PAM4 enzyme immunoassay alone and in combination with CA 19-9 for the detection of pancreatic adenocarcinoma




The monoclonal antibody PAM4 has high specificity for pancreatic ductal adenocarcinoma (PDAC), as well as its precursor lesions, but has not been found to be reactive with normal and benign pancreatic tissues. The objective of the current study was to evaluate a PAM4-based serum enzyme immunoassay alone and in combination with the carbohydrate antigen (CA) 19-9 assay for the detection of PDAC, with particular attention to early stage disease.


Sera from patients with confirmed PDAC (N = 298), other cancers (N = 99), benign disease of the pancreas (N = 120), and healthy adults (N = 79) were evaluated by a specific enzyme immunoassay for the concentration of PAM4 and CA 19-9 antigen levels by blinded analyses. All tests for statistical significance were 2-sided.


The overall sensitivity for PAM4 detection of PDAC was 76%, with 64% of patients with stage I disease also identified. The detection rate was considerably higher (85%) for patients with advanced disease. The assay demonstrated high specificity compared with benign pancreatic disease (85%), with a positive likelihood ratio of 4.93. CA 19-9 provided an overall sensitivity of 77%, and was positive in 58% of patients with stage I disease; however, the specificity was significantly lower for CA 19-9 (68%), with a positive likelihood ratio of 2.85 (P = .026 compared with PAM4). It is important to note that a combined PAM4 and CA 19-9 biomarker serum assay demonstrated an improved sensitivity (84%) for the overall detection of PDAC without a significant loss of specificity (82%) compared with either arm alone.


The PAM4 enzyme immunoassay identified approximately two-thirds of patients with stage I PDAC with high discriminatory power with respect to benign, nonneoplastic pancreatic disease. These results provide a rationale for testing patient groups considered to be at high risk for PDAC with a combined PAM4 and CA 19-9 biomarker serum assay for the detection of early stage PDAC. Cancer 2013. © 2012 American Cancer Society.


Although imaging methods play an important role in the detection of pancreatic cancer,1-4 especially in selecting a course of therapy, there are limitations with respect to the detection of small lesions,1 as well as for discriminating pancreatic cancer from pancreatitis and other benign lesions.2, 5, 6 Thus, at the current time, only 7% of all pancreatic cancers are detected early.7 With no effective treatment for advanced pancreatic cancer, the prognosis for the majority of patients is dismal.

Biomarkers potentially can provide a more cost-effective means for early detection and diagnosis, and a great deal of effort has been devoted to biomarker research.8-16 Such biomarkers also are in high demand for the detection and diagnosis of pancreatic cancer, especially early stage disease, but to the best of our knowledge no single assay to date has achieved this role.

We have been engaged in the preclinical and clinical evaluation of monoclonal antibodies (MoAbs) for the detection, diagnosis, imaging, and treatment of cancer, and have focused on the assessment of the PAM4 antibody, which binds to a large-size mucin17 for the detection and diagnosis of pancreatic ductal adenocarcinoma (PDAC),18-21 for imaging,22, 23 and for therapy as the clinical reagent 90Y-clivatuzumab tetraxetan.24, 25 Our data at the tissue level provide strong evidence that the reactivity of the PAM4 antibody is highly restricted to PDAC, and that the specific PAM4 epitope is expressed (or becomes accessible) at the earliest stages of PDAC development (ie, the precursor lesions pancreatic intraepithelial neoplasia [PanIN], intraductal papillary mucinous neoplasia, and mucinous cystic neoplasm).17, 18 The PAM4 antigen is absent from normal pancreatic tissues and nonneoplastic disease of the pancreas. The antigen was not detected within ducts or acinar cells, nor isolated acinar-ductal metaplasia (ADM) within surgical specimens obtained from > 50 patients with chronic pancreatitis (CP).20-22 However, the PAM4 antigen was expressed by invasive PDAC identified in 2 cases, and in PanIN precursor lesions found in several of the specimens.

We recently reported that a serum-based enzyme immunoassay (EIA) was able to correctly identify 82% of patients with known PDAC and, importantly, that this assay had promising sensitivity for the detection of early stage disease (62% of patients with stage I and 86% of patients with stage II disease).20 In the current study, we confirmed and extended these findings in a much larger patient population that included patients with both malignant and benign diseases of the pancreas and surrounding tissues. Importantly, we also compared and combined the PAM4 assay with carbohydrate antigen (CA) 19-9 assay results, the latter considered the standard for use in the management of pancreatic cancer, and demonstrated that the unique combination of these 2 biomarkers provides significantly greater sensitivity for the detection of PDAC with high diagnostic value.


Human Specimens

Sera from a total of 517 patients (assessed at each of the individual medical centers involved) with histologically or cytologically confirmed diagnoses of the pancreaticobiliary and periampullary tissues were received from the University Medical Center Gottingen (UMG; Gottingen, Germany); Johns Hopkins Medical Institutions (JHMI; Baltimore, Md), Immunomedics, Inc (IMMU; Morris Plains, NJ); and the Cooperative Human Tissue Network (CHTN; Philadelphia, Pa). All sera from UMG, JHMI, and CHTN were obtained from patients who had undergone surgical resection with stage of disease established according to the criteria set by the International Union Against Cancer (UICC). In total, patients with early stage I and stage II disease comprised 40% of the PDAC population within this study. In addition, 32 specimens from a control group of patients diagnosed with colon cancer were purchased from ZeptoMetrix Corporation (Buffalo, NY), and 79 specimens from healthy adults were purchased from ZeptoMetrix (29 specimens) and SeraCare Life Sciences (Milford, Mass) (50 specimens). Separate Institutional Review Boards approved this study, and the New England Institutional Review Board approved the protocol of the Center for Molecular Medicine and Immunology. All specimens were deidentified and the enzyme immunoassay were performed in a blinded fashion.

Enzyme Immunoassays

The PAM4 EIA was conducted using the procedure described in a prior report.20 The anonymous serum specimens (300 μL) were first extracted with an equal volume of n-butanol, followed by mixture with an equal volume of chloroform to invert the organic and aqueous layers. The aqueous layer was then evaluated in the PAM4 EIA. Nonlinear regression was performed with Prism 4.0 statistical software (GraphPad Software, La Jolla, Calif) to interpolate unknowns.

CA 19-9 enzyme immunoassay were performed with a commercial kit (BioCheck, Inc, Foster City, Calif) according to the manufacturer's procedure. A value > 37 U/mL was considered a positive test. For a combination of the PAM4 and CA 19-9 enzyme immunoassay results, we used the algorithm: Result = (PAM4) + 0.01*(CA 19-9); a value > 2.4 U/mL was considered to be a positive interpretation. This algorithm was chosen by review of receiver operating characteristic (ROC) curves for the maximum area under the curve.

Statistical Analyses

ROC curves were evaluated using MedCalc (version 7.5; MedCalc Software, Gent Belgium). Regression analyses, nonparametric log-rank comparisons, the Student t test, and analysis of variance for the comparison of test groups were evaluated using Prism statistical software (version 4.0; (GraphPad Software; San Diego, Calif.). All tests for statistical significance were 2-sided.


Performance Characteristics of the PAM4-Based EIA

To evaluate EIA performance, normal human serum (pool of 5 individuals) was spiked with the PAM4 antigen at nominal concentrations surrounding the critical cutoff point of 2.4 U/mL (8.26 U/mL, 3.30 U/mL, 1.32 U/mL, and 0 U/mL) and then assayed 14 times over a 2-month period. Linear regression of nominal versus measured antigen concentrations gave a trendline with a correlation coefficient (r2) of 0.997, indicating a consistent linear relation over the range of concentrations surrounding the cutoff value (Fig. 1). Values for the coefficients of variation (CV) were 11.30% and 12.76% for the 8.26 U/mL and 3.30 U/mL concentrations of spiked sera above the cutoff value, and 20.12% for the 1.32 U/mL value below the cutoff, which were well within the guidelines for reproducibility of 15% above the cutoff and 20% at the cutoff value.20 A cutoff value of 2.4 U/mL, determined by ROC analysis in a prior learning set,20 was used as an indicator of positive response.

Figure 1.

Accuracy of the quantitation of normal human serum spiked with the PAM4 antigen is shown. A consistent linear relation was observed for nominal versus measured antigen concentrations at or near the cutoff value of 2.4 U/mL. r2 indicates correlation coefficient.

PAM4 Antigen Levels in the Circulation

Overall sensitivity for the detection of PDAC (298 patients, with those with early stage I and II disease comprising 40% of cases) was 76%, with a median value of 10.40 U/mL at a specificity of 96% with respect to healthy adults (Table 1). The detection rate for patients with stage I disease was 64% (28 cases) and, as expected, was considerably higher for patients with advanced disease (85%). For the most part, sera from patients with pancreatic cancers arising from other tissues of origin (squamous, gastrointestinal stromal tumor, clear cell, etc) did not have detectable levels of the PAM4 antigen. Furthermore, several nonpancreatic cancers with metastases to the pancreas (11 cases) were among the group of sera specimens evaluated, but only 2, an ovarian cancer and a breast carcinoma specimen, were found to have elevated levels of the PAM4 antigen. PAM4-negative metastatic disease to the pancreas included cervical, prostate, and renal cancers, along with melanoma and lymphoma.

Table 1. PAM4 Antigen Levels in Patient Sera
DiseaseNo.Median, U/mLNo. of Positive CasesPercentage of Positive CasesPa
  • Abbreviations: mets, metastases; PC, pancreatic cancer.

  • a

    All P values are in comparison with pancreatic adenocarcinoma and were determined using the Mann-Whitney nonparametric U test.

Pancreatic adenocarcinoma29810.4022576 
Pancreatic neuroendocrine200.08210.0001
Pancreatic, other morphology70.51114.0001
Non-PC, mets to the pancreas110.00218.0001
Ampullary adenocarcinoma211.521048.0001
Biliary adenocarcinoma264.411350.0257
Duodenal adenocarcinoma72.80457.0001
All biliary and periampullary611.782948.0001
Colon carcinoma320.15516.0001
Chronic pancreatitis800.411823.0001
Benign cystadenoma150.1817.0001
Benign, other250.20520.0001
All benign disease1200.262420.0001
Healthy volunteers790.2734.0001

Approximately one-half of the patients with ampullary (48%) and extrahepatic biliary (50%) adenocarcinomas had positive levels of circulating antigen by this assay. Nine of the 13 PAM4-positive biliary adenocarcinomas (69%) were located within the distal bile duct and 4 were located at the junction of the left and right hepatic ducts (Klatskin tumor). Although small patient numbers were evaluated, sera from patients with duodenal adenocarcinomas were mostly positive for the PAM4 antigen, whereas sera from patients with intrahepatic cholangiocarcinomas were mostly negative. Patients with colon carcinoma demonstrated little reactivity in this EIA, with only 5 of 32 cases (16%) considered to be positive. PAM4 antigen levels were significantly higher in patients with PDAC than all other patient groups (P < .0001 for all groups, except those with extrahepatic biliary adenocarcinoma [P = .0257]).

Of 120 patients diagnosed with benign, nonneoplastic conditions of the pancreas, only 24 (20%) were found to be positive for the PAM4 antigen, and in particular, 18 of 80 patients with CP (23%) were positive. As shown in Figure 2, ROC curve analyses demonstrated a statistically significant difference between the PDAC and CP groups (P < .0001), with an area under the curve of 0.84 ± 0.02 (95% confidence interval [95% CI], 0.79-0.89). A similar finding was demonstrated for the comparison of PDAC with all benign specimens taken together (0.85 ± 0.02; 95% CI, 0.82-0.89). The positive and negative likelihood ratios for differentiating PDAC from benign conditions of the pancreas were 4.00 and 0.30, respectively, which was statistically significant (P < .0001).

Figure 2.

Receiver operating characteristic curves for performance of the PAM4-based enzyme immunoassay are shown. The pancreatic ductal adenocarcinoma (PDAC) versus chronic pancreatitis (CP) curve is shown on the left, and the PDAC versus all patients with diagnoses of benign pancreatic disease (including CP) curve is shown on the right. Values for the area under the curve (AUC) and 95% confidence interval (95% CI) are provided for each curve.

Circulating levels of CA 19-9 are known to be influenced by serum bilirubin.26, 27 Thus, we attempted to determine whether a relation existed between PAM4 antigen elevation and bilirubin levels in patients with PDAC. There was no such relation observed, with PAM4 antigen levels found to be elevated independent of the bilirubin concentration (N = 44 cases from a single institution; r2 = 0.0024 [P = .789]).

Comparison of PAM4 and CA 19-9 Enzyme Immunoassays

Of the 628 total serum samples, sufficient volume was available to evaluate both PAM4 and CA 19-9 antigens in 474 specimens. Overall sensitivity for the detection of PDAC was similar for the 2 assays (74% and 77%, respectively, for PAM4 and CA 19-9) (Table 2). The sensitivity for the detection of stage I disease, although somewhat higher for the PAM4 antigen, was also similar (65% and 58%, respectively, for PAM4 and CA 19-9; P = .578). However, specificity was significantly greater for the PAM4 antigen, particularly with regard to CP (86% and 68%, respectively, for the PAM4 and CA 19-9 assays; P = .014). In addition, as expected, CA 19-9 results demonstrated considerably higher detection rates for non-PDAC neoplasia, including patients with nonpancreatic cancers that metastasized to the pancreas. Thus, the positive likelihood ratio was significantly higher for the PAM4 assay (5.29) than the CA 19-9 assay (2.41).

Table 2. Comparison of PAM4 and CA 19-9 Antigen Levels in Patient Sera
  PAM4CA 19-9Combined
DiseaseNo.No. of Positive CasesPercentage of Positive CasesNo. of Positive CasesPercentage of Positive CasesNo. of Positive CasesPercentage of Positive Cases
  1. Abbreviations: CA 19-9, carbohydrate antigen 19-9; mets, metastases; PC, pancreatic cancer.

Pancreatic adenocarcinoma234173741807719684
Pancreatic neuroendocrine17212529212
Pancreatic, other morphology6117467233
Non-PC, mets to the pancreas11218764436
Ampullary adenocarcinoma13538754754
Biliary adenocarcinoma23135716701670
Duodenal adenocarcinoma3133133133
All biliary and periampullary42204825602560
Colon carcinoma31516929619
Chronic pancreatitis507141632918
Benign cystadenoma911100111
Benign, other24521625521
All benign disease83131622271518
Healthy volunteers50360036

Concentrations of circulating PAM4 and CA 19-9 antigens in patients with PDAC were independent of each other (r2 = 0.003; P = .410); the positive and negative interpretations were found to be concordant in only 68% of the cases. This led us to investigate the potential use of a combined biomarker serum assay that ultimately provided an improved overall detection rate for PDAC (84%) than either arm alone. It is important to note that this increased sensitivity did not appear to have a commensurate significant decrease in specificity. ROC curves and statistical comparisons are presented in Figure 3 and Table 3.

Figure 3.

Receiver operating characteristic (ROC) curves for the performance of the PAM4 enzyme immunoassay (bold black line), carbohydrate antigen (CA) 19-9 enzyme immunoassay (dashed black line), and combined PDAC and CA 19-19 biomarker serum assay (dotted gray line) are shown. (Left) ROC curves for the discrimination of pancreatic ductal adenocarcinoma (PDAC) versus chronic pancreatitis (CP) are shown. (Right) ROC curves for the discrimination of PDAC versus all patients with diagnoses of benign pancreatic disease (including CP) are shown. Descriptive statistical values are provided in Table 3. For each of the comparisons evaluated, the combined biomarker serum assay was found to have significantly greater sensitivity for the detection of pancreatic cancer when compared with either the PAM4 or CA 19-9 enzyme immunoassays alone (P < .0257 or better).

Table 3. Diagnostic Performance of PAM4 and CA 19-9 Biomarker Enzyme Immunoassays (ROC Curve Analyses)
  • Abbreviations: +, positive; −, negative; AUC, area under the curve; CA 19-9, carbohydrate antigen 19-9; CP, chronic pancreatitis; LR, likelihood ratio; PDAC, pancreatic ductal adenocarcinoma; ROC, receiver operating characteristic.

  • a

    All P values are for comparison with the PAM4 enzyme immunoassay. P values for the CA 19-9 enzyme immunoassay versus the combined PAM4 and CA 19-9 biomarker serum assay were identical to P values for the PAM4 enzyme immunoassay versus the combined serum assay.

PDAC vs CP      
 PAM474860.87 + 0.02.00015.290.3
 CA 19-977680.84 + 0.03.00732.410.34
 Combined84820.91 + 0.02 4.670.2
PDAC vs benign      
 PAM474850.87 + 0.02.00014.930.31
 CA 19-977730.85 + 0.02.02572.850.32
 Combined84830.91 + 0.02 4.940.19


There are several important findings from the current study. The primary conclusion is that the PAM4 enzyme immunoassay is able to identify approximately 67% of patients with stage I PDAC, and does so with high discriminatory power with regard to benign pancreatic disease. To the best of our knowledge, there are only a few reports to date that describe the use of a noninvasive biomarker assay to detect stage I disease, and the majority of these discuss the performance characteristics of CA 19-9.28-30 The sensitivity reported for CA 19-9 in patients with stage I PDAC ranges from 40% to 64%, with the results of the current study demonstrating a detection rate of 58%. However, the specificity reported for CA 19-9 in the literature14, 31, 32 is considerably lower than that reported for the PAM4 antigen, as is also true for the paired study described herein, particularly with regard to discrimination between PDAC and CP. Positive likelihood ratios were significantly higher for the PAM4 enzyme immunoassay.

The data from the current study suggest that the PAM4 antigen is not expressed by pancreatic tumors originating from nonepithelial tissues. However, PAM4 is expressed and released by biliary and periampullary adenocarcinomas. The detection of these latter cancers, although rare (approximately 3500 new cases in total are reported each year in the United States), is likely to prove to be of clinical value, with follow-up imaging studies providing confirmation of tumor mass and location. The finding that these latter cancers express the PAM4 antigen and are detectable by the PAM4 enzyme immunoassay was not unexpected, considering that these tissues are derived from closely related structures in early embryonic development. Indeed, many of the reported biomarkers for PDAC also are reactive with these tumors. The limited expression of PAM4 in the control colon cancer group confirms our prior serum assay and immunohistochemical studies indicating that PAM4 has limited elevation with other gastrointestinal and nongastrointestinal cancers.17-20

It is also important to note the ability of the PAM4 enzyme immunoassay to discriminate PDAC (and adjacent carcinomas) from benign, nonmalignant disease of the pancreas, and in particular the distinction between PDAC and CP. In a prior study,20 a discordance was reported between PAM4 antigen levels in the serum of patients with CP (29 patients with a 38% false positive rate) and immunohistochemical data from a separate group of patients with CP, in which only 2 of 19 specimens had evidence of weak, focal expression of the PAM4 antigen localized to ADM. In a follow-up immunohistochemical study of an additional 32 surgically resected specimens from patients with CP, PAM4 labeling was observed in 6 of 32 cases (19%); however, PAM4 reactivity was restricted to the PanIN precursor lesions associated with CP, and was not observed in nonneoplastic, inflamed pancreatic tissue.21 Taken together, the data from the immunohistochemical labeling of > 50 specimens of CP demonstrate that the PAM4 MoAb does not react with the inflamed, nonneoplastic tissues. Thus, the question of the biological and clinical significance of a positive serum PAM4 antigen level in this group of patients is of great importance. Are these assay results false-positive findings or do they provide evidence of incipient PDAC (and/or precursor lesions)? Unfortunately, clinical follow-up for this patient group was unavailable. However, at the very least, the data suggest the need to undertake a prospective investigation to identify patients with PAM4-positive CP and/or others considered to be at high risk for PDAC, for whom follow-up surveillance could be performed for the potential discovery of early PDAC.

Based on the limitations of the CA 19-9 assay (it cannot be performed in Lewis antigen-negative patients and is altered by bilirubin levels), we determined that a combined PAM4 and CA 19-9 biomarker serum assay would provide a superior detection and diagnostic tool than either assay alone. The overall sensitivity was improved without a loss of specificity. Thus, a positive result provides a rationale for proceeding to diagnostic imaging for the confirmation of disease. Furthermore, this combined biomarker serum assay could play a role in monitoring treatment and perhaps prove useful for the earlier detection of recurrent disease.

Due to the asymptomatic nature of PDAC, and its relatively low incidence, both medical and economic concerns have argued against screening of the general population at large.33-35 However, with recent studies providing the ability to identify specific groups of patients at high risk for PDAC, a rationale exists for longitudinal surveillance as a means to improve early detection specifically in these risk settings. Individuals with a family history of PDAC,2, 36, 37 those patients with long-standing CP,38, 39 patients with new-onset diabetes who meet certain other conditions,40, 41 or those with familial atypical multiple mole melanoma (FAMMM) syndrome,42 for example, could be evaluated on a long-term basis for the detection of early malignant changes through the use of the combined PAM4 and CA19-9 biomarker serum assay. Imaging has played an important role in these surveillance programs, the most significant being endoscopic ultrasound (EUS) scanning of the pancreas. EUS offers the additional ability to obtain fine-needle aspiration and/or fluid (pancreatic juice and cyst fluids) specimens that can be examined for specific morphologic and biomarker changes representative of malignancy. Several reports have identified increased levels of carcinoembryonic antigen cell adhesion molecules and CA 19-9 antigen,43-45 or have observed mutations in KRAS and other genes that are potentially indicative of cancer from these endoscopically retrieved materials.15, 46, 47 Likewise, it is of interest to evaluate these fluids for the PAM4 antigen. However, it is obvious that use of a serum-based biomarker to detect early stage PDAC would provide a clinically more valuable and cost-effective tool for monitoring patients considered to be at high risk for this disease.

In the current study, a blinded analysis of > 600 patient specimens demonstrated that the PAM4 assay could detect approximately two-thirds of patients with early PDAC, and did so with high specificity by discriminating PDAC from benign diseases of the pancreas. Furthermore, the inclusion of a second biomarker, CA 19-9, was found to significantly enhance the overall positive identification of patients with PDAC without loss of specificity. Although we have examined the CA 19-9 assay in combination with the PAM4 assay, we appreciate that other biomarkers may prove to be of equal or greater value in combination with PAM4. Thus, the results of the current study provide a basis for future studies of biomarker combinations with PAM4 for the surveillance of patients at high risk for PDAC, and as a potential means for monitoring patients with more advanced disease. Finally, the ability of the PAM4 enzyme immunoassay to identify a significant number of patients with biliary and periampullary adenocarcinomas, although relatively rare, may provide an additional means for improving the management of patients with these cancers.


Supported in part by grants from the National Cancer Institute (CA096924), the Canale Fund, and The Turpin Foundation.


Dr. Goldenberg has a financial interest in Immunomedics, Inc. Drs. Goldenberg and Gold are patent inventors.