Clinical Chemistry / CA19-9 Methods Comparison

The Pitfalls of CA19-9 Routine Testing and Comparison of Two Automated Immunoassays in a Reference Oncology Center
Rita Passerini, DSc,1 Maria C. Cassatella, DSc,1 Sara Boveri, MS,2 Michela Salvatici, DSc,1 Davide Radice, MSc,3 Laura Zorzino, DSc,1 Claudio Galli, MD, PhD,4 and Maria T. Sandri, MD1
Key Words: CA19-9; Standardization; Tumor markers; Automated immunoassays; Interference
DOI: 10.1309/AJCPOPNPLLCYR07H

Abstract
We evaluated CA19-9 as a marker of various malignancies and compared the results of 2 commercial immunoassays. The Abbott ARCHITECT i2000 and Roche cobas 410 immunoassays were used on 500 consecutive samples to evaluate the frequency of positive results by cancer type and the correlation between assays. The patients were tested before or after surgery and/or during chemotherapy. The rate of results exceeding conventional thresholds was 92.3% in pancreatic cancer, 36.8% in gastric cancer, and ranged from 3.0% to 35.9% in other tumors. Agreement (90.6%) and correlation (R2 = 0.865) between the 2 assays were good and the frequency of highly discordant results was low (6/500). In some cases, interference by heterophilic antibodies was demonstrated. The 2 methods were comparable in diagnostic accuracy and had good correlation but are not interchangeable. Patients should always be monitored for CA19-9 with the same method and it should be indicated in the report.

The carbohydrate antigen sialyl Lewis a, most commonly referred to as CA19-9 from the name of the monoclonal antibody that is currently used for its detection, is the most frequently used tumor marker for cancers of the digestive tract, after carcinoembryonic antigen.1-3 The rise of tissue and blood levels of this antigen is believed to depend on the tumorrelated hypoxia in locally advanced cancers that causes the transcription of several glycogenes involved in sialyl Lewis a synthesis. The expression of this determinant is therefore enhanced in more malignant, advanced cancers whose cells are more resistant to hypoxia, and is associated with a greater probability of the patient developing hematogenous metastasis.4 Although the maximum tissue expression of CA19-9 occurs in pancreatic cancer,5 this antigen is not tissue-specific, because it has been demonstrated in cancers involving other organs, such as the stomach, lung, colon, breast, ovary, and uterus.6-12 Assays for the quantitative detection of CA19-9 have been available for more than 2 decades, and almost all of them depend on the use of the monoclonal antibody 1116NS-19-9, which recognizes a carbohydrate epitope expressed on circulating antigen. The interpretation of results for CA19-9 is often hampered by nonspecific elevations, either because of associate morbidity, such as obstruction of the biliary tree or inflammation, or assay-dependant, both in diseased and healthy subjects. Furthermore, the levels of CA19-9 measured using different assays may show significant differences in individual patients or samples. The aims of our study were (1) to compare the results obtained by 2 widespread commercial methods and (2) to evaluate the levels of this antigen in patients with cancers of different organs for which CA19-9 can be used.
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Materials and Methods

Patients and Samples This study evaluated 500 consecutive serum samples that were sent to the Laboratory Division of the European Institute of Oncology (IEO) during a 3-month period (AprilJune 2010). Informed consent was obtained from all patients to perform additional testing on their residual samples, and no supplemental blood samples were drawn for the purpose of this study. All patients were anonymous and were identified by alphanumeric codes; a review of their clinical records allowed us to carry on a separate evaluation by cancer type and to consider also the stage of the disease (in particular presurgery, postsurgery, and/or during chemotherapy follow-up). CA19-9 Testing All samples, identified by a barcode, were tested for CA19-9 using 2 commercial assays: a chemiluminescent microparticle enzyme immunoassay on the ARCHITECT i2000 analyzer (Abbott Diagnostics, Wiesbaden, Germany) and an enzyme immunoassay on the cobas 410 instrument (Roche Diagnostic System, Basel, Switzerland). Both assays use the monoclonal antibody 1116-NS-19-9 and have been described elsewhere.13,14 Testing with both methods was carried out within 24 hours of collection. The results obtained with these 2 assays were initially evaluated from a comparative point of view, considering the absolute values and the percentage of samples exceeding the respective thresholds suggested by the manufacturers (37 U/mL for ARCHITECT i2000 and 39 U/mL for cobas 410). The quantitative results and the distribution of CA19-9 levels in different cancers were then evaluated. The results were considered as highly discrepant if, besides being positive by one method and negative by the other, the value was higher than 100 U/mL. This value was chosen as an arbitrary threshold, corresponding to definitely suspicious clinical results. On all specimens with a discrepant result, an additional procedure was used to explore the possible interference by heterophilic antibodies: samples were incubated in specific coated tubes (Scantibodies Laboratory Inc, Santee, CA) for 1 hour at room temperature and then retested with the method that gave the initial positive result; the variation of CA19-9 levels was then recorded. Statistical Analysis The CA19-9 assays were distributed in a box-plot according to the cancer site and method. Concordance between CA19-9 assays was calculated using the Deming regression analysis. Furthermore, the CA19-9 values were categorized and cross-tabulated by assay methods whose agreement was tested using a weighted k statistic and the symmetry test. The between-assay differences and age normality assumption were checked using the Shapiro-Wilk
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test; significance was calculated using the paired Student t test or the signed rank sum test or the 2-sample 2-sided Wilcoxon test as appropriate; and the differences between percentages were calculated with the c2 test. P < .05 was considered significant. Data were analyzed with SAS software version 9.2 (SAS Institute, Cary, NC).

Results
Five hundred serum specimens were obtained from 429 patients with various types of cancer. The distribution of different cancer types of the whole population is reported in Table 1. The age of patients suffering from cancer of the uterus was the only variable that showed a significant difference compared with all the other groups (P < .05). With the exception of ovarian cancer samples, 19 of which were also tested before surgery, all samples were taken during the follow-up after surgery and/or chemotherapy; the clinical diagnosis in all patients had been established 1 month to 11 years earlier. Results of the concordance between the 2 CA19-9 assays are reported in Table 2. The highest frequency of positive samples was recorded in pancreatic carcinoma (12/13, 92.3%), the second highest in gastric cancers (7/19, 36.8%), followed by endometrial cancer (35.9%), cancer of the uterus (29.8%), colorectal cancer (28.5%), breast cancer (24.3%), ovarian cancer (15.2%), and other malignancies (3%). The overall agreement between the 2 CA19-9 assays was 90.6%, with 378 concordant negative and 75 concordant positive results (k = 0.70; 95% CI = 0.62-0.78). Of the 47 discrepant specimens, 31 were above the diagnostic cut-off with cobas 410 and 16 with ARCHITECT i2000. The concordance between the 2 assays ranged from 79.6% to 100% among different cancer types. Of the 47 discrepant specimens, 6 were classified as highly discordant according to the following adopted criteria:

Table 1 Distribution of Different Cancer Types Among the Population

Cancer Site Pancreas Colon/rectum Stomach Ovary Breast Endometrium Uterus Other Total
*

No. (%) of Samples 13 (2.6) 130 (26.0) 19 (3.8) 105 (21.0) 70 (14.0) 39 (7.8) 57 (11.4) 67 (13.4) 500

No. (%) of Patients 7 (1.6) 105 (24.5) 17 (4.0) 85 (19.8) 67 (15.6) 34 (7.9) 47 (11.0) 67 (15.6) 429

Mean SD Age, y 69.7 4.7 66.0 10.0 61.0 12.8 55.8 12.6 55.9 12.4 60.1 11.8 48.2 12.1* 57.9 15.9 58.8 13.4

P < .05.

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Table 2 Concordance Between the CA19-9 Results Obtained With ARCHITECT i2000 and cobas 410
No. (%) Concordant Negative/ Cancer Site of Samples Positive Ratio Pancreas Colon/rectum Stomach Ovary Breast Endometrium Uterus Other Total 13 (2.6) 130 (26.0) 19 (3.8) 105 (21.0) 70 (14.0) 39 (7.8) 57 (11.4) 67 (13.4) 500 (100) 1:9 93:23 12:2 89:10 53:8 25:10 40:11 65:2 378:75 Positive Only ARCHITECT/ cobas Ratio Concordance (%) Agreement (k) 0:3 5:9 4:1 2:4 3:6 1:3 1:5 0:0 16:31 76.9 89.2 73.7 94.3 87.1 89.7 89.5 100 90.6 0.32 0.70 0.30 0.74 0.56 0.76 0.72 1.00 0.70 Overall Positivity (%) 92.3 28.5 36.8 15.2 24.3 35.9 29.8 3.0 24.4

5 of them were positive with ARCHITECT i2000, with values ranging from 119.09 to 487.8 U/mL and corresponding cobas 410 results ranged between 5.62 and 36.8 U/mL, and 1 was positive with cobas 410 at 113.7 U/mL and negative with ARCHITECT i2000 at 17.22 U/mL. For the remaining 41 samples, the average difference in CA19-9 levels between the 2 assays was 86% (median, 54.2%), and the values for 15 of them (36.6%) were within 10% of the proposed diagnostic thresholds. The Deming regression analysis of quantitative results Figure 1A demonstrated a good overall agreement between ARCHITECT i2000 and cobas 410, with a correlation coefficient of R2 = 0.86. When the analysis was restricted to samples yielding a result lower than 200 U/mL Figure 1B, the correlation was weaker (R2 = 0.56) because of the greater effect of discordant results. When data were analyzed separately for each cancer type, the distribution of CA19-9 levels

with both methods was similar, with levels being significantly higher in pancreatic cancer than in all other cancer types with both assays Figure 2A and Figure 2B. Nevertheless, the 2 assays had noticeable differences in measurement: median levels were almost identical in pancreatic cancer, with almost all samples testing positive, whereas they differed significantly in 4 of the other 7 groups (breast, P = .0145; ovary, P = .0004; colorectal and other cancers, P < .0001) Table 3. The differences in CA19-9 measurement between the 2 assays were also evident when the distribution of negative and positive results was considered Figure 3A and Figure 3B. Overall, the negative results were significantly less frequent with ARCHITECT i2000 than cobas 410 (Figure 3A, P < .001, symmetry test). Furthermore, the median negative value of ARCHITECT i2000 was significantly lower than that of cobas 410 (7.83 U/mL vs 12.22 U/mL; P < .0001). Conversely, among positive samples, the frequency of results was

A
4,500 4,000 cobas CA19-9 (U/mL) 3,500 3,000 2,500 2,000 1,500 1,000 500 0 0 1,000 2,000 3,000 4,000 5,000 6,000 7,000 8,000 9,000 ARCHITECT CA19-9 (U/mL)

B
200 175 cobas CA19-9 (U/mL) 150 125 100 75 50 25 0 0 25 50 75 100 125 150 175 200 ARCHITECT CA19-9 (U/mL)

Figure 1 A, Correlation (Pearson) and linear regression (Deming) analysis of CA19-9 assays using ARCHITECT i2000 and cobas 410 on 500 serum samples from patients with different types of cancer. B, A subrange plot for CA19-9 values less than or equal to 200 U/mL. A, R 2 = 0.86; y = 0.54 * x + 23.55. B, R2 = 0.56; y = 0.87 * x + 7.78.
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10,000 ARCHITECT CA19-9 (U/mL)

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va ry B En re as do t m et riu m U te ru s

va ry B En re as do t m et riu m U te ru s
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Figure 2 A, Notched outlier box-plots show 95% confidence intervals for ARCHITECT i2000. B, 95% confidence intervals for cobas 410. CA19-9 results are shown according to cancer type. Table 3 Differences in Mean CA19-9 Values (U/mL) Obtained With the ARCHITECT i2000 and the cobas 410 Assays*
Cancer Type ARCHITECT i2000 cobas 410 P Value

Pancreas 283.32 Colon/rectum 9.36 Stomach 9.88 Ovary 8.11 Breast 9.43 Endometrium 12.69 Uterus 15.75 Other 6.42
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268.8 .8403 16.33 <.0001 17.39 .5302 13.43 .0004 15.74 .0145 15.23 .1757 24.77 .0946 12.04 <.0001

Mean CA19-9 values are given in units per milliliter.

not significantly different between ARCHITECT i2000 and cobas 410 (Figure 3B, P = .758, symmetry test). Finally, 45 of the 47 discrepant specimens (2 were missing because of insufficient sample volume) were tested for a possible interference by heterophilic antibodies. Both assays appeared to be affected by this type of interference, because 14 of the 30 cobas 410positive samples and 6 of 15 ARCHITECT i2000positive samples (46.7% vs 40%; P = .671) became negative after retesting. Of note, 3 of the 5 highly discrepant positive samples with ARCHITECT i2000 became negative after this procedure. Table 4 shows

100 90 80 70 Percent 60 50 40 30 20 10 0

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100 90 80 70 Percent 60 50 40 30 20 10 0

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U/mL

Figure 3 Frequency distribution of negative (A) and positive (B) results for CA19-9 with ARCHITECT i2000 and cobas 410. Values are expressed in units per milliliter (U/mL). Because of the different thresholds, on negative samples, the label 31 cut-off includes values ranging from 31.0 to 36.9 U/mL with ARCHITECT i2000 and from 31.0 to 38.9 U/mL with cobas 410; on positive samples, the label cut-off 100 includes values ranging from 37.0 to 99.9 U/mL with ARCHITECT i2000 and from 39.0 to 99.9 U/mL with cobas 410. The difference between median values for negative results (7.83 U/mL with ARCHITECT i2000; 12.22 U/mL with cobas 410) was P < .0001 (signed rank sum test).
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comparative discrepant results after excluding interference by heterophilic antibodies. The patterns of signal decrease were markedly different between the 2 assays: all 30 cobas 410only positive samples showed a signal reduction between 11% and 50% (mean SD, 23.9 5.2%; median, 24.3%), a more than 10% reduction of levels was observed only on 5 ARCHITECT i2000only positive samples, whereas the remaining 10 samples showed a mean reduction of 2.5% (median, 1.2%), that may be ascribed entirely to the assay imprecision.

Table 4 Comparative Values of Discrepant Results Between the 2 Assays*

Patient Cancer Site Architect Cobas 43.00 60.31 59.81 44.03 40.43 41.80 39.36 42.55 43.29 83.25 69.19 44.03 42.20 44.18 46.48 50.00 57.01 59.60 105.8 86.94 66.96 40.21 44.89 70.31 72.40

Discussion
The current international guidelines state that, based on available evidence, the use of CA19-9 may be recommended only for the monitoring of pancreatic cancer.15 Similar to other cancer biomarkers, the potential usefulness of CA19-9 has been explored in screening and diagnosis; thus far no evidence supports its use for screening purposes , but its potential applications for the differential diagnosis are still debated.15,16 Although the degree of increase in CA19-9 levels may be useful in differentiating pancreatic adenocarcinomas from inflammatory conditions of the pancreas, the attainable sensitivity (70%-80%) and specificity (68%-91%) are not deemed sufficient. False-negative results will always be found in subjects with Lewis anegative genotype, representing about 5% to 10% of the white population, whereas no data on other races are available. However, false-positive findings may occur in several situations. Although false-positive results have often been reported in asymptomatic subjects and in other patients for whom this test should not be ordered,17-19 and indeed inadequate requests are quite frequent,20 substantial increases are commonly reported in symptomatic patients. These increases may be ascribed to pancreatic cancer, ie, obstruction from nonmalignant diseases, such as chronic and acute pancreatitis, cholangitis, and liver cirrhosis,15 with levels that have been shown to be even higher than 1,000 U/mL. Because levels may decrease to normal values after proper relief of the obstructive syndromes, CA19-9 testing should be repeated after treatment. In patients diagnosed as having pancreatic cancer, CA199 is valuable either alone or in association with other parameters21; a decline of CA19-9 levels after surgery to values of 90 to 200 U/mL is considered a strong predictor of overall survival.17,21 Current recommendations are that serum CA199 levels should be considered for risk stratification in patients with pancreatic cancer, though CA19-9 is only one of the multiple factors that affect prognosis and treatment planning. In advanced disease states, CA19-9 may be particularly useful for assessing the response to systemic treatment. In the monitoring of patients, a clinically significant difference
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ARCHITECT values negative, Cobas values positive NC Breast 36.34 BF Breast 36.02 PMA Breast 32.45 MR Endometrium 19.94 TMT Uterus 35.07 BML Uterus 27.01 NA Ovary 29.30 PE Ovary 23.43 PE Ovary 30.94 CF Pancreas 17.22 CF Pancreas 18.52 BG Pancreas 20.16 AM Colon 26.24 AM Colon 23.81 FMT Colon 25.51 FMT Colon 26.12 Cobas values negative, ARCHITECT values positive BM Breast 28.12 BM Breast 27.51 GG Endometrium 36.80 MTG Stomach 23.53 MS Stomach 31.16 RG Stomach 24.43 MP Colon 25.13 LF Colon 37.03 LF Colon 36.26
*

Discrepant results were compared after excluding interference by heterophilic antibodies. Values are given in units per milliliter.

between successive levels, ie, the critical difference, has been established taking into account both the intraindividual biologic variation and the analytic imprecision as evaluated on apparently normal subjects. The current opinion is that a change of 40% to 50% in CA19-9 serial levels is adequate for this purpose.22-24 In addition to pancreatic malignancies, CA19-9 can be increased in multiple types of advanced gastrointestinal and gynecologic cancers. An overview study found elevated CA19-9 levels in patients with bile duct cancer, esophageal cancer, and, most noticeably, in 41% and 34% of patients with gastric or colorectal cancer, respectively.25 The most recent National Academy of Clinical Biochemistry guidelines do not recommend the use of tumor markers, including CA19-9, for screening, differential diagnosis, or even for prognosis or monitoring response to treatment.26 However, several reports, mainly from Asia, have highlighted the usefulness of CA19-9 in detecting recurrences of gastric or colorectal cancer during monitoring.27-31 Moreover, although reports have shown that CA19-9 is commonly elevated in primary ovarian mucinous tumors, it cannot be used to distinguish among benign, borderline, or malignant tumors.32 In our experience, the percentages of patients showing both raised and median values of CA19-9 in cancers other
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than pancreatic cancer were similar among the different cancer types we analyzed. Because this was a cross-sectional observation over 3 months, we could not identify long-term changes of CA19-9 levels, or ascertain the clinical usefulness of these levels; however, this was not the aim of our study. The comparison of different assays for CA19-9 has been studied extensively over the last few years, including by our group.13,33-35 Our current study differs substantially from the previous ones in that we selected to enroll only patients with cancer, and thus all samples were clinically characterized. In contrast, 2 previous studies enrolled a specific number of patients with pancreatic cancer (n = 30 and n = 50),34,35 colorectal cancer (n = 43), or other malignancies (n = 33).34 Although our approach did not allow us to establish a true clinical specificity of the 2 assays, we did enroll a control group of 67 patients with other types of cancer for which the use of CA19-9 is definitely not indicated. In this group, both assays demonstrated a specificity of 97%, with absolute concordance (65 negative and 2 positive results). We aimed to verify the relevance of one of the possible causes of interference for all immunoassays,36-38 ie, the presence of heterophilic antibodies that was previously reported.38 Such interference seems to affect both immunoassays considered in our study, because a reduction of values below the proposed diagnostic cut-off was seen in 40% to 46% of discrepant specimens after these antibodies were removed. However, the pattern of interference was not similar, because the signal reduction was almost equal in all cobas 410reactive specimens and detectable only in 33.3% (5/15), but to a much higher degree, in the ARCHITECT i2000reactive specimens. Moreover, the issue of analytic specificity for CA19-9 assays, as well as for other tumor markers, is linked to the concept of threshold, or cut-off, that needs to be discussed, in our opinion. First, as stated by Duffy et al,15 the cut-off point for CA19-9 depends on the context or the question being addressed; no values can be suggested to accurately discriminate between benign and malignant disease. Second, reference values usually reported as the 95th or 97.5th percentile, are generally established in healthy subjects, and do not represent the real life population that will be tested for this marker. Third, for any assay, the reference population is not large enough to give a really robust estimate, and ethnic diversities are also taken into consideration. For instance, when the cut-off proposed by the manufacturers for the ARCHITECT i2000 CA19-9 assay (37 U/mL) was verified in other studies, the value was shown to be significantly lower, at 25.9 and 26.4 U/mL, respectively33,34; in our experience, the upper 95th percentile of negative results in patients with cancer was 28.4 U/mL with ARCHITECT i2000 and 32.6 U/ mL with cobas 410. Assay-specific ranges also reflect the differences in measurement of CA19-9 obtained with the different
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immunoassay methods; although all studies report a good to excellent concordance, similar to, or sometimes exceeding, the 90.6% value we report herein, the same studies very often show a dismal qualitative concordance that may be as low as 51% in patients with benign disease.34 This concordance rate rises to more than 80% for patients with pancreatic cancer or other malignances, as seen in the current study (90.6%). Indeed, in the 3 patients with 3 specimens examined during the study period, the trend of CA19-9 was identical with the 2 assays, with levels remaining stable in 2 and rapidly decreasing in the third one. In conclusion, our experience, as well as that of other studies and current guidelines in this field,13,15,16,26,33-35 allow us to exclude the interchangeability of results obtained with different assays for CA19-9. We then recommend that patients always be monitored for CA19-9 with the same assay and that the method used should always be indicated in the report.
From the 1Division of Laboratory Medicine, 2Preventive Gynecology Unit, 3Division of Epidemiology and Biostatistics, European Institute of Oncology, Milan, Italy; and 4Scientific Affairs, Abbott Diagnostics, Rome, Italy. Address reprint requests to Dr Passerini: Laboratory Medicine Division, European Institute of Oncology, Via Ripamonti 435, 20141 Milan, Italy; rita.passerini@ieo.it. Acknowledgments: We wish to thank Ermenenziana Soccio, laboratory technician, for performing the tests and the physicians and nurses who helped us in collecting the clinical information on the patients whose samples were used in this study.

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