Sensitivity and specificity of serum soluble interleukin-2 receptor for diagnosing sarcoidosis in a population of patients suspected of sarcoidosis.

BACKGROUND: The soluble interleukin 2 receptor (sIL-2R) has been proposed as a marker of disease activity in patients with sarcoidosis. However, no studies have evaluated whether serum sIL-2R measurement is of use in establishing the diagnosis of sarcoidosis in patients who are suspected of sarcoidosis among other diseases.
METHODS: A cohort study was conducted, consisting of new patients who visited the immunology outpatient clinic and whose serum sIL-2R levels were available before a definitive diagnosis was established between February 2011 and February 2016. All patients underwent standard diagnostic testing for sarcoidosis (e.g. laboratory tests, radiographic and/or nuclear imaging and/or affected site biopsy). This resulted either in the diagnosis of sarcoidosis or the exclusion of sarcoidosis with the diagnosis of another disease. Results of sIL-2R and angiotensin-converting enzyme (ACE) levels, radiographic and nuclear imaging and histology results were collected and definitive diagnoses were recorded. Sensitivity, specificity, the concordance statistic from the receiver operating characteristic curve and Youden's Index were calculated to assess the performance of sIL-2R in the diagnosis of sarcoidosis and were compared to ACE, currently one of the most used diagnostic biomarkers in the diagnosis of sarcoidosis.
RESULTS: In total 983 patients were screened for inclusion, of which 189 patients met the inclusion criteria. A total of 101 patients were diagnosed with sarcoidosis after diagnostic workup, of whom 79 were biopsy-proven. In 88 patients a diagnosis other than sarcoidosis was made. The sensitivity and specificity of serum soluble interleukin 2 receptor levels to detect sarcoidosis were 88% and 85%. The sensitivity and specificity of ACE were 62% and 76%. Receiver operating characteristic curve analysis revealed that sIL-2R receptor is superior to ACE (p<0.0001).
CONCLUSION: Serum sIL-2R is a sensitive biomarker and superior to ACE in establishing the diagnosis of sarcoidosis and can be used to rule out sarcoidosis in patients suspected of sarcoidosis.

1. Introduction

Sarcoidosis is a multisystem disease of unknown origin, characterized by non-caseating granulomas, which can affect almost any organ system. The diagnosis of sarcoidosis is based on clinical and radiographic manifestations and histopathologic detection of non-caseating granulomas in the affected organ, after exclusion of other diseases that may present similarly. [1] Diagnostic tests that can contribute to the diagnosis of sarcoidosis include serum angiotensin-converting enzyme (ACE), conventional chest radiograph, high-resolution chest computed tomography (CT), broncho-alveolar lavage and fluorodeoxyglucose-positron emission tomography (FDG-PET).[2] Undetected sarcoidosis can lead to substantial morbidity. [3, 4]Although ACE is one of the most used diagnostic biomarkers for sarcoidosis, it lacks sensitivity. [5] High sensitivity is needed when a test is used in the initial diagnostic workup, especially when this test is used to rule out the disease.

One of the main immunologic features of sarcoidosis is the influx of CD4+ T-helper (Th)1 cells at sites of active disease. [6] Th 1 cells secrete interleukin (IL) 2, which promotes T-cell proliferation and survival. [7] IL-2 acts via the IL-2 receptor, which consists of the common γ chain (CD132), a β chain (CD122) and an α chain (CD25). CD25 is overexpressed by activated T-cells and regulatory T-cells and can be secreted from the cell membrane in a soluble form; also referred to as soluble IL-2R (sIL-2R.) Hence, sIL-2R is a surrogate marker for T-cell activation. Peripheral blood sIL-2R levels thus reflect the level of T cell activation in an individual and elevated blood sIL-2R levels correlate with disease activity, for instance in patients with rheumatoid arthritis, systemic lupus erythematosus or IgG4-related disease, but also in sarcoidosis patients. [8-12]

There are various theories on the biological action of sIL-2R in the immunopathology of inflammatory diseases. One of the proposed mechanisms of action is that sIL-2R competes with activated T-cells for available IL-2 and thereby inhibits T-cell proliferation. [13, 14] Another proposed function is that sIL-2R binds IL-2, thereby prolonging IL-2 half-life, which enhances its immune-stimulatory effects. [15, 16] On the other hand, it has been proposed that IL-2 can be presented to CD4+ T-cells via the sIL-2R-IL-2 complex, thereby upregulating FOXP3 expression and differentiation into T regulatory cells that subsequently control immune activity. [17]

Up to now, it is unclear whether sIL-2R is produced to combat the sarcoidosis-associated immune activation or whether it has an active role in the pathogenesis of sarcoidosis. It is clear, however, that there is a correlation between sIL-2R and sarcoidosis. Earlier research showed a correlation between sIL-2R and disease activity, the extent of disease and response to treatment. [11, 18–23] In two studies which specifically examined patients with ocular sarcoidosis, serum sIL2-R has been proposed as a diagnostic biomarker. [24, 25] Another study, conducted in patients with sarcoid skin lesions demonstrated a correlation between serum sIL-2R levels and the number of involved organs. [26] However, no study has evaluated whether the measurement of serum sIL-2R can be useful in diagnosing systemic sarcoidosis in symptomatic patients, whose diagnosis is still uncertain. Thus it remains unknown, whether serum sIL-2R, apart from being a biomarker for disease activity, can be used as a diagnostic tool for sarcoidosis in clinical practice. We hypothesized that serum sIL-2R is a sensitive and specific diagnostic biomarker that can differentiate between sarcoidosis and non-sarcoidosis patients in a cohort of patients suspected of sarcoidosis.

To test this, we conducted a cohort study consisting of new patients in whom sarcoidosis was suspected, but not yet proven or disproven and whose sIL-2R levels were measured before a diagnosis of sarcoidosis or another disease was established. In this cohort, after diagnostic workup and after a definitive diagnosis (sarcoidosis or another disease) was established, we calculated the sensitivity and specificity of sIL-2R in the diagnosis of sarcoidosis and defined the optimal cut-off value and compared this to serum ACE levels.

2. Material and methods

2.1 Study population

In this study, we included patients visiting the immunology outpatient clinic, in which sarcoidosis, among other diseases, was a part of the differential diagnosis and of whom serum sIL-2R levels were measured before the definitive diagnosis of sarcoidosis or another disease was established. Sarcoidosis was suspected when a patient was referred to the Immunology department with one or more of these symptoms: 1) lung complaints, i.e. shortness of breath, persistent dry cough or chest pains; 2) uveitis or inflammation of the lacrimal glands; 3) skin disorders, i.e. erythema nodosum, lupus pernio, infiltration of scars or tattoo; 4) neurological complaints, i.e. hearing loss, facial nerve palsy or epilepsy; 5) polyarthritis; 6) incidental finding of lymphadenopathy on imaging.

Medical records of patients, whose serum sIL-2R level was assessed at the Erasmus MC between February 2011 and February 2016, were screened for potential inclusion in our study. The following inclusion criteria were applied: availability of medical records, sarcoidosis as part of the differential diagnosis, availability of serum sIL-2R value before a definitive diagnosis was established and a finished diagnostic workup at the immunology outpatient department (Fig 1). Exclusion criteria were: an established diagnosis of sarcoidosis before serum sIL-2R measurement and immunosuppressive medication within the two weeks prior to sIL-2R measurement or use of ACE inhibitors one month prior to ACE measurement. Patients who received immunosuppressive medication were excluded, because immunosuppressive medication can reduce sIL-2R levels. [11, 27] The diagnosis of sarcoidosis relies on consistent clinical and radiological features and histological demonstration of non-caseating granulomas in the affected organ(s), according to the American Thoracic Society/European Respiratory Society/World Association of Sarcoidosis and other Granulomatous Disorders (ATS/ERS/WASOG) guidelines. [28] In our Sarcoidosis Expertise Centre, sarcoidosis is diagnosed according to these criteria and the diagnosis is verified by at least one pulmonary specialist and at least one clinical immunologist. Patients without histological confirmation of sarcoidosis were, in addition to the evaluation by a pulmonary specialist and a clinical immunologist, evaluated by a, for sIL-2R and ACE blinded, pulmonary specialist who assessed clinical information, radiologic and nuclear imaging. When categorized as sarcoidosis or very probable sarcoidosis the patient was included in the group of patients with a definitive diagnosis of sarcoidosis. Otherwise, the patient was included as a non-sarcoidosis patient.

Fig 1

Flowchart of study design and inclusion of patients.

* sIL-2R: soluble interleukin 2 receptor. ** ROC: receiver operating characteristic curves

The ACE measurement of the date closest to the date of the first sIL-2R measurement was used. If the ACE measurement was performed more than one month prior to or more than one month after sIL-2R measurement, serum ACE was regarded as not available.

Demographic data and anatomical localization of sarcoidosis disease activity were recorded. Four anatomical localizations of sarcoidosis activity were distinguished in this study: sarcoidosis with predominantly lung, ocular, neurological or skin involvement. Multiple organ involvement could be present within one patient. Additionally, when sarcoidosis was an incidental finding on radiographic imaging, performed for other reasons, with no organ-related symptoms, none of these categories was chosen. Data on chest radiograph, chest CT and FDG-PET scan were collected. Chest radiographs were scored using the Scadding scale. [29]

The serum sIL-2R levels of 101 anonymous blood bank donors were used as healthy controls.

Strengthening the reporting of observational studies in epidemiology guidelines were used to structure the reporting of this observational study.[30] The use of laboratory investigation to gather non-identifiable data in this observational study adheres to the tenets of the Declaration of Helsinki. The medical ethical committee of Erasmus University Medical Center approved the protocol and the associated procedures.

2.2 Analysis of serum parameters

Serum sIL-2R measurements were performed (ELISA; Diaclone, Besancon Cedex, France) at the laboratory medical immunology at Erasmus MC. A serum sIL-2R value <2500 pg/mL is considered as not elevated within the Erasmus MC, which is based on values sIL-2R levels measured in 101 healthy blood donors. Serum ACE levels were determined by kinetic assay (Bühlmann Laboratories, Switzerland), with reference range ≤68 U/mL.

2.3 Sample size calculation

Hypothetical confidence intervals were calculated using Buderer’s formula. Reaching a confidence interval of approximately 5% on either side, with a power of 80%, would require 200 patients Therefore, we strived to include 200 patients.

2.4 Statistical analysis

The characteristics of patients with and without the definitive diagnosis of sarcoidosis were summarized using descriptive statistics, such as medians and percentages. Non-parametric tests were used to compare characteristics between the groups. We calculated the specificity, sensitivity and the Youden’s index, as well as the concordance statistic (C-statistic), which can be calculated from the receiver operating characteristic (ROC) curve for all patients. Additionally, we calculated the sensitivity, specificity, Youden’s index and C-statistic for the subgroup of biopsy-confirmed sarcoidosis patients.

Sensitivity and specificity can be used to calculate the Youden’s index. The Youden’s index reflects the performance of a dichotomous diagnostic test.

The ROC curve is designed to visualize and detect the optimal performance of a binary test with a varied cut-off point. The optimal cut-off for sIL-2R was calculated by maximizing sensitivity and specificity, using the values generated by a ROC curve. Kruskal-Wallis one-way analysis of variance of the data on the localization of sarcoidosis and the levels of sIL-2R was performed.

The statistical analyses were all done using Excel, IBM SPSS statistics 21.0.0 for Windows (SPSS inc., Chicago, IL, USA), and R, using the package pROC. [31]

3. Results

3.1 Patient characteristics

Of the 983 screened patients 189 were included and 794 were excluded (Fig 1). The exclusion was due to unavailable medical records (n = 446), sarcoidosis not in the differential diagnosis (thus not being suspected of sarcoidosis at all: n = 75), or undocumented differential diagnosis (n = 26). Patients were also excluded when the diagnosis was established prior to serum sIL-2R measurement (n = 218) or when the diagnostic workup was not finished or finished elsewhere (n = 7). Furthermore, 22 patients were excluded due to medication use: some patients due to immunosuppressive medication use prior to serum sIL-2R measurement (n = 14), others due to ACE inhibitor use prior to serum ACE measurement (n = 8). Of the 189 included patients, 101 patients were finally diagnosed with sarcoidosis, of which 79 were biopsy-confirmed sarcoidosis patients and another 22 patients without positive biopsy were classified as very probable sarcoidosis by the blinded reviewer. In 88 patients, sarcoidosis was excluded and another disease was diagnosed, including Sjögren’s syndrome, tuberculosis, multiple sclerosis or rheumatoid arthritis. A comprehensive list of all the diagnoses is provided in the footnotes of Table 1. ACE levels were available in 120 patients.

Table 1

Patient characteristics.

	Definitive sarcoidosis(n = 101)	Definitive diagnosis other than sarcoidosisb(n = 88)
Sex, female (% female)	51 (50%)	52 (59%)
Median age at diagnosis, years (IQRa)	43 (35–52)	44 (31–57)
Positive biopsy (n)	79	0
Angiotensin-converting enzyme
median	77	51
IQR a	44–109	31–69
Soluble interleukin 2 receptor
median	6100	2600
IQR a	4500–9850	1925–3300

a IQR: interquartile range

b Including uveitis of unknown origin (n = 19), Nonspecific interstitial pneumonia (n = 3), Systemic lupus erythematosus (n = 3), Asthma (n = 2), Chronic obstructive pulmonary disease (n = 2), Fatigue of unknown origin (n = 2), Fibromyalgia (n = 3), Multiple sclerosis (n = 2), Neuromyelitis optica (n = 2), Ocular vasculitis (n = 2), Rheumatoid arthritis (n = 2), Tuberculosis (n = 2), Sjögren’s syndrome (n = 2), Sudden deafness of unknown origin (n = 2), Alopecia (n = 1), Arthritis psoriatica (n = 2), Atypical parkinsonism (n = 1), Auto-immune encephalitis (n = 1), Cervical myelopathy (n = 1), Chronic cough due to reflux (n = 1), Dacryops (n = 1), Epstein Barr viral infection (n = 1), Erythema nodosum (n = 1), Exertional dyspnea (n = 1), Extrinsic allergic alveolitis (n = 1), Folliculitis (n = 1), Guillain-Barre syndrome (n = 1), Herpes simplex related apthosis (n = 1), Human immunodeficiency virus (n = 1), Hyper IgE syndrome (n = 1), Hypophysitis (n = 1), Idiopathic pulmonary fibrosis (n = 1), Increased intracranial pressure (n = 1), Klebsiella pneumoniae infection (n = 1), Langerhans cell histiocytosis (n = 1), Lipoma (n = 1), Neuroendocrine tumor (n = 1), Macular retinopathy (n = 1), Nontuberculous Mycobacterium infection (n = 1), Osteoarthritis (n = 1), Presumed ocular histoplasmosis (n = 1), Rosacea (n = 2), Schwannoma (n = 1), Scleritis (n = 1), Small fiber neuropathy (n = 1), Stills disease (n = 1), Systemic vasculitis (n = 1), Toxoplasmosis (n = 1), Ulcerative colitis (n = 1), Urticaria (n = 1), Vitreomacular traction syndrome (n = 1), Vocal cord paralysis (n = 1)

Demographic characteristics did not differ between the sarcoidosis and non-sarcoidosis groups (Table 1).

Serum sIL-2R levels are depicted in Fig 2 and presented in Table 1. The median serum sIL-2R level in patients with a definitive diagnosis of sarcoidosis (n = 101) was 6100 pg/mL, 2600 pg/mL in patients with a definitive diagnosis of another disease(n = 88) and 1515 pg/mL in healthy controls (n = 101, healthy blood bank donors). The sIL-2R levels were significantly higher in patients with a definitive diagnosis of sarcoidosis compared to patients with diseases other than sarcoidosis (P < 0.001). In patients with a definitive diagnosis of granulomatous diseases, lung diseases, uveitis, infectious diseases or other diseases, sIL-2R levels were higher than healthy controls (P < 0.001) but lower than sarcoidosis patients (P < 0.001).

Fig 2

Dot plot of serum soluble interleukin 2 receptor levels in various diseases and healthy controls.

*. Four patients have been assigned towards multiple groups: Two tuberculosis and one extrinsic allergic alveolitis patients were assigned towards lung diseases and granulomatous diseases. One Patient with toxoplasmosis was assigned towards granulomatous diseases as well as towards infectious diseases.

Of all definitive sarcoidosis patients, 65% had a positive chest radiograph, while 99% had a positive CT-scan. Of all sarcoidosis patients who underwent a PET-CT scan, 91% had a positive PET-CT scan. Thirty-five percent of sarcoidosis patients presented with sarcoidosis stage 0 on chest radiograph, while 28% showed stage I, 25% stage II, 10% stage III and only 2% showed stage IV fibrosis on the chest radiograph.

3.2 Sensitivity, specificity, Youden’s index, and C-statistic

The sensitivity of sIL-2R was superior to ACE (Table 2) in the diagnosis of sarcoidosis. The Youden’s index performed better for sIL-2R than for ACE (0.73 versus 0.38, respectively). The ROC curve (Fig 3) depicts the performance of both sIL-2R and ACE. The C-statistic, a measure of test performance which can be calculated from this curve, is significantly better for sIL-2R (C-statistic = 0,91, with 95% CI 0,87–0,96) versus ACE (C-statistic = 0,68 with 95% CI 0.58–0.78) (P < 0.001).

Table 2

Sensitivity, specificity, Youden’s index and concordance statistic in the diagnosis of sarcoidosis.

	Sensitivity (95% CIa)	Specificity(95% CIa)	Youden’sIndexb	Concordance statisticc (95% CIa)
sIL-2R, cutoff 2500 pg/mL	99% (95–100)	47% (36–57)	0.45	0.91 (0.87–0.96)
sIL-2R, cutoff 3550 pg/mL	88% (82–94)	85% (78–93)	0.73	0.91 (0.87–0.96)
ACE, cutoff 68 U/mL	62% (51–73)	76% (64–88)	0.38	0.68 (0.58–0.78)
sIL-2R, cutoff 3550 pg/mL, biopsy-confirmed sarcoidosis	87% (80–95)	85% (78–93)	0.73	0.93 (0.87–0.98)
ACE, cutoff 68 U/mL, biopsy-confirmed sarcoidosis	60% (48–72)	76% (64–88)	0.36	0.68 (0.50–0.79)

a CI: confidence interval

b Interpretation Youden’s index: A higher Youden’s index is more favorable.

c Interpretation concordance statistic: A higher concordance statistic is more favorable.

Fig 3

Receiver operating characteristic curves comparing soluble interleukin 2 receptor levels and angiotensin-converting enzyme in the diagnosis of sarcoidosis (N = 189).

* sIL-2R: soluble interleukin 2 receptor. ** ACE: angiotensin-converting enzyme

The area under the curve (AUC) is a measure of test performance. A larger area under the curve indicates a better test performance.

When calculating the sensitivity and specificity of serum sIL-2R in a subgroup of patients, consisting of patients with a definitive diagnosis of biopsy-confirmed sarcoidosis (n = 79) and patients with a definitive diagnosis other than sarcoidosis (n = 88), the associated sensitivity and specificity of sIL-2R and ACE similar to the sensitivity and specificity for the whole study group (Table 2). In this subgroup, the C-statistic remained significantly better for sIL-2R versus ACE (P = 0.003).

3.3 Correlation of sIL-2R with diagnostic parameters

There is a significant, but rather weak correlation between sIL-2R and chest radiograph stage (Spearman’s rho 0,24; P = 0.021). Median sIL-2R levels were lower in patients without parenchymal involvement (stages 0 and I) as compared to patients with parenchymal involvement (stages II, III and IV). (Table 3).

Table 3

Diagnostic parameters in sarcoidosis patients.

		Number of patients	Median sIL-2R levels (IQRb) in pg/mL
Chest radiographa	Stage 0normal chest radiograph	31	5500 (3700–7700)
	Stage 1hilar or mediastinal nodal enlargement	27	6300 (4854–9100)
	Stage 2nodal enlargement and parenchymal disease	19	10800 (5300–19368)
	Stage 3parenchymal disease without nodal enlargement	8	5947 (3700–8625)
	Stage 4pulmonary fibrosis	5	8487 (5620–15932)
Chest CT-scan	Negativeno sarcoidosis-associated findings	2	3100; 3700
	PositiveSarcoidosis-associated findings	74	6564 (4841–10800)
FDG-PET-scan	NegativeNo sarcoidosis-associated findings	2	3600, 4600
	PositiveSarcoidosis-associated findings	24	5700 (4075–7900)

a Chest radiograph staging: stage 2 is both nodal enlargement and parenchymal disease, while stage 3 is parenchymal disease without nodal enlargement. Therefore, stage 2 is widely regarded as more severe than stage 3.

b IQR: interquartile range

Only two patients with a definitive diagnosis of sarcoidosis had no sarcoidosis-associated signs on CT-scan. Therefore, a correlation between CT positive and negative patients was not performed. However, the two patients with a negative CT-scan, displayed markedly lower serum sIL-2R levels (3100 pg/mL and 3700 pg/mL), in comparison to the interquartile range of patients with a positive CT-scan (5700–7900 pg/mL). Although the sIL-2R value of the two FDG-PET negative sarcoidosis patients was lower (3600 pg/mL and 4600 pg/mL) than the median sIL-2R value of the FDG-PET positive sarcoidosis patients (5700 pg m/L), the values were near or within the interquartile range observed of the FDG-PET positive patients (4075–7900).

3.4 Determining the optimal cutoff of serum sIL-2R to facilitate the diagnosis of sarcoidosis

The current cut-off used in our hospital to indicate an elevated serum sIL-2R level in an individual is 2500 pg/mL and as such was not chosen to facilitate the diagnosis of a certain disease. This is clearly reflected by the unbalanced specificity sensitivity pattern for sarcoidosis when this sIL-2R cut-off was applied (Table 2). ROC analysis indicated that a serum sIL-2R value of 3550 pg/ml represents the optimal cut-off for diagnosing sarcoidosis with 88% sensitivity and 85% specificity and a Youden’s Index of 0.73, which is considered good.

Applying this optimal cut-off to the biopsy-confirmed subgroup resulted in similar sensitivity and specificity and a Youden’s index of 0.73. In order to make a fair comparison, we also calculated the optimal cut-off point for ACE, using the ROC curve. The optimal cut-off point for ACE was 68.75, which equals its current cut-off point.

3.5 Serum sIL-2R levels in sarcoidosis subgroups

A trend for a higher median sIL-2R level was observed in case of nervous system involvement, however, this was not statistically significant (Table 4).

Table 4

Soluble interleukin 2 receptor levels.

Group	Number of persons	Median sIL-2R level (pg/mL)	IQRa
Healthy controls	101	1515	1150–1880
Sarcoidosis	101	6100	4500–9850
Nervous system	6	16851	5027–11020
Ocular	40	6150	4866–7900
Skin	16	6050	4975–8750
Lung	48	7300	4739–11175
Granulomatous diseasesb	9	3300	3000–3550
Lung diseasesc	11	3000	2400–4700
Infectious diseasesd	7	2300	1900–3200
Uveitis	19	2300	1900–3200

a IQR: interquartile range

b Including Rheumatoid arthritis (n = 2), Tuberculosis (n = 2), Erythema nodosum (n = 1), Extrinsic allergic alveolitis (n = 1), Langerhans cell histiocytosis (n = 1), Systemic vasculitis (n = 1), Toxoplasmosis (n = 1)

c Including Nonspecific interstitial pneumonia (n = 3), Asthma (n = 2), Chronic obstructive pulmonary disease (n = 2), Tuberculosis (n = 2), Extrinsic allergic alveolitis (n = 1), Idiopathic pulmonary fibrosis (n = 1)

d Including Epstein Barr viral infection (n = 1), Herpes simplex related apthosis (n = 1), Human immunodeficiency virus (n = 1), Klebsiella pneumoniae infection (n = 1), Nontuberculous Mycobacterium infection (n = 1), Presumed ocular histoplasmosis (n = 1), Toxoplasmosis (n = 1)

4. Discussion

In this study we demonstrated that serum sIL-2R has high and superior sensitivity and specificity compared to ACE for diagnosing sarcoidosis in a group of patients, presenting with symptoms, which raised a suspicion of sarcoidosis as well as other diseases. In patients with a definitive diagnosis of sarcoidosis, sIL-2R is significantly elevated compared to patients with a definitive diagnosis of another disease. Thus, in a group of patients who have not yet received a definitive diagnosis, but are suspected of sarcoidosis, serum sIL-2R is able to discriminate between patients with and without sarcoidosis.

Sarcoidosis is a heterogeneous systemic disease and notoriously difficult to diagnose. Evaluation often requires extensive diagnostic testing and invasive tests, such as histological sampling of the affected tissue. [2, 32] The ACE, which is widely used as a diagnostic biomarker for sarcoidosis, has a low sensitivity, ranging from 22–80% that hampers its use in a diagnostic setting. [5, 24, 33, 34]The sensitivity of 60% observed for ACE in the current study is within this range. Our study, using robust and frequently used statistical methods such as the C-statistic and Youden’s index, demonstrates that serum sIL-2R is more sensitive and equally specific as ACE in diagnosis in patients suspected of sarcoidosis. [35-39] The demonstrated superiority of serum sIL-2R over ACE as a diagnostic tool for sarcoidosis underlines its use as a valuable clinical instrument in the workup of patients suspected for sarcoidosis and might have consequences for subsequent and potential unnecessary (radiological or invasive) evaluation. [40]

We also observed a significant correlation between serum sIL-2R levels and chest radiograph stage. However, this correlation was rather weak (spearman’s’ rho 0.24) and might therefore not be clinically relevant. Sarcoidosis stage III chest radiograph represents less severe disease (parenchymal involvement without nodal enlargement) compared to sarcoidosis stage II chest radiograph (parenchymal involvement in addition to nodal enlargement) and was also associated with lower levels of serum sIL-2R than radiographic stage II sarcoidosis. Stage IV disease, which is characterized by pulmonary fibrosis, showed high levels serum of sIL-2R. However, these levels were lower than serum sIL-2R levels found in patients with stage II disease, which is associated with nodal and parenchymal involvement. However, stage IV disease might not necessarily have correlated with disease activity at the time of assessment. Since lung fibrosis is irreversible, stage IV disease can still be visible on chest radiograph, while sarcoidosis is biochemically and clinically less active. This might explain why the highest values of sIL-2R were not found in stage IV disease, but rather in stage II.

In the tertiary hospital where this study took place, ACE is less expensive than sIL-2R. (€10,00 versus €53,57). However, since sIL-2R is very sensitive, one could speculate that other diagnostic tests, such as CT or FDG-PET might not be necessary for patients with low sIL-2R values. The associated costs (€250,00 and €800,00 respectively) could perhaps be avoided. We measured sIL-2R with ELISA, a relatively rapid (5–6 hours) method that requires standard laboratory equipment and can thus easily be implemented in most clinical laboratories. Alternatively, fully automated techniques are also available. [24] A detailed evaluation of the cost-effectiveness of diagnostic tests used in the diagnostic workup of sarcoidosis is beyond the scope of this study. Yet whether parameters, such as ACE and FDG-PET screening, are rendered redundant by sIL-2R assessment needs evaluation in a cost-effectiveness study

We demonstrate that the probability of having sarcoidosis with a serum sIL-2R level of < 3550 pg/mL is low. This optimal cut-off value of 3550 pg/mL results in a good test performance, both for sensitivity and specificity when used as a diagnostic biomarker for sarcoidosis. [41]

Although underpowered and not designed for subgroup analysis, variations in serum sIL-2R levels between subgroups based on anatomical localization of sarcoidosis disease activity were observed. This was most evident for patients with neurological involvement, who displayed the highest sIL-2R levels. Whether these high serum sIL-2R levels in neurosarcoidosis reflects the total burden of T-cell activation or is an incidental finding, remains unknown.

There is a high clinical variation in sarcoidosis patients. This might be explained by biochemical or genetic differences. It has been shown that certain human leukocyte antigen alleles are associated with variations in the clinical manifestations. For example, the DRB1*0301 allele is associated with Löfgren’s syndrome in a Dutch cohort.[42] Whether genetic variances of human leukocyte antigen alleles influence the sIL-2R levels is unknown. Further studies specifically designed for this purpose might further elucidate this observation.

A limitation of our study is the number of patients included. However, the number of included patients (189) did approach our initial sample size calculation of 200 patients. Furthermore, since the effect difference observed in this study between ACE and sIL-2R is larger than the estimated effect difference used in the sample size calculation, it is easier to detect a significant difference. Therefore, our study is adequately powered for our primary outcomes.

A strength of our study is the control group of non-sarcoidosis patients. All patients included in this study were suspected of sarcoidosis at presentation. Therefore, all diseases that could cause sarcoidosis-like symptoms, ranging from tuberculosis to arthritis psoriatica, are included in this study. Therefore, we strongly believe that our study approach most optimally reflects clinical practice

In this study, all sIL-2R levels were measured before the definitive diagnosis was established. Therefore, the outcome, i.e. the diagnosis of sarcoidosis, has not influenced the measurement of sIL-2R and prevents bias. [43] For ACE measurements, only one patient had ACE measured after the diagnosis of sarcoidosis. We feel that this one patient will not have biased the overall result.

There were no missing values for sIL-2R. There were, however, missing values for ACE. When we performed a sensitivity analysis in a subgroup of patients that had no missing values for ACE, sensitivity for sIL-2R was 91% and specificity 85% with a cut-off of >3550 pg/mL. These numbers are almost identical to those of the whole population (88% and 85% respectively). With this, we can safely assume that the missing data are not related to the outcome and therefore the whole study population can be used, including patients who have missing values for ACE.

The results of this study have limitations in their application to clinical practice, due to the population in this study, which does not reflect the general population, but rather the population in a tertiary hospital. The patients in this study were often referred to the tertiary hospital due to difficult to diagnose disease.

This study compared patients with a definitive diagnosis of sarcoidosis and patients with a definitive diagnosis of another disease, not with healthy controls. These patients were diagnosed with various diseases and thus represent a heterogeneous group. Comparing the use of sIL-2R within a diseased population is a comparison that mimics clinical practice more closely than comparing sarcoidosis patients and healthy controls.

5. Conclusion

In this cohort study consisting of patients in whom sarcoidosis is suspected but not yet proven or excluded, we demonstrate the superiority of serum sIL-2R above ACE in the screening for sarcoidosis. Therefore, serum sIL-2R may serve as a diagnostic, discriminating tool and as a biomarker for patients that are suspected for sarcoidosis. Due to its high sensitivity, sIL-2R can be a useful tool to rule out sarcoidosis in patients suspected of sarcoidosis.

Dataset of sarcoidosis and non-sarcoidosis patients.

(XLSX)

Click here for additional data file.

13 Aug 2019

PONE-D-19-20768

Sensitivity and specificity of the soluble interleukin-2 receptor in biopsy-proven sarcoidosis patients

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Reviewer #1: Sarcoidosis is a systemic granulomatous disease of unknown etiology. The polarization into T helper type-1 response and its associated cytokines have been reported to play key roles in pathogenesis of this disease. Until now, the diagnosis of sarcoidosis is still difficult and sometimes very problematic, almost bases on a combination of clinical and imaging findings, histological demonstration and exclusion of other capable diseases. So, exploring a marker with high sensitivity and specificity for diagnosis of sarcoidosis is very interesting and meaning with clinical practices. However, serum level of sIL-2R is not a new marker for sarcoidosis with many research studies before in which sensitivity and correlation between this serum level with disease activity and other diagnosis parameters were already confirmed. Because this serum correlates with the level of T-cell activation, identification for the specificity of sIL-2R required a bigger survey from many other diseases and I have a few questions that I would like to discuss below.

1. The new information from your study is identification for the specificity of sIL-2R that required a bigger survey from many non-sarcoidosis diseases. In your manuscript, the number patient is small, they are not representative for the disease populations. If you increase these numbers, your study may be more convincing.

2. In Figure 3, please change the format box-plot to dot-plot, and add the number of each group. It will help to recognize the number of patients involved in this study.

3. In the result 3.3: correlation of sIL-2R with diagnostic parameter, the author showed the significant correlation between sIL-2R and chest radiograph stage, but R=0.27 is not difference. May you check it? And it might be better if you show these data by correlation chart.

Reviewer #2: Review comment PONE-D-19-207

The authors examined the diagnostic utility of sIL-2R and ACE in patients with suspected sarcoidosis patients, and concluded that sIL-2R was useful for such biomarkers. Although the manuscript basically well written, this reviewer feels that there is serious concern that should be addressed.

Major

1. Study population:

To consider sensitivity and specificity of biomarker, study subjects (e.g. sarcoidosis, suspected sarcoidosis, other mimicking disease, healthy subjects) are very important.

A) Suspected of sarcoidosis patients: As mentioned in the introduction, sarcoidosis is systemic disease. Thus, there were many courses in suspected sarcoidosis patients (e.g. lung, heart, liver, skin, nervous). Were all the patients who have lung sarcoidosis in the present study? The authors should clearly define the patients who suspected sarcoidosis. To clarify study subject who suspected sarcoidosis, how about the authors consider to limit the study subjects who have lung sarcoidosis or lung disease.

B) As shown in Figure 2, patients with immunosuppressive medication <2 weeks before sIL-2R measurement or ACE inhibitors < 1 month before ACE were excluded. Are there any backgrounds in this setting period? This reviewer feels that all patients who prescribed immunosuppressive medication should be excluded in the study subjects.

C) In Figure 4, to examine sensitivity and specificity of sIL-2R and ACE, which subjects (e.g. included patients n=180, or include patients n=180 + healthy subjects n=101) did the authors use?

2. Results Figures 1 and 2

Please consider combine Figures 1 and 2, to easy to understand for readers.

Minor

3. Figure 3

Please add the numbers of each group in Figure 3.

**********

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Reviewer #1: Yes: Ikuko Ueda-Hayakawa

Reviewer #2: No

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16 Sep 2019

Dear editor,

Thank you for the opportunity to clarify the message of our manuscript. We have answered the remarks of the reviewers, and we feel that the manuscript, after applying the suggested changes, has improved significantly. In addition to this, we noticed that the objective of our study was not stated clearly enough. Therefore, in addition to our replies and corrections based on the advice of the reviewers, we have shifted the focus of the manuscript slightly to highlight our study population and our objective more clearly. Consequently, we have changed the title to “Sensitivity and specificity of serum soluble interleukin 2 receptor in a population of patients suspected of sarcoidosis”. We hope you will find the manuscript sufficiently improved and suitable for publishing in PlosOne.

Reviewer #1: Sarcoidosis is a systemic granulomatous disease of unknown etiology. The polarization into T helper type-1 response and its associated cytokines have been reported to play key roles in pathogenesis of this disease. Until now, the diagnosis of sarcoidosis is still difficult and sometimes very problematic, almost bases on a combination of clinical and imaging findings, histological demonstration and exclusion of other capable diseases. So, exploring a marker with high sensitivity and specificity for diagnosis of sarcoidosis is very interesting and meaning with clinical practices. However, serum level of sIL-2R is not a new marker for sarcoidosis with many research studies before in which sensitivity and correlation between this serum level with disease activity and other diagnosis parameters were already confirmed. Because this serum correlates with the level of T-cell activation, identification for the specificity of sIL-2R required a bigger survey from many other diseases and I have a few questions that I would like to discuss below.

1. The new information from your study is identification for the specificity of sIL-2R that required a bigger survey from many non-sarcoidosis diseases. In your manuscript, the number patient is small, they are not representative for the disease populations. If you increase these numbers, your study may be more convincing.

Thank you for your comment. We agree that increasing the number of patients, thus patients who got a definitive diagnosis of sarcoidosis or another disease than sarcoidosis, in our study would add statistical power and value to our study. Therefore, we did go back into the dataset and now added patients in whom a diagnosis (sarcoidosis or another disease) was now established (they were not included in the initially submitted manuscript, as, although serum sIL-2R levels were available at that time, no definitive clinical diagnosis was available then(for example, because histology results were still pending).

In total, we added 9 additional patients, of whom 4 got the definitive diagnosis of sarcoidosis and 5 patients who were diagnosed with another disease. The addition of these extra patients to our study implied recalculations for the whole manuscript, so you will find that all of our numbers have changed slightly, although not significantly. With the addition of these patients, the total number of patients in the revised manuscript now is 189. Prior to the analysis of this study, we have performed a power calculation, with a power of 80% and a confidence interval of approximately 5% on either side, with a modest estimated effect difference of 10%. This yielded a suggested sample size of 200 patients. The number of patients included in this study (189), is close to the suggested sample size. Importantly, since the effect difference appeared larger than the estimated effect difference used in the initial power calculations, it is easier to detect significant differences. Therefore, we feel our study is adequately powered for our primary outcome.

Adding more patients from another population to add power for our secondary outcomes might induce bias to this study. In this study, we selected all patients from the same pool: patients whose sIL-2R had been assessed because sarcoidosis was part of the differential diagnosis. Therefore, we are limited in the number of patients we can include by our population pool of patients that at that time of serum sIL-2R measurement had a differential diagnosis that included sarcoidosis among other diseases. If we would have selected patients from other departments, such as Rheumatology for example with high suspicion of rheumatoid arthritis yet without any suspicion of sarcoidosis to add to the non-sarcoidosis patient group, we include patients who were never suspected of sarcoidosis. For example, a female patient with a family history of rheumatoid arthritis, with a symmetrical polyarthritis of the hands, morning stiffness and a positive rheumatoid factor or anti-citrullinated protein antibody, would in practice never be tested for sarcoidosis. As the aim of our study was to explore whether serum sIL-2R measurements could be of added value to diagnose sarcoidosis in patients that present at the clinic with features that can be associated with sarcoidosis, as well as other diseases, we feel it would not be statistically sound to include serum the sIL-2R from patients who never have been suspected of sarcoidosis. This would clearly not reflect the daily clinical practice where we want to apply serum sIL-2R measurements in patients who are suspected of having sarcoidosis. Furthermore, it would not answer the research question: can sIL-2R be used in case of suspicion of sarcoidosis? Therefore, we only used this pool of patients suspected of sarcoidosis whose sIL-2R was assessed.

2. In Figure 3, please change the format box-plot to dot-plot, and add the number of each group. It will help to recognize the number of patients involved in this study.

We agree with the reviewer and have changed the initial box-plot into a dot-pot and added the group sizes to make this figure more insightful.

3. In the result 3.3: correlation of sIL-2R with diagnostic parameter, the author showed the significant correlation between sIL-2R and chest radiograph stage, but R=0.27 is not difference. May you check it? And it might be better if you show these data by correlation chart.

We thank the reviewer for addressing this point. Indeed, a correlation of 0.27, even though it might be statistically significant (P < 0.05) is not a strong correlation and presumably of low clinical value. Therefore, we have changed our wording in that paragraph to ‘There is a statistically significant, weak correlation between sIL-2R and chest radiograph stage’ and we have commented on this in the discussion as well. Since this finding is of low clinical value, we feel it might not be appropriate to use an additional graph for this finding.

Reviewer #2: Review comment PONE-D-19-207

The authors examined the diagnostic utility of sIL-2R and ACE in patients with suspected sarcoidosis patients, and concluded that sIL-2R was useful for such biomarkers. Although the manuscript basically well written, this reviewer feels that there is serious concern that should be addressed.

Major

1. Study population:

To consider sensitivity and specificity of biomarker, study subjects (e.g. sarcoidosis, suspected sarcoidosis, other mimicking disease, healthy subjects) are very important.

A) Suspected of sarcoidosis patients: As mentioned in the introduction, sarcoidosis is systemic disease. Thus, there were many courses in suspected sarcoidosis patients (e.g. lung, heart, liver, skin, nervous). Were all the patients who have lung sarcoidosis in the present study? The authors should clearly define the patients who suspected sarcoidosis. To clarify study subject who suspected sarcoidosis, how about the authors consider to limit the study subjects who have lung sarcoidosis or lung disease.

Thank you for this insightful comment. As you mentioned, there are many localizations of sarcoidosis and sarcoidosis patients may present with a broad variety of symptoms. This directly translates into a difficulty describing the suspicion of sarcoidosis. In our study, we included a wide range of patients who were diagnosed with sarcoidosis after the diagnosis workup, and not only those presenting with lung complaints. On the one hand, including only those patients presenting only with lung complaints, would have resulted in the inclusion of a more homogenous group. On the other hand, including all patients suspected of sarcoidosis, who eventually were diagnosed with sarcoidosis of any organ, enabled us to include more patients, (and thus gain more statistical power). Importantly, this also better reflects the usefulness of sIL-2R in establishing the diagnosis of sarcoidosis, since not all patients suspected of sarcoidosis will initially present themselves to the clinician with lung complaints.

Sarcoidosis was suspected when a patient was referred to the Immunology department with one or more of these criteria:

1) lung complaints i.e. shortness of breath, persistent dry cough or chest pains

2) uveitis or inflammation of the lacrimal glands

3) skin disorders i.e. erythema nodosum, lupus pernio, infiltration of scars or tattoos

4) neurological complaints i.e. hearing loss, facial nerve palsy or epilepsy

5) polyarthritis

6) incidental finding of lymphadenopathy on imaging

We have added these criteria to the manuscript to clearly describe our study population.

B) As shown in Figure 2, patients with immunosuppressive medication <2 weeks before sIL-2R measurement or ACE inhibitors < 1 month before ACE were excluded. Are there any backgrounds in this setting period? This reviewer feels that all patients who prescribed immunosuppressive medication should be excluded in the study subjects.

Thank you for this astute observation. We agree that patients who were prescribed immunosuppressive medication should be excluded from this study. To assess immunosuppressive medication use, the list of medication at first visit to the department was screened. The referral letter was also screened for use of any immunosuppressive medication. We cannot know whether the patient had any immunosuppressive medication prior to the referral letter. For example, one patient might have had corticosteroids prescribed to them by their general practitioner. If this medication has been stopped just before the referral and the referral letter, we might not be aware of this use. Therefore, we defined the immunosuppressive use as two weeks before the first visit and sIL-2R determination, since this is the minimum waiting time between referral and visit to the immunology department. Within this time frame, we are certain that patients have not used any immunosuppressive medication. However, for most patients, the time between the referral letter and the first visit to our clinic is longer, and thus increases the interval of time of which we are sure that they did not use immunosuppressive medication.

With regards to the use of ACE-inhibitors, we were able to employ a broader strategy. Since this medication is prescribed for long periods of time, and not in short courses as corticosteroids are, we were able to use data not only from the list of medication at first visit and the referral letter, but also from letters from other departments of our Medical Centre, such as the cardiology or ophthalmology department. Therefore we were able to achieve the certainty that patients did not use ACE-inhibitors one month prior to ACE measurement.

C) In Figure 4, to examine sensitivity and specificity of sIL-2R and ACE, which subjects (e.g. included patients n=180, or include patients n=180 + healthy subjects n=101) did the authors use?

Thank you for your comment. In this figure, we examined the sensitivity and specificity of sIL-2R and ACE in included patients (n = 189) that had a suspicion of sarcoidosis in the differential diagnosis. We choose to use this specific population, since this is the population who, in clinical practice, would undergo diagnostic testing for sarcoidosis. We feel that, if we include healthy controls as well, sIL-2R would over-perform in this setting and would not give realistic values that are applicable for the current medical practice. To clarify, we now have added the numbers patient to this Figure.

2. Results Figures 1 and 2

Please consider combine Figures 1 and 2, to easy to understand for readers.

We welcome this helpful comment and we have merged Figure 1 and 2 into one figure.

Minor

3. Figure 3

Please add the numbers of each group in Figure 3.

We have added the group size to make this figure more insightful.

2 Oct 2019

Sensitivity and specificity of serum soluble interleukin-2 receptor for diagnosing sarcoidosis in a population of patients suspected of sarcoidosis

PONE-D-19-20768R1

Dear Dr. Eurelings,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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With kind regards,

Masaki Mogi

Academic Editor

PLOS ONE

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Reviewers' comments:

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Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: N/A

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Authors answered the remarks of the reviewers, and they had improved their report significantly. The revised version of the manuscript is acceptable for publication.

Reviewer #2: This manuscript has now much improved.

I have no further comments.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

9 Oct 2019

PONE-D-19-20768R1

Sensitivity and specificity of serum soluble interleukin-2 receptor for diagnosing sarcoidosis in a population of patients suspected of sarcoidosis

Dear Dr. Eurelings:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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