Total Colonic Aganglionosis

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1 Total Colonic Aganglionosis Case Report, Practical Diagnostic Approach and Pitfalls Raja Rabah, MD N Hirschsprung disease remains a challenging diagnosis for many pathologists. The disease is characterized by a lack of ganglion cells in the myenteric and submucosal plexus, associated with increased numbers of acetylcholinesterasepositive nerve fibers. Hypertrophic nerve fibers are present in most but not all patients. Total colonic aganglionosis (TCA) is an uncommon form of Hirschsprung disease with clinical, histologic, and genetic differences and is even more difficult to diagnose and manage. This case illustrates some of the difficulties frequently faced by the pathologists dealing with total colonic aganglionosis. Suction rectal biopsy specimens often lack significant nerve hypertrophy and positive acetylcholinesterase staining, which aid in the diagnosis. Pathologists have to depend mainly on the lack of ganglion cells in adequate submucosa to establish the diagnosis. Transition zone is often long in total colonic aganglionosis and interpretation of frozen sections can be difficult. The presence of several uniformly distributed clusters of mature ganglion cells and lack of nerve hypertrophy are required to avoid connections at the transition zone. (Arch Pathol Lab Med. 2010;134: ) REPORT OF A CASE A 5-day-old full-term female neonate was brought to the emergency department with a complaint of emesis and absence of bowel movements. The patient was initially given bottle feedings but was unable to tolerate them and was switched to breast feedings. Her birth weight was 3.4 kg and development was appropriate for her age. We had additional medical history information that the father had Hirschsprung disease (HSCR) as a neonate. Barium enema showed a small-caliber descending left colon with a transition zone at the splenic flexure, with distended hepatic flexure and ascending colon. These findings were consistent with HSCR; however, they do not exclude functional immaturity. Rectal suction biopsy was performed. Accepted for publication May 25, From the Department of Pathology, University of Michigan, Ann Arbor. The author has no relevant financial interest in the products or companies described in this article. Presented at New Frontiers in Pathology: An Update for Practicing Pathologists, University of Michigan, Ann Arbor, Michigan, October 10, Reprints: Raja Rabah, MD, Department of Pathology, University of Michigan, 1500 E Medical Center Dr, Room 2G332, Ann Arbor, MI ( rajar@med.umich.edu). MICROSCOPIC DESCRIPTION Fifty serial sections were prepared from 2 fragments of rectal tissue and stained with hematoxylin-eosin (H&E). On microscopic examination, adequate amount of submucosa was present but no ganglion cells (GCs) were identified, and no significant nerve hypertrophy was present (Figure 1, a and b). Acetylcholinesterase (AChE) stain was performed on an additional frozen fragment but the results were inconclusive. Only few questionable nerve twigs were present in the muscularis mucosa and lamina propria (Figure 1c). Diagnosis of HSCR disease was made and the patient was taken to the operating room on the next day. Multiple seromuscular biopsy specimens were obtained during the procedure for frozen-section examination. A transverse colon biopsy specimen proximal to the presumed transition zone was negative for GCs (Figure 2, a). Biopsy specimens from the hepatic flexure and ascending colon revealed rare clusters of small GCs with pyknotic nuclei and some thick nerves between the muscular layers (Figure 2, b). The appendix did not show GCs (Figure 2, c). Biopsy specimens of distal terminal ileum and the proximal resection margin of the ileocolectomy showed only few clusters of GCs. An additional 2 cm of the ileum was resected and mature GCs appeared in good number in the margin (Figure 2, d). An ileocolectomy specimen was received, and representative sequential sections of the total length were submitted for histologic examination, which confirmed the diagnosis of distal aganglionosis up to the hepatic flexure (Figure 3, a). The ascending colon, cecum, and the distal ileum showed segmental areas of aganglionosis in the myenteric plexus alternating with hypoganglionic segments with scattered thick nerves. The GCs in these areas were abnormal, and some were noted in clusters but others were isolated. The nuclei were generally small and some were hyperchromatic. Many had degenerating ghost-cell appearance (Figure 3, b and c) and lacked the characteristic eccentric shape and conspicuous nucleolus expected for the normal myenteric plexus GCs. Adequate numbers of evenly distributed mature GCs were present in the proximal 2 cm of the ileum (Figure 3, d). This case is an example of total colonic aganglionosis with a long transition zone extending from the hepatic flexure to about 16 cm in the ileum. COMMENT Hirschsprung disease remains a challenging diagnosis for pediatric pathologists and especially for general Arch Pathol Lab Med Vol 134, October 2010 Total Colonic Aganglionosis Rabah 1467

2 pathologists who do not see these cases sufficiently to gain experience and are unfamiliar with optimal handling of specimens. There is also considerable variation in the approaches taken by surgeons submitting these samples and pathologists processing these cases; this adds confusion, making clear understanding of diagnostic objectives even more challenging. 1 This variation in approach extends to the recommended number of biopsies, site of sampling, number of sections necessary to be examined, and use of AChE stain. Some institutions base the diagnosis mainly on morphologic examination of serial sections of rectal biopsy specimens stained with H&E; others use AChE as the primary diagnostic tool and some use AChE as an adjunct tool to routine histology. 2 In Europe and Japan, some centers use a battery of histochemical stains on frozen sections of suction rectal biopsy specimens as their gold standard, and paraffin sections are not required. In all instances, the pathologist must be familiar with what constitutes an adequate sample, adequate number of sections, appropriate orientation, variation in the size and appearance of ganglion cells (GCs), and with interpretation of additional staining, if applicable. The standard approach for diagnosing HSCR is the rectal suction biopsy. The disease is defined by the absence of submucosal GCs and the presence of excessive cholinergic nerve fibers in an adequate biopsy specimen. 3,4 But what really defines an adequate biopsy specimen? Some factors related to adequacy are out of the pathologist s control, like the location, size, and number of pieces. Pathologists, however, should monitor appropriate handling of biopsy specimens in their laboratories, including orientation/embedding of biopsy samples and procurement of an adequate number of sections. There is a lack of consensus about where biopsy specimens should be obtained, 1 5 and the recommended sites in the surgical literature vary from 1 to 2 cm, 2 to 3 cm, or 4 cm from the pectinate line. Obtaining multiple biopsy samples at 1, 2, and 3 cm is suggested by others. The advantage of the latter approach is the recognition of ultrashort-segment HSCR. Most surgeons agree that biopsy samples should be obtained at least 2 to 3 cm above the pectinate line to safely avoid the physiologic hypoganglionic zone in the distal rectum. Biopsy specimens are considered inadequate for evaluation of HSCR if they contain squamous epithelium or striated muscle and lack GCs. However, these specimens should still be examined carefully, since finding GCs would exclude HSCR and obviate the need for repeated biopsy. At least 2 samples are needed for routine histology. 4 If AChE staining is to be performed, an additional piece is required and the tissue should be placed in saline wet gauze and sent to the laboratory immediately. The optimal size of the biopsy specimen is 3 to 3.5 mm in diameter and should include sufficient submucosa, with a minimum of 1 mm or one-third of the sample to be considered adequate. 4,5 Adequacy of submucosa is best evaluated microscopically by the presence r Figure 1. Suction rectal biopsy specimen with adequate amount of submucosa. No ganglion cells or nerve hypertrophy is identified in hematoxylin-eosin stained sections. a, Low-power view. b, High-power view. c, Acetylcholinesterase stain shows few thin nerve twigs (arrows) in the lamina propria (hematoxylin-eosin, original magnifications 3100 [a] and 3200 [b]; original magnification 3400 [c]) Arch Pathol Lab Med Vol 134, October 2010 Total Colonic Aganglionosis Rabah

3 Figure 2. Frozen sections stained with hematoxylin-eosin. a, No ganglion cells are identified between the muscular layers in this properly oriented seromuscular biopsy specimen from the aganglionic segment. b, Two small clusters (arrows) lacking characteristic features of mature ganglion cells are present in the transition zone. The nuclei are small and pyknotic and no cytoplasm is identified. c, Cross section of the appendix with no ganglion cells or thick nerves noted between the muscular layers. d, Section from the proximal ileal margin showing 3 clusters (large arrows) containing mature ganglion cells (small arrows) with characteristic eccentric nuclei and moderate amount of cytoplasm. Nucleoli are not usually well visualized in frozen sections (original magnifications 3200 [a and c] and 3400 [b and d]). of equal amount of submucosa (below the muscularis mucosa) to overlying mucosa in a well-oriented biopsy sample at most levels. Samples must be properly oriented on edge at embedding. A minimum of 50 well-oriented, not crushed, H&Estained serial sections with sufficient submucosa are required to exclude the presence of GCs and suggest the diagnosis of HSCR (Figure 4). Some centers recommend 75 to 100 levels. Pathologists may need to examine numerous levels or even exhaust the tissue if the amount of submucosa is borderline. If the findings are still inconclusive, a repeated biopsy should be recommended. In the neonatal period, submucosal GCs may not be easily recognized because they are small and immature and other cells such as lymphocytes, stromal cells, and endothelial cells can mimic GCs. Immature GCs are usually present in clusters called neural units, which are more readily recognized underneath the muscularis mucosa. They are best seen by examining the slide at low-power magnification (310), not looking for individ- Arch Pathol Lab Med Vol 134, October 2010 Total Colonic Aganglionosis Rabah 1469

4 Figure 3. Segment of ileum and colon proximal to the splenic flexure. Additional shorter segment of ileum is received separately (a). The cecum and ascending colon are relatively more dilated proximal to the hepatic flexure (arrow), marking the beginning of a long transition zone, which is characterized by segmental hypoganglionosis alternating with regions of aganglionosis and scattered thick nerves. The proximal additional 2 cm of ileum is more dilated than the rest of the ileum, giving an impression of a second transition zone (small arrows). This segment showed appropriate ganglionosis. b, Permanent section from the transition zone shows rare small clusters and individual cells with nuclear features of ganglion cells (arrow) and no appreciable cytoplasm. c, At high-power magnification, this section from transition zone shows an individual cell with cytoplasmic features of ganglion cell (arrow) and a thick nerve (stars). d, Representative permanent section from the additional proximal ileal segment with 3 clusters (arrows) containing mature ganglion cells with prominent nucleoli and moderate amount of cytoplasm (hematoxylin-eosin, original magnifications 3400 [b and d] and 3600 [c]). ual GCs (Figure 5, a). These units are composed of central, pale, neurofibrillary region with surrounding mixture of small cells including immature GCs and supporting Schwann cells. 3 Immature GCs have smaller and darker nuclei, with lower nuclear to cytoplasmic ratio when compared to GCs in older children and adults. They also have inconspicuous nucleoli and eccentric, pear-shaped, gray-blue cytoplasm without the apparent stippled Nissl substance seen in mature GCs. Nucleoli are often not well recognized in the neonate. The characteristic nuclear and cytoplasmic features may not all be present in each cell at each level. High-power examination at 320 and 340 and following these clusters in sequential levels will help to confirm the presence of bona fide GCs (Figure 5, b through f). When biopsy samples have adequate submucosa and are properly oriented and stained, identification of GCs is easy, and reviewing such samples does not usually require a long time. Often, GCs are found in the first few levels. However, when GCs are not present, especially in samples lacking hypertrophic nerves and/or in samples with inadequate amount of submucosa, additional levels and special stains will be needed and 1470 Arch Pathol Lab Med Vol 134, October 2010 Total Colonic Aganglionosis Rabah

5 Figure 4. Photograph of a slide tray with hematoxylin-eosin stained, properly oriented serial sections of a suction rectal biopsy specimen. longer examination times of up to 30 to 60 minutes may be required to establish the diagnosis of HSCR. Identification of even sparse numbers of GCs, even if immature, in a suction rectal biopsy specimen is sufficient to exclude HSCR. The mucosa should always be evaluated for inflammation, eosinophilic infiltrates, features of cystic fibrosis, or other pathologic changes. The presence of hypertrophic nerve fibers in the submucosa is helpful but nerve hypertrophy alone is not sufficient to establish the diagnosis of HSCR. 3,4 Most cases with classic HSCR show numerous prominent nerves (.40 mm diameter) in the submucosa (Figure 6, a). The degree of nerve hypertrophy is variable and it increases with age. Hypertrophic nerves may not be present in ultrashort- or long-segment HSCR. In suboptimal biopsy samples owing to insufficient submucosa, crush artifact, poor orientation, or other factors, AChE staining, which highlights increased activity in the parasympathetic nerve fibers, and absence of nerve cells can be valuable. This histochemical staining can only be done on cryosections of frozen tissue; it therefore requires an additional specimen. It is a technically challenging staining procedure that is not readily available in nonpediatric institutions. False-negative and false-positive results are frequently encountered, thereby requiring experienced, careful interpretation of the results. Most general laboratories, as mentioned above, do not use this stain routinely in their practice and the ones that do use it in conjunction with H&E and not as a replacement. 1,3,5 Biopsy specimens from patients with HSCR show AChE-positive thick fibers in the muscularis mucosa extending to a variable length in the lamina propria (Figure 6, b), and biopsy samples from ganglionic bowel may show only few or absent cholinergic fibers. Intensity of acetylcholinesterase staining apparently increases with age. Immunostaining for various neuronal and nerve markers such as neuron-specific enolase, synaptophysin, and S100 has been used by some investigators but is generally not found to be of any added value by most practicing pathologists. 5 For many pathologists who regularly examine rectal biopsy specimens, it is not difficult to find Figure 5. Microscopic examination of ganglion cells. a, Several neural units are seen in low power in the submucosa (arrows). b and c, At higher power, bona fide ganglion cells are confirmed (arrows). d through f, Same cluster of ganglion cells is followed at 3 different levels to identify all characteristic nuclear and cytoplasmic features (hematoxylin-eosin, original magnifications 3100 [a], 3200 [b], 3400 [c], and 3600 [d, e, and f]). Arch Pathol Lab Med Vol 134, October 2010 Total Colonic Aganglionosis Rabah 1471

6 Figure 6. a, Several hypertrophic nerves (.40 mm, arrows) are noted in the submucosa of this suction rectal biopsy specimen. b, Acetylcholinesterase-positive coarse nerve twigs are present in the muscularis mucosa and lamina propria (arrows) in this aganglionic biopsy specimen. c, Suction rectal biopsy specimen from ganglionic bowel stained with calretinin. Nerves in the lamina propria and muscularis mucosa are highlighted in addition to a neural unit in the submucosa (arrow). d, Suction rectal biopsy specimen stained with calretinin from patient with Hirschsprung disease. Complete absence of staining in the muscularis mucosa and lamina propria (hematoxylin-eosin, original magnification 3400 [a]; acetylcholinesterase, original magnification 3400 [b]; calretinin, original magnifications 3400 [c] and 340 [d]). GCs and thick nerves in well-stained H&E sections. When the biopsy samples are low, have insufficient submucosa, or are poorly oriented, the best stain would be a marker that is specific for HSCR, which is not yet available. Recent studies suggest that calretinin immunohistochemistry might be helpful in the workup of patients with HSCR.5 7 Calretinin is expressed in a subset of submucosal GCs and nerves and it also highlights thin nerves in the muscularis mucosa and lamina propria in normal colonic tissue (Figure 6, c). In HSCR cases, staining is completely absent (Figure 6, d). Preliminary reports and our own experience suggest that it is very promising and indeed may replace AChE in routine practice. This staining procedure can be performed on fixed tissue, eliminating the need for 1472 Arch Pathol Lab Med Vol 134, October 2010 additional pieces, is available in most laboratories, is reproducible, and is much easier to interpret. Our case is an example of total colonic aganglionosis (TCA), which is estimated to comprise about 2% to 10% of all HSCR cases. Total colonic aganglionosis has clinical, genetic, and histologic features that are different from the classic form of HSCR.8,9 It has been defined as aganglionosis that affects the entire colon, to at least the ileocecal valve, and a variable segment of ileum, not exceeding 50 cm. It tends to affect females and males nearly equally, as compared to the male predominance in the classic short-segment form. There is greater familial incidence and higher association with other malformations and abnormalities in the Ret gene. Total Colonic Aganglionosis Rabah

7 The TCA form is more difficult to manage and to diagnose. Suction rectal biopsy specimens often lack significant nerve hypertrophy and positive AChE staining, which aid in the diagnosis. Pathologists have to depend mainly on the lack of GCs in adequate submucosa to establish the diagnosis, and in some cases, repeated biopsies are needed for confirmation. Handling intraoperative seromuscular biopsies for ileostomy/colostomy or definitive pull-through can be especially challenging to the pathologist. Many biopsies are usually needed to identify the appropriate ganglionic segment. Total colonic aganglionosis is often associated with a long transition zone characterized by hypoganglionosis and the presence of abnormal immature cells individually and in clusters. 9 At the time of frozen section it may be very difficult to differentiate between hypoganglionic transition zone and normal ganglionic bowel, especially in small seromuscular biopsies. It is even more challenging for the pathologist, once GC clusters are seen in the frozen tissue, to appreciate the degree of maturity. A higher incidence of postsurgical obstruction is associated with TCA than with classic short-segment disease, which often requires reoperation and is thought to be related to transition zone pullthrough or ostomy. 9 Therefore, pathologists are required not only to find GCs intraoperatively but also to evaluate the quality and density of GCs to appropriately guide the surgical team. Surgeons also must be aware of the limitations of the intraoperative seromuscular biopsies and help pathologists by submitting adequate biopsy specimens at least 1 cm in length, with a depth of 3 to 5 mm, to include enough of the 2 muscular layers. 4 Proper orientation, embedding the biopsy on edge, and examining several levels will help. Recognizing GCs is usually not difficult but inflammation may sometimes obscure them. Adequate seromuscular biopsy samples from the ganglionic bowel should contain several relatively uniformly distributed clusters of GCs and should lack nerves between the muscular layers. Surgeons usually resect 2 to 3 cm proximally to the reported adequate biopsy sites. Some authors recommend frozen section examination of the proximal margin of the resected bowel to assure that GCs are present around the entire circumference. In summary, regardless of the approach used to diagnose HSCR, the pathologist must be aware of what constitutes adequate specimens, orientation, and staining quality in order to avoid pitfalls in diagnosis of HSCR. It is extremely important to be able to recognize biopsy samples that are not acceptable or borderline and clearly communicate the results to the surgeon and ask for repeated biopsy when indicated. It cannot be overemphasized that GCs or neural units must exhibit conclusive features to be bona fide. Ganglion cells show great variations in sizes and shapes, and other cells may mimic GCs. Establishing the diagnosis of HSCR should be strongly avoided during intraoperative procedures on suction biopsies owing to the produced artifact, time limitation, and inability to obtain the appropriate number of levels to examine. In emergent situations, frozen sections of adequate-size seromuscular biopsies may be helpful. Frozen sections for leveling ostomies or pullthroughs should be evaluated for GC quality, density, and distribution and hypertrophic nerves. The presence of few and even immature GCs in the submucosa would exclude HSCR in a suction rectal biopsy specimen; however, for leveling ostomies and pull-throughs, the pathologist should require the presence of several relatively uniformly distributed clusters of mature bona fide GCs and no nerves between the muscular layers to avoid the transition zone. References 1. Qualman SJ, Jaffe R, Bove KE, Monforte-Munoz H. Diagnosis of Hirschsprung disease using the rectal biopsy: multi-institutional survey. Pediatr Dev Pathol. 1999;2(6): Martucciello G. Hirschsprung s disease, one of the most difficult diagnoses in pediatric surgery: a review of the problems from clinical practice to the bench. Eur J Pediatr Surg. 2008;18(3): Gilbert-Barness E, Kapur RP, Laurier Oligny L, Siebert JR, eds. Potter s Pathology of the Fetus, Infant and Child. 2nd ed. St Louis, MO: Mosby/Elsevier; Knowles CH, De Giorgio R, Kapur RP, et al. Gastrointestinal neuromuscular pathology: guidelines for histological techniques and reporting on behalf of the Gastro 2009 International Working Group. Neuropathology. 2009;118(2): Kapur RP, Reed RC, Finn LS, Patterson K, Johanson J, Rutledge JC. Calretinin immunohistochemistry versus acetylcholinesterase histochemistry in the evaluation of suction rectal biopsies for Hirschsprung disease. Pediatr Dev Pathol. 2009;12(1): Guinard-Samuel V, Bonnard A, De Lagausie P, et al. Calretinin immunohistochemistry: a simple and efficient tool to diagnose Hirschsprung disease. Mod Pathol. 2009;22(10): Barshack I, Fridman E, Goldberg I, Chowers Y, Kopolovic. The loss of calretinin expression indicates aganglionosis in Hirschsprung s disease. J Clin Pathol. 2004;57(7): Moore SW. Total colonic aganglionosis and Hirshsprung s disease: shades of the same or different? Pediatr Surg Int. 2009;25(8): Moore SW, Zaahl M. Clinical and genetic differences in total colonic aganglionosis in Hirschsprung s disease. J Pediatr Surg. 2009;44(10): Arch Pathol Lab Med Vol 134, October 2010 Total Colonic Aganglionosis Rabah 1473