Molecular diagnosis of orbital inflammatory disease

https://doi.org/10.1016/j.yexmp.2015.01.009Get rights and content

Highlights

  • Orbital inflammation has a broad differential diagnosis.

  • An international team quantified specific RNA transcripts in orbital biopsies.

  • This information enhanced the diagnostic accuracy of biopsies.

Abstract

Orbital inflammatory diseases include thyroid eye disease (TED), granulomatosis with polyangiitis (GPA), sarcoidosis, and nonspecific orbital inflammation (NSOI). Histopathological diagnosis usually relies on the clinical context and is not always definitive. Gene expression profiling provides diagnostic and therapeutic information in several malignancies, but its role in evaluating nonmalignant disease is relatively untested. We hypothesized that gene expression profiling could provide diagnostic information for NSOI. We collected formalin-fixed, paraffin-embedded orbital biopsies from 10 institutions and 83 subjects including 25 with thyroid eye disease, 25 nonspecific orbital inflammation, 20 healthy controls, 6 with granulomatosis with polyangiitis, and 7 with sarcoidosis. Tissues were divided into discovery and validation sets. Gene expression was quantified using Affymetrix U133 Plus 2.0 microarrays. A random forest statistical algorithm based on data from 39 probe sets identified controls, GPA, or TED with an average accuracy of 76% (p = 0.02). Random forest analysis indicated that 52% of tissues from patients with nonspecific inflammation were consistent with a diagnosis of GPA. Molecular diagnosis by gene expression profiling will augment clinical data and histopathology in differentiating forms of orbital inflammatory disease.

Introduction

Molecular diagnosis using transcriptomics has demonstrated tremendous utility in malignant diseases (Hoadley et al., 2014) such as forms of lymphoma (Bohen et al., 2003, Dave et al., 2006) or ocular melanoma (van Gils et al., 2008). The added diagnostic value of profiling gene expression in nonmalignant diseases is less certain, but utility has been reported in inflammatory disorders such as myocarditis (Lassner et al., 2014), synovitis (Yeremenko et al., 2013), and esophagitis (Wen et al., 2013).

Orbital inflammation is an important cause of morbidity that results in pain, diplopia, and sometimes visual loss. Orbital biopsy helps to distinguish malignancy or infection from inflammation. Graves disease is the most common cause of orbital inflammation. It is often referred to as thyroid eye disease (TED). Additional systemic disease associations with orbital disease include sarcoidosis, granulomatosis with polyangiitis (GPA) (formerly Wegener's granulomatosis), sarcoidosis, Crohn's disease, IgG4-related disease, and histiocytosis. Many patients suffer from an inflammatory process that is difficult to categorize. These patients are labeled with terms such as nonspecific orbital inflammation (NSOI), orbital pseudotumor, or idiopathic orbital inflammation. Many of these diagnoses are suggested by clinical context and difficult to make based on histopathology alone. For example, the inflammatory infiltrate in TED can be scant such that, when TED affects orbital fat, it is sometimes difficult to distinguish TED from normal orbital adipose tissue. The diagnosis of GPA requires a vasculitis affecting a medium size vessel, but vessels of this size are rarely obtained on an orbital biopsy. Many patients suffer from an inflammatory process that is difficult to categorize. These patients are labeled with terms such as nonspecific orbital inflammation (NSOI), orbital pseudotumor, or idiopathic orbital inflammation.

While orbital inflammation is not rare, many patients do not undergo biopsy which entails some morbidity and expense. In order to understand nonspecific orbital inflammation, we organized an international consortium of orbital surgeons and pathologists. We have recently reported on the transcripts expressed by tissue representing the four most common diagnoses: TED, sarcoidosis, GPA, and NSOI (Rosenbaum et al., manuscript submitted). In this report, we test the hypothesis that a diagnostic algorithm based on a limited number of transcripts could complement observations made by experienced pathologists.

Section snippets

Centers, biopsies, database

This study was approved by the Institutional Review Board (IRB) at Oregon Health & Science University (IRB00006301) and at each of the other contributing centers. The research adhered to the tenets of the Declaration of Helsinki. Formalin-fixed, paraffin-embedded (FFPE) samples and relevant demographic and clinical data were obtained from 10 institutions. The diagnoses of NSOI, sarcoidosis, GPA, TED, and normal were based on the clinical and histopathological information obtained and submitted

Results

NSOI is heterogeneous in symptoms, histopathology, and response to treatment. The extreme variability of NSOI histopathology is illustrated in Fig. 1. Tissue can vary markedly in terms of the degree of inflammation, the extent of uninflamed adipose tissue, the amount of fibrosis, and the presence of granuloma.

Tissues were divided based on the time of collection into a discovery set and a validation set. We previously reported the heterogeneity of gene expression in NSOI tissues and the

Discussion

While molecular diagnosis has demonstrated marked success in subdividing malignant diseases, it has had fewer applications in inflammatory disease. Orbital inflammatory disease is ideal to test the potential of molecular diagnosis. First, there is a broad differential diagnosis. Second, the various diagnoses often have therapeutic implications. Standard therapy, for example, for GPA differs from standard therapy for sarcoidosis. Third, evaluation by histopathology alone does not always allow a

Acknowledgments

RNA extraction and microarray assays were performed in the OHSU Gene Profiling Shared Resource. We are grateful to Kristina Vartanian for her excellent technical support for the microarray work.

Financial support was from NIH Grants EY020249, EY010572 and RR024140, the Research to Prevent Blindness, the William and Mary Bauman Foundation, the Mas Family Foundation, and the Stan and Madelle Rosenfeld Family Trust.

Potential Conflict of Interest: James Rosenbaum has in the past consulted for

References (13)

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    We then applied this algorithm to orbital adipose biopsies that were diagnosed as healthy, thyroid eye disease, or GPA on the basis of pathology and clinical information from highly regarded ophthalmology centers. The computerized algorithm demonstrated better accuracy (76%) than either of the expert pathologists who scored an accuracy 49% and 58% respectively (Rosenbaum et al., 2015d). Accuracy is defined as the average of sensitivity and specificity.

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    Even among patients who are eventually diagnosed with GPA, biopsy of certain tissue sites such as the orbit, sinus, nasal mucosa, or sub-lottis provides definitive diagnostic information in a minority of cases (Borner et al., 2012; Del Buono and Flint, 1991; Devaney et al., 1990; Kalina et al., 1992; Raynaud et al., 2005). We reported that an algorithm based on our data is more accurate than histopathology at identifying the cause of orbital inflammation (Rosenbaum et al., 2015). We predict that molecular diagnosis will enhance the ability to diagnose GPA in mucosal sites.

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1

For members of the consortium, please see Appendix A.

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