An overlap of antineutrophil cytoplasmic antibody-associated vasculitis and IgG4-related disease: distinct clinicopathologic clues for precise diagnosis

Background: Both antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) and IgG4-related disease (IgG4-RD) are multi-system inammatory disorders. The coexistent of both diseases present the possibility of a new overlap syndrome which leads to different treatment and outcome. In this study, we aim to investigate the possibility and clinicopathologic clues to the diagnosis of this overlap syndrome. Methods: A case of a 67-year-old man in our hospital who exhibited the clinicopathologic characteristics of both AAV and IgG4-RD was presented. The serial alterations in serum parameters and treatment response of the case were followed up for the next 15 months. Then, a systematic literature review of the overlap syndrome was performed on PUBMED database from 1976 until January 2020. Results: Forty-two patients fully met both AAV and IgG4-RD criteria in the literature. The median age was 60 years ranged from 31 to 73 years at diagnosis. Twenty-eight (66.7%) patients were men. Serum IgG4 concentration increased in 41 (97.6%) patients (median:395 mg/dl; range:177-876mg/dl). Forty-one patients (97.6%) tested positive for ANCA with 37 (90.2%) patients showing a specicity for MPO. Kidney histology of 23 (69.7%) patients presented pauci-immune necrotizing or crescentic glomerulonephritis and IgG4-related tubulointerstitial nephritis. Glucocorticoids combined with cyclophosphamide therapy was commonly prescribed with a high remission rate within 3 months. Four common clinicopathologic features of the overlap syndrome were identied from the case and literature. Conclusion: AAV may overlap with IgG4-RD while presenting atypical manifestations. Four common clinicopathologic characteristics could be used as specic clues to the diagnosis of overlap syndrome.

Both AAV and IgG4-RD are autoimmune-mediated multi-system disorders, suggesting a potential association between these two disease entities. Recently, increased IgG4 + cells had been seen in tissue biopsies of GPA patients [5] and ANCA positive IgG4-RD has been observed. The coexistent of AAV and IgG4-RD with clinical and/or histological evidence of both diseases had been proposed as a new overlap syndrome by a few researches since 2017 [6,7]. However, this suggestion has received a myriad of different opinions since IgG4-RD shares a large spectrum of features with AAV, which can clinically and histopathologically mimic IgG4-RD [8]. Yoo et al argued that elevated serum IgG4 may simply re ect the activity and in ammatory burden of AAV during diagnosis in a retrospective study including 139 patients with MPA and GPA but only 33.1% with results on serum IgG4 [9]. Meanwhile, Erden et al proposed that the existing ndings that these two diseases overlapped was perhaps a synergy in the laboratory rather than an overlap [10]. Therefore, whether the concomitance presented between AAV and IgG4-RD is actually the manifestation of an overlap syndrome remains controversial. As treatment and prognosis are different between both diseases, distinction of the overlap syndrome remains essential. Especially when AAV is missed due to an overlapped diagnosis of IgG4-RD, it will probably cause signi cantly poor outcomes.
Hence, we present a case with coexistence of biopsy-proven AAV manifested as MPO-ANCA-associated crescentic glomerulonephritis and IgG4-related tubulointerstitial nephritis, in which serial alterations in serum IgG4 and MPO-ANCA were traced. Furthermore, we also provide a comprehensive literature review on the common clinicopathologic characteristics of the overlap syndrome, to nd out speci c clues to the diagnosis providing better understanding for the special and rare entity.

Patient and data collection
A 67-year-old man that exhibited the characteristics of both AAV and IgG4-RD was admitted in our hospital on September 7, 2018. The clinical and laboratory data of the patient was collected, and the treatment response and outcomes were followed up for 15 months. A systematic literature review base on biopsy was then performed using PUBMED database covering the time period from 1976 until January 2020. The patients who met both AAV and IgG4-RD criteria as overlap syndrome would be included. AAV was de ned by the 2012 Chapel Hill Consensus Conference nomenclature criteria [1]; while IgG4-RD diagnosis was based on the comprehensive diagnostic criteria [11] and IgG4-TIN was de ned by the criteria set by Mayo Clinic [12]. According to the criteria, the de nite diagnosis of IgG4-RD was de ned by the following 3 criteria: (1) clinical examination showing characteristic diffuse or localized swelling or masses in single or multiple organs; (2) elevated serum IgG4 concentrations (> 135 mg/dL); (3) histopathological examination showing: (I) marked lymphocyte and plasmacyte in ltration and brosis; (II) in ltration of IgG4 + plasma cells: ratio of IgG4+/IgG + plasma cells > 40% and > 10 IgG4 + plasma cells per high-power eld (HPF). Results were restricted to studies in human and articles written in English language. Articles without histopathological information or detail data were excluded. This study was approved by the Ethics Committee of the Sun Yat-sen Memorial Hospital, Sun Yat-sen University (SYSEC-KY-KS-2020-080-001).

Statistical analysis
Statistical analysis was performed with SPSS 19.0 (IBM, Armonk, NY, USA). Continuous variables were expressed as a mean ± standard deviation, and categorical variables were expressed as a number (percentage).

Case presentation
A 67-year-old man was admitted to our hospital complaining of repeated cough, hemoptysis, anorexia and fatigue. He has a signi cant medical history for hypertension and was a heavy smoker. Five months prior to admission, the patient was diagnosed with pneumonia at the local hospital on the basis of cough, hemoptysis and diffuse lesions in both lungs that were observed through the chest computed tomography (CT) scan. At that time, his serum IgG was 16.35 g/L (normal range: 7-16 g/L), while his hemoglobin, serum creatinine and C-reactive protein (CRP) levels were in the normal range. In addition, urinalysis yielded negative results for proteinuria and hematuria. Two months before admission, he experienced ear stu ness due to mixed hearing loss, after which a CT scan revealed bilateral middle ear mastoiditis, bilateral ethmoid sinusitis. The month before admission, he complained about anorexia and fatigue, and developed severe anemia with hemoglobin concentration at only 59 g/L. Rapid deterioration of renal function was observed with serum creatinine level at 717.2 µmol/L. The test for serum anti-MPO antibody showed positive result, while anti-PR3 antibody was weakly positive. The diagnosis of AAV was made and methylprednisolone 40 mg/d was prescribed. Serum creatinine level decreased to 548.1µmol/L 6 days after treatment.
Later on, the patient was admitted to our hospital. Doppler ultrasound examination revealed that the mean length of the left kidney amounted to 90.4mm, and the right kidney 104.4 mm. CT scan showed right maxillary sinusitis and multiple subpleural small nodules (approximately 2 to 3 mm in diameter) could be seen in both lungs (shown in Fig. 1. A, B). The right upper lung had shown in ammation, while bilateral ground-glass opacity was displayed by the lower lung (shown in Fig. 1. C). Serum IgG was 19.7 g/L (normal range:7-16 g/L), IgG4 was 4.07 g/L (normal range: 0.030-2.010 g/L), and the IgG4: IgG ratio was 20.7%. Serum IgE was 406 IU/mL (normal range: 0-100 IU/mL) and CRP was 12.6 mg/L (normal range: < 5.0 mg/L). No hypocomplementemia was apparent, with C3 and C4 levels at 790 and 147 mg/L, respectively. The levels of serum creatinine was 481 µmol/L (normal range: 44-133 µmol/L), urinary red blood cells 6 to 10/high-power eld, urine microalbumin 129.0 mg/L and 24-hour urine protein quantitation at 0.16 to 0.32g. Peripheral white blood cells were 8.97 x10 9 /L with normal eosinophils, and hemoglobin was 89 g/L. A test for serum perinuclear-ANCA had shown positive result, and MPO-ANCA level was 275.27 U/mL (normal range 0-15 U/mL). The PR3-ANCA level was 2.181U/mL (normal range 0-15 U/mL). A renal biopsy was then performed with the following results: immuno uorescence examination showed brinoid necrosis, IgM (+) and C3 (+/-), tubular basement membrane (TBM) deposits showed granular staining for IgG (shown in Fig. 2. A); under light microscopy, fteen glomeruli could be seen in the specimen, among which two were observed with cellular crescents and eight with cell brotic crescents (shown in Fig. 3. A); multifocal in ammatory cell in ltration was evident in the tubulointerstitial area (shown in Fig. 3. B). Immunohistochemical staining showed that IgG4 positive plasma cells were more than 10/HPF, and the ratio of IgG4-positive to IgG-positive plasma cells was more than 40% (shown in Fig. 3. C, D). Ultrastructural examination reveals electron-dense deposits, without substructure, in the TBM (shown in Fig. 2.

B, C)
The patient was nally diagnosed with IgG4-RD based on the IgG4-positive plasma cell-enriched TIN, increased serum IgG4, TBM deposits and pulmonary nodules. The ndings of pauci-immune crescentic GN, ground-glass opacities in the lower lungs, middle ear mastoiditis, paranasal sinusitis and increased MPO-ANCA led to a concurrent diagnosis of systemic ANCA-associated vasculitis in the form of MPA (shown in Table 1). The patient received methylprednisolone pulse therapy of 0.5 g/d for 3 consecutive days followed by 40 mg/d intravenous drip. Later, oral prednisone was tapered to 20 mg/d due to complication of pulmonary in ammation. The patient responded partially to this regime of treatment. His serum creatinine and IgG level decreased to 319 µmol/L and 10.4 g/L respectively; while his serum IgG4 and IgG4: IgG ratio dropped to 1.53 g/L and 14.71% respectively.
Four months later, re-examination through CT showed improvement in bilateral interstitial changes (shown in Fig. 1. D), but ground-glass nodules were still seen in the right middle lobe with multiple subpleural small nodules. However, the bronchial lavage uid examination revealed no evidence of fungus or tuberculosis. Changes in the lungs were thought to be caused by IgG4-RD, but biopsy could not be performed due to the location of upper lobe and the high risk it bears. After 6 months of treatment, the level of serum IgG4 was 0.323 g/L, and serum creatinine was maintained at 230 µmol/L (shown in Fig. 4). Five months ago, the patient received intravenous cyclophosphamide at 0.2-0.4 g every two weeks in local hospital. His latest serum creatinine level was at 187.2 µmol/L. Regular follow-up is still ongoing.

Literature review
Clinical profile Through the literature review (from 1976 to 2020), 52 patients who met IgG4-RD and AAV criteria based on histopathological investigation were identi ed, which were de ned as having overlap syndrome according to the previous researches [6,7]. 10 patients without detailed data were excluded [6] and 42 patients were included. Among them, 34 cases showed concurrent IgG4-RKD and AAV, and 29 cases (85.3%) were IgG4-RKD concomitant AAV associated glomerulonephritis (ANCA-GN) with mild to moderate renal impairment (shown in Table 2-3).
Among the 42 patients ranged in age from 31 to 73 years old with a median age of 60 at diagnosis, 14 (33.3%) patients were women, and 28 (66.7%) patients were men. Serological tests of CRP were showed in 20 patients. 16 of them (80.0%) has an elevated level of CRP ranging from 2.11 to 17.9 mg/dl. Serum IgG4 concentration increased in 41 (97.6%) patients (median:395 mg/dl; range:177-876mg/dl). Forty-one patients (97.6%) tested positive for ANCA with 37 (90.2%) patients showing a speci city for MPO and 4 (9.8%) patients with a speci city for PR3. The identi cation of IgG subclasses of MPO-ANCA was performed in three studies involving 17 patients [15][16][17] and all patients were positive for IgG4-MPO-ANCA in overlap syndrome. The main organs involved in both AAV and IgG4-RD are kidney and lung. The ear, nose and throat (ENT) and peripheral nervous system (PNS) involvement were prevalent in AAV (21.4%, 19.0%, respectively), while pancreas involvement appeared mainly in IgG4-RD cases (9.5%) (shown in Table 2-3).
In the 34 patients with concurrent IgG4-RKD and AAV, the average of serum creatinine level was 302.7±193.5 µmol/L. In 15 patients with recorded proteinuria level, 14 exhibited ~2 g/day proteinurea level, with no nephrotic syndrome. Microhematuria was either absent or low in most patients (0-15 erythrocytes/high power field in light microscopy of urine sediments) ( Table 3).

Pathological profile
A total of 48 biopsies were performed in 42 patients. The biopsy site with the highest frequency was the kidneys (75.0%), followed by the lungs (8.3%). Evidence of necrotizing vasculitis lesions, leukocytoclastic vasculitis lesions and necrotizing granulomatous in ammation was respectively observed in 3 patients, while necrotizing glomerulonephritis in 9 patients, and crescentic glomerulonephritis in 12 patients. Lymphoplasmacytic in ltrate was noted in all IgG4-RD patients, with an IgG4/IgG ratio of > 40% in 8 (19.0%) patients and the number of IgG4-positive plasma cells >10 per HPF was observed in 33 (78.6%) patients. Storiform brosis was present in 23 (54.8%) patients (shown in Table 2-4).

Treatment and outcome
From the data, 42 patients received immunosuppressive therapy, among which 7 (16.7%) patients were treated with oral glucocorticoids alone, 30 (71.4%) patients with glucocorticoids in combination with cyclophosphamide, 3 (7.1%) patients with glucocorticoids in combination with rituximab, and t 2 (4.8%) patients w with oral glucocorticoids in combination with azathioprine. Additionally, 22 (52.4%) patients received intravenous methylprednisolone pulse therapy and 5 (11.9%) patients received additional plasmapheresis. 3 (7.1%) patients experienced relapse of AAV or treatment resistance during treatment with glucocorticoids in combination with cyclophosphamide or azathioprine, and were then being treated with rituximab and achieved remission thereafter. During follow-up, 36 (85.7%) out of 42 patients achieved remission within 3 months, while relapse occurred in 2 (4.8%) cases. Treatment resistance occurred in 6 (14.3%) patients, with 5 of them proceeded to routine hemodialysis. No fatality was recorded. In regards to IgG4-RKD, 30 (88.2%) out of 34 patients achieved renal improvement with lower serum creatinine levels as serum IgG4 concentration decreased (shown in Table 2-3).

Discussion
In this study, we describe a patient with biopsy-proven pauci-immune crescentic GN due to MPO-ANCA positive AAV can also have IgG4-RKD based on histopathology, which is de ned as overlap syndrome according to the criteria for both AAV and IgG4-RD diseases. It's worth noting that serum IgG4 elevation and IgG4-positive cell in ltration in the tissue could be seen in some cases of crescentic glomerulonephritis of AAV highlighted by Kawashima et al [7]. This is consistent with a view of that serum IgG4 might not be associated with concurrent IgG4-RD but showed activity and in ammatory burden of MPA and GPA proposed by Yoo et al [9]. It should be pointed out that no patients ful lled comprehensive diagnostic criteria for IgG4-RD requiring IgG4+/IgG + cells > 40% or IgG4 + cells/HPF > 10 on the tissues in their AAV research [9]. The absence of TBM immune complex deposits by immuno uorescence will help underline the AAV when compared with IgG4-RKD [29]. Despite these considerations, our case and others shown in the literature review found some patients could meet the Chapel Hill Consensus Conference nomenclature criteria for AAV and comprehensive diagnostic criteria for IgG4-RD or Mayo Clinic proposed criteria for IgG4-TIN, which were classed as overlap syndrome.
Although AAV and IgG4-RD can be distinguished based on characteristic ndings in many cases, the diagnosis can be unclear in some cases, in which the possible coexistence of AAV and IgG4-RD should be considered. From the comprehensive literature review on biopsy-proven co-existing AAV and IgG4-RD, we identify four common clinicopathologic features of the overlap of AAV and IgG4-RD.
First of all, atypical clinical and laboratory manifestations were characteristics of this entity. As shown in Table 1 obtained from our case, atypical organ involvement, such as ENT and PNS in IgG4-RD, and lacrimal or salivary gland and pancreas in AAV, would often indicate the other commitment disease. On the other hand, our case and 14 out of 15 patients in the literature review exhibited negligible to moderate proteinurea level (0 to 2 g/day), with absent or low microhematuria in AAV. It suggested that unusual manifestations such as absent or mild urine sediments in AAV associated crescentic GN, or granulomatous in ammation in IgG4-RD, would also usually point to the coexistence of another disease. As for in ammation marker, elevated serum CRP was identi ed in the case study and 80% of the patients from the literature review demonstrated a tendency of slightly higher CRP level as compared to typical IgG4-RD cases, which is consistent with a previous study [2]. Atypical clinical and laboratory manifestations are often the rst clue for clinicians when performing further workup.
Secondly, positive MPO-ANCA are more common in the overlap syndrome. Our literature review found that most patients had positive ANCA, and up to 89.8% of patients showed a speci city for MPO, while 9.8% patients with a speci city for PR3, which was consistent with our case and previous research [15,16]. The IgG isotypes are predominant in ANCA, particularly represented by IgG1, IgG3 and IgG4 subclasses [30]. IgG4-ANCA was assumed to contribute to both the correlation between serum IgG4 and the in ammatory markers, and between serum IgG4 and the activity of MPA and GPA [9]. It was hypothesized that IgG4-MPO-ANCA produced by in ltrating IgG4-positive plasma cells might be responsible for the necrotizing crescentic GN [17].
Thirdly, histological patterns could overlap in the same tissue samples when kidneys are involved. Biopsy is important for the diagnosis of the overlap syndrome. AAV histological ndings is characteristic of vasculitis and/or granulomatous in ammation, along with brosis and/or lymphoplasmacytic in ltration up to a certain level [1]. While the key features of IgG4-RD histology include the presence of dense lymphoplasmacytic in ltrate, storiform brosis and obliterative phlebitis [4]. In our case, pauci-immune crescentic GN and IgG4-positive plasma cell-enriched TIN pointed the characteristics of the histological patterns of both AAV and IgG4-RD on the biopsy specimen from the kidney. Consistent with the literature, we found that TIN is the most common renal pathological manifestation of IgG4-RKD in the overlap syndrome. Meanwhile, pauci-immune necrotizing and crescentic glomerulonephritis were the main pathological manifestation of AAV.
Finally, treatment often requires the combination of glucocorticoids and immunosuppressive therapy targeting both conditions. Glucocorticoids are currently the rst-line treatment for IgG4-RD [4]. Glucocorticoids alone was prescribed in our patient due to Infectious complications for 6 months. Although the renal function was improved quickly in 1 month as the levels of MPO-ANCA and IgG4 decreased, it was only a partial improvemet. In the literature review, most of the patients required glucocorticoids combined with cyclophosphamide, azathioprine or rituximab targeting AAV. Fortunately, most of them resulted in a remission within 3 months, which is similar to the treatment response for IgG4-RD. Furthermore, 88.2% patients with IgG4-RKD could improve renal function with lower serum creatinine levels as serum IgG4 concentration decreased.
It is acknowledged that there are some limitations to this work. There was a lack of histological evaluation of the lung lesion in the case study due to the high risk of biopsy. Besides, the limited availability of detailed data in some literature might also lead to biased perceptions. Future follow-up on imaging changes of lung in the patient should be continued.

Conclusion
In conclusion, through a case study and the literature review, four common clinicopathologic characteristics will help to alert the clinicians to look for the overlap of both conditions. This study suggests that AAV may overlap with IgG4-RD while presenting atypical manifestations. A common etiology and pathogenesis for both IgG4-RD and AAV should be further explored.

Declarations Ethics approval and consent to participate
This study was approved by the Ethics Committee of the Sun Yat-sen Memorial Hospital, Sun Yat-sen University (SYSEC-KY-KS-2020-080-001).

Consent for publication
Written consent was obtained from the patient.

Availability of data and materials
All data generated or analysed during this study are included in this published article.

Competing interests
The authors declare no competing interest.  Tables   Table 1. Comparison of features of AAV vs. IgG4-RD vs. the case.