3 Serum diagnostic tests for pancreatic cancer J O N A T H A N M. R H O D E S CHI KONG CHING
DEFINING AN APPROPRIATE ROLE
Despite the availability of several serological tests for pancreatic cancer their adoption into clinical practice has been slow. These tests have been criticized because they fail to detect pancreatic cancer at a treatable stage and tend to be less specific in the presence of jaundice. Before assessing the individual tests we should decide what role we can reasonably expect them to perform. EARLY DIAGNOSIS--AN UNATTAINABLE ROLE The poor overall results of current treatment for pancreatic cancer (Carter, 1990) contrast with a 5-year survival of 37% following resection of tumours with a diameter under 2 cm (Tsuchiya et al, 1986). This suggests that better results might be achieved if patients could be identified earlier. On the other hand, the size of the tumour does not always correlate with biological behaviour. Thus only 44% of patients with tumours of diameter l.5g/ml). Although the CA19-9 antibody was generated against a colorectal cancer cell line, mucin bearing its epitope is found more frequently in the sera of patients with pancreatic carcinoma than in colorectal or stomach carcinoma (Herlyn et al, 1982). This probably reflects the propensity of this tumour to cause back-secretion of mucin into the blood rather than differential expression of the epitope. This is supported by the finding that CA19-9 binding mucins are found in similar concentration in pancreatic juice from patients with a normal pancreas and from those with pancreatic cancer (Schmiegel et al, 1985; Tatsuta et al, 1985; Kalthoff et al, 1986). There is, however, some evidence that the 2-3 sialylated Lewisa that is recognized by CA19-9 is present in higher concentration in malignant pancreatic cells than in normal cells in contrast to 2-6 sialylated Lewisa which is equally represented in benign and malignant pancreatic cells (Itai et al, 1988). Concentrations of CA19-9 binding activity have been found to be only slightly higher in portal blood than in peripheral venous blood suggesting that there is little first-pass clearance of the mucin by the liver (Talbot et al, 1989) possibly because the mucin is so heavily sialylated (Klug et al, 1988). Radioimmunoassay for serum CA19-9 binding has been reported now in many studies and shown to have a sensitivity for diagnosis of pancreatic cancer of 68-93% (Herlyn et al, 1982; Del Villano et al, 1983; Jalanko et al, 1984; Savarino et al, 1984; Gupta et al, 1985; Satake et al, 1985; Schmiegel et al, 1985; Tatsuta et al, 1985; Yoshikawa et al, 1985; Haglund et al, 1986a; Steinberg et al, 1986; Sail et al, 1987; Fabris et al, 1988; Harmenberg et al, 1988; Hayakawa et al, 1988; Paganuzzi et al, 1988; Ching and Rhodes, 1989; Pleskow et al, 1989; Yamamura et al, 1989; Kiriyama et al, 1990). False negative results will be obtained in any patient with pancreatic cancer who is Lewis negative (Lea-b-) which pertains in about 5% of Caucasians. Specificity is good, most studies reporting values of 80-93 % but elevated CA19-9 binding has been reported in up to 28% of patients with acute or chronic pancreatitis or benign obstructive jaundice. This specificity is too low, however, to allow its use as a screening test (Frebourg et al, 1988) (Figures 1 and 2). Elevated serum CA19-9 binding activity may also be found in some patients (about 30%) with mucin-secreting tumours of other tissues such as colon, stomach and bronchus (Koprowski et al, 1981; Yoshikawa et al, 1985; Ching and Rhodes, 1989). This probably does not constitute a major problem to the clinician as the clinical presentation of these conditions will usually be sufficiently different to avoid confusion. Serum concentrations of CA19-9 binding activity do not discriminate well between localized and advanced disease (Ching and Rhodes, 1989, Pleskow et al, 1989) but may be be valuable in monitoring patients for recurrence following resection. Beretta and colleagues (1987) reported that serum CA19-9 binding values fell to normal in 7/14 patients following resection and that all these patients
838
J. M. RHODES AND C, K. CHING
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Prevalence of disease (%) Figure 1. The application of Bayes theorem to the results of CA19-9 testing in patients with suspected pancreatic cancer showing the effect of the prevalence of the disease in the population under study on the usefulness of the test. Filled circles indicate positive CA19-9 values, open circles indicate negative CA19-9 values. In this study patients with a pancreatic mass and liver metastases demonstrated on CT scan had a prevalence of >80% for pancreatic cancer; at this level of prevalence a negative CA19-9 test is of little diagnostic value as the post-test probability of the patient having the disease is still over 50%. The overall prevalence of pancreatic cancer in the symptomatic patients studied was 21%. At this level of prevalence it can be seen that the CA19-9 assay discriminates much better between patients with the disease and those without (relative risk ratio for a positive CA19-9 value at this level of prevalence is 7.3). It can be seen that the value of the test also falls off sharply at very low levels of prevalence. From Pleskow et al 1989, with permission. s u r v i v e d m o r e t h a n 7 m o n t h s w h e r e a s all the p a t i e n t s with p e r s i s t e n t e l e v a t i o n of CA19-9 b i n d i n g died within this time. A l l s e v e n p a t i e n t s s u b s e q u e n t l y d e v e l o p e d r e c u r r e n t disease over the next 16 m o n t h s a n d in each case an increase in CA19-9 b i n d i n g p r e c e d e d u l t r a s o u n d d e t e c t i o n of r e c u r r e n c e by at least 2 m o n t h s . A c o m m e r c i a l r a d i o i m m u n o a s s a y kit is available b u t this is r a t h e r e x p e n sive a n d has a short shelf-life. This p r o b l e m can to a large e x t e n t be a v o i d e d by using a n e n z y m e - l i n k e d assay which has recently b e c o m e available.
839
SERUM DIAGNOSTIC TESTS
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Figure 2. Results of CA19-9 radioimmunoassay in pancreatic cancer and controls. Ill: Histological diagnosis; O: radiological diagnosis. From Ching and Rhodes 1989, with permission. Thomsen-Friedenreich
(T) b l o o d g r o u p e x p r e s s i o n b y s e c r e t e d m u c i n
The conventional approach to the identification of new tumour markers has been to develop antibodies by immunizing animals with tumour extracts. Since several of the more useful tumour markers have been shown to be carried in serum on mucus glycoproteins, an alternative approach has been to determine whether these mucins could be detected in serum more directly (Ching and Rhodes, 1988). Using a variety of conjugated lectins it was found that peanut agglutinin (PNA) which binds the Thomsen-Friedenreich (T) blood group antigen had the greatest sensitivity for detecting mucin in pancreatic cancer sera (Figure 3). Further studies showed that a similar PNA binding mucin could be extracted from both benign and malignant pancreatic tissue (Ching and Rhodes, 1990a). Partial characterization of the mucin oligosaccharide side-chains demonstrated that the pancreatic cancer related serum mucins variably expressed binding sites for PNA or CA19-9 or both. Moreover, it was possible to predict the presence of at least four different side-chain structures on the mucin molecule (Ching and Rhodes, 1990b; Figure 4).
840
J . M . RHODES AND C. K. CHING
Using the lectin blotting technique for PNA a high specificity for pancreatic cancer was found, although the sensitivity was too low (50%) for general use. A number of normal serum glycoproteins of lower molecular weight as well as CA19-9 were found to bind PNA (Ching and Rhodes, 1988). Since it seemed likely that the mucin would account for a high proportion of the total serum PNA binding activity, an enzyme-linked lectin assay (ELLA) was
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Figure 3. Peanut agglutinin (PNA) blotting of serum from patients with pancreatic cancer and controls with benign obstructive jaundice. The sera have been electrophoresed under reducing conditions on a 2-16% polyacrylamide gel, electroblotted on to nitrocellulose paper and incubated with peroxidase-tagged lectin. As well as a number of normal serum glycoproteins which bind the lectin, there is a very high-molecular-weight glycoprotein (arrowed) present only in the pancreatic cancer sera. Subsequent analysis has confirmed this as a mucin. From Ching and Rhodes 1988, with permission.
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DEFINING AN APPROPRIATE ROLE
Despite the availability of several serological tests for pancreatic cancer their adoption into clinical practice has been slow. These tests have been criticized because they fail to detect pancreatic cancer at a treatable stage and tend to be less specific in the presence of jaundice. Before assessing the individual tests we should decide what role we can reasonably expect them to perform. EARLY DIAGNOSIS--AN UNATTAINABLE ROLE The poor overall results of current treatment for pancreatic cancer (Carter, 1990) contrast with a 5-year survival of 37% following resection of tumours with a diameter under 2 cm (Tsuchiya et al, 1986). This suggests that better results might be achieved if patients could be identified earlier. On the other hand, the size of the tumour does not always correlate with biological behaviour. Thus only 44% of patients with tumours of diameter l.5g/ml). Although the CA19-9 antibody was generated against a colorectal cancer cell line, mucin bearing its epitope is found more frequently in the sera of patients with pancreatic carcinoma than in colorectal or stomach carcinoma (Herlyn et al, 1982). This probably reflects the propensity of this tumour to cause back-secretion of mucin into the blood rather than differential expression of the epitope. This is supported by the finding that CA19-9 binding mucins are found in similar concentration in pancreatic juice from patients with a normal pancreas and from those with pancreatic cancer (Schmiegel et al, 1985; Tatsuta et al, 1985; Kalthoff et al, 1986). There is, however, some evidence that the 2-3 sialylated Lewisa that is recognized by CA19-9 is present in higher concentration in malignant pancreatic cells than in normal cells in contrast to 2-6 sialylated Lewisa which is equally represented in benign and malignant pancreatic cells (Itai et al, 1988). Concentrations of CA19-9 binding activity have been found to be only slightly higher in portal blood than in peripheral venous blood suggesting that there is little first-pass clearance of the mucin by the liver (Talbot et al, 1989) possibly because the mucin is so heavily sialylated (Klug et al, 1988). Radioimmunoassay for serum CA19-9 binding has been reported now in many studies and shown to have a sensitivity for diagnosis of pancreatic cancer of 68-93% (Herlyn et al, 1982; Del Villano et al, 1983; Jalanko et al, 1984; Savarino et al, 1984; Gupta et al, 1985; Satake et al, 1985; Schmiegel et al, 1985; Tatsuta et al, 1985; Yoshikawa et al, 1985; Haglund et al, 1986a; Steinberg et al, 1986; Sail et al, 1987; Fabris et al, 1988; Harmenberg et al, 1988; Hayakawa et al, 1988; Paganuzzi et al, 1988; Ching and Rhodes, 1989; Pleskow et al, 1989; Yamamura et al, 1989; Kiriyama et al, 1990). False negative results will be obtained in any patient with pancreatic cancer who is Lewis negative (Lea-b-) which pertains in about 5% of Caucasians. Specificity is good, most studies reporting values of 80-93 % but elevated CA19-9 binding has been reported in up to 28% of patients with acute or chronic pancreatitis or benign obstructive jaundice. This specificity is too low, however, to allow its use as a screening test (Frebourg et al, 1988) (Figures 1 and 2). Elevated serum CA19-9 binding activity may also be found in some patients (about 30%) with mucin-secreting tumours of other tissues such as colon, stomach and bronchus (Koprowski et al, 1981; Yoshikawa et al, 1985; Ching and Rhodes, 1989). This probably does not constitute a major problem to the clinician as the clinical presentation of these conditions will usually be sufficiently different to avoid confusion. Serum concentrations of CA19-9 binding activity do not discriminate well between localized and advanced disease (Ching and Rhodes, 1989, Pleskow et al, 1989) but may be be valuable in monitoring patients for recurrence following resection. Beretta and colleagues (1987) reported that serum CA19-9 binding values fell to normal in 7/14 patients following resection and that all these patients
838
J. M. RHODES AND C, K. CHING
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Prevalence of disease (%) Figure 1. The application of Bayes theorem to the results of CA19-9 testing in patients with suspected pancreatic cancer showing the effect of the prevalence of the disease in the population under study on the usefulness of the test. Filled circles indicate positive CA19-9 values, open circles indicate negative CA19-9 values. In this study patients with a pancreatic mass and liver metastases demonstrated on CT scan had a prevalence of >80% for pancreatic cancer; at this level of prevalence a negative CA19-9 test is of little diagnostic value as the post-test probability of the patient having the disease is still over 50%. The overall prevalence of pancreatic cancer in the symptomatic patients studied was 21%. At this level of prevalence it can be seen that the CA19-9 assay discriminates much better between patients with the disease and those without (relative risk ratio for a positive CA19-9 value at this level of prevalence is 7.3). It can be seen that the value of the test also falls off sharply at very low levels of prevalence. From Pleskow et al 1989, with permission. s u r v i v e d m o r e t h a n 7 m o n t h s w h e r e a s all the p a t i e n t s with p e r s i s t e n t e l e v a t i o n of CA19-9 b i n d i n g died within this time. A l l s e v e n p a t i e n t s s u b s e q u e n t l y d e v e l o p e d r e c u r r e n t disease over the next 16 m o n t h s a n d in each case an increase in CA19-9 b i n d i n g p r e c e d e d u l t r a s o u n d d e t e c t i o n of r e c u r r e n c e by at least 2 m o n t h s . A c o m m e r c i a l r a d i o i m m u n o a s s a y kit is available b u t this is r a t h e r e x p e n sive a n d has a short shelf-life. This p r o b l e m can to a large e x t e n t be a v o i d e d by using a n e n z y m e - l i n k e d assay which has recently b e c o m e available.
839
SERUM DIAGNOSTIC TESTS
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Figure 2. Results of CA19-9 radioimmunoassay in pancreatic cancer and controls. Ill: Histological diagnosis; O: radiological diagnosis. From Ching and Rhodes 1989, with permission. Thomsen-Friedenreich
(T) b l o o d g r o u p e x p r e s s i o n b y s e c r e t e d m u c i n
The conventional approach to the identification of new tumour markers has been to develop antibodies by immunizing animals with tumour extracts. Since several of the more useful tumour markers have been shown to be carried in serum on mucus glycoproteins, an alternative approach has been to determine whether these mucins could be detected in serum more directly (Ching and Rhodes, 1988). Using a variety of conjugated lectins it was found that peanut agglutinin (PNA) which binds the Thomsen-Friedenreich (T) blood group antigen had the greatest sensitivity for detecting mucin in pancreatic cancer sera (Figure 3). Further studies showed that a similar PNA binding mucin could be extracted from both benign and malignant pancreatic tissue (Ching and Rhodes, 1990a). Partial characterization of the mucin oligosaccharide side-chains demonstrated that the pancreatic cancer related serum mucins variably expressed binding sites for PNA or CA19-9 or both. Moreover, it was possible to predict the presence of at least four different side-chain structures on the mucin molecule (Ching and Rhodes, 1990b; Figure 4).
840
J . M . RHODES AND C. K. CHING
Using the lectin blotting technique for PNA a high specificity for pancreatic cancer was found, although the sensitivity was too low (50%) for general use. A number of normal serum glycoproteins of lower molecular weight as well as CA19-9 were found to bind PNA (Ching and Rhodes, 1988). Since it seemed likely that the mucin would account for a high proportion of the total serum PNA binding activity, an enzyme-linked lectin assay (ELLA) was
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Figure 3. Peanut agglutinin (PNA) blotting of serum from patients with pancreatic cancer and controls with benign obstructive jaundice. The sera have been electrophoresed under reducing conditions on a 2-16% polyacrylamide gel, electroblotted on to nitrocellulose paper and incubated with peroxidase-tagged lectin. As well as a number of normal serum glycoproteins which bind the lectin, there is a very high-molecular-weight glycoprotein (arrowed) present only in the pancreatic cancer sera. Subsequent analysis has confirmed this as a mucin. From Ching and Rhodes 1988, with permission.
841
SERUM DIAGNOSTIC TESTS
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