ORIGINAL STUDY
Adipophilin Expression in Necrobiosis Lipoidica, Granuloma Annulare, and Sarcoidosis Joshua M. Schulman, MD and Philip E. LeBoit, MD
Abstract: Necrobiosis lipoidica (NL), granuloma annulare (GA), and sarcoidosis usually are distinguished by clinical presentation and routine microscopy, but their distinction can sometimes be challenging. Historically, a clue to diagnosing NL or GA has been the identification of lipid droplets in the areas of altered collagen, but such studies have required fresh frozen tissue, making them impractical. Here, we present the first report of immunohistochemical staining to detect adipophilin, a membrane protein in lipid droplets, in NL (n = 12), GA (n = 19), sarcoidosis (n = 12), and, as a control for nonspecific tissue damage, nongranulomatous cutaneous necrosis (n = 13). Four patterns of labeling were identified: (1) extracellular, within zones of altered collagen; (2) both intracellular and extracellular, after the distribution of palisaded or scattered histiocytes; (3) intracellular, within clustered histiocytes; and (4) periadnexal. All cases of NL demonstrated pattern 1; nearly all cases of GA (18/19) demonstrated pattern 2; most sarcoidosis (10/12) demonstrated pattern 3; and nongranulomatous necrosis demonstrated either pattern 4 (6/13) or did not stain (6/13), confirming that the antibody to adipophilin did not adhere nonspecifically to the damaged tissue. An additional set of 3 biopsies with overlapping or partially sampled features of NL, GA, and/or sarcoidosis subsequently confirmed the potential utility of adipophilin staining in diagnostically challenging cases. We conclude that the pattern of adipophilin expression is a useful adjunct in the evaluation of granulomatous dermatitis. Key Words: necrobiosis lipoidica, granuloma annulare, sarcoidosis, adipophilin, granuloma, granulomatous dermatitis (Am J Dermatopathol 2015;37:203–209)
INTRODUCTION Necrobiosis lipoidica (NL), granuloma annulare (GA), and sarcoidosis are granulomatous conditions of unclear etiologies, which typically can be distinguished based on their clinical and histopathological features. NL usually presents on the anterior lower legs of adults as shiny atrophic plaques, characterized histopathologically by histiocytic infiltrates arrayed around zones of degenerated collagen in the mid-to-deep dermis. GA may present in a variety of forms, but in its localized variant, it most classically appears as one or more erythematous annular plaques, histopathologically From the Departments of Pathology and Dermatology, University of California, San Francisco, CA. The authors declare no conflicts of interest. Reprints: Philip E. LeBoit, MD, University of California, 1701 Divisadero St, Suite 280, San Francisco, CA 94115 (e-mail: [email protected]). Copyright © 2014 Wolters Kluwer Health, Inc. All rights reserved.
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demonstrating histiocytic infiltrates palisaded around hypocellular zones containing altered collagen and increased dermal mucin. Cutaneous sarcoidosis has widely variable clinical appearances but is defined histopathologically by clusters of epithelioid histiocytes lacking significant necrosis or surrounding lymphocytic infiltrates. In many cases, however, these 3 conditions may demonstrate overlapping clinical and histopathological features, presenting a diagnostic challenge.1,2 Diagnosis may further be stymied by partial biopsies that reveal only focal granulomatous inflammation or subtle alterations to the connective tissue. Historically, a clue to the diagnosis of NL or GA was the identification of small lipid droplets within an altered dermis.3–8 Staining these lipid droplets required the use of fresh frozen tissue, and this limitation led to the eventual abandonment of such studies. Immunohistochemical staining for adipophilin has been adopted as a method for labeling lipid droplets in formalin-fixed paraffin-embedded tissue.9 The purpose of our study was to evaluate whether adipophilin staining could identify lipid droplets in NL and GA. Additionally, we evaluated adipophilin expression in cutaneous sarcoidosis, a potential histopathologic mimicker of NL and GA. Lastly, we sought to determine whether the pattern of adipophilin expression could assist in distinguishing between these 3 relatively common granulomatous dermatitis.
MATERIALS AND METHODS Formalin-fixed paraffin-embedded tissue samples were retrieved from the archives of the Dermatopathology Service of the University of California, San Francisco. The initial study group included 12 specimens of NL, 19 of GA (13 with a predominantly palisaded pattern and 6 with an interstitial pattern), and 12 of sarcoidosis diagnosed between 2004 and 2013. Three of the cases of sarcoidosis had a history of systemic disease at the time of biopsy. A control group of 13 cases of nongranulomatous cutaneous necrosis was also identified. A secondary study group of diagnostically challenging cases was identified from our archives, based on a search of cases coded in our archival database as having overlapping features of NL, GA, and/or sarcoidosis. This secondary study group included 3 cases. One case, biopsied from a solitary plaque on the lower leg, was diagnosed as “palisaded granulomatous dermatitis, consistent with necrobiosis lipoidica,” although GA was raised as a diagnostic consideration. The second case was initially diagnosed as NL at the time of biopsy but was reinterpreted as sarcoidosis after subsequent biopsies from the patient revealed classic sarcoidal www.amjdermatopathology.com |
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granulomas. The third case was a superficial shave biopsy from the thigh diagnosed as NL. Hematoxylin and eosin–stained sections were reviewed to confirm the diagnoses. Immunohistochemical staining was performed with a predilute polyclonal antibody to adipophilin (Cell Marque, Rocklin, CA) using a Dako Autostainer platform (Carpinteria, CA). Optimal conditions for detecting adipophilin included a 1-hour pretreatment in Dako pH9 target retrieval solution and a 30-minute antibody incubation time. Positive adipophilin staining was defined by the presence of discrete vacuoles with moderate-to-strong labeling intensity, whereas weak diffuse staining and absent staining were both interpreted as a negative result. To make sure that such staining was not nonspecific adhesion of the antibody to the areas of necrobiosis or necrosis, we compared the staining pattern in the study cases with the pattern observed in the control cases in which there was epidermal or dermal necrosis.
RESULTS Adipophilin expression was identified in our initial study set in all cases of NL, in 18 cases (95%) of GA, and in 10 (83%) of sarcoidosis. Of the 13 control cases of nongranulomatous necrosis, 7 (54%) demonstrated some degree of adipophilin labeling, but the pattern of adipophilin expression tended to differ between the various granulomatous conditions and the control cases (Figs. 1–5). In total, 4 patterns of adipophilin expression were identified: (1) primarily extracellular, centered within zones of altered collagen; (2) both intracellular and extracellular, after the distribution of palisaded or scattered histiocytes; (3) intracellular, within clustered histiocytes; and (4) deposited in the periadnexal dermis. All cases of NL demonstrated pattern
1, whereas 6 cases (50%) of NL concomitantly demonstrated pattern 3 as a minor component. Eighteen cases (95%) of GA demonstrated pattern 2, whereas 4 (21%) concomitantly demonstrated pattern 1. The cases of GA that demonstrated pattern 2 included 6 cases with palisaded granulomas in the middermis, 5 with an exclusively interstitial pattern, 2 with features of actinic GA, and 1 with granulomas extending into the subcutis. One case of interstitial GA did not label with adipophilin. Ten cases (83%) of sarcoidosis demonstrated pattern 3, whereas the remaining cases did not demonstrate adipophilin labeling. The cases of sarcoidosis that labeled with adipophilin included 7 with granulomas limited to the dermis and 3 with granulomas extending into the subcutis. Of the control cases, 6 (46%) demonstrated pattern 4, 6 did not stain with adipophilin, and 1 (8%) demonstrated pattern 2. These results are summarized in Table 1. After identifying the patterns of adipophilin expression seen in the initial study set, the secondary set of diagnostically challenging cases was then evaluated. The case diagnosed as “palisaded granulomatous dermatitis, consistent with necrobiosis lipoidica” revealed an adipophilin-labeling pattern that was best classified as pattern 1, supporting the diagnosis of NL (Fig. 6). The case initially diagnosed as NL but later reclassified as sarcoidosis demonstrated pattern 3, supporting the diagnosis of sarcoidosis (Fig. 7). The superficial shave biopsy diagnosed as NL demonstrated pattern 1, which again supported the diagnosis of NL (Fig. 8).
DISCUSSION The deposition of lipid droplets has long been known to occur in NL and GA, and its identification has historically been used as a clue to the diagnosis of these conditions.
FIGURE 1. NL. A, A medium-power view demonstrates a zone of degenerated collagen flanked by granulomatous infiltrates (hematoxylin and eosin, original magnification ·100). B, Adipophilin immunostaining is positive within the zone of altered collagen (original magnification ·100). C, A higher-power view demonstrates labeling with most lipid droplets deposited extracellularly (adipophilin, original magnification ·600). D, A magnified view confirms vacuolar adipophilin labeling.
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Adipophilin Expression in NL, GA, and Sarcoidosis
FIGURE 2. Palisaded GA. A, At scanning magnification, a dense histiocytic infiltrate fills the upper dermis in a vaguely palisaded fashion (hematoxylin and eosin, original magnification ·40). B, The pattern of adipophilin immunostaining is distinctly palisaded. The intensity of staining is greater in this example than in several other cases of GA studied, but the pattern is common to most of our cases of palisaded GA (original magnification ·40). C, A higher-power view demonstrates both intracellular and extracellular deposition of lipid droplets (adipophilin, original magnification ·600). D, A magnified view confirms vacuolar adipophilin labeling.
Indeed, the importance of lipid in NL goes back to its first description in 1929, when Oppenheim named the condition “dermatitis atrophicans lipoides”; the first use of “necrobiosis lipoidica diabeticorum” by Urbach followed 3 years later.3 An early report of NL by Nicholas4 provides a more precise description of the lipid deposition, stating that “within the collagenous bundles that had undergone necrobiosis. numerous excessively small fat globules were uniformly distributed.” By the late 1940s, this observation had been applied
more commonly, with Laymon and Fisher5 claiming that the “most striking special feature of NL diabeticorum was the presence of varying amounts of lipids in the areas of necrobiosis. deposited both between and within the fibers.” A decade later, Wood and Beerman6 noted that the “presence of lipid deposits in the lesions of necrobiosis has been assigned a high degree of importance in identifying this process,” suggesting the widespread diagnostic utility of this finding. The characterization of lipid deposits in GA followed thereafter,
FIGURE 3. Interstitial GA. A, Histiocytes intercalate between collagen bundles, and a subtle increase in dermal mucin is also apparent (hematoxylin and eosin, original magnification ·200). B, There is focal adipophilin immunopositivity, which follows an interstitial distribution (original magnification ·200). C, A higher-power view demonstrates both intracellular and extracellular deposition of lipid droplets (adipophilin, original magnification ·600). D, A magnified view confirms vacuolar adipophilin labeling. Copyright 2014 Wolters Kluwer Health, Inc. All rights reserved.
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FIGURE 4. Sarcoidosis. A, Aggregates of epithelioid histiocytes reside in the dermis (hematoxylin and eosin, original magnification ·200). B, Adipophilin immunostaining is focally positive within the clustered histiocytes (original magnification ·200). C, A higher-power view demonstrates intracytoplasmic labeling (adipophilin, original magnification ·600). D, A magnified view confirms vacuolar adipophilin labeling.
with Dabski and Winkelmann8 reporting staining with Oil Red O in 15 of 33 cases of GA, noting that “staining was localized in fine scattered droplets that. was not seen outside the cellular aggregates of a granuloma.” More recently, however, studies to identify lipid droplets in NL and GA have fallen out of clinical practice, primarily because of the
challenge of directly staining lipid, which is dissolved during routine tissue processing. Traditional lipid markers, such as Oil Red O or Sudan IV therefore require fresh tissue and frozen sections. Lipid is also preserved with the fixatives used in processing with electron microscopy, but that technique is time consuming, expensive, and is increasingly less available.
FIGURE 5. Nongranulomatous necrosis. A, Degenerated collagen and elastic fibers surround a distorted follicular infundibulum (hematoxylin and eosin, original magnification ·200). B, Sparse labeling with adipophilin is present near the distorted follicle (original magnification ·200). C, A higher-power view demonstrates scant labeling amidst necrotic debris (adipophilin, original magnification ·600). D, A magnified view suggests both granular and vacuolar adipophilin labeling.
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Adipophilin Expression in NL, GA, and Sarcoidosis
TABLE 1. Patterns of Adipophilin Expression
NL (n = 12) GA (n = 19) Sarcoidosis (n = 12) Nongranulomatous necrosis (n = 13)
Mostly Extracellular, Within Altered Collagen, n (%)
Both Intracellular and Extracellular, Accompanying Histiocytic Infiltrates, n (%)
Mostly Intracellular, Within Clustered Histiocytes, n (%)
Periadnexal, n (%)
Negative, n (%)
12 (100) 4 (21) — —
— 18 (95) — 1 (8)
6 (50) — 10 (83) —
— — — 6 (46)
— 1 (5) 2 (17) 6 (46)
Staining for adipophilin provides an alternative method for identifying lipid deposits. Adipophilin is a protein expressed on the surface of lipid droplets, and it is thus preserved during tissue processing.10 Consequently, it is a suitable target for immunohistochemical staining. Its primary use in dermatopathology currently lies in diagnosing neoplasms of sebaceous lineage.9 Its expression has not previously been documented in granulomatous disorders. We found that adipophilin is expressed in NL, GA, and sarcoidosis, and the pattern of expression differs among these 3 conditions. In all cases of NL, the predominant pattern of adipophilin expression was that of diffuse deposition centered within the zones of altered collagen (the zones of “necrobiosis”). On high magnification, the staining was confirmed to highlight the periphery of tiny vacuoles, but fine, extracellular granular staining was also observed. In half of the cases examined, adipophilin also labeled small vacuoles within clustered histiocytes. This latter observation raises the possibility that the pathogenesis of NL may involve the deposition of intracellular lipid droplets onto altered collagen bundles; alternatively, extracellular lipid may be phagocytized by histiocytes.
Nearly all of our cases of GA also labeled for adipophilin, and the pattern corresponded to the distribution of the lesional histiocytes. That is, cases of palisaded GA tended to show a palisaded pattern of adipophilin staining, and cases of interstitial GA tended to show an interstitial pattern of staining. High magnification confirmed that the staining was both finely vacuolar and granular, with both intracellular and extracellular staining observed. In general, cases of interstitial GA demonstrated more focal adipophilin expression than cases of palisaded GA. In sarcoidosis, adipophilin labeled intracytoplasmic vacuoles within clustered histiocytes in the majority of cases, although in many cases only a small number of histiocytic clusters demonstrated staining. Despite the focality of staining, the pattern of adipophilin expression was clearly distinct from the patterns seen in NL or GA, most notably for the near-complete absence of extracellular labeling. Cases of nongranulomatous cutaneous necrosis were studied as controls to verify that adipophilin is not expressed in a nonspecific manner in the setting of altered or degenerated collagen. In roughly half of those cases, adipophilin was
FIGURE 6. A diagnostically challenging example of NL. A, At scanning magnification, there is a zone of granulomatous inflammation in the superficial dermis with minimal involvement of the deep reticular dermis (hematoxylin and eosin, original magnification ·40). B, Clusters of histiocytes are arranged in a roughly palisaded pattern around a paucicellular area with collagen degeneration (hematoxylin and eosin, original magnification ·200). C, Adipophilin labeling localizes to the area of collagen degeneration and not to the histiocytic infiltrate, supporting a diagnosis of NL (original magnification ·200). Copyright 2014 Wolters Kluwer Health, Inc. All rights reserved.
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FIGURE 7. A diagnostically challenging example of sarcoidosis. A, Scanning magnification reveals collections of histiocytes in a rather sclerotic dermis (hematoxylin and eosin, original magnification ·40). B, A cluster of histiocytes is seen at medium power (hematoxylin and eosin, original magnification ·200). C, Adipophilin staining is present within the clustered histiocytes (box) without extracellular labeling, supporting a diagnosis of sarcoidosis (original magnification ·200).
focally expressed near distorted or necrotic hair follicles, which presumably represents labeling of sebocyte remnants. One case displayed a broad arc of adipophilin expression that approximated a palisaded pattern, but it lacked the circumferential labeling that was typically observed in the cases of palisaded GA. The remaining cases did not demonstrate adipophilin expression. Some of these specimens showed fibrin deposition, and although some immunostains can nonspecifically adhere to fibrin, we did not observe this. The 3 diagnostically challenging cases were included in our study to demonstrate a possible role for adipophilin staining in evaluating biopsies that are not easily diagnosed on routine sections alone. In the first example (Fig. 6), the
clinical history of a solitary plaque on the lower leg suggested NL, but the superficial nature of the granulomatous infiltrate raised the histopathologic possibility of GA. Adipophilin staining localized to the areas of degenerated collagen rather than to the zones populated densely by histiocytes, and this distribution conforms to pattern 1, as seen in all of our initial cases of NL. In the second example (Fig. 7), conventional microscopy at first suggested the sclerotic phase of NL, until subsequent biopsies from the patient revealed classic findings of sarcoidosis, at which time the original diagnosis was reevaluated. This case demonstrated adipophilin expression within clustered histiocytes and lacked the diffuse extracellular staining pattern observed in all of our cases of NL, which
FIGURE 8. Superficial shave biopsy of NL. A, Only a portion of the dermis can be evaluated (hematoxylin and eosin, original magnification ·40). B, There is a vague palisade of histiocytes around a paucicellular area with collagen degeneration (hematoxylin and eosin, original magnification ·200). C, Adipophilin labeling is mostly present in the area of collagen degeneration and not within the histiocytic infiltrate, supporting a diagnosis of NL (original magnification ·200).
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supports the correct diagnosis of sarcoidosis. Examples of sarcoidosis mimicking the clinical and histopathological features of NL have previously been reported, and this current example reinforces the importance of identifying additional clues that may distinguish these diagnoses.1,2 Lastly, the example of a partial biopsy of NL (Fig. 8) again showed extracellular adipophilin labeling in the areas of altered collagen, which supports the diagnosis of NL and illustrates the potential utility of adipophilin staining in specimens where architectural features cannot be fully evaluated. The etiologies of NL, GA, and sarcoidosis remain unknown, but metabolic associations have been identified. NL has been linked to diabetes mellitus in a subset of patients, and individuals with GA have been found to be at an increased risk of dyslipidemia compared with the general population.11,12 Some cases of sarcoidosis have also been associated with hypertriglyceridemia and low levels of high-density lipoprotein cholesterol.13 It is therefore plausible that localized defects in lipid metabolism might contribute to the cutaneous findings in these conditions. Such defects could explain the presence of anomalous lipid droplets in lesional tissue, as identified with adipophilin staining. In summary, the presence of adipophilin expression and the pattern of staining provide diagnostic clues in the evaluation of NL, GA, and sarcoidosis. Adipophilin staining may be particularly useful when overlapping features of these conditions are observed in a single biopsy, or when there is discordance between the clinical and histopathological features. Additionally, the observation of adipophilin expression in NL, GA, and sarcoidosis may shed light on the underlying mechanisms of these granulomatous disorders. Indeed, far from being an historical curiosity, the identification of lipid
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Adipophilin Expression in NL, GA, and Sarcoidosis
droplets in granulomatous dermatitis continues to be relevant to modern dermatopathology. REFERENCES 1. Ball NJ, Kho GT, Martinka M. The histologic spectrum of cutaneous sarcoidosis: a study of twenty-eight cases. J Cutan Pathol. 2004;31:160– 168. 2. Chiba T, Takahara M, Nakahara T, et al. Cutaneous sarcoidosis clinically mimicking necrobiosis lipoidica in a patient with systemic sarcoidosis. Ann Dermatol. 2012;24:74–76. 3. Michelson HE, Laymon CW. Necrobiosis lipoidica diabeticorum (Urbach): dermatitis atrophicans lipoides diabetica (Oppenheim). J Am Med Assoc. 1934;103:163–169. 4. Nicholas L. Necrobiosis lipoidica diabeticorum with xanthoma cells. Arch Derm Syphilol. 1943;48:606–611. 5. Laymon CW, Fisher I. Necrobiosis lipoidica (diabeticorum?). Arch Derm Syphilol. 1949;59:150–167. 6. Wood MG, Beerman H. Necrobiosis lipoidica, granuloma annulare, and rheumatoid nodule. J Invest Dermatol. 1960;34:139–147. 7. Gibson LE, Reizner GT, Winkelmann RK. Necrobiosis lipoidica diabeticorum with cholesterol clefts in the differential diagnosis of necrobiotic xanthogranuloma. J Cutan Pathol. 1988;15:18–21. 8. Dabski K, Winkelmann RK. Generalized granuloma annulare: histopathology and immunopathology. J Am Acad Dermatol. 1989;20:28–39. 9. Muthusamy K, Halbert G, Roberts F. Immunohistochemical staining for adipophilin, perilipin and TIP47. J Clin Pathol. 2006;59:1166–1170. 10. Heid HW, Moll R, Schwetlick I, et al. Adipophilin is a specific marker of lipid accumulation in diverse cell types and disease. Cell Tissue Res. 1998;294:309–321. 11. Reid SD, Ladizinski B, Lee K, et al. Update on necrobiosis lipoidica: a review of etiology, diagnosis, and treatment options. J Am Acad Dermatol. 2013;69:783–791. 12. Wu W, Robinson-Bostom L, Kokkotou E, et al. Dyslipidemia in granuloma annulare. Arch Dermatol. 2012;148:1131–1136. 13. Ivanisevic J, Kotur-Stevuljevic J, Stefanovic A, et al. Dyslipidemia and oxidative stress in sarcoidosis patients. Clin Biochem. 2012;45: 677–682.
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Adipophilin Expression in Necrobiosis Lipoidica, Granuloma Annulare, and Sarcoidosis Joshua M. Schulman, MD and Philip E. LeBoit, MD
Abstract: Necrobiosis lipoidica (NL), granuloma annulare (GA), and sarcoidosis usually are distinguished by clinical presentation and routine microscopy, but their distinction can sometimes be challenging. Historically, a clue to diagnosing NL or GA has been the identification of lipid droplets in the areas of altered collagen, but such studies have required fresh frozen tissue, making them impractical. Here, we present the first report of immunohistochemical staining to detect adipophilin, a membrane protein in lipid droplets, in NL (n = 12), GA (n = 19), sarcoidosis (n = 12), and, as a control for nonspecific tissue damage, nongranulomatous cutaneous necrosis (n = 13). Four patterns of labeling were identified: (1) extracellular, within zones of altered collagen; (2) both intracellular and extracellular, after the distribution of palisaded or scattered histiocytes; (3) intracellular, within clustered histiocytes; and (4) periadnexal. All cases of NL demonstrated pattern 1; nearly all cases of GA (18/19) demonstrated pattern 2; most sarcoidosis (10/12) demonstrated pattern 3; and nongranulomatous necrosis demonstrated either pattern 4 (6/13) or did not stain (6/13), confirming that the antibody to adipophilin did not adhere nonspecifically to the damaged tissue. An additional set of 3 biopsies with overlapping or partially sampled features of NL, GA, and/or sarcoidosis subsequently confirmed the potential utility of adipophilin staining in diagnostically challenging cases. We conclude that the pattern of adipophilin expression is a useful adjunct in the evaluation of granulomatous dermatitis. Key Words: necrobiosis lipoidica, granuloma annulare, sarcoidosis, adipophilin, granuloma, granulomatous dermatitis (Am J Dermatopathol 2015;37:203–209)
INTRODUCTION Necrobiosis lipoidica (NL), granuloma annulare (GA), and sarcoidosis are granulomatous conditions of unclear etiologies, which typically can be distinguished based on their clinical and histopathological features. NL usually presents on the anterior lower legs of adults as shiny atrophic plaques, characterized histopathologically by histiocytic infiltrates arrayed around zones of degenerated collagen in the mid-to-deep dermis. GA may present in a variety of forms, but in its localized variant, it most classically appears as one or more erythematous annular plaques, histopathologically From the Departments of Pathology and Dermatology, University of California, San Francisco, CA. The authors declare no conflicts of interest. Reprints: Philip E. LeBoit, MD, University of California, 1701 Divisadero St, Suite 280, San Francisco, CA 94115 (e-mail: [email protected]). Copyright © 2014 Wolters Kluwer Health, Inc. All rights reserved.
Am J Dermatopathol Volume 37, Number 3, March 2015
demonstrating histiocytic infiltrates palisaded around hypocellular zones containing altered collagen and increased dermal mucin. Cutaneous sarcoidosis has widely variable clinical appearances but is defined histopathologically by clusters of epithelioid histiocytes lacking significant necrosis or surrounding lymphocytic infiltrates. In many cases, however, these 3 conditions may demonstrate overlapping clinical and histopathological features, presenting a diagnostic challenge.1,2 Diagnosis may further be stymied by partial biopsies that reveal only focal granulomatous inflammation or subtle alterations to the connective tissue. Historically, a clue to the diagnosis of NL or GA was the identification of small lipid droplets within an altered dermis.3–8 Staining these lipid droplets required the use of fresh frozen tissue, and this limitation led to the eventual abandonment of such studies. Immunohistochemical staining for adipophilin has been adopted as a method for labeling lipid droplets in formalin-fixed paraffin-embedded tissue.9 The purpose of our study was to evaluate whether adipophilin staining could identify lipid droplets in NL and GA. Additionally, we evaluated adipophilin expression in cutaneous sarcoidosis, a potential histopathologic mimicker of NL and GA. Lastly, we sought to determine whether the pattern of adipophilin expression could assist in distinguishing between these 3 relatively common granulomatous dermatitis.
MATERIALS AND METHODS Formalin-fixed paraffin-embedded tissue samples were retrieved from the archives of the Dermatopathology Service of the University of California, San Francisco. The initial study group included 12 specimens of NL, 19 of GA (13 with a predominantly palisaded pattern and 6 with an interstitial pattern), and 12 of sarcoidosis diagnosed between 2004 and 2013. Three of the cases of sarcoidosis had a history of systemic disease at the time of biopsy. A control group of 13 cases of nongranulomatous cutaneous necrosis was also identified. A secondary study group of diagnostically challenging cases was identified from our archives, based on a search of cases coded in our archival database as having overlapping features of NL, GA, and/or sarcoidosis. This secondary study group included 3 cases. One case, biopsied from a solitary plaque on the lower leg, was diagnosed as “palisaded granulomatous dermatitis, consistent with necrobiosis lipoidica,” although GA was raised as a diagnostic consideration. The second case was initially diagnosed as NL at the time of biopsy but was reinterpreted as sarcoidosis after subsequent biopsies from the patient revealed classic sarcoidal www.amjdermatopathology.com |
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granulomas. The third case was a superficial shave biopsy from the thigh diagnosed as NL. Hematoxylin and eosin–stained sections were reviewed to confirm the diagnoses. Immunohistochemical staining was performed with a predilute polyclonal antibody to adipophilin (Cell Marque, Rocklin, CA) using a Dako Autostainer platform (Carpinteria, CA). Optimal conditions for detecting adipophilin included a 1-hour pretreatment in Dako pH9 target retrieval solution and a 30-minute antibody incubation time. Positive adipophilin staining was defined by the presence of discrete vacuoles with moderate-to-strong labeling intensity, whereas weak diffuse staining and absent staining were both interpreted as a negative result. To make sure that such staining was not nonspecific adhesion of the antibody to the areas of necrobiosis or necrosis, we compared the staining pattern in the study cases with the pattern observed in the control cases in which there was epidermal or dermal necrosis.
RESULTS Adipophilin expression was identified in our initial study set in all cases of NL, in 18 cases (95%) of GA, and in 10 (83%) of sarcoidosis. Of the 13 control cases of nongranulomatous necrosis, 7 (54%) demonstrated some degree of adipophilin labeling, but the pattern of adipophilin expression tended to differ between the various granulomatous conditions and the control cases (Figs. 1–5). In total, 4 patterns of adipophilin expression were identified: (1) primarily extracellular, centered within zones of altered collagen; (2) both intracellular and extracellular, after the distribution of palisaded or scattered histiocytes; (3) intracellular, within clustered histiocytes; and (4) deposited in the periadnexal dermis. All cases of NL demonstrated pattern
1, whereas 6 cases (50%) of NL concomitantly demonstrated pattern 3 as a minor component. Eighteen cases (95%) of GA demonstrated pattern 2, whereas 4 (21%) concomitantly demonstrated pattern 1. The cases of GA that demonstrated pattern 2 included 6 cases with palisaded granulomas in the middermis, 5 with an exclusively interstitial pattern, 2 with features of actinic GA, and 1 with granulomas extending into the subcutis. One case of interstitial GA did not label with adipophilin. Ten cases (83%) of sarcoidosis demonstrated pattern 3, whereas the remaining cases did not demonstrate adipophilin labeling. The cases of sarcoidosis that labeled with adipophilin included 7 with granulomas limited to the dermis and 3 with granulomas extending into the subcutis. Of the control cases, 6 (46%) demonstrated pattern 4, 6 did not stain with adipophilin, and 1 (8%) demonstrated pattern 2. These results are summarized in Table 1. After identifying the patterns of adipophilin expression seen in the initial study set, the secondary set of diagnostically challenging cases was then evaluated. The case diagnosed as “palisaded granulomatous dermatitis, consistent with necrobiosis lipoidica” revealed an adipophilin-labeling pattern that was best classified as pattern 1, supporting the diagnosis of NL (Fig. 6). The case initially diagnosed as NL but later reclassified as sarcoidosis demonstrated pattern 3, supporting the diagnosis of sarcoidosis (Fig. 7). The superficial shave biopsy diagnosed as NL demonstrated pattern 1, which again supported the diagnosis of NL (Fig. 8).
DISCUSSION The deposition of lipid droplets has long been known to occur in NL and GA, and its identification has historically been used as a clue to the diagnosis of these conditions.
FIGURE 1. NL. A, A medium-power view demonstrates a zone of degenerated collagen flanked by granulomatous infiltrates (hematoxylin and eosin, original magnification ·100). B, Adipophilin immunostaining is positive within the zone of altered collagen (original magnification ·100). C, A higher-power view demonstrates labeling with most lipid droplets deposited extracellularly (adipophilin, original magnification ·600). D, A magnified view confirms vacuolar adipophilin labeling.
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FIGURE 2. Palisaded GA. A, At scanning magnification, a dense histiocytic infiltrate fills the upper dermis in a vaguely palisaded fashion (hematoxylin and eosin, original magnification ·40). B, The pattern of adipophilin immunostaining is distinctly palisaded. The intensity of staining is greater in this example than in several other cases of GA studied, but the pattern is common to most of our cases of palisaded GA (original magnification ·40). C, A higher-power view demonstrates both intracellular and extracellular deposition of lipid droplets (adipophilin, original magnification ·600). D, A magnified view confirms vacuolar adipophilin labeling.
Indeed, the importance of lipid in NL goes back to its first description in 1929, when Oppenheim named the condition “dermatitis atrophicans lipoides”; the first use of “necrobiosis lipoidica diabeticorum” by Urbach followed 3 years later.3 An early report of NL by Nicholas4 provides a more precise description of the lipid deposition, stating that “within the collagenous bundles that had undergone necrobiosis. numerous excessively small fat globules were uniformly distributed.” By the late 1940s, this observation had been applied
more commonly, with Laymon and Fisher5 claiming that the “most striking special feature of NL diabeticorum was the presence of varying amounts of lipids in the areas of necrobiosis. deposited both between and within the fibers.” A decade later, Wood and Beerman6 noted that the “presence of lipid deposits in the lesions of necrobiosis has been assigned a high degree of importance in identifying this process,” suggesting the widespread diagnostic utility of this finding. The characterization of lipid deposits in GA followed thereafter,
FIGURE 3. Interstitial GA. A, Histiocytes intercalate between collagen bundles, and a subtle increase in dermal mucin is also apparent (hematoxylin and eosin, original magnification ·200). B, There is focal adipophilin immunopositivity, which follows an interstitial distribution (original magnification ·200). C, A higher-power view demonstrates both intracellular and extracellular deposition of lipid droplets (adipophilin, original magnification ·600). D, A magnified view confirms vacuolar adipophilin labeling. Copyright 2014 Wolters Kluwer Health, Inc. All rights reserved.
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FIGURE 4. Sarcoidosis. A, Aggregates of epithelioid histiocytes reside in the dermis (hematoxylin and eosin, original magnification ·200). B, Adipophilin immunostaining is focally positive within the clustered histiocytes (original magnification ·200). C, A higher-power view demonstrates intracytoplasmic labeling (adipophilin, original magnification ·600). D, A magnified view confirms vacuolar adipophilin labeling.
with Dabski and Winkelmann8 reporting staining with Oil Red O in 15 of 33 cases of GA, noting that “staining was localized in fine scattered droplets that. was not seen outside the cellular aggregates of a granuloma.” More recently, however, studies to identify lipid droplets in NL and GA have fallen out of clinical practice, primarily because of the
challenge of directly staining lipid, which is dissolved during routine tissue processing. Traditional lipid markers, such as Oil Red O or Sudan IV therefore require fresh tissue and frozen sections. Lipid is also preserved with the fixatives used in processing with electron microscopy, but that technique is time consuming, expensive, and is increasingly less available.
FIGURE 5. Nongranulomatous necrosis. A, Degenerated collagen and elastic fibers surround a distorted follicular infundibulum (hematoxylin and eosin, original magnification ·200). B, Sparse labeling with adipophilin is present near the distorted follicle (original magnification ·200). C, A higher-power view demonstrates scant labeling amidst necrotic debris (adipophilin, original magnification ·600). D, A magnified view suggests both granular and vacuolar adipophilin labeling.
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Adipophilin Expression in NL, GA, and Sarcoidosis
TABLE 1. Patterns of Adipophilin Expression
NL (n = 12) GA (n = 19) Sarcoidosis (n = 12) Nongranulomatous necrosis (n = 13)
Mostly Extracellular, Within Altered Collagen, n (%)
Both Intracellular and Extracellular, Accompanying Histiocytic Infiltrates, n (%)
Mostly Intracellular, Within Clustered Histiocytes, n (%)
Periadnexal, n (%)
Negative, n (%)
12 (100) 4 (21) — —
— 18 (95) — 1 (8)
6 (50) — 10 (83) —
— — — 6 (46)
— 1 (5) 2 (17) 6 (46)
Staining for adipophilin provides an alternative method for identifying lipid deposits. Adipophilin is a protein expressed on the surface of lipid droplets, and it is thus preserved during tissue processing.10 Consequently, it is a suitable target for immunohistochemical staining. Its primary use in dermatopathology currently lies in diagnosing neoplasms of sebaceous lineage.9 Its expression has not previously been documented in granulomatous disorders. We found that adipophilin is expressed in NL, GA, and sarcoidosis, and the pattern of expression differs among these 3 conditions. In all cases of NL, the predominant pattern of adipophilin expression was that of diffuse deposition centered within the zones of altered collagen (the zones of “necrobiosis”). On high magnification, the staining was confirmed to highlight the periphery of tiny vacuoles, but fine, extracellular granular staining was also observed. In half of the cases examined, adipophilin also labeled small vacuoles within clustered histiocytes. This latter observation raises the possibility that the pathogenesis of NL may involve the deposition of intracellular lipid droplets onto altered collagen bundles; alternatively, extracellular lipid may be phagocytized by histiocytes.
Nearly all of our cases of GA also labeled for adipophilin, and the pattern corresponded to the distribution of the lesional histiocytes. That is, cases of palisaded GA tended to show a palisaded pattern of adipophilin staining, and cases of interstitial GA tended to show an interstitial pattern of staining. High magnification confirmed that the staining was both finely vacuolar and granular, with both intracellular and extracellular staining observed. In general, cases of interstitial GA demonstrated more focal adipophilin expression than cases of palisaded GA. In sarcoidosis, adipophilin labeled intracytoplasmic vacuoles within clustered histiocytes in the majority of cases, although in many cases only a small number of histiocytic clusters demonstrated staining. Despite the focality of staining, the pattern of adipophilin expression was clearly distinct from the patterns seen in NL or GA, most notably for the near-complete absence of extracellular labeling. Cases of nongranulomatous cutaneous necrosis were studied as controls to verify that adipophilin is not expressed in a nonspecific manner in the setting of altered or degenerated collagen. In roughly half of those cases, adipophilin was
FIGURE 6. A diagnostically challenging example of NL. A, At scanning magnification, there is a zone of granulomatous inflammation in the superficial dermis with minimal involvement of the deep reticular dermis (hematoxylin and eosin, original magnification ·40). B, Clusters of histiocytes are arranged in a roughly palisaded pattern around a paucicellular area with collagen degeneration (hematoxylin and eosin, original magnification ·200). C, Adipophilin labeling localizes to the area of collagen degeneration and not to the histiocytic infiltrate, supporting a diagnosis of NL (original magnification ·200). Copyright 2014 Wolters Kluwer Health, Inc. All rights reserved.
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FIGURE 7. A diagnostically challenging example of sarcoidosis. A, Scanning magnification reveals collections of histiocytes in a rather sclerotic dermis (hematoxylin and eosin, original magnification ·40). B, A cluster of histiocytes is seen at medium power (hematoxylin and eosin, original magnification ·200). C, Adipophilin staining is present within the clustered histiocytes (box) without extracellular labeling, supporting a diagnosis of sarcoidosis (original magnification ·200).
focally expressed near distorted or necrotic hair follicles, which presumably represents labeling of sebocyte remnants. One case displayed a broad arc of adipophilin expression that approximated a palisaded pattern, but it lacked the circumferential labeling that was typically observed in the cases of palisaded GA. The remaining cases did not demonstrate adipophilin expression. Some of these specimens showed fibrin deposition, and although some immunostains can nonspecifically adhere to fibrin, we did not observe this. The 3 diagnostically challenging cases were included in our study to demonstrate a possible role for adipophilin staining in evaluating biopsies that are not easily diagnosed on routine sections alone. In the first example (Fig. 6), the
clinical history of a solitary plaque on the lower leg suggested NL, but the superficial nature of the granulomatous infiltrate raised the histopathologic possibility of GA. Adipophilin staining localized to the areas of degenerated collagen rather than to the zones populated densely by histiocytes, and this distribution conforms to pattern 1, as seen in all of our initial cases of NL. In the second example (Fig. 7), conventional microscopy at first suggested the sclerotic phase of NL, until subsequent biopsies from the patient revealed classic findings of sarcoidosis, at which time the original diagnosis was reevaluated. This case demonstrated adipophilin expression within clustered histiocytes and lacked the diffuse extracellular staining pattern observed in all of our cases of NL, which
FIGURE 8. Superficial shave biopsy of NL. A, Only a portion of the dermis can be evaluated (hematoxylin and eosin, original magnification ·40). B, There is a vague palisade of histiocytes around a paucicellular area with collagen degeneration (hematoxylin and eosin, original magnification ·200). C, Adipophilin labeling is mostly present in the area of collagen degeneration and not within the histiocytic infiltrate, supporting a diagnosis of NL (original magnification ·200).
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supports the correct diagnosis of sarcoidosis. Examples of sarcoidosis mimicking the clinical and histopathological features of NL have previously been reported, and this current example reinforces the importance of identifying additional clues that may distinguish these diagnoses.1,2 Lastly, the example of a partial biopsy of NL (Fig. 8) again showed extracellular adipophilin labeling in the areas of altered collagen, which supports the diagnosis of NL and illustrates the potential utility of adipophilin staining in specimens where architectural features cannot be fully evaluated. The etiologies of NL, GA, and sarcoidosis remain unknown, but metabolic associations have been identified. NL has been linked to diabetes mellitus in a subset of patients, and individuals with GA have been found to be at an increased risk of dyslipidemia compared with the general population.11,12 Some cases of sarcoidosis have also been associated with hypertriglyceridemia and low levels of high-density lipoprotein cholesterol.13 It is therefore plausible that localized defects in lipid metabolism might contribute to the cutaneous findings in these conditions. Such defects could explain the presence of anomalous lipid droplets in lesional tissue, as identified with adipophilin staining. In summary, the presence of adipophilin expression and the pattern of staining provide diagnostic clues in the evaluation of NL, GA, and sarcoidosis. Adipophilin staining may be particularly useful when overlapping features of these conditions are observed in a single biopsy, or when there is discordance between the clinical and histopathological features. Additionally, the observation of adipophilin expression in NL, GA, and sarcoidosis may shed light on the underlying mechanisms of these granulomatous disorders. Indeed, far from being an historical curiosity, the identification of lipid
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Adipophilin Expression in NL, GA, and Sarcoidosis
droplets in granulomatous dermatitis continues to be relevant to modern dermatopathology. REFERENCES 1. Ball NJ, Kho GT, Martinka M. The histologic spectrum of cutaneous sarcoidosis: a study of twenty-eight cases. J Cutan Pathol. 2004;31:160– 168. 2. Chiba T, Takahara M, Nakahara T, et al. Cutaneous sarcoidosis clinically mimicking necrobiosis lipoidica in a patient with systemic sarcoidosis. Ann Dermatol. 2012;24:74–76. 3. Michelson HE, Laymon CW. Necrobiosis lipoidica diabeticorum (Urbach): dermatitis atrophicans lipoides diabetica (Oppenheim). J Am Med Assoc. 1934;103:163–169. 4. Nicholas L. Necrobiosis lipoidica diabeticorum with xanthoma cells. Arch Derm Syphilol. 1943;48:606–611. 5. Laymon CW, Fisher I. Necrobiosis lipoidica (diabeticorum?). Arch Derm Syphilol. 1949;59:150–167. 6. Wood MG, Beerman H. Necrobiosis lipoidica, granuloma annulare, and rheumatoid nodule. J Invest Dermatol. 1960;34:139–147. 7. Gibson LE, Reizner GT, Winkelmann RK. Necrobiosis lipoidica diabeticorum with cholesterol clefts in the differential diagnosis of necrobiotic xanthogranuloma. J Cutan Pathol. 1988;15:18–21. 8. Dabski K, Winkelmann RK. Generalized granuloma annulare: histopathology and immunopathology. J Am Acad Dermatol. 1989;20:28–39. 9. Muthusamy K, Halbert G, Roberts F. Immunohistochemical staining for adipophilin, perilipin and TIP47. J Clin Pathol. 2006;59:1166–1170. 10. Heid HW, Moll R, Schwetlick I, et al. Adipophilin is a specific marker of lipid accumulation in diverse cell types and disease. Cell Tissue Res. 1998;294:309–321. 11. Reid SD, Ladizinski B, Lee K, et al. Update on necrobiosis lipoidica: a review of etiology, diagnosis, and treatment options. J Am Acad Dermatol. 2013;69:783–791. 12. Wu W, Robinson-Bostom L, Kokkotou E, et al. Dyslipidemia in granuloma annulare. Arch Dermatol. 2012;148:1131–1136. 13. Ivanisevic J, Kotur-Stevuljevic J, Stefanovic A, et al. Dyslipidemia and oxidative stress in sarcoidosis patients. Clin Biochem. 2012;45: 677–682.
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