Glycan Motif Profiling Reveals Plasma Sialyl-Lewis X Elevations in Pancreatic Cancers That Are Negative for Sialyl-Lewis A *

Abstract

The sialyl-Lewis A (sLeA) glycan forms the basis of the CA19–9 assay and is the current best biomarker for pancreatic cancer, but because it is not elevated in ∼25% of pancreatic cancers, it is not useful for early diagnosis. We hypothesized that sLeA-low tumors secrete glycans that are related to sLeA but not detectable by CA19–9 antibodies. We used a method called motif profiling to predict that a structural isomer of sLeA called sialyl-Lewis X (sLeX) is elevated in the plasma of some sLeA-low cancers. We corroborated this prediction in a set of 48 plasma samples and in a blinded set of 200 samples. An antibody sandwich assay formed by the capture and detection of sLeX was elevated in 13 of 69 cancers that were not elevated in sLeA, and a novel hybrid assay of sLeA capture and sLeX detected 24 of 69 sLeA-low cancers. A two-marker panel based on combined sLeA and sLeX detection differentiated 109 pancreatic cancers from 91 benign pancreatic diseases with 79% accuracy (74% sensitivity and 78% specificity), significantly better than sLeA alone, which yielded 68% accuracy (65% sensitivity and 71% specificity). Furthermore, sLeX staining was evident in tumors that do not elevate plasma sLeA, including those with poorly differentiated ductal adenocarcinoma. Thus, glycan-based biomarkers could characterize distinct subgroups of patients. In addition, the combined use of sLeA and sLeX, or related glycans, could lead to a biomarker panel that is useful in the clinical diagnosis of pancreatic cancer.Précis: This paper shows that a structural isomer of the current best biomarker for pancreatic cancer, CA19–9, is elevated in the plasma of patients who are low in CA19–9, potentially enabling more comprehensive detection and classification of pancreatic cancers. The sialyl-Lewis A (sLeA) glycan forms the basis of the CA19–9 assay and is the current best biomarker for pancreatic cancer, but because it is not elevated in ∼25% of pancreatic cancers, it is not useful for early diagnosis. We hypothesized that sLeA-low tumors secrete glycans that are related to sLeA but not detectable by CA19–9 antibodies. We used a method called motif profiling to predict that a structural isomer of sLeA called sialyl-Lewis X (sLeX) is elevated in the plasma of some sLeA-low cancers. We corroborated this prediction in a set of 48 plasma samples and in a blinded set of 200 samples. An antibody sandwich assay formed by the capture and detection of sLeX was elevated in 13 of 69 cancers that were not elevated in sLeA, and a novel hybrid assay of sLeA capture and sLeX detected 24 of 69 sLeA-low cancers. A two-marker panel based on combined sLeA and sLeX detection differentiated 109 pancreatic cancers from 91 benign pancreatic diseases with 79% accuracy (74% sensitivity and 78% specificity), significantly better than sLeA alone, which yielded 68% accuracy (65% sensitivity and 71% specificity). Furthermore, sLeX staining was evident in tumors that do not elevate plasma sLeA, including those with poorly differentiated ductal adenocarcinoma. Thus, glycan-based biomarkers could characterize distinct subgroups of patients. In addition, the combined use of sLeA and sLeX, or related glycans, could lead to a biomarker panel that is useful in the clinical diagnosis of pancreatic cancer. Précis: This paper shows that a structural isomer of the current best biomarker for pancreatic cancer, CA19–9, is elevated in the plasma of patients who are low in CA19–9, potentially enabling more comprehensive detection and classification of pancreatic cancers. A patient with an uncertain lesion of the pancreas typically is referred to a specialist for dedicated scans of the pancreas and, if available, additional procedures such as endoscopic imaging with fine-needle aspiration to obtain material for cytology. The diagnostic challenges include differentiating benign from neoplastic conditions and determining the type and potential aggressiveness of a neoplasm (1Abraham S.C. Wilentz R.E. Yeo C.J. Sohn T.A. Cameron J.L. Boitnott J.K. Hruban R.H. Pancreaticoduodenectomy (whipple resections) in patients without malignancy: Are they all “chronic pancreatitis”?.Am. J. Surg. Pathol. 2003; 27: 110-120Crossref PubMed Scopus (300) Google Scholar, 2Adsay N.V. Bandyopadhyay S. Basturk O. Othman M. Cheng J.D. Klöppel G. Klimstra D.S. Chronic pancreatitis or pancreatic ductal adenocarcinoma?.Semin. Diagn. Pathol. 2004; 21: 268-276Crossref PubMed Scopus (45) Google Scholar, 3Kennedy T. Preczewski L. Stocker S.J. Rao S.M. Parsons W.G. Wayne J.D. Bell R.H. 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Molecular classification of neoplasms of the pancreas.Hum. Pathol. 2009; 40: 612-623Crossref PubMed Scopus (71) Google Scholar), as they could provide objective and detailed information about each patient's condition. But molecular markers to diagnose incipient pancreatic cancer are not available despite decades of research; the current best marker for pancreatic cancer, the CA19–9 test, was discovered in 1979 (7Koprowski H. Steplewski Z. Mitchell K. Herlyn M. Herlyn D. Fuhrer P. Colorectal carcinoma antigens detected by hybridoma antibodies.Somatic Cell Genet. 1979; 5: 957-971Crossref PubMed Scopus (1172) Google Scholar, 8Herlyn M. Steplewski Z. Herlyn D. Koprowski H. Colorectal carcinoma-specific antigen: Detection by means of monoclonal antibodies.Proc. Natl. Acad. Sci. U.S.A. 1979; 76: 1438-1442Crossref PubMed Scopus (560) Google Scholar). CA19–9 is elevated in about 75% of pancreatic cancers (9Goonetilleke K.S. Siriwardena A.K. Systematic review of carbohydrate antigen (CA 19–9) as a biochemical marker in the diagnosis of pancreatic cancer.Eur. J. Surg. Oncol. 2007; 33: 266-270Abstract Full Text Full Text PDF PubMed Scopus (627) Google Scholar), which is useful for certain purposes, such as monitoring response to treatment, but not for diagnosis. The antigen detected by the CA19–9 test is a glycan, a tetrasaccharide known as the sialyl-Lewis A (sLeA) antigen. The discovery that CA19–9 antibodies recognize a glycan (10Magnani J.L. Brockhaus M. Smith D.F. Ginsburg V. Blaszczyk M. Mitchell K.F. Steplewski Z. Koprowski H. A monosialoganglioside is a monoclonal antibody-defined antigen of colon carcinoma.Science. 1981; 212: 55-56Crossref PubMed Scopus (307) Google Scholar, 11Magnani J.L. Nilsson B. Brockhaus M. Zopf D. Steplewski Z. Koprowski H. Ginsburg V. A monoclonal antibody-defined antigen associated with gastrointestinal cancer is a ganglioside containing sialylated lacto-n-fucopentaose ii.J. Biol. Chem. 1982; 257: 14365-14369Abstract Full Text PDF PubMed Google Scholar) further revealed the prevalent nature of glycosylation alterations in cancer. Researchers have uncovered other glycans that show up with high abundance in cancer (12Dube D.H. Bertozzi C.R. Glycans in cancer and inflammation–potential for therapeutics and diagnostics.Nat. Rev. Drug Discov. 2005; 4: 477-488Crossref PubMed Scopus (1346) Google Scholar, 13Adamczyk B. Tharmalingam T. Rudd P.M. Glycans as cancer biomarkers.Biochim. Biophys. Acta. 2012; 1820: 1347-1353Crossref PubMed Scopus (381) Google Scholar), some of which contribute to cancer cell function and carry information about cell differentiation. Glycans, therefore, have good potential to serve as biomarkers of cancer. But for the diagnosis of pancreatic cancer, glycan-based markers are not yet effective because we do not have markers to detect the cancers that are low in sLeA. A strategy for improving upon the CA19–9 test is to identify biomarkers that are elevated in the patients who are low in sLeA. Previous research suggested that other glycans besides sLeA are overproduced in some cancers that are low in sLeA. All antibodies used in the CA19–9 assays primarily detect the sLeA glycan, which has the sequence Siaα2,3Galβ1,3(Fucα1,4)GlcNAc (where Sia is sialic acid, Gal is galactose, Fuc is fucose, and GlcNAc is N-acetylglucosamine), but some also detect other glycans (14Rye P.D. Bovin N.V. Vlasova E.V. Molodyk A.A. Baryshnikov A. Kreutz F.T. Garinther W.I. Schultes B.C. A.A. J. Nilsson O. Nilsson K. K. L. Bell H. J. on the of monoclonal antibodies A and related Biol. PubMed Scopus Google Scholar, K. R.E. monoclonal antibodies the antigen show in with for clinical 2012; PubMed Scopus Google Scholar). The available CA19–9 assays for patients K. R.E. monoclonal antibodies the antigen show in with for clinical 2012; PubMed Scopus Google Scholar, A. of for and markers from an J. Chem. Google Scholar, K. P. H. of for Acta. 2009; PubMed Scopus Google Scholar), the of the from the antibody S.J. of pancreatic by monoclonal 1982; Google Scholar), which a of sLeA called sialyl-Lewis S. M. H. S. T. H. K. of and and PubMed Scopus Google Scholar) detection shows in some pancreatic cancers that do not sLeA K. R.E. monoclonal antibodies the antigen show in with for clinical 2012; PubMed Scopus Google Scholar, H. E. P.M. on of and in and in of pancreatic cancer Google Scholar). The the that of the in antibodies could discovery of glycans that are in pancreatic cancers. 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PubMed Scopus Google Scholar). we could from antibodies samples and use the information about the of the antibodies to about the glycan that are in each We an for that called motif prediction D. H. K. S. J. M. of glycan of on by pancreatic cancer PubMed Scopus Google Scholar). 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Furthermore, this research a novel to for glycan-based called motif The method detailed of antibody available from glycan to predict patients in glycan that not based on a of the antibody We that the useful antibodies or with or for information because such without a The on the that the cancers glycans that are related to sLeA. The used monoclonal antibodies CA19–9, were by with cancer of CA19–9 with antibodies such as T. S. M. H. K. S. M. T. of and CA19–9 in the diagnosis of pancreatic J. PubMed Scopus Google Scholar), T. S. M. H. K. S. M. T. of and CA19–9 in the diagnosis of pancreatic J. PubMed Scopus Google Scholar), S. M. O. K. H. H. K. S. T. of and CA19–9 for the diagnosis of pancreatic J. PubMed Scopus Google Scholar), and others, CA19–9 the antibody for this was that some of the patients show in H. E. P.M. on of and in and in of pancreatic cancer Google Scholar, S. H. T. M. S. M. T. of in pancreatic cancer patients for J. 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The provide a for high sensitivity and high each of which detection of a of pancreatic cancer. In to further improve a strategy to for markers elevated in the pancreatic cancers not elevate sLeA or An of the was not in the of the but we some the nature of the tumors each type of marker All patients with high plasma sLeA for which was available or differentiated ductal with the staining for sLeA. In the patients with low plasma sLeA, some of which also high plasma sLeX, or poorly differentiated or The high ductal staining and elevated plasma a the and also that the ductal the and that is because the do not typically have to the pancreatic from a also this The molecular of the material from the that from the tumors H. H. of as pancreatic Google Scholar). of the tumors with low plasma sLeA high sLeA staining in the cancer that tumors not secrete the marker to detected in the The of differentiated ductal potentially such the current to the that glycans characterize distinct subgroups of cancers. this we a to the and plasma of sLeA and Previous also of sLeX in some pancreatic tumors but not in the plasma in to cancers that are low in sLeA. In an of patients with pancreatic cancer, sLeX staining was in of to sLeA staining in of patients P.M. H. E. Steplewski Z. of antigens and in pancreatic cancer in with the patient's Google Scholar). of and was in of that of patients staining in sLeX This of sLeA and sLeX staining with but the not of and of sialyl-Lewis A H. E. P.M. on of and in and in of pancreatic cancer Google Scholar, S. M. ganglioside in pancreatic cancer with the PubMed Scopus Google Scholar), but the not the to or the with The glycans that the cancer could about the in the cell and about with in to and by sLeX on cancer in to but also by cell S. M. of X in and by J. PubMed Scopus Google Scholar). The of sLeX in is with M. K. M. H. S. H. M. of a carbohydrate of the on high in as Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). A is also a for K. H. M. H. A. H. T. T. of and in the of pancreatic carcinoma to J. PubMed Scopus Google Scholar), the of sLeX and sLeA could cancer cell disease and more the glycan and the of of sLeX have for M. K. M. H. S. H. M. of a carbohydrate of the on high in as Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In we show that a of patients with low plasma sLeA have in plasma A novel assay formed by the capture of sLeA and detection of sLeX was useful for sLeA-low cancers with a low of The sLeA and sLeX glycans in and differentiated ductal but in other that do not elevated plasma sLeA, the of glycans as of of The that further of of glycans could the detection of a of pancreatic cancers than possible This could have early such as determining who to and disease use of CA19–9 could from additional biomarkers that are to tumors that do not secrete high of sLeA. We the and for the of the and the the for for the glycan and of for the with sialyl-Lewis A sialyl-Lewis X of