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Muscularis mucosae versus muscularis propria in gallbladder, cystic duct, and common bile duct: smoothelin and desmin immunohistochemical study
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Annals of Diagnostic Pathology 14 (2010) 408 – 412
Muscularis mucosae versus muscularis propria in gallbladder, cystic duct, and common bile duct: smoothelin and desmin immunohistochemical study☆ Kirtee Raparia, MD, Qihui J. Zhai, MD, Mary R. Schwartz, MD, Steven S. Shen, MD, Alberto G. Ayala, MD, Jae Y. Ro, MD⁎ Department of Pathology, The Methodist Hospital, Weill Medical College, Cornell University, Houston, TX 77030, USA
Abstract
Keywords:
The muscle layer in the cystic duct and common bile duct is not well defined, and it is unresolved whether it represents muscularis mucosae or muscularis propria. Smoothelin is a novel smooth muscle–specific contractile protein expressed only in fully differentiated smooth muscle cells of the muscularis propria and not in proliferative or noncontractile smooth muscle cells of the muscularis mucosae. In this study, we characterize the histologic aspects of the muscle layer in gallbladder, cystic duct, and common bile duct by evaluation of routine histologic sections and the utilization of immunohistochemistry using desmin and smoothelin. Formalin-fixed, paraffin-embedded sections of the gallbladder (15 cases), cystic duct (11 cases), and common bile duct (10 cases) were stained for smoothelin and desmin. Staining intensity was evaluated as weak or strong. The staining pattern score was evaluated as follows: 0 or negative = less than or equal to 5% positivity, +1 or focal = 6% to 10% positivity, +2 or moderate = 11% to 50% positivity, and +3 = greater than 50% muscle cells positivity. With desmin, strong and diffuse (+3) staining was observed in all gallbladder cases (15/15, 100%), highlighting one continuous muscle layer. The muscle layer was discontinuous and interrupted in all cystic duct cases and in most common bile ducts, highlighted by the desmin stain. Smoothelin intensely stained (at least +2) muscle fibers in the gallbladder in 11 (73%) of 15 cases similar to that observed with desmin staining. In contrast, common bile ducts predominantly had absent or weak and focal immunostaining (0 or +1 staining) with smoothelin (7/10, 70%), with only a few cases (3/10, 30%) having +2 staining (no cases with +3). Cystic ducts also showed absent or weak and focal immunostaining with smoothelin, with 5 (44%) of 11 cases showing 2+ immunostaining with smoothelin (no cases with 3+). Based on our findings, we conclude that, in the gallbladder wall, the muscle layer is muscularis propria and there is no muscularis mucosae present. In the cystic duct and common bile duct, only an attenuated and incomplete muscle layer of muscularis mucosae is present; because there is no muscularis propria, there probably is limited contractile function. Differentiating these anatomical muscle structures may be important for the pathologic staging of carcinoma in these organs. © 2010 Elsevier Inc. All rights reserved. Muscularis propria; Muscularis mucosae; Smoothelin; Desmin; Cancer staging
1. Introduction The organs of the gastrointestinal tract and urinary system have 2 distinct muscle layers: muscularis mucosae (MM) and
☆ This work was presented at the 98th Annual Meeting of the United States and Canadian Academy of Pathology, Boston, MA, March 2009. ⁎ Corresponding author. Tel.: +1 713 441 2263; fax: +1 713 793 1603. E-mail address: [email protected] (J.Y. Ro).
1092-9134/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.anndiagpath.2010.05.013
muscularis propria (MP). The MM in the urinary bladder is usually incomplete, and the muscle bundles are usually thin and different from those of the MP. Thickened hyperplastic MM can occur occasionally in the wall of the urinary bladder and create difficulty in diagnosis and staging, particularly in transurethral resections of bladder tumors [1]. The normal histology of the gallbladder and biliary tree is different from the remainder of the gastrointestinal tract, with the presence of only one muscle layer and lack of MM in gallbladder and a single discontinuous layer in the cystic
K. Raparia et al. / Annals of Diagnostic Pathology 14 (2010) 408–412
409
duct and common bile duct (CBD). The scattered muscle fibers in cystic duct and CBD are not well characterized. Smoothelin is a novel smooth muscle–specific contractile protein expressed only in fully/terminally differentiated smooth muscle cells. Its expression is absent or weak in proliferative or noncontractile smooth muscle cells or myofibroblasts [2,3]. In a recent study, the immunohistochemical staining pattern of smoothelin in MP and MM (including its hyperplastic forms) in urinary bladder proved to be an attractive marker to be incorporated in the evaluation of transurethral resection specimens when distinguishing between the 2 layers was difficult [4,5]. To resolve unanswered questions and to determine the potential clinical significance about the characterization of the muscle layers in gallbladder, cystic duct, and CBD, we studied the muscle layers of these organs (36 cases in total) by immunohistochemistry using desmin and smoothelin. To the best of our knowledge, this is the first study in the literature that tries to delineate the basic histologic characteristics of muscle layers of these organs, which may be important for the pathologic staging of carcinoma in these organs.
2. Materials and methods 2.1. Specimens Archival formalin-fixed, paraffin-embedded tissue blocks of the gallbladder (15 cases), cystic duct (11 cases), and CBD (10 cases) from The Methodist Hospital, Houston, TX, were randomly retrieved for the immunohistochemical study. The gallbladder and cystic duct specimens were selected from chronic cholecystitis cases of the same patients. The CBD specimens were from Whipple specimens removed for pancreatic cancer. Because the muscle layer is different depending on the level of CBD [6], the terminal portion of CBD was selected. These pancreatic cancer cases showed no carcinomatous involvement of the CBD. 2.2. Immunohistochemistry After deparaffinization and rehydration of tissue sections, heat-induced epitope retrieval was performed in 10-mmol/L citrate buffer (pH 6.0); and heating for 3 times was performed before immunostaining. The following antibodies were used: smoothelin (R4A; 1:100 dilution; Abcam Inc, Cambridge, MA) and desmin (1:200; Dako, Carpinteria, CA). Tissue sections were incubated with primary antibody for 30 minutes at room temperature, washed with phosphate-buffered saline, and incubated with a secondary antibody conjugated to horseradish peroxidase (Benchmark IHC/ISH module; Ventana, Tucson, AZ). Hematoxylin was used as a counter stain. The interpretation of immunoreactivity was performed in a semiquantitative manner by analyzing the extent of the staining positivity of the muscle cells. Staining intensity was evaluated as weak
Fig. 1. (A) Smooth muscle layer of gallbladder showing one layer of smooth muscle fibers without any interruptions. (B) Staining of muscle layer of the gallbladder. Desmin highlights the single discontinuous muscle layer with strong and diffuse immunostaining. (C) Smoothelin also intensely stains the muscle layer of the gallbladder, suggesting it to be MP-type muscle.
410
K. Raparia et al. / Annals of Diagnostic Pathology 14 (2010) 408–412
Fig. 2. (A) Smooth muscle layer of cystic duct showing discontinuous and interrupted smooth muscle fibers. (B) Staining of muscle layer of the cystic duct. Desmin immunostaining highlights the muscle layer as discontinuous and interrupted in all cases of cystic ducts. (C) Cystic duct showed absent or weak immunostaining with smoothelin, suggesting the muscle layer in cystic duct to be MM type.
Fig. 3. (A) Smooth muscle layer of CBD showing discontinuous and interrupted smooth muscle fibers. (B) Staining of muscle layer of the CBD. Desmin immunostaining highlights the muscle layer as discontinuous and interrupted in all cases of CBDs. (C) Common bile duct showed absent or weak immunostaining with smoothelin, suggesting the muscle layer in CBD to be MM type.
K. Raparia et al. / Annals of Diagnostic Pathology 14 (2010) 408–412
411
or strong. The staining was evaluated as follows: 0 or negative = less than or equal to 5% positivity, +1 or focal = 6% to 10% positivity, +2 or moderate = 11% to 50% positivity, and +3 or diffuse = greater than 50% muscle cells positivity. Strong intensity included +2 and +3. Colonic wall sections were used as positive control for smoothelin and desmin, and omission of the primary antibodies was used as negative control.
Table 2 Comparative immunohistochemical staining of muscle layer of gallbladder, cystic duct, and CBD with smoothelin
3. Results
showed +2 immunostaining with this marker. No case of the cystic ducts exhibited +3 immunostaining.
3.1. Smooth muscle layers in gallbladder, cystic duct, and CBD on hematoxylin and eosin staining All 15 cases of gallbladder showed a discontinuous smooth muscle layer without any interruption (Fig. 1A). The smooth muscle layer of cystic duct (Fig. 2A) and CBD (Fig. 3A) was discontinuous and interrupted in all 11 cystic duct cases and in all 10 CBD cases. 3.2. Desmin staining of muscle layers in gallbladder, cystic duct, and CBD With desmin, strong and diffuse (+3) cytoplasmic staining was observed in all gallbladders (15/15, 100%), highlighting a single discontinuous layer without areas devoid of smooth muscle fibers (Fig. 1B, Table 1). In contrast, the desmin staining highlighted a discontinuous and interrupted muscle layer in all cystic ducts and CBDs. Strong and diffuse (+3) stain with desmin was seen in all cystic (11/11) (Fig. 2B) and in 9 of 10 CBDs (Fig. 3B). The remaining 1 case of CBD stained +2. 3.3. Smoothelin staining of muscle layers in gallbladder, cystic duct and CBD Smoothelin intensely stained (+2 or +3) muscle fibers in the gallbladder wall similar to that observed with desmin staining (11/15, 73%) (Fig. 1C, Table 2). Weak or absent immunostaining was seen in 4 of 11 cases of the gallbladder sections (Fig. 2C). In contrast, the smoothelin expression in CBD was predominantly absent or focally weak (7/10, 70%; 0 or +1 staining), with only a few cases (3/10, 30%) having + 2 staining (Fig. 3C) and no case with +3 immunostaining. Cystic ducts also showed absent or focal immunostaining with smoothelin (6/11 cases, 55%). Five of these 11 cases
Table 1 Comparative immunohistochemical staining of muscle layer of gallbladder, cystic duct, and CBD with desmin
Desmin Negative Weak Strong
Score
Gallbladder
Cystic duct
CBD
0 +1 +2 +3
0/15 0/15 0/15 15/15
0/11 0/11 0/11 11/11
0/10 0/10 1/10 9/10
Smoothelin Negative Weak Strong
Score
Gallbladder
Cystic duct
CBD
0 1+ 2+ 3+
0/15 4/15 7/15 4/15
3/11 3/11 5/11 0/11
4/10 3/10 3/10 0/10
4. Discussion The normal histology of the muscle layers of gallbladder, cystic duct, and CBD is different from the remaining gastrointestinal tract; and the published descriptions are very limited. The layers of the gallbladder include the mucosa, one smooth muscle layer, perimuscular subserosal connective tissue, and serosa [6]. The smooth muscle layer of gallbladder consists of loosely arranged bundles of circular, longitudinal, and oblique fibers. The thickness of the muscle layer is quite variable, which may reflect variable contractile states of the gallbladder in different specimens. The smooth muscle layer of the gallbladder has been reported to be closely associated to “MP” with lack of MM and submucosa [6]. The wall of the cystic duct is histologically similar to that of the gallbladder. There is luminal mucosal epithelium, beneath which there are thin groups of smooth muscle fibers. The smooth muscle fibers are believed to prevent both overdistension and collapse of the cystic duct when it is subjected to changes in pressure as well to promote local stirring at the mucosal surface, to improve secretion and the absorption of nutrients. Although this layer might represent MM because of its function and thickness, its true nature has not been fully defined. The CBD that serves as conduit for the flow of the bile is lined by a single layer of tall columnar epithelial cells with an underlying dense connective tissue layer. The distribution of the smooth muscle fibers varies throughout the level of the CBD. There are scattered or no muscle fibers in the upper one third, whereas there is a continuous or interrupted pattern of thin to thick smooth muscle bundles throughout the lower portion [7]. These muscle fibers are frequently longitudinally aligned in the lower portion. Some authors have concluded that the supraduodenal portion of the CBD lacks smooth muscle [8]. Others found that scattered bundles of smooth muscle are present in small but significant amounts [9,10]. In a study of urinary bladder cases stained, the smooth muscle bundles/fibers of the MM either were absent (19/42, 45%) or showed weak and focal (18/42, 43%) staining with smoothelin. However, the bundles of smooth muscle of MP
412
K. Raparia et al. / Annals of Diagnostic Pathology 14 (2010) 408–412
showed strong and diffuse staining (36/42, 86%) with smoothelin [4]. The authors concluded that relatively distinct immunohistochemical staining patterns of smoothelin between MP and MM (including its hyperplastic forms) highlight its usefulness in the challenging cases of staging bladder urothelial carcinoma in the transurethral resection of bladder tumor specimens [4]. In a more recent study, the authors confirmed the relatively distinct staining pattern of smoothelin between MM and MP; however, they observed overlap of intensity in a few cases [11]. In our study, we found strong and diffuse (+3) immunostaining with desmin in all gallbladders (15/15, 100%), highlighting one layer of smooth muscle layer, which is discontinuous but without any interruptions. In contrast, the smooth muscle layer in all cystic ducts and CBDs was thin, discontinuous, and interrupted, but was still highlighted by the desmin stain. Smoothelin intensely stained (+2 or +3) the smooth muscle fibers in the gallbladder wall similar to that observed with desmin staining (11/15, 73%). This finding indicated that the muscle layer in the gallbladder is “MP” rather than “MM.” To accommodate the contractile function of bile accumulation, the gallbladder needs fully developed contractile smooth muscle fibers. The CBD has a thin and discontinuous muscle layer with absent or focally weak immunostaining (0 or +1 staining) with smoothelin (7/10, 70%), and only a few cases (3/10, 30%) have +2 staining. Similarly, cystic ducts also show absent or focally weak immunostaining with smoothelin, with 5 of 11 cases (44%) showing +2 immunostaining with smoothelin. Therefore, the muscle layer in cystic duct and CBD is not fully developed and is more compatible with MM than with MP. Functionally, unlike gallbladder, the cystic duct and CBD are not required to have a contractile function for bile but for the passage of the bile. The presence or absence of MM not only is of academic interest, but also has clinical significance in tumor staging. Thus, the distinction between MM and MP provides basis for the correct assessment of the depth of invasion in carcinomas of the gallbladder, cystic duct, or CBD. Although depth of invasion is an important prognostic factor for carcinomas of the CBD [12], the level of invasion is often difficult to evaluate because the muscle layer of the CBD is indistinct, especially in the proximal aspect of it. The current TNM staging system of the CBD defines T1 as tumor invading the subepithelial connective tissue or fibromuscular layer (tumor confined to the bile duct
histologically) and T2 as tumor invading beyond the wall of the bile duct [13]. The presence of smooth muscle fibers can be used as a landmark to delineate the layers and further to subclassify the tumor staging. In summary, the gallbladder contains a relatively thick, discontinuous layer of smooth muscle bundle that appears to be MP, rather than MM. The cystic duct and CBD contain only an attenuated and incomplete muscle layer that appears to be MM, suggesting no or limited contractile function. These anatomical muscle structures may be important for the correct pathologic staging of carcinoma of these organs. References [1] Vakar-Lopez F, Shen SS, Zhang S, Tamboli P, Ayala AG, Ro JY. Muscularis mucosae of the urinary bladder revisited with emphasis on its hyperplastic patterns: a study of a large series of cystectomy specimens. Ann Diagn Pathol 2007;11:395-401. [2] Kramer J, Aguirre-Arteta AM, Thiel C, et al. A novel isoform of the smooth muscle cell differentiation marker smoothelin. J Mol Med 1999;77:294-8. [3] van der Loop FT, Schaart G, Timmer ED, Ramaekers FC, van Eys GJ. Smoothelin, a novel cytoskeletal protein specific for smooth muscle cells. J Cell Biol 1996;134:401-11. [4] Paner GP, Shen SS, Lapetino S, et al. Diagnostic utility of antibody to smoothelin in the distinction of muscularis propria from muscularis mucosae of the urinary bladder: a potential ancillary tool in the pathologic staging of invasive urothelial carcinoma. Am J Surg Pathol 2009;33:91-8. [5] Council L, Hameed O. Differential expression of immunohistochemical markers in bladder smooth muscle and myofibroblasts, and the potential utility of desmin, smoothelin, and vimentin in staging of bladder carcinoma. Mod Pathol 2009;22:639-50. [6] Mills SE. Histology for pathologists. 3rd ed. Lippincott Williams & Wilkins; 2006. [7] Hong SM, Kang GH, Lee HY, Ro JY. Smooth muscle distribution in the extrahepatic bile duct: histologic and immunohistochemical studies of 122 cases. Am J Surg Pathol 2000;24:660-7. [8] Mahour GH, Wakim KG, Soule EH, Ferris DO. Structure of the common bile duct in man: presence or absence of smooth muscle. Ann Surg 1967;166:91-4. [9] Burden VG. Observations on the histologic and pathologic anatomy of the hepatic, cystic, and common bile ducts. Ann Surg 1925;82: 584-97. [10] Ludwick JR. Observations on the smooth muscle and contractile activity of the common bile duct. Ann Surg 1966;164:1041-50. [11] Miyamoto H, Sharma RB, Illei PB, Epstein JI. Pitfalls in the use of smoothelin to identify muscularis propria invasion by urothelial carcinoma. Am J Surg Pathol 2010;34:418-22. [12] Tsunoda T, Eto T, Koga M, et al. Early carcinoma of the extrahepatic bile duct. Jpn J Surg 1989;19:691-8. [13] AJCC cancer staging handbook. 7th ed. New York, NY: Springer; 2010. p. 255-76.
Annals of Diagnostic Pathology 14 (2010) 408 – 412
Muscularis mucosae versus muscularis propria in gallbladder, cystic duct, and common bile duct: smoothelin and desmin immunohistochemical study☆ Kirtee Raparia, MD, Qihui J. Zhai, MD, Mary R. Schwartz, MD, Steven S. Shen, MD, Alberto G. Ayala, MD, Jae Y. Ro, MD⁎ Department of Pathology, The Methodist Hospital, Weill Medical College, Cornell University, Houston, TX 77030, USA
Abstract
Keywords:
The muscle layer in the cystic duct and common bile duct is not well defined, and it is unresolved whether it represents muscularis mucosae or muscularis propria. Smoothelin is a novel smooth muscle–specific contractile protein expressed only in fully differentiated smooth muscle cells of the muscularis propria and not in proliferative or noncontractile smooth muscle cells of the muscularis mucosae. In this study, we characterize the histologic aspects of the muscle layer in gallbladder, cystic duct, and common bile duct by evaluation of routine histologic sections and the utilization of immunohistochemistry using desmin and smoothelin. Formalin-fixed, paraffin-embedded sections of the gallbladder (15 cases), cystic duct (11 cases), and common bile duct (10 cases) were stained for smoothelin and desmin. Staining intensity was evaluated as weak or strong. The staining pattern score was evaluated as follows: 0 or negative = less than or equal to 5% positivity, +1 or focal = 6% to 10% positivity, +2 or moderate = 11% to 50% positivity, and +3 = greater than 50% muscle cells positivity. With desmin, strong and diffuse (+3) staining was observed in all gallbladder cases (15/15, 100%), highlighting one continuous muscle layer. The muscle layer was discontinuous and interrupted in all cystic duct cases and in most common bile ducts, highlighted by the desmin stain. Smoothelin intensely stained (at least +2) muscle fibers in the gallbladder in 11 (73%) of 15 cases similar to that observed with desmin staining. In contrast, common bile ducts predominantly had absent or weak and focal immunostaining (0 or +1 staining) with smoothelin (7/10, 70%), with only a few cases (3/10, 30%) having +2 staining (no cases with +3). Cystic ducts also showed absent or weak and focal immunostaining with smoothelin, with 5 (44%) of 11 cases showing 2+ immunostaining with smoothelin (no cases with 3+). Based on our findings, we conclude that, in the gallbladder wall, the muscle layer is muscularis propria and there is no muscularis mucosae present. In the cystic duct and common bile duct, only an attenuated and incomplete muscle layer of muscularis mucosae is present; because there is no muscularis propria, there probably is limited contractile function. Differentiating these anatomical muscle structures may be important for the pathologic staging of carcinoma in these organs. © 2010 Elsevier Inc. All rights reserved. Muscularis propria; Muscularis mucosae; Smoothelin; Desmin; Cancer staging
1. Introduction The organs of the gastrointestinal tract and urinary system have 2 distinct muscle layers: muscularis mucosae (MM) and
☆ This work was presented at the 98th Annual Meeting of the United States and Canadian Academy of Pathology, Boston, MA, March 2009. ⁎ Corresponding author. Tel.: +1 713 441 2263; fax: +1 713 793 1603. E-mail address: [email protected] (J.Y. Ro).
1092-9134/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.anndiagpath.2010.05.013
muscularis propria (MP). The MM in the urinary bladder is usually incomplete, and the muscle bundles are usually thin and different from those of the MP. Thickened hyperplastic MM can occur occasionally in the wall of the urinary bladder and create difficulty in diagnosis and staging, particularly in transurethral resections of bladder tumors [1]. The normal histology of the gallbladder and biliary tree is different from the remainder of the gastrointestinal tract, with the presence of only one muscle layer and lack of MM in gallbladder and a single discontinuous layer in the cystic
K. Raparia et al. / Annals of Diagnostic Pathology 14 (2010) 408–412
409
duct and common bile duct (CBD). The scattered muscle fibers in cystic duct and CBD are not well characterized. Smoothelin is a novel smooth muscle–specific contractile protein expressed only in fully/terminally differentiated smooth muscle cells. Its expression is absent or weak in proliferative or noncontractile smooth muscle cells or myofibroblasts [2,3]. In a recent study, the immunohistochemical staining pattern of smoothelin in MP and MM (including its hyperplastic forms) in urinary bladder proved to be an attractive marker to be incorporated in the evaluation of transurethral resection specimens when distinguishing between the 2 layers was difficult [4,5]. To resolve unanswered questions and to determine the potential clinical significance about the characterization of the muscle layers in gallbladder, cystic duct, and CBD, we studied the muscle layers of these organs (36 cases in total) by immunohistochemistry using desmin and smoothelin. To the best of our knowledge, this is the first study in the literature that tries to delineate the basic histologic characteristics of muscle layers of these organs, which may be important for the pathologic staging of carcinoma in these organs.
2. Materials and methods 2.1. Specimens Archival formalin-fixed, paraffin-embedded tissue blocks of the gallbladder (15 cases), cystic duct (11 cases), and CBD (10 cases) from The Methodist Hospital, Houston, TX, were randomly retrieved for the immunohistochemical study. The gallbladder and cystic duct specimens were selected from chronic cholecystitis cases of the same patients. The CBD specimens were from Whipple specimens removed for pancreatic cancer. Because the muscle layer is different depending on the level of CBD [6], the terminal portion of CBD was selected. These pancreatic cancer cases showed no carcinomatous involvement of the CBD. 2.2. Immunohistochemistry After deparaffinization and rehydration of tissue sections, heat-induced epitope retrieval was performed in 10-mmol/L citrate buffer (pH 6.0); and heating for 3 times was performed before immunostaining. The following antibodies were used: smoothelin (R4A; 1:100 dilution; Abcam Inc, Cambridge, MA) and desmin (1:200; Dako, Carpinteria, CA). Tissue sections were incubated with primary antibody for 30 minutes at room temperature, washed with phosphate-buffered saline, and incubated with a secondary antibody conjugated to horseradish peroxidase (Benchmark IHC/ISH module; Ventana, Tucson, AZ). Hematoxylin was used as a counter stain. The interpretation of immunoreactivity was performed in a semiquantitative manner by analyzing the extent of the staining positivity of the muscle cells. Staining intensity was evaluated as weak
Fig. 1. (A) Smooth muscle layer of gallbladder showing one layer of smooth muscle fibers without any interruptions. (B) Staining of muscle layer of the gallbladder. Desmin highlights the single discontinuous muscle layer with strong and diffuse immunostaining. (C) Smoothelin also intensely stains the muscle layer of the gallbladder, suggesting it to be MP-type muscle.
410
K. Raparia et al. / Annals of Diagnostic Pathology 14 (2010) 408–412
Fig. 2. (A) Smooth muscle layer of cystic duct showing discontinuous and interrupted smooth muscle fibers. (B) Staining of muscle layer of the cystic duct. Desmin immunostaining highlights the muscle layer as discontinuous and interrupted in all cases of cystic ducts. (C) Cystic duct showed absent or weak immunostaining with smoothelin, suggesting the muscle layer in cystic duct to be MM type.
Fig. 3. (A) Smooth muscle layer of CBD showing discontinuous and interrupted smooth muscle fibers. (B) Staining of muscle layer of the CBD. Desmin immunostaining highlights the muscle layer as discontinuous and interrupted in all cases of CBDs. (C) Common bile duct showed absent or weak immunostaining with smoothelin, suggesting the muscle layer in CBD to be MM type.
K. Raparia et al. / Annals of Diagnostic Pathology 14 (2010) 408–412
411
or strong. The staining was evaluated as follows: 0 or negative = less than or equal to 5% positivity, +1 or focal = 6% to 10% positivity, +2 or moderate = 11% to 50% positivity, and +3 or diffuse = greater than 50% muscle cells positivity. Strong intensity included +2 and +3. Colonic wall sections were used as positive control for smoothelin and desmin, and omission of the primary antibodies was used as negative control.
Table 2 Comparative immunohistochemical staining of muscle layer of gallbladder, cystic duct, and CBD with smoothelin
3. Results
showed +2 immunostaining with this marker. No case of the cystic ducts exhibited +3 immunostaining.
3.1. Smooth muscle layers in gallbladder, cystic duct, and CBD on hematoxylin and eosin staining All 15 cases of gallbladder showed a discontinuous smooth muscle layer without any interruption (Fig. 1A). The smooth muscle layer of cystic duct (Fig. 2A) and CBD (Fig. 3A) was discontinuous and interrupted in all 11 cystic duct cases and in all 10 CBD cases. 3.2. Desmin staining of muscle layers in gallbladder, cystic duct, and CBD With desmin, strong and diffuse (+3) cytoplasmic staining was observed in all gallbladders (15/15, 100%), highlighting a single discontinuous layer without areas devoid of smooth muscle fibers (Fig. 1B, Table 1). In contrast, the desmin staining highlighted a discontinuous and interrupted muscle layer in all cystic ducts and CBDs. Strong and diffuse (+3) stain with desmin was seen in all cystic (11/11) (Fig. 2B) and in 9 of 10 CBDs (Fig. 3B). The remaining 1 case of CBD stained +2. 3.3. Smoothelin staining of muscle layers in gallbladder, cystic duct and CBD Smoothelin intensely stained (+2 or +3) muscle fibers in the gallbladder wall similar to that observed with desmin staining (11/15, 73%) (Fig. 1C, Table 2). Weak or absent immunostaining was seen in 4 of 11 cases of the gallbladder sections (Fig. 2C). In contrast, the smoothelin expression in CBD was predominantly absent or focally weak (7/10, 70%; 0 or +1 staining), with only a few cases (3/10, 30%) having + 2 staining (Fig. 3C) and no case with +3 immunostaining. Cystic ducts also showed absent or focal immunostaining with smoothelin (6/11 cases, 55%). Five of these 11 cases
Table 1 Comparative immunohistochemical staining of muscle layer of gallbladder, cystic duct, and CBD with desmin
Desmin Negative Weak Strong
Score
Gallbladder
Cystic duct
CBD
0 +1 +2 +3
0/15 0/15 0/15 15/15
0/11 0/11 0/11 11/11
0/10 0/10 1/10 9/10
Smoothelin Negative Weak Strong
Score
Gallbladder
Cystic duct
CBD
0 1+ 2+ 3+
0/15 4/15 7/15 4/15
3/11 3/11 5/11 0/11
4/10 3/10 3/10 0/10
4. Discussion The normal histology of the muscle layers of gallbladder, cystic duct, and CBD is different from the remaining gastrointestinal tract; and the published descriptions are very limited. The layers of the gallbladder include the mucosa, one smooth muscle layer, perimuscular subserosal connective tissue, and serosa [6]. The smooth muscle layer of gallbladder consists of loosely arranged bundles of circular, longitudinal, and oblique fibers. The thickness of the muscle layer is quite variable, which may reflect variable contractile states of the gallbladder in different specimens. The smooth muscle layer of the gallbladder has been reported to be closely associated to “MP” with lack of MM and submucosa [6]. The wall of the cystic duct is histologically similar to that of the gallbladder. There is luminal mucosal epithelium, beneath which there are thin groups of smooth muscle fibers. The smooth muscle fibers are believed to prevent both overdistension and collapse of the cystic duct when it is subjected to changes in pressure as well to promote local stirring at the mucosal surface, to improve secretion and the absorption of nutrients. Although this layer might represent MM because of its function and thickness, its true nature has not been fully defined. The CBD that serves as conduit for the flow of the bile is lined by a single layer of tall columnar epithelial cells with an underlying dense connective tissue layer. The distribution of the smooth muscle fibers varies throughout the level of the CBD. There are scattered or no muscle fibers in the upper one third, whereas there is a continuous or interrupted pattern of thin to thick smooth muscle bundles throughout the lower portion [7]. These muscle fibers are frequently longitudinally aligned in the lower portion. Some authors have concluded that the supraduodenal portion of the CBD lacks smooth muscle [8]. Others found that scattered bundles of smooth muscle are present in small but significant amounts [9,10]. In a study of urinary bladder cases stained, the smooth muscle bundles/fibers of the MM either were absent (19/42, 45%) or showed weak and focal (18/42, 43%) staining with smoothelin. However, the bundles of smooth muscle of MP
412
K. Raparia et al. / Annals of Diagnostic Pathology 14 (2010) 408–412
showed strong and diffuse staining (36/42, 86%) with smoothelin [4]. The authors concluded that relatively distinct immunohistochemical staining patterns of smoothelin between MP and MM (including its hyperplastic forms) highlight its usefulness in the challenging cases of staging bladder urothelial carcinoma in the transurethral resection of bladder tumor specimens [4]. In a more recent study, the authors confirmed the relatively distinct staining pattern of smoothelin between MM and MP; however, they observed overlap of intensity in a few cases [11]. In our study, we found strong and diffuse (+3) immunostaining with desmin in all gallbladders (15/15, 100%), highlighting one layer of smooth muscle layer, which is discontinuous but without any interruptions. In contrast, the smooth muscle layer in all cystic ducts and CBDs was thin, discontinuous, and interrupted, but was still highlighted by the desmin stain. Smoothelin intensely stained (+2 or +3) the smooth muscle fibers in the gallbladder wall similar to that observed with desmin staining (11/15, 73%). This finding indicated that the muscle layer in the gallbladder is “MP” rather than “MM.” To accommodate the contractile function of bile accumulation, the gallbladder needs fully developed contractile smooth muscle fibers. The CBD has a thin and discontinuous muscle layer with absent or focally weak immunostaining (0 or +1 staining) with smoothelin (7/10, 70%), and only a few cases (3/10, 30%) have +2 staining. Similarly, cystic ducts also show absent or focally weak immunostaining with smoothelin, with 5 of 11 cases (44%) showing +2 immunostaining with smoothelin. Therefore, the muscle layer in cystic duct and CBD is not fully developed and is more compatible with MM than with MP. Functionally, unlike gallbladder, the cystic duct and CBD are not required to have a contractile function for bile but for the passage of the bile. The presence or absence of MM not only is of academic interest, but also has clinical significance in tumor staging. Thus, the distinction between MM and MP provides basis for the correct assessment of the depth of invasion in carcinomas of the gallbladder, cystic duct, or CBD. Although depth of invasion is an important prognostic factor for carcinomas of the CBD [12], the level of invasion is often difficult to evaluate because the muscle layer of the CBD is indistinct, especially in the proximal aspect of it. The current TNM staging system of the CBD defines T1 as tumor invading the subepithelial connective tissue or fibromuscular layer (tumor confined to the bile duct
histologically) and T2 as tumor invading beyond the wall of the bile duct [13]. The presence of smooth muscle fibers can be used as a landmark to delineate the layers and further to subclassify the tumor staging. In summary, the gallbladder contains a relatively thick, discontinuous layer of smooth muscle bundle that appears to be MP, rather than MM. The cystic duct and CBD contain only an attenuated and incomplete muscle layer that appears to be MM, suggesting no or limited contractile function. These anatomical muscle structures may be important for the correct pathologic staging of carcinoma of these organs. References [1] Vakar-Lopez F, Shen SS, Zhang S, Tamboli P, Ayala AG, Ro JY. Muscularis mucosae of the urinary bladder revisited with emphasis on its hyperplastic patterns: a study of a large series of cystectomy specimens. Ann Diagn Pathol 2007;11:395-401. [2] Kramer J, Aguirre-Arteta AM, Thiel C, et al. A novel isoform of the smooth muscle cell differentiation marker smoothelin. J Mol Med 1999;77:294-8. [3] van der Loop FT, Schaart G, Timmer ED, Ramaekers FC, van Eys GJ. Smoothelin, a novel cytoskeletal protein specific for smooth muscle cells. J Cell Biol 1996;134:401-11. [4] Paner GP, Shen SS, Lapetino S, et al. Diagnostic utility of antibody to smoothelin in the distinction of muscularis propria from muscularis mucosae of the urinary bladder: a potential ancillary tool in the pathologic staging of invasive urothelial carcinoma. Am J Surg Pathol 2009;33:91-8. [5] Council L, Hameed O. Differential expression of immunohistochemical markers in bladder smooth muscle and myofibroblasts, and the potential utility of desmin, smoothelin, and vimentin in staging of bladder carcinoma. Mod Pathol 2009;22:639-50. [6] Mills SE. Histology for pathologists. 3rd ed. Lippincott Williams & Wilkins; 2006. [7] Hong SM, Kang GH, Lee HY, Ro JY. Smooth muscle distribution in the extrahepatic bile duct: histologic and immunohistochemical studies of 122 cases. Am J Surg Pathol 2000;24:660-7. [8] Mahour GH, Wakim KG, Soule EH, Ferris DO. Structure of the common bile duct in man: presence or absence of smooth muscle. Ann Surg 1967;166:91-4. [9] Burden VG. Observations on the histologic and pathologic anatomy of the hepatic, cystic, and common bile ducts. Ann Surg 1925;82: 584-97. [10] Ludwick JR. Observations on the smooth muscle and contractile activity of the common bile duct. Ann Surg 1966;164:1041-50. [11] Miyamoto H, Sharma RB, Illei PB, Epstein JI. Pitfalls in the use of smoothelin to identify muscularis propria invasion by urothelial carcinoma. Am J Surg Pathol 2010;34:418-22. [12] Tsunoda T, Eto T, Koga M, et al. Early carcinoma of the extrahepatic bile duct. Jpn J Surg 1989;19:691-8. [13] AJCC cancer staging handbook. 7th ed. New York, NY: Springer; 2010. p. 255-76.