The IgG/IgG4 mRNA Ratio By Quantitative PCR Accurately Diagnoses IgG4-Related Disease and Predicts Treatment Response

Lowiek Hubers¹, Marieke Doorenspleet², Paulus Klarenbeek³, Emma Culver⁴, Lucas Maillette de Buy Wenniger⁵, Roger Chapman⁴, Stan Van de Graaf⁶, Joanne Verheij⁷, Thomas Van Gulik⁸, Frank Baas⁹, Ellie Barnes⁴, Ulrich Beuers¹ and Niek De Vries³, ¹Dept. of Gastroenterology & Hepatology and Tytgat Institute of Liver and Intestinal Research, Dept. of Gastroenterology & Hepatology and Tytgat Institute of Liver and Intestinal Research, Academic Medical Center/University of Amsterdam, Amsterdam, Netherlands, ²Dept. of Clinical Immunology & Rheumatology, Amsterdam Rheumatology and immunology Center | Academic Medical Center/University of Amsterdam, Amsterdam, Netherlands, ³Dept. of Clinical Immunology & Rheumatology, Amsterdam Rheumatology and immunology Center, Academic Medical Center/University of Amsterdam, Amsterdam, Netherlands, ⁴Translational Gastroenterology Unit and NDM Oxford University, Translational Gastroenterology Unit and NDM Oxford University, John Radcliffe Hospital/Oxford University, Oxford, United Kingdom, ⁵1Dept. of Gastroenterology & Hepatology and Tytgat Institute of Liver and Intestinal Research, Dept. of Gastroenterology & Hepatology and Tytgat Institute of Liver and Intestinal Research, Academic Medical Center/University of Amsterdam, Amsterdam, Netherlands, ⁶Dept. of Gastroenterology & Hepatology and Tytgat Institute of Liver and Intestinal Research,, Dept. of Gastroenterology & Hepatology and Tytgat Institute of Liver and Intestinal Research, Academic Medical Center/University of Amsterdam, Amsterdam, Netherlands, ⁷Dept. of Pathology, Dept. of Pathology, Academic Medical Center/University of Amsterdam, Amsterdam, Netherlands, ⁸Dept. of Surgery, Dept. of Surgery, Academic Medical Center/University of Amsterdam, Amsterdam, Netherlands, ⁹Dept. of Genome Analysis, Academic Medical Center/University of Amsterdam, Amsterdam, Netherlands

Meeting: 2015 ACR/ARHP Annual Meeting

Date of first publication: September 29, 2015

Keywords: B cells, Biomarkers, IgG4 Related Disease, immunoglobulin (IG) and inflammation

Session Information

Date: Monday, November 9, 2015

Title: B cell Biology and Targets in Rheumatolid Arthritis and other Autoimmune Disease Poster

Session Type: ACR Poster Session B

Session Time: 9:00AM-11:00AM

Background/Purpose : IgG4-associated cholangitis (IAC) and autoimmune pancreatitis (AIP) are major manifestations of IgG4-related disease (IRD). Misdiagnosis and inadequate treatment are common since IAC and AIP mimic other inflammatory and malignant pancreatobiliary diseases and accurate diagnostic biomarkers are lacking. Moreover, relapse after tapering of immunosuppressive therapy is seen in 50% of patients, underscoring the need for biomarkers monitoring disease activity. Recently, using Next-Generation Sequencing, we found that dominant IgG4+ B-cell receptor clones in peripheral blood distinguish patients with active IAC/AIP from primary sclerosing cholangitis (PSC) and pancreatobiliary malignancies (CA) [Hepatology 2013;57:2340]. Therefore, we aimed to develop a quantitative PCR (qPCR) protocol for diagnosing IAC/AIP and monitoring disease activity before and after treatment.

Methods : 15 patients with IAC and/or AIP according to HISORt criteria, 7 patients with PSC and 8 with CA formed the test cohort. Intra- and extramural replication cohorts consisted of 16 IAC/AIP, 5 PSC and 13 CA patients (Dutch cohort), and 8 IAC/AIP and 8 PSC patients (British cohort). From 20 Dutch IAC/AIP patients, follow-up samples after 4 and 8 weeks of corticosteroid therapy were available. RNA was isolated, and a forward primer amplifying all IgG subtypes, together with a generic reverse primer for all IgG subtypes and a specific reverse primer for IgG4 were used to amplify the constant region of the B-cell receptor. The ratio total IgG/IgG4 mRNA was calculated and expressed as ΔC_T.

Results : ΔC_T as measure of IgG/IgG4 mRNA expression in peripheral blood of the test cohort was 2.8±1.1 (mean+SD) in IAC/AIP patients, compared to 6.8±1.6 in PSC and 7.6±1.4 in CA (Figure 1A, p<0.0001). ROC analysis revealed a ΔC_T cut-off value of 5.1 in the test cohort distinguishing all cases from controls. In the replication cohort sensitivity was 95% and specificity 100% (p=8.6*10^-21). ΔC_T increased after 4 weeks (5.1±0.8) and 8 weeks (7,1±0.7) of corticosteroid treatment, compared to pre-treatment (mean 3.1±1.4, p<0.0001, Figure 1B).

Conclusion : IgG4-related disease of the biliary tree and pancreas can accurately be distinguished from PSC or pancreatobiliary malignancies by ΔC_T based on an affordable qPCR test. ΔC_T can also be used as a marker for treatment response and disease activity in IgG4-related disease.

Acknowledgement: This study was supported by grants from German and American PSC patient organizations (to UB).

Figure 1

Disclosure: L. Hubers, None; M. Doorenspleet, Patent on “Method for diagnosing IgG4 related diseases”, application number PCT/ EP2013/070384, publication number WO/2014/049177, 9; P. Klarenbeek, None; E. Culver, None; L. Maillette de Buy Wenniger, Patent on “Method for diagnosing IgG4 related diseases”, application number PCT/ EP2013/070384, publication number WO/2014/049177, 9; R. Chapman, None; S. Van de Graaf, None; J. Verheij, None; T. Van Gulik, None; F. Baas, None; E. Barnes, None; U. Beuers, Patent on “Method for diagnosing IgG4 related diseases”, application number PCT/ EP2013/070384, publication number WO/2014/049177, 9,Research grants from German and American PSC patient organizations, 2; N. De Vries, Patent on “Method for diagnosing IgG4 related diseases”, application number PCT/ EP2013/070384, publication number WO/2014/049177, 9.

To cite this abstract in AMA style:

Hubers L, Doorenspleet M, Klarenbeek P, Culver E, Maillette de Buy Wenniger L, Chapman R, Van de Graaf S, Verheij J, Van Gulik T, Baas F, Barnes E, Beuers U, De Vries N. The IgG/IgG4 mRNA Ratio By Quantitative PCR Accurately Diagnoses IgG4-Related Disease and Predicts Treatment Response [abstract]. Arthritis Rheumatol. 2015; 67 (suppl 10). https://acrabstracts.org/abstract/the-iggigg4-mrna-ratio-by-quantitative-pcr-accurately-diagnoses-igg4-related-disease-and-predicts-treatment-response/. Accessed .

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