ORIGINAL ARTICLE
Villin Immunohistochemistry Is a Reliable Method for Diagnosing Microvillus Inclusion Disease Nick M. Shillingford, MD,* Monica L. Calicchio, MS,* Lisa A. Teot, MD,* Theonia Boyd, MD,* Kyle C. Kurek, MD,* Jeffrey D. Goldsmith, MD,* Athos Bousvaros, MD,w Antonio R. Perez-Atayde, MD,* and Harry P.W. Kozakewich, MD*
Abstract: Microvillus inclusion disease (MVID) is a rare congenital disorder that manifests early in infancy as intractable watery diarrhea. The entity is characterized morphologically by a deficient brush border and apical cytoplasmic inclusions within absorptive cells (enterocytes) due to misplaced assembly of brush border proteins. The diagnosis is based upon histopathology, special stains, immunohistochemistry (IHC), and ultimately upon electron microscopy. Currently, the periodic acid-Schiff stain (PAS) and CD10 IHC are commonly used as adjuncts, but in addition to brush border structures, they stain a variety of apical cytoplasmic inclusions and organelles, thereby interfering with recognition of microvillus inclusions. Villin is a protein that specifically binds to the actin core bundle of microvilli. We utilized villin IHC in formalin-fixed paraffin-embedded gastrointestinal biopsies from 6 patients with MVID, 5 with celiac disease, and 17 children with normal intestinal biopsies and compared the results with those obtained with CD10 IHC and PAS staining. All MVID cases had confirmatory electron microscopy at the time of diagnosis. Villin immunoreactivity was restricted to the brush border in the control groups. In MVID, villin IHC showed attenuation or loss of the surface brush border and also highlighted the cytoplasmic microvillus inclusions with clarity. In MVID, CD10 IHC and the PAS stain also showed attenuation or loss of the surface brush border, but staining of a variety of cytoplasmic structures largely obscured the microvillus inclusions. In sum, villin IHC is a reliable and superior adjunct in the diagnosis of MVID. Study of additional cases will determine whether villin IHC would obviate the need for electron microscopic confirmation. Key Words: intractable diarrhea, infancy, celiac disease (Am J Surg Pathol 2015;39:245–250)
From the *Department of Pathology; and wDivision of Gastroenterology, Boston Children’s Hospital, Harvard Medical School, Boston, MA. Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article. Antonio R. Perez-Atayde is co-senior author (e-mail: antonio. [email protected]). Correspondence: Harry P.W. Kozakewich, MD, Department of Pathology, Boston Children’s Hospital, 300 Longwood Ave, Boston, MA 02115 (e-mail: [email protected]). Copyright r 2014 Wolters Kluwer Health, Inc. All rights reserved.
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Volume 39, Number 2, February 2015
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icrovillus inclusion disease (MVID) is a rare autosomal recessive disorder characterized by profuse watery diarrhea beginning shortly after birth. The disease is progressive and fatal unless intestinal transplantation is performed. Intestinal biopsies show villous atrophy with variable absence of the brush border of enterocytes, increase in lysosome-like inclusions, and characteristic microvillus inclusions containing misplaced brush border structures. The extensive absence or misplacement of surface microvilli leads to the inability to absorb even the simplest of nutrients.1,2 Since the first report of MVID in 1978, light and electron microscopic evaluation of intestinal biopsies have been traditionally used for the diagnosis, although special stains, histochemistry, and immunohistochemistry (IHC) have aided in establishing the diagnosis. Currently, the periodic acid-Schiff (PAS) staining and CD10 IHC are the most commonly used adjuncts, followed by confirmatory ultrastructural examination.2,3 Villin, an actin-binding protein of the enterocyte brush border, is thought to participate in the assembly of other brush border proteins.4–6 Villin IHC demonstrating microvillus inclusions in MVID was first reported in a review article on neonatal enteropathies in 2004, but there have been no additional studies of its utility.7 We sought to further explore villin IHC in the diagnosis of MVID and to compare it with other currently used methodologies.
MATERIALS AND METHODS This study was undertaken after approval of the Institutional Review Board of Boston Children’s Hospital. We reviewed the medical records and endoscopic biopsies of 6 children with a confirmed diagnosis of MVID (8 duodenal, 5 gastric, 4 colonic, 1 terminal ileal), 5 with celiac disease (all duodenal), and 17 with nondescript abdominal pain without discernible origin and normal endoscopic biopsies (17 duodenal, 9 gastric, 5 colonic, 5 terminal ileal). The 6 infants with MVID were the only patients in the files of the Pathology Department who were diagnosed with the condition between 1993 and 2012. All cases of MVID had confirmatory electron microscopy. In only 1 patient genetic studies had been carried out and were found to harbor 2 variant mutations in the MYO5B gene: c.947G > T and c.1906-1G > A. www.ajsp.com |
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All the biopsies were stained with antibodies to villin, and all the biopsies from patients with MVID, celiac disease, and selected representative normal controls were stained with CD10 and PAS. The antibodies utilized were villin (CWWB1) (Ventana, Tucson, AZ), and CD10 (SP67) (Ventana), mouse monoclonal and rabbit monoclonal, respectively, using the Ventana Discovery XT automated IHC slide processing platform according to the manufacturer’s instructions (Ventana Medical Systems). After optimization of the antibodies, the best results were obtained as follows: 30 minutes EDTA pretreatment and 16 minutes incubation for the prediluted villin antibody and 30 minutes EDTA pretreatment and 32 minutes incubation for the prediluted CD10 antibody. As CD10 IHC highlighted not only brush border and the microvillus inclusions but also cytoplasmic organelles, we attempted to diminish the intensity of staining by further diluting the antibody and decreasing the incubation time; this produced an overall similar staining pattern but less intense with only faint staining of the microvillus inclusions.
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For quantifying the number of microvillus inclusions observed with villin IHC, 1 section on the slide was evaluated; when multiple sections or biopsies were present, only the profile with the greatest surface length was examined.
RESULTS Hematoxylin and eosin–stained sections from the duodenal and terminal ileal biopsies from the patients with MVID revealed the characteristic features of the disease with varying degrees of villous atrophy, preservation of the columnar shape of enterocytes, and diminished or absent brush border. Sections from the stomach and colon did not show any significant pathologic changes. Villin IHC of duodenal, ileal, and colonic biopsies from the normal controls and celiac disease showed an intensely stained microvillus brush border and only a faint cytoplasmic blush (Figs. 1, 2). The brush border was also seen in the gastric biopsies, although it appeared diminished (Fig. 1A). The brush border in celiac disease appeared less uniform and slightly shortened (Fig. 2C). In normal
FIGURE 1. Villin IHC in normal controls. Distinct delineation of brush border is observed in (A) stomach, (B) duodenum, (C) ileum, and (D) colon. In stomach and colon the brush border is shorter. A faint cytoplasmic blush in absorptive cells is present, but cytoplasmic organelles and goblet cells are negative.
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Villin IHC in Diagnosis of MVID
FIGURE 2. Villin and CD10 IHC in normal controls and celiac disease. A, Villin IHC in normal villous tip showing uniform, welldemarcated brush border and no significant cytoplasmic staining. B, CD10 IHC in normal villous tip showing uniform, welldemarcated brush border and numerous intensely stained cytoplasmic structures including the supranuclear Golgi complex. C, Villin IHC in celiac disease showing an irregular and shortened brush border. D, CD10 IHC in celiac disease similarly showing an irregular and shortened brush border as well as numerous intensely stained cytoplasmic structures including the supranuclear Golgi complex.
controls and celiac disease, CD10 IHC also highlighted the brush border with clarity; however, there was significant nonspecific staining of cytoplasmic structures (Figs. 2B, D). With the PAS, the staining pattern was similar to that obtained with CD10 IHC. In all duodenal, ileal, and colonic biopsies from patients with MVID, villin immunostaining was distinct, with the linear surface staining interrupted by stretches where it was markedly diminished or absent, corresponding to areas deficient in or devoid of microvilli, and with crisply delineated cytoplasmic microvillus inclusions, the latter seen only in duodenal and ileal biopsies (Fig. 3). No other cytoplasmic structures were immunoreactive for villin. The staining pattern with CD10 IHC was similar with focal deficient or absent surface immunopositivity as well as strong staining of microvillus inclusions within the cytoplasm. However, because CD10 IHC also stained cytoplasmic structures other than microvillus inclusions, the latter were more difficult to discern (Fig. 3B). PAS staining Copyright
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was similar to CD10 IHC, and mucin was also positive; nevertheless, some microvillus inclusions were discernible (Fig. 3C). In addition, at highest magnification, individual microvilli within the cytoplasmic microvillus inclusions were clearly visualized with villin IHC but not with PAS staining or CD10 IHC (Fig. 4). In the duodenal biopsies, there was a considerable range in the number of microvillus inclusions in the single selected profile. The respective counts per profile (biopsy surface length in millimeters shown in parenthesis) were: 44 (3.2 mm), 18 (2.8 mm), 16 (2.7 mm), 10 (2.4 mm), 5 (3.0 mm), 2 (2.3 mm), and 2 (0.3 mm). Microvillus inclusions were unevenly distributed, often clustered, and were not observed deep in the crypts. There was a suggestion that loss of surface microvilli in a given cell correlated with the presence of microvillus inclusions in that cell; however, a conclusion in this regard could not be reached, as the plane of sectioning did not always depict the entire profile of the cell. Gastric biopsies showed weak surface immunopositivity for villin, www.ajsp.com |
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and microvillus inclusions were not identified. There were no convincing microvillus inclusions observed in colonic biopsies with villin IHC, although occasional cytoplasmic immunoreactive structures raised that possibility. CD10 IHC and PAS staining showed a pattern similar to that in small bowel with nonspecific staining and no discernible microvillus inclusions. Electron microscopy of duodenal endoscopic biopsies in all cases of MVID showed diagnostic features with patchy loss or markedly shortened microvilli on the surface of absorptive cells and the characteristic apical cytoplasmic microvillus inclusions (Fig. 4C).
DISCUSSION
FIGURE 3. Villin and CD10 IHC and PAS staining of MVID in adjacent sections of the duodenal biopsy. A, Villin IHC depicting markedly shortened and focally absent brush border as well as numerous well-delineated, ring-like microvillous inclusions. B, CD10 IHC also reveals diminished or absent brush border, but in contrast to villin IHC, the microvillus inclusions are indistinguishable from other similarly stained cytoplasmic structures. C, PAS stain showing diminished brush border and microvillus inclusions partly obscured by intense staining of other apical cytoplasmic structures and goblet cells.
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MVID is a rare familial, progressive, and, if untreated, uniformly lethal disorder involving primarily the intestinal tract and manifesting as intractable secretory diarrhea.2,8 Affected infants are currently treated with total parenteral nutrition, followed in due course by intestinal transplantation. The major histopathologic findings in MVID are villous atrophy involving small intestinal mucosa, shortened or absent microvilli, and cytoplasmic microvillus inclusions in absorptive cells. These microvillus inclusions are best visualized by electron microscopy, which has traditionally been required for a definitive diagnosis of MVID.9–11 These inclusions have also been described in epithelial cells lining the renal tubules, stomach, and gallbladder.8,12,13 Over the years a number of histochemical and IHC stains have been used as adjuncts in the diagnosis of MVID. PAS staining was introduced in 1985 to demonstrate the diminished, absent, or misplaced brush border.10 Histochemical staining for alkaline phosphatase, an enzyme present within the glycocalyx, was utilized in 1988 by Lake.9 As these stains highlight the brush border, areas of microvillus attenuation or loss are readily recognized, in addition to misplaced clumps of activity within the apical cytoplasm of enterocytes.3,14 Polyclonal carcinoembryonic antigen IHC was first used by Groisman et al15 in 1993 reporting the absence of linear staining of the glycocalyx of surface and upper crypt cells and prominent cytoplasmic reactivity, likely corresponding to microvillus inclusions. Polyclonal antibody to carcinoembryonic antigen, however, also stains intracytoplasmic organelles and mucin in goblet cells, thereby obscuring the microvillus inclusions. In 2002, CD10 IHC was described by the same group as another useful marker in diagnosing MVID.16 However, immunopositivity is also present in the Golgi apparatus and other structures interfering with identification of the microvillus inclusions. Sherman and colleagues in 2004 showed for the first time that villin IHC demonstrated the microvillus inclusions but did not elaborate on its utility or compare the results with those obtained by other stains.2,7 In 2012, an antibody against the actin-binding protein Rab11 was utilized.17 The Rab family of small GTPases in certain recycling systems interacts with myosin Vb motor protein, Copyright
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FIGURE 4. Villin IHC and ultrastructure of duodenal biopsy in MVID. A, Microvillus ring-like inclusions. B, Magnified view of (A) showing radially oriented individual microvilli within inclusions. C, Electron photomicrograph depicting subtotal loss of brush border microvilli and cytoplasmic variably formed microvillus inclusions.
which is the major actin-bundling protein found predominantly in microvilli of enterocytes and renal epithelial cells. Mutations in the MYO5B gene that encodes Copyright
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Villin IHC in Diagnosis of MVID
this motor protein account for nearly all infants with MVID.2,18,19 Rab11 IHC has a staining pattern identical to that of PAS and CD10, with diffuse apical cytoplasmic staining in enterocytes. Recently, mutations in STX3 gene, which encodes syntaxin 3, an apical receptor involved in membrane fusion of enterocyte vesicles, have been described in infants with MVID lacking mutations in MYOB5.20 IHC absence of syntaxin 3 was shown in duodenal biopsies of infants with this MVID variant. In current practice, PAS staining and CD10 IHC are the most common adjuncts in diagnosing MVID, followed by confirmatory electron microscopy. Villin, a 92.5-kDa epithelial cell–specific actin-binding protein, is expressed in differentiated epithelium with a microvillus brush border such as intestinal, renal, oviductal, and gustatory, as well as in osteocytes. Additionally, villin is expressed in cells devoid of microvillus structures such as intestinal crypt and M cells. Microvilli are found on the adluminal surface of these cells and contain a core of crosslinked actin filaments. Villin is the major protein associated with actin filaments in the microvillus core and within the terminal web. It is believed to participate in the capping, bundling, and severing of actin filaments in microvilli.4–6 Other biological functions of villin include the regulation of cell morphology, signal transduction, cell migration, and apoptosis.6 Villin IHC has been reported to be useful in oncologic pathology. Villin is commonly expressed in gastrointestinal, pancreatic, and biliary carcinomas, particularly when glandular differentiation is present. Pulmonary, renal, and endometrioid carcinomas, as well as neuroendocrine tumors may also be positive.21,22 Villin IHC is especially useful in certain circumstances, such as when breast carcinoma is considered in the differential diagnosis of metastatic disease, as this carcinoma is consistently negative for villin. Other types of neoplasia that are immunonegative for villin include ovarian serous adenocarcinoma, urothelial carcinoma, prostatic adenocarcinoma, and mesothelioma. In this study, we describe the IHC expression of villin compared with that of CD10 IHC and PAS staining in MVID specimens. The staining pattern with villin IHC clearly demonstrated the microvillus inclusions and the brush border of enterocytes delineating regions of the surface mucosa with diminished or absent microvilli. The villin expression was restricted to the brush border and microvillus inclusions without the confounding staining of cytoplasmic organelles or other cytoplasmic constituents encountered with CD10 IHC and PAS staining. In summary, villin IHC is an optimal adjunct in the diagnosis of MVID and appears as effective as electron microscopy in the diagnostic confirmation of the disease. Further, in contrast to electron microscopy, IHC assesses a larger tissue sample than electron microscopy, is more expeditious, less laborious, and less costly. ACKNOWLEDGMENT The authors would like to thank Mr Howard Mulhern for his excellent technical work in electron microscopy. www.ajsp.com |
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REFERENCES 1. Davidson GP, Cutz E, Hamilton JR, et al. Familial enteropathy: a syndrome of protracted diarrhea from birth, failure to thrive, and hypoplastic villus atrophy. Gastroenterology. 1978;75:783–790. 2. van der Velde KJ, Dhekne HS, Swertz MA, et al. An overview and online registry of microvillus inclusion disease patients and their MYO5B mutations. Hum Mutat. 2013;34:1597–1605. 3. Cutz E, Rhoads JM, Drumm B, et al. Microvillus inclusion disease: an inherited defect of brush-border assembly and differentiation. N Engl J Med. 1989;320:646–651. 4. Friederich E, Vancompernolle K, Louvard D, et al. Villin function in the organization of the actin cytoskeleton. Correlation of in vivo effects to its biochemical activities in vitro. J Biol Chem. 1999; 274:26751–26760. 5. George SP, Wang Y, Mathew S, et al. Dimerization and actinbundling properties of villin and its role in the assembly of epithelial cell brush borders. J Biol Chem. 2007;282:26528–26541. 6. Khurana S, George SP. Regulation of cell structure and function by actin-binding proteins: villin’s perspective. FEBS Lett. 2008;582: 2128–2139. 7. Sherman PM, Mitchell DJ, Cutz E. Neonatal enteropathies: defining the causes of protracted diarrhea of infancy. J Pediatr Gastroenterol Nutr. 2004;38:16–26. 8. Rund CR, Carmichael B, Harris AA, et al. Small bowel biopsy in a 6-week-old infant. Microvillous inclusion disease of the small intestine. Arch Pathol Lab Med. 2006;130:e19–e21. 9. Lake BD. Microvillus inclusion disease: specific diagnostic features shown by alkaline phosphatase histochemistry. J Clin Pathol. 1988; 41:880–882. 10. Phillips AD, Jenkins P, Raafat F, et al. Congenital microvillous atrophy: specific diagnostic features. Arch Dis Child. 1985;60:135–140. 11. Pohl JF, Shub MD, Trevelline EE, et al. A cluster of microvillous inclusion disease in the Navajo population. J Pediatr. 1999;134:103–106.
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12. Schofield DE, Agostini RM Jr, Yunis EJ. Gastrointestinal microvillus inclusion disease. Am J Clin Pathol. 1992;98:119–124. 13. Thomas N, Pulimood AB, Kumar M, et al. Microvillous inclusion disease diagnosed by gastric biopsy. Indian Pediatr. 2012;49:58–60. 14. Perez-Atayde AR, Katz A, Flores A, et al. Diarrheal syndrome characterized by defective assembly of absorptive cell brush borders. Lab Invest. 1987;56:59A. 15. Groisman GM, Ben-Izhak O, Schwersenz A, et al. The value of polyclonal carcinoembryonic antigen immunostaining in the diagnosis of microvillous inclusion disease. Hum Pathol. 1993;24:1232–1237. 16. Groisman GM, Amar M, Livne E. CD10: a valuable tool for the light microscopic diagnosis of microvillous inclusion disease (familial microvillous atrophy). Am J Surg Pathol. 2002;26: 902–907. 17. Talmon G, Holzapfel M, DiMaio DJ, et al. Rab11 is a useful tool for the diagnosis of microvillous inclusion disease. Int J Surg Pathol. 2012;20:252–256. 18. Erickson RP, Larson-Thome K, Valenzuela RK, et al. Navajo microvillous inclusion disease is due to a mutation in MYO5B. Am J Med Genet A. 2008;146A:3117–3119. 19. Muller T, Hess MW, Schiefermeier N, et al. MYO5B mutations cause microvillus inclusion disease and disrupt epithelial cell polarity. Nat Genet. 2008;40:1163–1165. 20. Wiegerinck CL, Janecke AR, Schneeberger K, et al. Loss of syntaxin 3 causes variant microvillus inclusion disease. Gastroenterology. 2014;147:65–68. 21. Bacchi CE, Gown AM. Distribution and pattern of expression of villin, a gastrointestinal-associated cytoskeletal protein, in human carcinomas: a study employing paraffin-embedded tissue. Lab Invest. 1991;64:418–424. 22. Zhang PJ, Harris KR, Alobeid B, et al. Immunoexpression of villin in neuroendocrine tumors and its diagnostic implications. Arch Pathol Lab Med. 1999;123:812–816.
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Villin Immunohistochemistry Is a Reliable Method for Diagnosing Microvillus Inclusion Disease Nick M. Shillingford, MD,* Monica L. Calicchio, MS,* Lisa A. Teot, MD,* Theonia Boyd, MD,* Kyle C. Kurek, MD,* Jeffrey D. Goldsmith, MD,* Athos Bousvaros, MD,w Antonio R. Perez-Atayde, MD,* and Harry P.W. Kozakewich, MD*
Abstract: Microvillus inclusion disease (MVID) is a rare congenital disorder that manifests early in infancy as intractable watery diarrhea. The entity is characterized morphologically by a deficient brush border and apical cytoplasmic inclusions within absorptive cells (enterocytes) due to misplaced assembly of brush border proteins. The diagnosis is based upon histopathology, special stains, immunohistochemistry (IHC), and ultimately upon electron microscopy. Currently, the periodic acid-Schiff stain (PAS) and CD10 IHC are commonly used as adjuncts, but in addition to brush border structures, they stain a variety of apical cytoplasmic inclusions and organelles, thereby interfering with recognition of microvillus inclusions. Villin is a protein that specifically binds to the actin core bundle of microvilli. We utilized villin IHC in formalin-fixed paraffin-embedded gastrointestinal biopsies from 6 patients with MVID, 5 with celiac disease, and 17 children with normal intestinal biopsies and compared the results with those obtained with CD10 IHC and PAS staining. All MVID cases had confirmatory electron microscopy at the time of diagnosis. Villin immunoreactivity was restricted to the brush border in the control groups. In MVID, villin IHC showed attenuation or loss of the surface brush border and also highlighted the cytoplasmic microvillus inclusions with clarity. In MVID, CD10 IHC and the PAS stain also showed attenuation or loss of the surface brush border, but staining of a variety of cytoplasmic structures largely obscured the microvillus inclusions. In sum, villin IHC is a reliable and superior adjunct in the diagnosis of MVID. Study of additional cases will determine whether villin IHC would obviate the need for electron microscopic confirmation. Key Words: intractable diarrhea, infancy, celiac disease (Am J Surg Pathol 2015;39:245–250)
From the *Department of Pathology; and wDivision of Gastroenterology, Boston Children’s Hospital, Harvard Medical School, Boston, MA. Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article. Antonio R. Perez-Atayde is co-senior author (e-mail: antonio. [email protected]). Correspondence: Harry P.W. Kozakewich, MD, Department of Pathology, Boston Children’s Hospital, 300 Longwood Ave, Boston, MA 02115 (e-mail: [email protected]). Copyright r 2014 Wolters Kluwer Health, Inc. All rights reserved.
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M
icrovillus inclusion disease (MVID) is a rare autosomal recessive disorder characterized by profuse watery diarrhea beginning shortly after birth. The disease is progressive and fatal unless intestinal transplantation is performed. Intestinal biopsies show villous atrophy with variable absence of the brush border of enterocytes, increase in lysosome-like inclusions, and characteristic microvillus inclusions containing misplaced brush border structures. The extensive absence or misplacement of surface microvilli leads to the inability to absorb even the simplest of nutrients.1,2 Since the first report of MVID in 1978, light and electron microscopic evaluation of intestinal biopsies have been traditionally used for the diagnosis, although special stains, histochemistry, and immunohistochemistry (IHC) have aided in establishing the diagnosis. Currently, the periodic acid-Schiff (PAS) staining and CD10 IHC are the most commonly used adjuncts, followed by confirmatory ultrastructural examination.2,3 Villin, an actin-binding protein of the enterocyte brush border, is thought to participate in the assembly of other brush border proteins.4–6 Villin IHC demonstrating microvillus inclusions in MVID was first reported in a review article on neonatal enteropathies in 2004, but there have been no additional studies of its utility.7 We sought to further explore villin IHC in the diagnosis of MVID and to compare it with other currently used methodologies.
MATERIALS AND METHODS This study was undertaken after approval of the Institutional Review Board of Boston Children’s Hospital. We reviewed the medical records and endoscopic biopsies of 6 children with a confirmed diagnosis of MVID (8 duodenal, 5 gastric, 4 colonic, 1 terminal ileal), 5 with celiac disease (all duodenal), and 17 with nondescript abdominal pain without discernible origin and normal endoscopic biopsies (17 duodenal, 9 gastric, 5 colonic, 5 terminal ileal). The 6 infants with MVID were the only patients in the files of the Pathology Department who were diagnosed with the condition between 1993 and 2012. All cases of MVID had confirmatory electron microscopy. In only 1 patient genetic studies had been carried out and were found to harbor 2 variant mutations in the MYO5B gene: c.947G > T and c.1906-1G > A. www.ajsp.com |
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All the biopsies were stained with antibodies to villin, and all the biopsies from patients with MVID, celiac disease, and selected representative normal controls were stained with CD10 and PAS. The antibodies utilized were villin (CWWB1) (Ventana, Tucson, AZ), and CD10 (SP67) (Ventana), mouse monoclonal and rabbit monoclonal, respectively, using the Ventana Discovery XT automated IHC slide processing platform according to the manufacturer’s instructions (Ventana Medical Systems). After optimization of the antibodies, the best results were obtained as follows: 30 minutes EDTA pretreatment and 16 minutes incubation for the prediluted villin antibody and 30 minutes EDTA pretreatment and 32 minutes incubation for the prediluted CD10 antibody. As CD10 IHC highlighted not only brush border and the microvillus inclusions but also cytoplasmic organelles, we attempted to diminish the intensity of staining by further diluting the antibody and decreasing the incubation time; this produced an overall similar staining pattern but less intense with only faint staining of the microvillus inclusions.
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For quantifying the number of microvillus inclusions observed with villin IHC, 1 section on the slide was evaluated; when multiple sections or biopsies were present, only the profile with the greatest surface length was examined.
RESULTS Hematoxylin and eosin–stained sections from the duodenal and terminal ileal biopsies from the patients with MVID revealed the characteristic features of the disease with varying degrees of villous atrophy, preservation of the columnar shape of enterocytes, and diminished or absent brush border. Sections from the stomach and colon did not show any significant pathologic changes. Villin IHC of duodenal, ileal, and colonic biopsies from the normal controls and celiac disease showed an intensely stained microvillus brush border and only a faint cytoplasmic blush (Figs. 1, 2). The brush border was also seen in the gastric biopsies, although it appeared diminished (Fig. 1A). The brush border in celiac disease appeared less uniform and slightly shortened (Fig. 2C). In normal
FIGURE 1. Villin IHC in normal controls. Distinct delineation of brush border is observed in (A) stomach, (B) duodenum, (C) ileum, and (D) colon. In stomach and colon the brush border is shorter. A faint cytoplasmic blush in absorptive cells is present, but cytoplasmic organelles and goblet cells are negative.
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2014 Wolters Kluwer Health, Inc. All rights reserved.
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Villin IHC in Diagnosis of MVID
FIGURE 2. Villin and CD10 IHC in normal controls and celiac disease. A, Villin IHC in normal villous tip showing uniform, welldemarcated brush border and no significant cytoplasmic staining. B, CD10 IHC in normal villous tip showing uniform, welldemarcated brush border and numerous intensely stained cytoplasmic structures including the supranuclear Golgi complex. C, Villin IHC in celiac disease showing an irregular and shortened brush border. D, CD10 IHC in celiac disease similarly showing an irregular and shortened brush border as well as numerous intensely stained cytoplasmic structures including the supranuclear Golgi complex.
controls and celiac disease, CD10 IHC also highlighted the brush border with clarity; however, there was significant nonspecific staining of cytoplasmic structures (Figs. 2B, D). With the PAS, the staining pattern was similar to that obtained with CD10 IHC. In all duodenal, ileal, and colonic biopsies from patients with MVID, villin immunostaining was distinct, with the linear surface staining interrupted by stretches where it was markedly diminished or absent, corresponding to areas deficient in or devoid of microvilli, and with crisply delineated cytoplasmic microvillus inclusions, the latter seen only in duodenal and ileal biopsies (Fig. 3). No other cytoplasmic structures were immunoreactive for villin. The staining pattern with CD10 IHC was similar with focal deficient or absent surface immunopositivity as well as strong staining of microvillus inclusions within the cytoplasm. However, because CD10 IHC also stained cytoplasmic structures other than microvillus inclusions, the latter were more difficult to discern (Fig. 3B). PAS staining Copyright
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was similar to CD10 IHC, and mucin was also positive; nevertheless, some microvillus inclusions were discernible (Fig. 3C). In addition, at highest magnification, individual microvilli within the cytoplasmic microvillus inclusions were clearly visualized with villin IHC but not with PAS staining or CD10 IHC (Fig. 4). In the duodenal biopsies, there was a considerable range in the number of microvillus inclusions in the single selected profile. The respective counts per profile (biopsy surface length in millimeters shown in parenthesis) were: 44 (3.2 mm), 18 (2.8 mm), 16 (2.7 mm), 10 (2.4 mm), 5 (3.0 mm), 2 (2.3 mm), and 2 (0.3 mm). Microvillus inclusions were unevenly distributed, often clustered, and were not observed deep in the crypts. There was a suggestion that loss of surface microvilli in a given cell correlated with the presence of microvillus inclusions in that cell; however, a conclusion in this regard could not be reached, as the plane of sectioning did not always depict the entire profile of the cell. Gastric biopsies showed weak surface immunopositivity for villin, www.ajsp.com |
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and microvillus inclusions were not identified. There were no convincing microvillus inclusions observed in colonic biopsies with villin IHC, although occasional cytoplasmic immunoreactive structures raised that possibility. CD10 IHC and PAS staining showed a pattern similar to that in small bowel with nonspecific staining and no discernible microvillus inclusions. Electron microscopy of duodenal endoscopic biopsies in all cases of MVID showed diagnostic features with patchy loss or markedly shortened microvilli on the surface of absorptive cells and the characteristic apical cytoplasmic microvillus inclusions (Fig. 4C).
DISCUSSION
FIGURE 3. Villin and CD10 IHC and PAS staining of MVID in adjacent sections of the duodenal biopsy. A, Villin IHC depicting markedly shortened and focally absent brush border as well as numerous well-delineated, ring-like microvillous inclusions. B, CD10 IHC also reveals diminished or absent brush border, but in contrast to villin IHC, the microvillus inclusions are indistinguishable from other similarly stained cytoplasmic structures. C, PAS stain showing diminished brush border and microvillus inclusions partly obscured by intense staining of other apical cytoplasmic structures and goblet cells.
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MVID is a rare familial, progressive, and, if untreated, uniformly lethal disorder involving primarily the intestinal tract and manifesting as intractable secretory diarrhea.2,8 Affected infants are currently treated with total parenteral nutrition, followed in due course by intestinal transplantation. The major histopathologic findings in MVID are villous atrophy involving small intestinal mucosa, shortened or absent microvilli, and cytoplasmic microvillus inclusions in absorptive cells. These microvillus inclusions are best visualized by electron microscopy, which has traditionally been required for a definitive diagnosis of MVID.9–11 These inclusions have also been described in epithelial cells lining the renal tubules, stomach, and gallbladder.8,12,13 Over the years a number of histochemical and IHC stains have been used as adjuncts in the diagnosis of MVID. PAS staining was introduced in 1985 to demonstrate the diminished, absent, or misplaced brush border.10 Histochemical staining for alkaline phosphatase, an enzyme present within the glycocalyx, was utilized in 1988 by Lake.9 As these stains highlight the brush border, areas of microvillus attenuation or loss are readily recognized, in addition to misplaced clumps of activity within the apical cytoplasm of enterocytes.3,14 Polyclonal carcinoembryonic antigen IHC was first used by Groisman et al15 in 1993 reporting the absence of linear staining of the glycocalyx of surface and upper crypt cells and prominent cytoplasmic reactivity, likely corresponding to microvillus inclusions. Polyclonal antibody to carcinoembryonic antigen, however, also stains intracytoplasmic organelles and mucin in goblet cells, thereby obscuring the microvillus inclusions. In 2002, CD10 IHC was described by the same group as another useful marker in diagnosing MVID.16 However, immunopositivity is also present in the Golgi apparatus and other structures interfering with identification of the microvillus inclusions. Sherman and colleagues in 2004 showed for the first time that villin IHC demonstrated the microvillus inclusions but did not elaborate on its utility or compare the results with those obtained by other stains.2,7 In 2012, an antibody against the actin-binding protein Rab11 was utilized.17 The Rab family of small GTPases in certain recycling systems interacts with myosin Vb motor protein, Copyright
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Am J Surg Pathol
Volume 39, Number 2, February 2015
FIGURE 4. Villin IHC and ultrastructure of duodenal biopsy in MVID. A, Microvillus ring-like inclusions. B, Magnified view of (A) showing radially oriented individual microvilli within inclusions. C, Electron photomicrograph depicting subtotal loss of brush border microvilli and cytoplasmic variably formed microvillus inclusions.
which is the major actin-bundling protein found predominantly in microvilli of enterocytes and renal epithelial cells. Mutations in the MYO5B gene that encodes Copyright
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Villin IHC in Diagnosis of MVID
this motor protein account for nearly all infants with MVID.2,18,19 Rab11 IHC has a staining pattern identical to that of PAS and CD10, with diffuse apical cytoplasmic staining in enterocytes. Recently, mutations in STX3 gene, which encodes syntaxin 3, an apical receptor involved in membrane fusion of enterocyte vesicles, have been described in infants with MVID lacking mutations in MYOB5.20 IHC absence of syntaxin 3 was shown in duodenal biopsies of infants with this MVID variant. In current practice, PAS staining and CD10 IHC are the most common adjuncts in diagnosing MVID, followed by confirmatory electron microscopy. Villin, a 92.5-kDa epithelial cell–specific actin-binding protein, is expressed in differentiated epithelium with a microvillus brush border such as intestinal, renal, oviductal, and gustatory, as well as in osteocytes. Additionally, villin is expressed in cells devoid of microvillus structures such as intestinal crypt and M cells. Microvilli are found on the adluminal surface of these cells and contain a core of crosslinked actin filaments. Villin is the major protein associated with actin filaments in the microvillus core and within the terminal web. It is believed to participate in the capping, bundling, and severing of actin filaments in microvilli.4–6 Other biological functions of villin include the regulation of cell morphology, signal transduction, cell migration, and apoptosis.6 Villin IHC has been reported to be useful in oncologic pathology. Villin is commonly expressed in gastrointestinal, pancreatic, and biliary carcinomas, particularly when glandular differentiation is present. Pulmonary, renal, and endometrioid carcinomas, as well as neuroendocrine tumors may also be positive.21,22 Villin IHC is especially useful in certain circumstances, such as when breast carcinoma is considered in the differential diagnosis of metastatic disease, as this carcinoma is consistently negative for villin. Other types of neoplasia that are immunonegative for villin include ovarian serous adenocarcinoma, urothelial carcinoma, prostatic adenocarcinoma, and mesothelioma. In this study, we describe the IHC expression of villin compared with that of CD10 IHC and PAS staining in MVID specimens. The staining pattern with villin IHC clearly demonstrated the microvillus inclusions and the brush border of enterocytes delineating regions of the surface mucosa with diminished or absent microvilli. The villin expression was restricted to the brush border and microvillus inclusions without the confounding staining of cytoplasmic organelles or other cytoplasmic constituents encountered with CD10 IHC and PAS staining. In summary, villin IHC is an optimal adjunct in the diagnosis of MVID and appears as effective as electron microscopy in the diagnostic confirmation of the disease. Further, in contrast to electron microscopy, IHC assesses a larger tissue sample than electron microscopy, is more expeditious, less laborious, and less costly. ACKNOWLEDGMENT The authors would like to thank Mr Howard Mulhern for his excellent technical work in electron microscopy. www.ajsp.com |
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REFERENCES 1. Davidson GP, Cutz E, Hamilton JR, et al. Familial enteropathy: a syndrome of protracted diarrhea from birth, failure to thrive, and hypoplastic villus atrophy. Gastroenterology. 1978;75:783–790. 2. van der Velde KJ, Dhekne HS, Swertz MA, et al. An overview and online registry of microvillus inclusion disease patients and their MYO5B mutations. Hum Mutat. 2013;34:1597–1605. 3. Cutz E, Rhoads JM, Drumm B, et al. Microvillus inclusion disease: an inherited defect of brush-border assembly and differentiation. N Engl J Med. 1989;320:646–651. 4. Friederich E, Vancompernolle K, Louvard D, et al. Villin function in the organization of the actin cytoskeleton. Correlation of in vivo effects to its biochemical activities in vitro. J Biol Chem. 1999; 274:26751–26760. 5. George SP, Wang Y, Mathew S, et al. Dimerization and actinbundling properties of villin and its role in the assembly of epithelial cell brush borders. J Biol Chem. 2007;282:26528–26541. 6. Khurana S, George SP. Regulation of cell structure and function by actin-binding proteins: villin’s perspective. FEBS Lett. 2008;582: 2128–2139. 7. Sherman PM, Mitchell DJ, Cutz E. Neonatal enteropathies: defining the causes of protracted diarrhea of infancy. J Pediatr Gastroenterol Nutr. 2004;38:16–26. 8. Rund CR, Carmichael B, Harris AA, et al. Small bowel biopsy in a 6-week-old infant. Microvillous inclusion disease of the small intestine. Arch Pathol Lab Med. 2006;130:e19–e21. 9. Lake BD. Microvillus inclusion disease: specific diagnostic features shown by alkaline phosphatase histochemistry. J Clin Pathol. 1988; 41:880–882. 10. Phillips AD, Jenkins P, Raafat F, et al. Congenital microvillous atrophy: specific diagnostic features. Arch Dis Child. 1985;60:135–140. 11. Pohl JF, Shub MD, Trevelline EE, et al. A cluster of microvillous inclusion disease in the Navajo population. J Pediatr. 1999;134:103–106.
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12. Schofield DE, Agostini RM Jr, Yunis EJ. Gastrointestinal microvillus inclusion disease. Am J Clin Pathol. 1992;98:119–124. 13. Thomas N, Pulimood AB, Kumar M, et al. Microvillous inclusion disease diagnosed by gastric biopsy. Indian Pediatr. 2012;49:58–60. 14. Perez-Atayde AR, Katz A, Flores A, et al. Diarrheal syndrome characterized by defective assembly of absorptive cell brush borders. Lab Invest. 1987;56:59A. 15. Groisman GM, Ben-Izhak O, Schwersenz A, et al. The value of polyclonal carcinoembryonic antigen immunostaining in the diagnosis of microvillous inclusion disease. Hum Pathol. 1993;24:1232–1237. 16. Groisman GM, Amar M, Livne E. CD10: a valuable tool for the light microscopic diagnosis of microvillous inclusion disease (familial microvillous atrophy). Am J Surg Pathol. 2002;26: 902–907. 17. Talmon G, Holzapfel M, DiMaio DJ, et al. Rab11 is a useful tool for the diagnosis of microvillous inclusion disease. Int J Surg Pathol. 2012;20:252–256. 18. Erickson RP, Larson-Thome K, Valenzuela RK, et al. Navajo microvillous inclusion disease is due to a mutation in MYO5B. Am J Med Genet A. 2008;146A:3117–3119. 19. Muller T, Hess MW, Schiefermeier N, et al. MYO5B mutations cause microvillus inclusion disease and disrupt epithelial cell polarity. Nat Genet. 2008;40:1163–1165. 20. Wiegerinck CL, Janecke AR, Schneeberger K, et al. Loss of syntaxin 3 causes variant microvillus inclusion disease. Gastroenterology. 2014;147:65–68. 21. Bacchi CE, Gown AM. Distribution and pattern of expression of villin, a gastrointestinal-associated cytoskeletal protein, in human carcinomas: a study employing paraffin-embedded tissue. Lab Invest. 1991;64:418–424. 22. Zhang PJ, Harris KR, Alobeid B, et al. Immunoexpression of villin in neuroendocrine tumors and its diagnostic implications. Arch Pathol Lab Med. 1999;123:812–816.
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