation occurs. Thus, in Europe and North America, for a patient present with radiologic or symptomatic evidence of a liver tumor of unknown origin, the AFP level is not particularly helpful in establishing a diagnosis of HCC. Although HCC has a higher AFP positivity rate, the overall percentage of patients with HCC in these areas will be very low. In areas with high incidence of HCC, a raised AFP level will carry more weight because a much higher percentage of liver tumors will be HCCs. Benign Liver Diseases

Modest elevations of AFP levels (between 10 and 500 ng/mL and occasionally up to 1000 ng/mL) may also be seen in adult patients with hepatitis of any type or liver cirrhosis. The frequency of elevation (> 10 ng/mL) has been reported as around 20% in chronic hepatitis and as 40% in cirrhosis.", 24, ~ 5 51 , Alpha-fetoprotein elevations may be associated with the seroconversion from HBe antigenemia to HBe antibody positivity accompanied by bridging necrosis in the liver.33These elevations seem to occur either when there is a high degree of inflammatory activity within the liver or towards the end of an acute hepatitis when the liver function is recovering. Although these observations have often led to the suggestion that aberrant AFP secretion is related to cell proliferation or hepatic regeneration, this is not always the case. For example, after hepatic resection (in the nondiseased liver), the classic initiator of liver cell regeneration, there is no increase in AFP level^.^ Alpha-fetoprotein levels between 10 ng/mL and 1000 ng/mL therefore represent a grey area, because both benign conditions such as chronic hepatitis and cirrhosis and small HCCs may give values within this range (Fig. 2). Because

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Figure 2. The range of AFP levels seen in normal subjects, patients with chronic liver disease, and those with hepatocellular carcinoma. The range between the upper limit of the reference range (10 ng/mL) and 500 ng/mL is a gray area. An AFP value falling in this range does not allow the clinician to make a confident diagnosis of hepatocellular carcinoma (HCC).

the clinician is often faced with the problem of determining if HCC has arisen in a patient with cirrhosis, the fact that AFP can be elevated in patients with chronic liver disease alone presents a major clinical problem. Precise figures for the frequency of AFP elevations in benign liver disease should be taken with some caution for three reasons. First, minor elevations close to the lower limits of the assay’s limit of sensitivity can cause major changes in the reported frequency. Second, there may be considerable fluctuation during the course of particular chronic liver diseases. For example, there is probably a stage during most cases of acute and chronic hepatitis that AFP becomes transiently positive. Finally, and most importantly, serum AFP may be raised during the preclinical phase of HCC, before a tumor can be detected by imaging techniques. For this reason most series probably underestimate the true specificity of AFP for HCC.22 Factors influencing Serum Alpha-Fetoprotein Levels in Patients with Hepatocellular Carcinoma

Gene expression is controlled mainly at the level of transcription. At presentation, levels can range from within the reference range up to 10,000,000 ng/mL (10 g/L) (Fig. 2). This range of up to 7 log scales is wider than seen with any other marker and comes close to the range of the number of cells seen in human tumors. About 40% of patients with HCC have AFP levels above 1000 11g/mL.5~ It has been reported that well-differentiated small HCCs (<2 cm) seldom express detectable serum AFP.27Because such tumors have been increasingly detected by ultrasound screening since the 1970s, the number of AFP-negative tumors has increased, and the sensitivity of the test for HCC has fallen. Small tumors tend to express lower levels of AFP, although there is no clear

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direct relation between AFP levels and tumor size (Fig. 3),47because individual tumors have different capacities for AFP synthesis. Also, it is difficult to measure viable tumor cell mass accurately and thereby investigate the true correlation between viable tumor mass and AFP level. Tumors that appear large on imaging procedures may in fact be necrotic and contain only a small number of viable cells. Men with HCCs tend to have slightly higher serum AFP levels than women.37,44 It has also been observed that there is a general increase in mean 42 serum AFP levels in younger ~atients.3~.

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Tumor Size Figure 3. The range of AFP levels in patients with small, medium, and large HCCs. Although there is no direct relationship between tumor size and AFP levels, there is a general trend for larger tumors to have higher AFP levels. (From Sawabu N, Hattori N: Serological tumor markers in hepatocellular carcinoma. In Okuda K, lshak KG (eds): Neoplasms of the Liver. Tokyo, Springer-Verlag, 1987, p 227)

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Hepatitis B surface antigen (HBsAg)-seropositive patients with HCC have a greater frequency of high AFP levels, but they also overlap to a greater extent with the AFP levels in HBsAg-positive patients with cirrhosis or chronic active hepatitis. The specificity of AFP for the detection of HCC in this serologically defined subgroup is thus decreased?] Several studies have found a significantly higher AFP positivity rate among HCC patients with underlying cirrhosis than in those without cirrhosis.41, DIAGNOSIS OF HEPATOCELLULAR CARCINOMA The sensitivity and specificity of AFP as a diagnostic marker for HCC depends on the cut-off level chosen. An evaluation on 239 cases of chronic hepatitis, 277 cases of cirrhosis, and 95 cases of HCC at varying clinical stages demonstrated a sensitivity of 78.9% and a specificity of 78.1% when the cut-off level was greater than 20 ng/mL for the diagnosis of HCC.53When the cut-off level was increased to greater than 200 ng/mL, the specificity of the test rose to 99.6%, but the sensitivity fell to 52.6%. Recognizing that modestly raised AFP levels occur in patients with chronic liver disease, a cut-off of 400 or 500 ng/mL has been widely used in clinical practice for the diagnosis of HCC. As noted previously, serum AFP levels may rise before the tumor can be seen by imaging techniques, so the finding of a raised AFP level without evidence of a tumor is not necessarily a false-positive result. To calculate the specificity accurately, data should be derived from cirrhotic patients followed up for more than 1year without evidence of HCC. Typical figures for sensitivity and specificity, based on such a patient group and at different cut-off levels, are shown in Table 1. In general, the finding of a level of greater than 500 ng/mL in patients known to have chronic liver disease is an indication for further detailed investigation to track down the lesion. In the presence of a hepatic mass detected by an imaging technique and chronic liver disease, an AFP level of greater than 500 ng/mL is virtually diagnostic of HCC. There are no clear management guidelines for a patient with chronic liver disease and an AFP level below 500 ng/mL. Detailed investigation of all such patients will undoubtedly lead to some early and useful diagnoses of HCC, but there will also be numerous false-positive results. Most workers in the field will take the finding of a raised AFP level more seriously if it is not associated with

Table 1. CHANGES IN THE SENSITIVITY AND SPECIFICITY OF ALPHAFETOPROTEIN FOR THE DIAGNOSIS OF HEPATOCELLULAR CARCINOMA IN PATIENTS WITH CHRONIC LIVER DISEASE USING VARIOUS CUT-OFF POINTS* Diagnostic criteria

AFP AFP AFP AFP AFP

> 615 ng/mL > 530 ng/mL > 445 ng/mL > 100 ng/mL > 20 ng/mL

Sensitivity

Specificity

56.4 56.4 56.4 72.6 87.1

96.4 94.5 94.5 70.9 30.9

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*These data were calculated using patients with chronic liver disease who had been followed up for at least 1 year to ensure that subclinical HCC was not present. Reference range = < 20 ng/mL. (Data courtesy of Dr. Terence CW Poon PhD.) AFP = Alpha-fetoprotein; HCC = hepatocellular carcinoma

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evidence of hepatic inflammation (i.e., a markedly raised activity of serum aspartate aminotransferase [AST]). Otherwise, a brief period of observation is in order. A continued rise in AFP level suggests HCC. Conversely, if levels fall or fluctuate, HCC is less likely, but it should be noted that marked reductions in AFP levels have also been observed in the terminal stage of HCC patients. The option of careful follow-up in such patients is not without problems. Lok et a134reported that six patients with chronic liver disease had persistent or progressive increase in AFP levels and were confirmed to have HCC. In a further six patients in the same series, however, HCC was not identified despite AFP elevations exceeding 200 ng/mL for more than 6 months. This experience led to the conclusion that monitoring time-trends of AFP levels alone is not an entirely satisfactory method for early diagnosis of HCC. Steadily increasing levels of serum AFP above 1000 ng/mL are, however, generally accepted as an indication of AFP-producing tumors.49These problems in the grey area of AFP elevations have led to attempts to discover tumor-specific isoforms of AFP. Improving Diagnostic Specificity-The Disease-specificlsoforms

Search for

Several approaches to identify tumor-specificisoforms and, thereby improve the specificity of AFP as a diagnostic test for HCC have been investigated. The most successful has been based on the difference in affinity of binding of the AFP molecules from different sources to various lectin~.~, 6, 13, 17, *,56 (Lectins are proteins of animal or vegetable origin that possess multiple binding sites that are highly specific for particular sugars.) Alpha-fetoprotein from patients with HCC was found to bind to the lectin Concanavalin A, whereas AFP from yolk-sac tumors did so to a much lesser extent. On the other hand, lentil lectin bound to AFP from HCC in preference to AFP from benign liver disease. The use of additional lectins, particularly Lens culinnrisa agglutinin (LCA-A) and erythroagglutinating phytohemagglutinin (E-PHA), resulted in characteristic patterns of binding that could differentiate between AFP of cord-blood type, HCC type, gastrointestinal tumor type, and yolk sac type.” Commercially available assays based on this approach are now available and have been shown to be useful in the early detection of HCC in patients with hepatitis and liver cirrhosis. More recently, isoelectric focusing (IEF) has been used to identify isoforms of AFP dire~t1y.l~. 21, 22 Of the three main bands observed, the AFP I1 band and AFP+III band seem relatively specific for HCC and nonseminomatous germ cell tumors (NSGCT), respectively. A further band ( +I) is found in the AFP that arises in patients with chronic liver disease without any evidence of malignancy. A preliminary study that followed a large cohort of patients with chronic liver disease for the development of HCC showed that the band +II. hepatomaspecific AFP could often be detected several months before ultrasound scanning could find the tumor.22A simple qualitative assessment of the tumor-specific band led to an increase in positive predictive value from 41.5%with the conventional assay for total AFP to 73.1%with the specific isoform. The sensitivity and specificity were 86% and 77%, respectively. 553

+

The Structural Basis of Disease-specific lsoforms

The early studies by Yoshima et aP3and Yamashita et a1@described the basic structures of the asparagine (at position 232) N-linked sugar chain molecule. The

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charge heterogeneity of AFP and the molecular basis of the specific lectin binding were related to difference in terminal sialylation and fucosylation. Specifically, it seemed that the HCC-related AFP isoform has one terminal sialic acid and the proximal N-acetyl glucosamine molecule is fucosylated (see Fig. l).50, 62 As noted previously, these structures have recently been confirmed by direct sequencing of the AFP glycans from the serum of patients with HCC and NSGCT, but it has been found that there are, in fact, several other glycans, including some that are 0-linked to the protein backbone. It has also been shown that the hepatoma-specific band seen on IEF represents a series of glycans that are all characterized by being monosialyated. The author and co-workers have suggested that not only fucosylated monosialo biantennary complex-type N-glycan but also non-fucosylated monosialo biantennary complex-type N-glycans are the major carbohydrate structures of the HCC-specific AFP i s o f o r m ~ . ~ ~ Both approaches, lectin binding and isoelectric focusing, seem to be potentially clinically valuable. Depending, as they do, however, on some form of electrophoresis, they have not become widely used in clinical practice. Until a simple, cheap, and robust assay of tumor-specific AFP becomes available, this situation is unlikely to change. The minor changes that account for the difference between tumor-related and non-tumor-related AFP make the development of such a routine assay a technically difficult task. ROLE OF ALPHA-FETOPROTEIN IN MONITORING RESPONSE TO SURGICAL AND MEDICAL THERAPY

Serial estimation of serum AFP has been widely used to monitor response to therapy. Until recently this practice was most appropriately applied to surgical treatment, because nonsurgical approaches were considered ineffective. Even when decreases in AFP levels were observed after chemotherapy, it was difficult to be sure whether they were really related to a decrease in tumor cell mass. Surgery

Complete excision of HCCs either by resection or orthotopic liver transplantation leads to an immediate fall in serum levels of AFP with a half-life of 3.5 to 4 days. Poor survival rates were observed in patients showing longer halflives.25*48 The achievement of a normal level, however, does not necessarily imply complete clearance of the disease, as was demonstrated in several patients with recurrence of tumor after transplantation whose AFP levels had initially returned to 25, 61 Presumably, tkis recurrence results from micrometastases that are too small to secrete sufficient AFP to raise the serum AFP level above the reference range. Also an important observation was that, even if resection appeared complete, if the AFP did not reach the reference range, residual, nondetectable tumor was invariably present and presaged clinical tumor recurrence. Reelevation of the AFP level in any situation is usually a strong indication of recurrence, although there are always exceptions: gradual rise in serum AFP levels has been observed in seven patients without any evidence of postoperative recurrence.'* Conversely, patients having residual disease may occasionally show no rise even when they were initially AFP-positive."j The important lesson from surgical therapy is that although the factors influencing the absolute level of AFP in patients with HCC are still not clear,

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changes in serum AFP in the individual patient seem to mirror changes in tumor mass. These observations in patients undergoing surgical treatments form the basis of the belief that changes in serum AFP may accurately reflect response to other forms of therapy. Nonsurgical Therapy

Until recently, chemotherapy has been considered largely ineffective for HCC, and systemic therapy did, indeed, cause only minor and transient decreases in serum AFP levels. Some authors, however, found a broad correlation between AFP changes and tumor response. Patients classified as achieving a significant, prolonged fall of AFP tended to survive longer than those with a transient fall, who in turn did better than those who had a continuously rising 38

More effective systemic therapies such as the PIAF regimen are now becoming available and offer some important insights.32Such treatment does not lead to complete remissions, and the rate of partial remissions is modest (26%). The AFP level decreases dramatically, however, in 75% of cases, and some of these attain levels in the reference range (Fig. 4). Furthermore, in about 20% of cases (most of those with partial responses), initially inoperable cases became operable. Detailed histopathologic examination of the resected specimen showed complete histologic remission or only minimal residual disease. When the changes in AFP level in such cases were studied, it was found that the AFP had fallen to within or close to the reference range (Figs. 5A and B). On the other hand, there was clearly progressive disease in the patients whose AFP levels continued to rise.

Figure 4. Changes in serum AFP in patients undergoing systemic chemoth$yapy with the cis-platinum, interferon, adriamycin, 5-fluorouracil (PIAF) regimen. The vertical axis is the percentage change in serum AFP with treatment. Note that the majority of patients show a fall in levels. Many of those in whom levels fell by 90-100% subsequently went on to surgical resection at which time the lesions were found to be completely necrotic or to contain only minimal residual disease. (Courtesy of Dr. Thomas Leung, MD.)

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Figure 5. CT scan before (A) and after (6) 6 courses of PIAF therapy. Note that although the tumor has shrunk in response to therapy, the response is only partial. Illustration continued on following page

Similar experience has been reported with radiotherapy for primary and secondary liver tumors. Response rates as measured by the decrease in tumor markers were much higher than those based on changes in tumor size or Also, the reduction in tumor volumes volumes as determined by CT appeared much later than the fall in tumor marker levels. In a number of patients with hepatic metastases, no change in the size of the liver defects on CT scanning was noted, yet at the time of exploration no viable tumor cells were found in the liver." The author and co-workers have had a very similar

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Figure 5 (Continued). C, The serum AFP level over the same period, fell to within the reference range. Histopathological examination of the lesion after surgical resection revealed no viable tumor cells. (Courtesy of Dr. Thomas Leung, MD.)

experience in patients treated by selective internal radiation (SIR) therapy using yttrium-90 micro sphere^.^^ Change in Serum Alpha-Fetoprotein is a Useful Indicator of Response

These observations demonstrate that monitoring serum AFP may be a useful method of assessing response to nonsurgical treatments. The discrepancy between the tumor marker levels and CT images has been attributed to residual fibrosis and necrotic tissue after the treatment! It has been suggested that the size of tumor nodules seen on CT scans after treatment depends on a number of factors other than cell kill, including the rate of the tumor cell death and the reparative process and the extent to which the repair and reorganization of cells is visible on CT images.43It is, therefore, not surprising that although tumor marker production ceases completely following the killing or damage of the tumor cells, abnormalities may persist on CT imaging. Indeed, previous clinical trials that relied on conventional radiologic criteria to assess response may have missed active agents. The possibility that the treatments described previously may have eradicated the clone of tumor cells producing the tumor marker and that the residual tumor shown on the CT scan represents marker-negative clones can not always be ruled out. It would be interesting to compare serum tumor markers such as AFP with an imaging modality such as positron emission tomography that is capable of differentiating viable tumor from necrotic tissue by measuring metabolic utilization of radio-labeled glucose. SUMMARY

Forty years after its discovery, estimation of serum AFP remains a useful test for clinicians involved in management of patients with HCC or chronic liver

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disease. The test, when used with the conventional cut-off point of 500 ng/mL, has a sensitivity of about 50% and a specificity of more than 90% in detecting the presence of HCC in a patient with coexisting liver disease. New tests that can significantly increase the specificity at lower levels (i.e., between 10 and 500 ng/mL) are available but have, to date, been too complex to be widely applied in clinical practice. Serum AFP estimation may also be useful in monitoring response to therapy, particularly as more effective systemic regimens are becoming available. Indeed, there is preliminary evidence that changes in serum AFP may be a more accurate and sensitive way of determining the degree of response to treatment than conventional imaging procedures that rely on physical determination of tumor size. It may, perhaps, be time to add changes in serum AFP to the conventional imaging criteria for assessing response in clinical trials. References 1. Abelev GI, Perova SD, Khramkova NI, et al: Production of embryonal a-globulin by transplantable mouse hepatomas. Transplant Bulletin 1:174, 1963 2. Alpert E, Drysdale JW, Isselbacher KJ, et al: Human a-fetoprotein: Isolation, characterization, and demonstration of microheterogeneity. J Biol Chem 2473792,1972 3. Alpert E, Feller E R Alpha-fetoprotein (AFP) in benign liver disease: evidence that normal liver regeneration does not induce AFP synthesis. Gastroenterology 74856, 1978 4. Alpert E, Hershberg R, Schur PH, et al: Alpha-fetoprotein in human hepatoma: Improved detection in serum and quantitative studies using a new sensitive technique. Gastroenterology 61:137, 1971 5. Andomo E, Salizzoni M, Schieroni R, et al: Role of serum alpha-fetoprotein in pre- and post-orthotopic liver transplantation (OLT) for malignant disease. Journal of Nuclear Medicine Allied Sciences 33 (suppl):132, 1989 6. Aoyagi Y, Isemura M, Suzuki Y, et al: Fucosylated alphafetoprotein as a marker of early hepatocellular carcinoma. Lancet 2:1353, 1985 7. Aoyagi Y, Suzuki Y, Isemuna M, et al: Differential reactivity of alpha-fetoprotein with lectins and evaluation of its usefulness in the diagnosis of hepatocellular carcinoma. Gann Mannograph 75:809, 1984 8. Barone RM, Byfield JE, Goldfarb PB, et al: Intra-arterial chemotherapy using an implantable infusion pump and liver irradiation for treatment of hepatic metastases. Cancer 502350, 1982 9. Belanger I, Roy S, Allard D New albumin gene 3’ adjacent to the alpha 1-fetoprotein locus. J Biol Chem 269:5481, 1994 10. Bergstrand CG, Czar B: Demonstration of new protein fraction in serum from the human fetus. Scand J Clin Lab Invest 8:174, 1956 11. Bloomer JR, Waldmann TA, McIntire KR, et al: Serum alpha-fetoprotein levels in patients with non-neoplastic liver diseases. Gastroenterology 65:530, 1973 12. Bloomer JR, Waldmann TA, McIntire KR, et al: Alpha-fetoprotein in nonneoplastic hepatic disorders. JAMA 233:38, 1975 13. Buamah PK, Harris R, James DFW, et al: Lentil lectin-reactive alphafetoprotein in the differential diagnosis of benign and malignant liver disease. Clin Chem 329083, 1986 14. Burditt LJ, Johnson MM, Johnson PJ, et a1 Detection of hepatocellular carcinomaspecific alpha-fetoprotein by isoelectric focusing. Cancer 7425, 1994 15. Chaivialle JAP, Ganguli PC: Radioimmunoassay of alpha-fetoprotein in human plasma. Lancet 1:1355, 1973 16. Curtin JP, Rubin SC, Hoskins WK, et al: Second-look laparotomy in endodermal sinus tumour: a report of two patients with normal levels of alpha-fetoprotein and residual tumour at re-exploration. Obstet Gynecol 73:893, 1989 17. Du MQ, Hutchinson WL, Johnson PJ, et al: Differential alpha-fetoprotein lectin binding in hepatocellular carcinoma. Cancer 67476, 1991

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Address reprint requests to Philip J. Johnson, MD, FRCP Department of Clinical Oncology Chinese University of Hong Kong Prince of Wales Hospital Shatin, Hong Kong SAR, China