**3.3. Laboratory tests and biomarkers**

Since the spectrum of ILD associated with CTD is broad, careful evaluation for autoantibodies or other serologic tests in conjunction with clinical features of autoimmune disease is crucial [19, 20]. Because of the variable incidence and outcome of ILD in CTD, biomarkers including autoantibodies are critical for diagnosis, prognosis, patient subtyping, and predicting response to treatment. Major autoantibodies and serologic tests commonly available for the evaluation of CTD-ILDs include antinuclear antibody (ANA), anti-double-stranded DNA, anti-ribonucleoprotein (anti-RNP) antibody, anti-Smith (anti-Sm) antibody, anti-scleroderma-70 (Scl-70) antibody, anti-Ro (SS A), anti-La (SS B), anti-Jo-1 antibody, rheumatoid factor (RF), and anti-cyclic citrullinated peptide antibody (ACPA) [5].

positive ANA but does not fulfill any rheumatology classification criteria for specific CTDs [30, 31]. Mosca et al. reported that 60% of patients with UCTD remain undifferentiated. When evolution to defined CTD occurs, it usually does within the first 5 years of disease. UCTD may develop into any of the CTDs, most often into SLE [29]. There are a large number of patients, in whom the IP appears to be the lone part for the clinically predominant manifestation of an occult CTD with subtle clinical features that suggest an autoimmune process but not meet established criteria for CTD, raising a controversy over the strategies for identification and classification of these patients. Well-organized prospective studies have been needed to better understand this entity of the lung disease and distinguish it from the ILD with well-defined CTD or IIP. Proposed terminology to classify such patients includes "undifferentiated CTD" [31], "lung-dominant CTD" [15], and "autoimmune-featured ILD" [32]. Recently, the "ATS/ ERS Task Force on Undifferentiated Forms of CTD-associated ILD" created consensus regarding the nomenclature and classification criteria for patients with IIP and features of autoimmunity and proposed the term "interstitial pneumonia with autoimmune features" (IPAF). The classification criteria require evidence of IP and are organized around three central domains: a clinical domain consisting of specific extra-thoracic features, a serologic domain consisting of specific autoantibodies, and a morphologic domain consisting of specific chest imaging, histopathologic, or pulmonary physiologic features [33]. Currently, it is not yet clear whether IPAF is a distinct phenotype of ILD or simply a part of IIP. Adopting IPAF classification may provide platform for the future study of a more uniform cohort, and prospective survey will be needed in determining efficacy of therapy and outcomes for the patients [34].

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**4. Pulmonary histopathology in connective tissue disease**

tively better prognosis than UIP.

The underlying pathology in CTD-associated ILD can be dominated by inflammation or fibrosis or by a combination of both with distinct radiologic and histopathologic patterns [12]. Classification of histological and radiological patterns developed for IIPs is applied to CTD-ILD [1, 5, 11]. The radiological and corresponding histological patterns defining each entity of CTD-associated IP are summarized in **Table 2** [11]. Although there is substantial histological overlap among the pulmonary manifestations of different CTDs and with other etiologies, certain histologic patterns may favor one CTD over another, and occasionally distinctive histologic clues may be present [35, 36]. It is possible in many cases to confirm CTD-ILD and guide patient management using histologic features. Pulmonary histopathology is thus helpful, and surgical lung biopsy remains the gold standard for evaluation of CTD-associated ILD [35]. ILD can present acutely or chronically, with acute presentations being more common in SLE and PM/DM. Histological patterns of CTD-associated IP include, most frequently, nonspecific interstitial pneumonia (NSIP), usual interstitial pneumonia (UIP), organizing pneumonia (OP), diffuse alveolar damage (DAD), and lymphocytic interstitial pneumonia (LIP). By contrast, desquamative interstitial pneumonia (DIP) and respiratory bronchiolitisassociated interstitial pneumonia (RB-ILD) are uncommon forms of IP in CTD. Both, typically affecting cigarette smokers, share overlapping clinicopathological features and have a rela-

Some of the established biomarkers include lung epithelium-specific proteins [20]. Evidence indicates that repetitive injuries to alveolar epithelial cells (AEC) and airway Club cells trigger an exaggerated wound healing response. During the process, while AEC type I cells undergo apoptosis, regenerated hyperplastic AEC type II cells produce a vast array of cytokines, growth factors, and release surfactant proteins and mucins [21]. Surfactant proteins (SP-A and SP-D) and KL-6 in the serum are useful biomarkers, which have been well established for various ILDs. SP-D and SP-A, secreted by AEC II and airway Club cells, are surfactant lipoproteins and phospholipids which stabilize alveolar surface tension, playing an important role in the lung host defense system [22]. SP-D serum levels are more sensitive than SP-A in detecting ILD as defined by CT but less specific [23]. KL-6 is a high-molecular-weight mucinlike glycoprotein, now classified as MUC1, which is highly expressed by AEC II and bronchiolar epithelial cells and increases following cellular injury and/or regeneration [24]. KL-6 has profibrotic and antiapoptotic effects on lung fibroblasts [25]. Serum KL-6 has been shown to be elevated not only in IIP but also in CTD-ILD, as well as hypersensitivity pneumonitis, drug-induced pneumonitis, etc. [24, 26].

There are a number of principal autoantibodies which have been validated for the clinical use. Antinuclear antibody (ANA) determined by an immunofluorescence assay is most versatile, presenting with several major patterns; mainly homogeneous (associated with ANAs against double strand (ds) DNA in SLE and histones), speckled/peripheral (less specific), and nuclear (most often associated with limited scleroderma). ANA titer higher than 1–160 is regarded as significant in most laboratories [27]. When using enzyme immunoassay (EIA) and enzymelinked immunosorbent assay (ELISA) for ANAs, we can detect single autoantigens such as dsDNA, Smith antigen, scleroderma (Scl-70) (also termed topoisomerase-1), SSA/Ro, SSB/La, etc. [27]. Some of the recent, newly developed autoantibodies with distinct clinical and immunological characteristics will be explained later.
