Highly Accurate Identification of Cystic Precursor Lesions of Pancreatic Cancer Through Targeted Mass Spectrometry: A Phase IIc Diagnostic Study

Abstract

Purpose Pancreatic cystic lesions are common incidental findings on imaging, but up to half may be forerunners of pancreatic cancer. Therefore, accurate differential diagnosis is crucial for correct patient management. Unfortunately, currently available diagnostic methods cannot robustly identify premalignant and malignant pancreatic cystic lesions. Methods Cyst fluid samples obtained by routine endoscopic ultrasound-guided aspiration were used for the analyses. In a cohort of 24 patients, eight biomarker candidates for malignant potential and high-grade dysplasia/cancer were identified by an explorative proteomic approach. Subsequently, a quantitative analysis, using 30 heavy-labeled peptides from the biomarkers and parallel reaction monitoring mass spectrometry, was devised, tested in a training cohort of 80, and prospectively evaluated in a validation cohort of 68 patients. End points were surgical pathology diagnosis/clinical follow-up. Diagnostic assessments were blinded to mass spectrometry results. Results The optimal set of markers for detecting malignant potential was a panel of peptides from mucin-5AC and mucin-2, which could discriminate premalignant/malignant lesions from benign with an accuracy of 97% (95% CI, 89% to 99%) in the validation cohort. This result compared favorably with the accuracy of standard analyses: cyst fluid carcinoembryonic antigen (61%; 95% CI, 46% to 74%; P < .001) and cytology (84%; 95% CI, 71% to 92%; P = .02). A combination of proteins mucin-5AC and prostate stem-cell antigen could identify high-grade dysplasia/cancer with an accuracy of 96% (95% CI, 90% to 99%), and detected 95% of malignant/severely dysplastic lesions, compared with 35% and 50% for carcinoembryonic antigen and cytology ( P < .001 and P = .003, respectively). Conclusion Targeted mass spectrometry analysis of just three cyst fluid biomarkers provides highly accurate identification and assessment of cystic precursors to pancreatic adenocarcinoma. Additional studies should determine whether the method can facilitate timely cancer diagnosis, successful intervention, and prevention.

Fig 1.

Flow diagrams of patient inclusion/exclusion. (A) Training cohort and (B) validation cohort. IPMN, intraductal papillary mucinous neoplasm; SCN, serous cystic neoplasm.

Fig 2.

Mucin-5AC (MUC5AC) and carcinoembryonic antigen (CEA) concentrations in benign, premalignant, and malignant/severely dysplastic pancreatic cystic lesions. Boxes represent the 25th to 75th percentile, the line represents the median, and the whiskers represent the 10th to 90th percentile. (A) MUC5AC concentrations in (1) intrinsically benign pancreatic cystic lesions, (2) premalignant lesions (ie, IPMNs or mucinous cystic neoplasms with low-grade or intermediate-grade dysplasia), and (3) lesions with high-grade dysplasia (HGD) or invasive cancer. Three high outliers for the group with HGD/cancer do not appear in the figure (1,631, 1,312 and 341 fmol/µL). Two outliers high in MUC5AC among the premalignant lesions, which were wrongly classified as HGD/cancer by targeted mass spectrometry, are included in the figure. In both cases, histology of the surgical specimen revealed IPMNs with main duct involvement and intermediate-grade dysplasia. Taken together, this means that the risk of future malignant progression would have been high and that surgery was the correct treatment option. The P value for the comparison of the three groups is statistically significant: < .001 (Kruskal-Wallis test with Bonferroni adjustment of the significance threshold to .017). (B) Carcinoembryonic antigen (CEA) concentrations in (1) intrinsically benign pancreatic cystic lesions; (2) premalignant lesions (ie, IPMNs or mucinous cystic neoplasms with low-grade or intermediate-grade dysplasia); and (3) lesions with high-grade dysplasia (HGD)/invasive cancer. Two high outliers for the HGD/cancer group do not appear in the figure (11,246 and 33,700 ng/mL). There is a substantial overlap between patient groups.

Fig 3.

Identification of cystic lesions with malignant potential. Malignant potential is defined as either premalignancy or malignancy. Cutoff values were mucin-5AC (MUC5AC) plus mucin-2 (MUC2), 0.01 fmol/µL cyst fluid (summed protein concentration levels); MUC5AC, 0.01 fmol/µL; and carcinoembryonic antigen (CEA), 192 ng/mL. A positive result for cytology was defined as either presence of mucus in the sample or evidence of dysplasia. The results are from the validation cohort. (A) Performance characteristics for the identification of cystic lesions with malignant potential; inconclusive results not included (validation cohort). The accuracy for MUC5AC plus MUC2 was statistically significantly higher than that of CEA (P < .001) and cytology (P = .02). Comparative statistical analysis was performed by Fisher´s exact test with Bonferroni correction for multiple comparisons (threshold for significance, .025). Error bars represent 95% CIs, calculated using the Wilson procedure,²³ with correction for continuity. (B) Diagnostic outcomes for the identification of malignant potential in a cystic lesion (validation cohort). Percentages are displayed on the y-axis, and the number of individuals are displayed in labels on the stacked bar chart. The preferred panel for the identification of malignant potential, MUC5AC plus MUC2, gave a statistically significantly higher proportion of correct results than CEA (P < .001) and cytology (P < .001). Comparative statistical analysis was performed by Fisher´s exact test with Bonferroni correction for multiple comparisons (threshold for significance, .025). NPV, negative predictive value; PPV, positive predictive value.

Fig 4.

Identification of cystic lesions with high-grade dysplasia (HGD) or invasive cancer. Cutoff values were mucin-5AC (MUC5AC) plus prostate stem-cell antigen (PSCA), 12 fmol/µL cyst fluid (summed protein concentration levels); MUC5AC, 7.6 fmol/µL; and carcinoembryonic antigen (CEA), 1,000 ng/mL. PSCA levels were only considered if MUC5AC was present in the sample. One hundred five patients could be assessed for the presence of HGD/cancer, 70 from the training cohort and 35 from the validation cohort. The results are from the entire study population (training and validation cohorts). (A) Performance characteristics for the identification of HGD/malignant lesions; inconclusive results not included (entire study population). The sensitivity of MUC5AC plus PSCA was higher than that of CEA (P = .008) and cytology (P = .007; Fisher´s exact test). Error bars represent 95% CIs, calculated using the Wilson procedure,²³ with correction for continuity. (B) Diagnostic outcomes for the identification of malignant/severely dysplastic cystic lesions (entire study population). Percentages are displayed on the y-axis and the number of individuals in labels on the stacked bar chart. The preferred panel for the identification of HGD/cancer, MUC5AC plus PSCA, gave a statistically significantly higher proportion of correct results than CEA (P < .001) and cytology (P < .001). Comparative statistical analysis was performed by Fisher´s exact test with Bonferroni correction for multiple comparisons (threshold for significance, 0.025). Supportive biomarkers for the identification of HGD/cancer, along with their positive and negative predictive values, are provided in a text box. (C) Proportion of cystic lesions with HGD/cancer identified by the different diagnostic methods. MUC5AC plus PSCA identified a statistically significantly higher proportion of HGD/malignant lesions than CEA (P < .001) and cytology (P = .003; Fisher´s exact test with Bonferroni correction). The results are from the entire study population (105 patients). CLCA1, calcium-activated chloride channel regulator 1; DMBT1, deleted in malignant brain tumors 1; FCGBP, IgGFc-binding protein; MUC1, mucin-1; NPV, negative predictive value; PPV, positive predictive value.