ORIGINAL ARTICLE
Immunohistochemical localization of autophagosomal membrane-associated protein LC3in granular cell tumor and schwannoma
Masayuki Shintaku
Received:16March 2011/Revised:26April 2011/Accepted:30May 2011/Published online:15June 2011#The Author(s)2011.This article is published with open access at Springerlink
Abstract Granular cell tumor (GCT)is a neoplasm derived from Schwann cell or (in cases arising in the neurohypoph-ysis)pituicyte and is characterized by abundant cytoplasm filled with numerous eosinophilic granules,which have been considered autophagolysosomes on the basis of their ultrastructure.T o confirm that the formation of these granules is related to an autophagy phenomenon,12cases of GCT (including two cases of GCT of the neurohypoph-ysis)were studied immunohistochemically using an anti-body against LC3(microtubule-associated protein 1light chain 3,a specific marker of autophagy).All cases of GCT showed granular immunoreactivity for LC3in the cyto-plasm of tumor cells,indicating that the formation of intracytoplasmic granules in GCT is closely related to an autophagy phenomenon.For elucidation of the relationship between GCT and schwannoma,20ca
ses of schwannoma were similarly studied using the anti-LC3antibody.In eight of 20cases,a small number of tumor cells showed granular immunoreactivity for LC3,suggesting an increased auto-phagic activity in some schwannomas and further reinforc-ing the close relationship between GCT and schwannoma.Keywords Granular cell tumor .Schwannoma .Autophagy .Immunohistochemistry .LC3
Introduction
Granular cell tumor (GCT)is a neoplasm with distinctive histopathological and ultrastructural characteristics.It com-monly arises in the skin and subcutaneous tissue,various visceral organs,especially the breast and upper aerodigestive tracts,and in the neurohypophysis [1].Regarding the histogenesis of GCT ,cases of GCT arising in the skin,subcutaneous tissue,and visceral organs are widely believed to originate from Schwann cells [2–7].The histogenesis of GCT in the neurohypophysis remains controversial,but derivation from pituicyte has been proposed [8,9].Regard-less of their localization or origin,the histopathology of GCT is quite uniform.It consists of a proliferation of large polyhedral cells with abundant cytoplasm which is loaded with numerous eosinophilic granules.Although these gran-ules have been considered autophagosomes or autophagoly-sosomes based on their ultrastructural features [2,3,10],their exact nature and the process of their formation remain to be elucidated.It is also unknown why a large number of these granules are formed specificall
reactive materials studiesy in GCT .
In recent years,various genes and proteins related to autophagy (autophagocytosis)have been identified,and it has become possible to investigate the autophagy phenom-enon by immunohistochemical methods,employing anti-bodies against these autophagy-related proteins [11,12].W e investigated cases of GCT immunohistochemically employ-ing an antibody that was raised against microtubule-associated protein 1light chain 3(LC3)[11].This protein is considered to be specifically localized to the membranes of autophagosomes,and immunohistochemical studies employing this antibody have demonstrated its usefulness in the elucidation of the autophagy phenomenon [11,12].
M.Shintaku (*)
Department of Pathology ,Osaka Red Cross Hospital,T ennoji,
Osaka 543-8555,Japan e-mail:masa-s@sings.jp
Virchows Arch (2011)459:315–319DOI 10.1007/s00428-011-1104-z
T o clarify further the histogenetic relationship between GCT and schwannoma,we also examined case
s of schwannoma for LC3immunoreactivity.
Materials and methods
The surgical pathology files at Osaka Red Cross Hospital during the recent6years were searched for cases of GCT.T en consecutive cases of GCT were retrieved for the study,and in all of these cases,the tumors exhibited a typical histopathological appearance of GCT.The clinicopathological features of individual cases are presented in T able1.These consisted of four cases from the skin or subcutaneous tissue,five cases from the digestive tract,and one case from the trachea.T wo GCT s of the neurohypophysis which were found incidentally at autopsies were also examined similarly.T wenty cases of schwannoma,which are listed in T able2,were also retrieved from the surgical pathology files.These com-prised10cases in which the tumors arose in the skin or subcutaneous tissue and another10cases in which the tumors arose in the cranial nerves or spinal nerve roots. Only tumors demonstrating histopathological features that were typical of conventional schwannoma were used for the study,and questionable or problematic cases were excluded.All tumors examined in this study had been fixed in10%neutral buffered formalin and embedded in paraffin.
Immunohistochemical study was performed on paraffin sections using the Envision Plus detection syst
em(Dako, Glostrup,Denmark),and polyclonal or monoclonal primary antibodies against the following substances were employed: LC3(polyclonal;Medical&Biological Laboratories, Nagoya,Japan;1:1,000),CD68(clone KP1,Dako;1:100),and S-100protein(clone ER-PR8,Dako;1:500). T wo GCT s of the neurohypophysis were also examined for the expression of glial fibrillary acidic protein(GF AP, polyclonal;Dako;1:100).Heat-induced epitope retrieval using a hot bath was performed before applying immunos-tains for LC3,CD68,and S-100protein.
Results
A summary of the results of immunohistochemical study is presented in T ables1and2.In all cases of GCT,tumors consisted of an alveolar,trabecular,or diffuse proliferation of large polyhedral cells that had abundant cytoplasm loaded with numerous eosinophilic granules of variable sizes(Fig.1a).These granules were strongly or weakly positive for periodic acid-Schiff reaction.The cytoplasm of tumor cells exhibited intense,coarsely or finely granular immunoreactivity for LC3in all cases(Fig.1b).The cytoplasm was similarly immunoreactive for CD68and S-100protein.T wo GCT s of the neurohypophysis(Fig.2a) also showed granular immunoreactivity of the cytoplasm for LC3(Fig.2b).Both tumors were also immunoreactive for CD68and S-100protein,but only one of the two tumors exhibited focal immunoreactivity for GF AP.
In8of20schwannomas examined,a small number of large polyhedral cells loaded with eosinophilic granules(“granular cells”)were scattered within the tumors(Fig.3a).These cells were easily overlooked or often difficult to distinguish from reactive macrophages on hematoxylin–eosin sections,but they showed intense granular immunoreactivity and stood out clearly on sections immunostained for LC3(Fig.3b). These LC3-immunoreactive cells within schwannoma were also immunoreactive for CD68and S-100protein on serial sections.However,immunohistochemistry for these two markers was not useful for the detection of“granular cells,”because almost all tumor cells were immunoreactive for S-100protein,and CD68was intensely immunoreactive in reactive macrophages within the tumors.Most schwannomas contained both Antoni A and B areas in variable proportions within a given tumor,and the distribution of“granular cells”tended to be more prevalent in Antoni A area than in Antoni B area.
Discussion
Abundant intracytoplasmic granules that accumulate in tumor cells of GCT have been considered autophagosomes or autophagolysosomes on the basis of their ultrastructural features[2,3,10].However,as pointed out by some investigators[13],the electron microscopic identification of intracytoplasmic inclusions as autophagy-related structures
T able1Clinicopathological findings and results of LC3immunohis-tochemistry in granular cell tumors
Case Age/gender Site/diameter(mm)LC3
154/F abdomen/40+ 276/F face/18+ 360/M inguinal region/20+ 435/F chest wall/10+ 556/F cecum/7+ 635/F stomach/10+ 766/M esophagus/5+ 841/M esophagus/5+ 942/M esophagus/5+ 1013/F trachea/15+ 1174/M neurohypophysis/1+ 1259/F neurohypophysis/4+
is occasionally not straightforward,and some structures unrelated to autophagy can be misinterpreted as autophago-somes.It is also unknown why autophagolysosomes are accumulated in a large amount specifically in tumor cells of GCT .Some intrinsic metabolic abnormalities within tumor cells of GCT probably result in focal cytoplasmic degradation and an accumulation of numerous autophagolysosomes.Monoclonal antibodies against CD68molecules,such as KP1and PGM-1,have been employed as a marker for lysosome or phagolysosome [14–16],and several investiga-tors demonstrated the immunoreactivity of GCT for these antibodies [14–16],which was also confirmed in our present study .W e further applied an anti-LC3antibody for the investigation of GCT and demonstrated that all GCT s examined showed immunoreactivity for LC3.LC3is a protein located on the inner and outer membranes of
autophagosomes [11,12],and the findings of our study provide additional evidence for the autophagoso
mal origin of intracytoplasmic granules in tumor cells of GCT .Although the specificity of LC3as a marker for autophagosome at the light microscopic level leaves some room for reservation because LC3has been shown to be incorporated into protein aggregates independent of autophagy [12],the immunohis-tochemical reactivity of GCT for LC3can be reasonably interpreted as representing an autophagy phenomenon in the light of their well-known ultrastructural features [2,3,10].Concerning the histogenesis of GCT ,the Schwann cell origin has been widely accepted in recent years [2–7].The immunoreactivity of GCT for S-100protein has been well known [5–7],and a recent study concerning many cases of peripheral nerve sheath tumors arising in the gastrointesti-nal tract demonstrated the immunoreactivity for
CD56,
Fig.1a A subcutaneous GCT .Large polyhedral cells having abundant cytoplasm loaded with numerous eosinophilic granules proliferated diffusely (hematoxylin –eosin stain).
b The intracytoplasmi
c granules in GCT showe
d an
immunoreactivity for LC3(immunoperoxidase method)
Case Age/gender Site/diameter (mm)Antoni A &B Granular cells LC3151/F shoulder/25
A=B ++248/F retroperitoneum/38A>B −−392/F orbit/25A>B ++446/F hand/13A>B −−529/F forearm/17A>B −−658/M forearm/29A<B −−736/M finger/9A<B −−861/M knee/13A>B −−960/F thigh/40
A>B −−1052/F upper arm/15A>B ++1164/M cauda equina/4A ++1242/M thoracic root/18A<B −−1362/
F lumbar root/18A>B ++1423/F acoustic nerve/15A>B −−1576/M acoustic nerve/12A ++1675/F acoustic nerve/20A<B −−1771/F cauda equina/10A ++1833/F acoustic nerve/19A ++1975/M lumbar root/23A>B −−20
58/F
acoustic nerve/27
A
−
−
T able 2Clinicopathological findings and results of LC3immunohistochemistry in schwannomas
calretinin,and α-inhibin (as additional markers of Schwann cell differentiation)in GCT [7].Immunohistochemical studies employing antibodies against various proteinaceous components of the myelin sheath have shown that intra-cytoplasmic granules in GCT contain degradation products of the myelin sheath,thus emphasizing further the close relationship between Schwann cell and GCT [6].
However,the relationship between GCT and ordinary schwannoma remains unclear.In an ultrastructural study of schwannoma,Sian and Ryan reported the occurrence of many cells containing intracytoplasmic osmiophilic granules in the Antoni B area and pointed out the similarity of these granules to those found in GCT [17].A few cases of schwannoma have been reported in which scattered or aggregated “granular cells ”were found [4,18].In the present study ,we examined schwannoma for the presence of LC3-immunoreactive granules in the cytoplasm.In 8of 20schwannoma cases,a small number of tumor cells showed cytoplasmic swelling,and the swollen cytoplasm contained numerous granules that were immunoreactive for LC3.This finding probably corresponds to the immunoreactivity for CD68of autophagosomes in Schwann cells in cases of degeneration of nerve fibers and also in schwannoma and neurofibroma [15,16].The immunohistochemistry for LC3is superior to that for CD68in detection of “granular cells ”within schwannoma,because,while not only “granular cells ”but also reactive macrophages showed immunoreactivity for CD68,only the former was highlighted by immunohisto-chemistry for LC3.The presence of “granular cells ”immunoreactive for LC3indicates increased autophagic activity in some cases of schwannoma and further reinforces the close relationship between GCT and schwannoma.
Some neoplasms other than schwannoma,for example smooth muscle tumors [19],astrocytoma [20],gli
oblastoma [21],oligodendroglioma [22],and anaplastic medulloblas-toma [23],are known to rarely exhibit a “granular cell change ”of the cytoplasm.Mentzel et al.studied the “granular cell change ”in smooth muscle tumors but found that the change was not related topographically to areas showing degenerative features such as necrosis,hemor-rhage,hyalinization,or myxoid change [19].Although it remains to be clarified whether these tumors also show immunoreactivities for proteins specific to the autophagy-related processes,this finding has something in common with findings we obtained in schwannoma,that is,the occurrence of “granular cells ”was not always associated with degenerative changes represented by the Antoni B area in schwannoma.The role of autophagy is diverse and includes the adaptation to nutrient starvation by producing amino acids and the constitutive removal or turnover of cytosolic proteins and redundant or damaged organelles [11,12,24].Autophagy phenomenon therefore does not necessarily indicate degenerative changes of the cells,but it is a dynamic adaptive response to sublethal stress [24].In conclusion,we demonstrated immunoreactivity for LC3,an autophagy-specific marker,in tumor cells in
all
Fig.2a A GCT of the neuro-hypophysis.Large polyhedral cells having abundant granular cytoplasm formed a small nodule (hematoxylin –eosin stain).b The intracytoplasmic granules showed an
immunoreactivity for LC3(immunoperoxidase
method)
Fig.3a A subcutaneous schwan-noma.A small number of large polyhedral cells with swollen cytoplasm filled with numerous,eosinophilic fine granules
(“granular cells ”)were scattered within the tumor (hematoxylin –eosin stain).b The intracytoplas-mic granules in these
“granular cells ”were immunore-active for LC3(immunoperoxi-dase method)
cases of GCT.Eight of20cases of schwannoma contained LC3-immunoreactive“granular cells.”These results indi-cate that intracytoplasmic granules in tumor cells of GCT are actually autophagosomes or autophagolysosomes and the activity of autophagy is increased in some cases of schwannoma,supporting a close histogenetic relationship between GCT and schwannoma.
Acknowledgment W e are grateful to Ms.T omoko Honda for her skillful technical assistance in performing the immunohistochemical studies.
Conflict of interest statement W e declare that we have no conflict of interest.
Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which per-mits any noncommercial use,distribution,and reproduction in any medium,provided the original author(s)and source are credited. References
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