Impact of Age on Submucosal Nerve Measurements in Rectal Biopsies from Patients

Home , Adventitia, Anal columns, External anal sphincter, Internal anal sphincter, Muscularis mucosae, Splanchnic nerves

with Hirschsprung Disease

THESIS

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in

the Graduate School of The Ohio State University

Sarah Beach, B.S.

Graduate Program in Anatomy

The Ohio State University

2020

Thesis Committee

Melissa Quinn, PhD, Academic Advisor

Miriam Conces, MD, Research Advisor

Christopher Pierson, MD, PhD

James Cray, PhD

Copyrighted by

Sarah Beach

2020

Abstract

Hypertrophic submucosal nerves, defined as ≥ 40 microns in diameter, are considered supportive of a diagnosis of Hirschsprung disease (HSCR), but the effect of age on nerve diameter has not been well-studied. We sought to characterize the distribution of nerve diameter in ganglionated rectal biopsies and the significance of hypertrophic submucosal nerves in the diagnosis of Hirschsprung disease based on age.

Rectal biopsies were performed at Nationwide Children’s Hospital, Columbus,

Ohio, for the evaluation of Hirschsprung disease. The biopsies from 2017-2018 were retrospectively collected and reviewed. Hirschsprung disease status was determined by the presence or absence of ganglion cells. The diameter of the largest submucosal nerve was measured and compared between age groups.

Within the two-year period, 179 rectal biopsies with adequate submucosa were identified. Ganglion cells were present in 151 biopsies, and 28 aganglionic biopsies were diagnosed as Hirschsprung disease. Submucosal nerve diameter range was 17.5-101.5 microns in non-Hirschsprung disease biopsies (non-HSCR) and 14-98 microns in HSCR biopsies. Across all ages, hypertrophic submucosal nerves were significantly associated with Hirschsprung disease [HSCR = 25/28 (83%) vs non-HSCR = 59/151 (39.1%), p=<0.0001] and showed a sensitivity of 89.29% and specificity of 60.93%. Stratified by

ii age, the submucosal nerve diameter remained statistically significant for HSCR in patients <1 year of age [HSCR = 22/24 (91.7%) vs. non-HSCR 19/91 (20.9%), p=<0.0001] with sensitivity of 91.67% and specificity of 79.12%. Hypertrophic submucosal nerves were not statistically significant in patients ≥ 1 year of age [HSCR = 3/4 (75%) vs. non-

HSCR = 40/60 (66.7%), p=1] and showed reduced sensitivity (75%) and specificity

(33.33%) for the diagnosis for HSCR. Based on a receiver operating characteristic curve, a nerve diameter of 45 microns demonstrates the greatest sensitivity (88%) and specificity (83%) for HSCR.

The average submucosal nerve diameter in non-HSCR rectal biopsies increases with age. Hypertrophic submucosal nerves are significantly associated with HSCR in patients <1 year of age but may have limited utility for the diagnosis of HSCR in patients >1 year of age. However, our data includes very few patients with HSCR that are >1 year, and additional studies are needed to evaluate biopsies from older children.

iii

Dedication

This document is dedicated to my friends and family.

Acknowledgements

Without the help of each of my committee members, I would not have been able to perform this research project for my master’s thesis.

I would like to thank Dr. Miriam Conces for being a wonderful research coordinator and mentor. She set aside hours of her time in the past year to work on this project with me, from confirming every single nerve measurement to editing drafts of this document. She helped me learn so much about a disease I previously did not know much about, and she was always there to answer my questions. I would not have been able to conduct the research without her support and guidance.

I would like to thank Dr. Chris Pierson for being genuinely interested in helping his students and setting them up for success in their future careers. Without his help, I would not have found my research project – one with a heavy emphasis on anatomy while still suiting my interests in gastroenterology.

I would like to thank Dr. James Cray for reading draft after draft of this thesis paper and consistently giving me advice to improve. His experience in organizing and writing research papers has been very helpful.

I would like to thank Dr. Melissa Quinn for being an outstanding anatomy professor and guiding me on the right path. She was also there to support me in finding a project for my thesis and always had an open door to discuss my options.

Vita

2014……………………………………………………………Notre Dame-Cathedral Latin,

Chardon, Ohio

2018………………………………………………………….…B.S. Food and Nutrition Sciences,

Ohio University

2018 to present…………………………………………..Graduate Student, Division of Anatomy,

The Ohio State University

Field of Study

Major Field: Anatomy

Table of Contents

Abstract………………………………………………………………………………………………………………………ii

Dedication………………………………………………………………………………………………………………….iv

Acknowledgements…………………………………………………………………………………………………….v

Vita…………………………………………………………………………………………………………………………….vi

List of Tables …………………………………………………………………………………………………………..…ix

List of Figures……………………………………………………………………………………………………………..x

Chapter 1: Introduction………………………………………………………………………………………………1

1.1: Hirschsprung Disease……………………………………………………………………………….1

Pathogenesis……………………………………………………………………………………….2

Diagnosis…………………………………………………………………………………………….3

Treatment…………………………………………………………………………………………..8

Emerging Therapies……………………………………………………………………………10

Chapter 2: Anatomy of Hirschsprung Disease – Large Intestine…………………………………12

2.1: Normal Anatomy of the Large Intestine and Anal Canal …………………………12

Embryological Development of the Large Intestine and Anal Canal……12

Histology of the Large Intestine and Anal Canal…………………………………15

Gross Anatomy of the Large Intestine and Anal Canal………………………..19

Innervation of the Large Intestine and Anal Canal……………………………..21

vii 2.2: Abnormal Anatomy of the Large Intestine and Anal Canal in Hirschsprung

Disease…………………………………………………………………………………………………...24

Chapter 3: Purpose of Study……………………………………………………………………………………..25

Chapter 4: Methods………………………………………………………………………………………………….27

4.1 Study approval and gathering of the cases...... 27

4.2 Measuring the submucosal nerve diameters...... 29

4.3 Comparing non-HSCR and HSCR nerve diameters across ages...... 30

4.4 Determining an optimal nerve cutoff point supportive of HSCR...... 31

Chapter 5: Results……………………………………………………………………………………………………..33

5.1 Distribution of nerve diameter in non-HSCR patients……………………………….33

5.2 Comparison between nerve diameters of non-HSCR and HSCR patients….35

5.3 Determining optimal cutoff of nerve diameter in HSCR patients………….….43

Chapter 6: Discussion………………………………………………………………………………………………..45

References………………………………………………………………………………………………………………..48

viii

List of Tables

Table 1. Number of non-HSCR and HSCR cases across ages……………………………………….34

Table 2. Non-HSCR average nerve diameter with standard deviation, maximum nerve

diameter, and minimum nerve diameter for age groups.………………………………34

Table 3. HSCR average nerve diameter with standard deviation, maximum nerve diameter, and minimum nerve diameter for age groups.…………………………………………..36

Table 4. Average submucosal nerve diameters in micrometers for non-HSCR and HSCR

patients across ages with standard deviations.………………………………………………37

Table 5. Hypertrophic nerves in non-HSCR and HSCR biopsies by age, sensitivities and

specificities for HSCR, and p-values across all ages ………………………………………..40

Table 6. HSCR patients separated as the number of false negative and true positive cases

across ages ……….…………………………………………………………………………………………..41

Table 7. Non-HSCR patients separated as the number of false negative and true positive

cases across ages.……….………………………………………………………………………………….42

ix List of Figures

Figure 1. Layers in a full-thickness rectal biopsy…………………………………………………………..6

Figure 2. Inclusion and exclusion criteria ……………………………………………………………………28

Figure 3. Average submucosal nerve diameter in non-HSCR patients ………………………..35

Figure 4. Comparison of average submucosal nerve diameters in HSCR and non-HSCR

patients across ages ……………………………………………………………………………………….38

Figure 5. Comparison of submucosal nerve diameters in non-HSCR and HSCR

patients...... 39

Figure 6. Receiver operating characteristic curve...... 44

Chapter 1: Introduction

1.1 Hirschsprung Disease

Hirschsprung disease (HSCR) is characterized by a lack of ganglion cells in the submucosal and myenteric plexuses at variable lengths of the distal gastrointestinal tract. The length of the aganglionic segment in HSCR may vary from the distal one to two cm just proximal to the dentate line (ultrashort segment HSCR) to the entire large intestine (total colonic HSCR) (Szylberg and Marszalek, 2014). The most common type of

HSCR, accounting for seventy-five to eighty percent of all cases, is rectosigmoid HSCR, in which parts of the rectum and sigmoid colon are aganglionic (Das and Mohanty, 2017).

Long segment HSCR, accounting for ten to fifteen percent of HSCR cases, affects a greater length of the large intestine and extends from the sigmoid colon to the descending or transverse colon (Das and Mohanty, 2017). However, there is still a component of the large intestine that is ganglionic in long segment HSCR. The rarest form of HSCR is total colonic aganglionosis (TCA). TCA, present in five to seven percent of HSCR cases, is characterized by the absence of ganglion cells throughout the entire length of the large intestine (Das and Mohanty, 2017). Rare cases of TCA extend proximally to involve part or all of the small intestine. While the lengths of the aganglionic segments may vary, all cases of HSCR affect the distalmost rectum. HSCR has a prevalence of one in five thousand live births (Kapur, 2016). HSCR has a male to

1 female ratio of approximately four to one (Szylberg and Marzalek, 2014; Muise et al.,

2016).

Pathogenesis

During embryonic development, neural crest cells form from the edges of neural folds. There are different types of neural crest cells: cranial, cardiac, truncal, and enteric neural crest (Xi and Lui, 2019). The enteric neural crest is responsible for the formation of the enteric nervous system in the gastrointestinal tract (Xi and Lui, 2019). Vagal neural crest cells, the precursors to the enteric neural crest, are responsible for migrating to form the majority of the enteric ganglia (Carlson, 2018, Ch 12 pages 242-

254). Other cells, including sacral neural crest cells and Schwann cell precursors, also contribute to the formation of the enteric nervous system (Carlson, 2018, Ch 12 pages

242-254).

There are various hypotheses of the pathogenesis of Hirschsprung disease. The most popular hypothesis is the lack of migration of neural crest cells to the distal large intestine (Szylberg and Marszalek, 2014). HSCR is often caused by a mutation in the RET signaling pathway. A lack of RET signaling, which is normally expressed by neural crest cells, may lead to the development of HSCR (Nagy and Goldstein, 2017; Heuckeroth,

2018). Other hypotheses suggest that neural crest cells migrate throughout the large intestine and form ganglion cells, but the ganglion cells degenerate and become necrotic (Szylberg and Marszalek, 2014).

2 It is most likely that when neural crest cells fail to migrate to the distal large intestine, the result is aganglionosis and HSCR. Without the formation of the intrinsic nervous system, comprised of the submucosal and myenteric plexuses, there is abnormal motility of the gastrointestinal tract. Once digested food reaches the beginning of the aganglionic segment, it stops and is unable to move any further. The region of bowel that lacks innervation remains contracted (Szylberg and Marszalek,

2014). The feces builds up, and the part of the colon proximal to the aganglionic region becomes large and distended.

Diagnosis

Clinical history and physical examination can provide important clues to raise suspicion for a diagnosis of HSCR (De La Torre and Langer, 2010). Symptoms of

Hirschsprung disease include constipation, abdominal distention, enterocolitis, and delayed passage of meconium in newborns (Das and Mohanty, 2017). Clinical symptoms are evident within the first few days after birth in seventy to ninety percent of patients with HSCR (Szylberg and Marszalek, 2014). The literature suggests that fifty to ninety percent of infants with HSCR have a delayed passage of meconium (Heuckeroth, 2018;

Das and Mohanty, 2017). The obstruction of stool in the large intestine leads to further intestinal obstruction in the large intestine proximal to the contracted, aganglionic region. While most cases are typically identified during the infant stage, some children have very mild symptoms at birth and may present later in childhood with the

3 symptoms of chronic constipation. These instances in which HSCR is diagnosed during childhood or adulthood are rare (Muise et al., 2016).

Hirschsprung-associated enterocolitis is a life-threatening complication that may present with abdominal distention, fever, and diarrhea (Heuckeroth, 2018). Enterocolitis can start suddenly and progress rapidly and must be treated quickly. If the inflamed bowel becomes too distended and perforates, the bacteria in the stool can cause sepsis

(Heuckeroth, 2018). If a patient presents with enterocolitis, the colon should be decompressed to remove the compacted feces and antibiotics should be administered

(Heuckeroth, 2018).

If the patient history and physical examination suggest HSCR, imaging studies may be conducted including an abdominal radiograph and a water-soluble contrast

(barium) enema (De La Torre and Langer, 2010). An abdominal radiograph can show dilated bowel. A contrast enema is an X-ray that uses a contrast agent to visualize the structures of the large intestine and is used to see where the transition zone is from the dilated, ganglionic colon to the contracted, aganglionic colon.

Anorectal manometry may also be conducted to test the pressures in the rectum and to determine if there is an abnormal anorectal inhibitory reflex (De La Torre and

Langer, 2010). Anorectal manometry allows for the pressures in the rectum and anus to be gathered and evaluated. During a normal anorectal reflex, when the rectum is distended with feces, the internal anal sphincter relaxes, allowing the feces to exit the body. In children with HSCR, the internal anal sphincter remains contracted when the

4 rectum is distended, causing constipation (De La Torre and Langer, 2010; Szylberg and

Marszalek, 2014).

The gold standard for diagnosing HSCR is a rectal biopsy. Diagnostic biopsies may either be suction rectal biopsies (SRB) or full thickness rectal biopsies (FTRB) (Muise et al. 2016). A SRB contains the mucosa (epithelium, lamina propria, and muscularis mucosae) and submucosa. A SRB is commonly performed in newborns since it does not require general anesthesia, can be performed at the bedside, and does not require surgical sutures (Szylberg and Marszalek, 2014). The histologic evaluation of SRBs provides less tissue than FTRBs which may make evaluation more challenging. As a result, SRBs may require more biopsies to be taken compared to FTRBs (Bjorn et al.,

2018). For a biopsy to be adequate, it must be taken approximately 2 cm proximal to the dentate line; it must have sufficient submucosa present, defined greater than 50% by biopsy depth (Muise et al., 2016); it must measure 2 to 3 mm in diameter and 1 mm in depth (Qualman et al., 1999), and it must not contain squamous epithelium. A SRB in an older child or young adult may not be sufficient due to lack of adequate amount of submucosa (De La Torre and Langer, 2010). These children and young adults will have a

FTRB, which contain all of the layers of the wall of the colon (Figure 1). A FTRB is performed under general anesthesia and requires sutures to stop the bleeding (Muise et al., 2016).

Figure 1. Layers in a full-thickness rectal biopsy. An image of a full-thickness rectal biopsy at 10x, which shows the mucosa (epithelium, lamina propria, muscularis mucosae), submucosa, and muscularis externa (inner circular and outer longitudinal layers).

Diagnostic biopsies must be taken at least two cm above the dentate line

(Szylberg and Marszalek, 2014). This is important because the dentate line is a physiological aganglionic zone (Das and Mohanty, 2017). Taking a biopsy in the aganglionic region of the anal canal is inadequate and often is repeated to decrease the

6 incidence of a false diagnosis of HSCR. The pathologist can determine if the biopsy was inappropriately harvested in the anal canal if there is squamous mucosa present.

Pathologists determine the presence or absence of ganglion cells and hypertrophic nerves by examining H&E stained biopsies (De La Torre and Langer, 2010).

For a HSCR diagnosis, there needs to be at least 100 sections examined (Qualman et al.,

1999) and there be no ganglion cells present.

In HSCR there are hypertrophic nerves in the submucosal and myenteric plexuses, which are found in the submucosa and muscularis externa layers, respectively

(Das and Mohanty, 2017). According to Monforte-Munoz and colleagues, submucosal nerve diameters of 40 microns or larger are strongly correlated with HSCR (Monforte-

Munoz, 1998). In 2002, a study published by Sangkhathat and colleagues reinforced the aforementioned study, also showing that 40 microns or greater is supportive of a HSCR diagnosis (Sangkhathat et al., 2002). Ninety percent of SRBs from patients with HSCR contain nerve diameters of 40 microns or greater (Kapur, 2006; Qualman et al., 1999).

However, in cases with TCA, the submucosal nerves are typically not hypertrophic, unlike the other forms of HSCR (Das and Mohanty, 2017; Qualman et al., 1999).

Pathologists may wish to confirm a HSCR diagnosis with special staining techniques including acetylcholinesterase enzyme histochemistry (AChE) and calretinin immunohistochemistry (Das and Mohanty, 2017). Acetylcholinesterase stains extrinsic nerve fibers (i.e. sympathetic, parasympathetic, and visceral afferent fibers) and requires frozen sections (Guinard-Samuel et al., 2009). A positive AChE staining pattern

7 supports a HSCR diagnosis (Szylberg and Marszalek, 2014). Calretinin is a vitamin D- dependent calcium-binding protein that is expressed in ganglion cells and intrinsic nerves in the intestines (Das and Mohanty, 2017). The absence of calretinin staining (i.e. negative calretinin staining) in the submucosal plexus supports a HSCR diagnosis.

Normal cases have normal calretinin staining (i.e. positive calretinin staining) (Guinard-

Samuel et al., 2009).

Treatment

Surgical management of Hirschsprung disease involves removing the part of the bowel that is aganglionic and reattaching the functional bowel to the distal rectum.

During surgery in the operating room, the surgeon will initially conduct a mapping procedure to determine where the bowel transitions from aganglionic to ganglionic.

Intraoperative frozen sections are sent to pathology to confirm the absence and presence of ganglion cells. Once the most distal segment of normal bowel is identified, a doughnut section will be cut for examination by a pathologist (Das and Mohanty, 2017).

The circumference of the doughnut is examined to make sure the entirety of the colon is normally innervated and has ganglion cells present. A pathologist may note that the resection was taken at the transition zone if there is partial circumferential aganglionosis, myenteric hypoganglionosis, and/or hypertrophy of submucosal nerves

(Kapur, 2016). If a patient continues to have obstruction and dysmotility problems

8 postoperatively, it may be due to these features of the transition zone that failed to be resected (Kapur, 2016).

There are three standard transanal pull-through procedures for Hirschsprung disease: Suave, Swenson, and Duhamel (Das and Mohanty, 2017). Evidence is not sufficient to say if one procedure is more efficacious than another and thus, the surgical procedure safest for the patient is the procedure that the surgeon has been trained to do (Langer, 2013). For long segment HSCR, a laparotomy is necessary (Das and Mohanty,

2017). The transanal approach of managing HSCR has advantages over the open surgery because there is less pain, shorter hospital stay, and better cosmetic results (De La Torre and Langer, 2010). The transanal approach can be done by any pediatric gastroenterology surgeon, does not require a colostomy, and can be performed on newborn infants (Lukac et al., 2016). Laparoscopy may be used during these procedures but requires the surgeon to have laparoscopy training (De La Torre and Langer, 2010).

A major goal of surgery is to avoid damaging the anal canal and the anal sphincters (Wester and Granström, 2017). If these structures are damaged, this could lead to fecal incontinence (Wester and Granström, 2017). Because the procedure is typically a transanal approach, this makes the goal difficult.

Frozen sections are taken and sent to pathology for immediate reports of whether the region is aganglionic or ganglionic. If the biopsy does not show ganglion cells, another biopsy is taken about 5 centimeters proximal until ganglion cells are identified (Smith et al., 2020). The aganglionic region is pulled out, and a varying amount

9 of colon is also taken out proximal to the normal ganglionic region to ensure that the resection does not include the transition zone (Xu et al., 2008). An anastomosis is then made between the normal ganglionic region and the distal rectum.

There are differences in the three types of pull-through procedures performed.

The Swenson procedure removes the segment of aganglionic colon and connects the ganglionic colon with the anus (Heuckeroth, 2018). In the Soave procedure innervated ganglionic colon is brought through a cuff of rectal muscle (Heuckeroth, 2018). The mucosa of the aganglionic rectum is stripped away from the underlying muscle, and the ganglionic bowel is pulled through the muscular cuff (Langer, 2013). In the Duhamel procedure healthy bowel is re-attached to the posterior aspect of the diseased rectum

(Heuckeroth, 2018; Duhamel, 1960; Langer, 2013).

Emerging Therapies

The possibility of stem cell transplantation to regenerate the aganglionic portion of the enteric nervous system remains a topic of interest in the scientific community

(Lukac et al., 2016; Langer, 2013). Injection of neuronal stem cells could potentially become an alternative for surgery in the future (Heuckeroth, 2018).

A database known as the Hirschsprung Disease Research Collaborative is being developed which lists patients with HSCR (Langer, 2013). The database will include as much information from each patient as possible, including genetic mutations, pathological findings, and clinical outcomes (Langer, 2013). This will allow medical

10 professionals to develop a better understanding of HSCR. This database aims to combine the expertise of an interprofessional team to find the underlying genetics of

HSCR. With knowledge of the genes responsible for HSCR, there are hopes to find ways to prevent, manage, and treat HSCR.

Chapter 2: Anatomy of Hirschsprung Disease – Large Intestine and Anal Canal

2.1: Normal Anatomy of the Large Intestine and Anal Canal

Embryological Development of the Large Intestine and Anal Canal

During the third week of development the process of gastrulation occurs, which forms three germ layers: ectoderm, mesoderm, endoderm. The ectoderm thickens, forming the neural plate (Sadler, 2014, page 71). The sides of the neural plate elevate, forming neural folds. The neural folds continue to elevate until they come together to form the neural tube (Sadler, 2014, page 71). Cells at the edge of the neural folds specialize and become neural crest cells. The neural crest cells migrate, undergoing an ectodermal-to-mesenchymal transition, and differentiate into a wide spectrum of structures including melanocytes in the skin, dorsal root ganglia, connective tissue of the face, and parasympathetic ganglia of the gastrointestinal tract (Sadler, 2014, page

77). The ectoderm becomes the neural tube, which develops into the central nervous system comprised of the brain and spinal cord. The ectoderm also forms the peripheral nervous system and the surface ectoderm forms the epidermis, hair, and nails (Sadler,

2014, page 77).

12 The mesoderm becomes three different types: paraxial, intermediate, and lateral plate. The paraxial mesoderm becomes the dermis for the skin of the back, tendons and ligaments, skeletal muscle, cartilage, bone, ribs, and the vertebral column (Sadler, 2014, pages 82, 92). The intermediate mesoderm will become the urogenital system (kidneys and gonads) (Sadler, 2014, pages 82-83). The lateral plate mesoderm has two components: the parietal (somatic) mesoderm and the visceral (splanchnic) mesoderm

(Sadler, 2014, pages 83, 96).

The endoderm becomes the gastrointestinal tract (Sadler, 2014, page 93). The cranial end of the endoderm will form the foregut, while the caudal end of the endoderm will form the hindgut. The midgut is formed in between the foregut and hindgut. The endoderm also forms the lining epithelium of the respiratory tract, tissues of the thyroid, pancreas, and liver, and visceral layer covering the organs of the abdomen, heart, lungs, and the internal lining of the bladder and urethra (Sadler, 2014, pages 88, 93, 247).

The three parts of the gastrointestinal tract are the foregut, midgut, and hindgut.

The organs of the foregut include the spleen, stomach, liver, gallbladder, pancreas, and proximal duodenum. These organs are supplied by the celiac trunk. The organs of the midgut include the distal portion of the duodenum and the jejunum, ileum, cecum, vermiform appendix, ascending colon, and proximal two-thirds of the transverse colon.

The midgut is supplied by the superior mesenteric artery (Sadler, 2014, page 239). The organs of the hindgut include the distal one-third of the transverse colon and the

13 descending colon, sigmoid colon, rectum, and upper part of the anal canal (Sadler, 2014, page 247). The inferior mesenteric artery supplies the components of the hindgut.

The midgut is made up of the primary intestinal loop (Sadler, 2014, page 239).

The primary intestinal loop has a cephalic limb, which develops into the distal duodenum, jejunum, and proximal ileum, and a caudal limb, which develops into the distal ileum, cecum, appendix, ascending colon, and proximal two-thirds of transverse colon (Sadler, 2014, pages 239-240). As the organs in the abdomen grow, much of the space needed for growth of the intestines is taken up by other viscera, especially the liver. The intestines undergo a physiologic herniation around week 6 of development, in which they herniate into the umbilical cord (Carlson, 2018, Ch 15 pages 318-357; Sadler,

2014, page 240). As the intestines protrude into the umbilical cord, they rotate around the superior mesenteric artery, which serves as the axis of rotation (Sadler, 2014, page

240). The primary intestinal loop undergoes a 90-degree counterclockwise rotation as it elongates and moves into the umbilical cord. During week 10 of development, there is adequate space in the abdominal cavity and the intestinal loops migrate back inside, undergoing another 180-degree counterclockwise rotation in the process (Sadler, 2014, pages 240-241). Thus, in total the intestinal loops undergo a 270-degree counterclockwise rotation.

The hindgut enters the posterior region of the cloaca at the anorectal canal

(Sadler, 2014, page 247). The allantois enters the anterior region of the cloaca at the urogenital sinus (Sadler, 2014, page 247). The cloaca is the end point for both digestive

14 and urogenital systems (Carlson, 2018, Ch 15 pages 318-357). The cloaca has endoderm on the inner portion and surface ectoderm on the outer portion. Separating the endoderm and surface ectoderm is the cloacal membrane (Sadler, 2014, page 247). The urorectal septum migrates towards the cloacal membrane, creating a separation of the cloaca into the urogenital sinus anteriorly and the anorectal canal posteriorly

(Schoenwolf et al., 2015, Ch 14 pages 341-374). The cloacal membrane eventually ruptures, creating an opening for the urogenital sinus and the anus (Sadler, 2014, page

247). The perineal body is the division between the urogenital system and anus (Carlson,

2018, Ch 15 pages 318-357). Mesenchymal proliferation occurs around the anal canal, forming the proctodeum (Schoenwolf et al., 2015, Ch 14 pages 341-374). The superior two-thirds of the anal canal is derived from endoderm of the distal portion of the hindgut, while the inferior one-third of the anal canal is derived from ectoderm from the proctodeum (Schoenwolf et al., 2015, Ch 14 pages 341-374) (Sadler, 2014, page 247).

The dentate line in the anal canal is the junction point between the hindgut and proctodeum (Schoenwolf et al., 2015, Ch 14 pages 341-374; Sadler, 2014, page 247).

Histology of the Large Intestine and Anal Canal

The alimentary canal has a similar structural organization from the beginning to end. There are four layers, which include the mucosa, submucosa, muscularis externa, and serosa or adventitia (Ross and Pawlina, 2016, page 568). For the purposes of this project, we will specifically focus on the histology of the large intestine and anal canal.

15 The mucosa of the large intestine has a simple columnar epithelium, important for mucus secretion and water and electrolyte absorption (Ross and Pawlina, 2016, page

595). The surface of the large intestine mucosa lacks villi and plicae circulares (Lowe et al., 2019, Ch 11 pages 177-208; Ross and Pawlina, 2016, page 594). Crypts of Lieberkühn are present in the large intestine, and they have many types of cells in the large intestine including the following: absorptive cells of the colon known as colonocytes, goblet cells, enteroendocrine cells, and stem cells. The colonocytes function to reabsorb water and electrolytes. Boblet cells secrete mucin to keep the fecal matter moving through the large intestine (Ross and Pawlina, 2016, page 596). The number of goblet cells increase as you move distally through the gastrointestinal tract (Ross and Pawlina,

2016, pages 595). The stem cells in the large intestine are located at the bottom of the tubular intestinal glands and function to produce epithelial cells to replace older and damaged cells (Lowe et al., 2019, Ch 11 pages 177-208; Ross and Pawlina, 2016, page

596).

Under the epithelial layer of the large intestine is the lamina propria. The lamina propria is part of the mucosa and is comprised of loose connective tissue. The lamina propria contains lymphatic vessels, which are important for the absorption of lipids, and blood vessels, which are important for taking the nutrient-rich blood and distributing it to the avascular epithelial cells above (Ross and Pawlina, 2016, page 570). The lamina propria of the large intestine contains lymphoid cells and lymphoid nodules that are part of the GALT (Ross and Pawlina, 2016, page 597). The development of the GALT in the

16 large intestine may be due to the immune system keeping the bacterial populations in check (Ross and Pawlina, 2016, page 597).

Deep to the lamina propria is the muscularis mucosae, comprised of inner circular and outer longitudinal layers of smooth muscle (Ross and Pawlina, 2016, page

570). Innervation of this smooth muscle layer allows the mucosa to have some movement (Ross and Pawlina, 2016). The crypts of Lieberkühn do not extend past this layer.

The submucosa of the large intestine is comprised of dense irregular connective tissue (Ross and Pawlina, 2016, page 570). This layer has blood vessels, lymphatic vessels, and the submucosal nerve plexus (Meissner’s plexus), which allows for secretion of glands and movement of the muscularis mucosae layer. The absence of ganglion cells in the submucosal nerve plexus in the large intestine is important for diagnosing

Hirschsprung disease.

The muscularis externa of the large intestine has two layers of smooth muscle: the inner circular layer and the outer longitudinal layer. The inner circular layer forms the sphincters and valves located throughout the digestive tract, including the internal anal sphincter. The outer longitudinal layer of the muscularis externa thickens and forms three bands that run along most of the length of the colon, known as taeniae coli (Ross and Pawlina, 2016, page 597). The taeniae coli function in peristalsis by contracting and shortening the tube to move the contents along. Taeniae coli are present in the cecum, ascending colon, transverse colon, and descending colon (Ross and Pawlina, 2016, page

17 597). Parts of muscle from the taeniae coli penetrate into the inner circular layer of the muscularis externa, forming haustra. Haustra are sacculations of the colon.

The outer layer of the large intestine may be either serosa or adventitia, depending on the parts of the colon that are intraperitoneal or retroperitoneal. Organs that are intraperitoneal are covered by serosa, a simple squamous epithelium with underlying connective tissue (Ross and Pawlina, 2016, page 571). Organs that are retroperitoneal have adventitia, which is only composed of connective tissue (Ross and

Pawlina, 2016, page 571). The ascending colon, descending colon, and rectum are retroperitoneal, and are fixed against the body wall (Drake et al., 2015, page 322). Thus, these parts of the large intestine have adventitia. The cecum, transverse colon and sigmoid colon are intraperitoneal, and thus have serosa (Drake et al., 2015, page 322).

Omental appendages are projections of the serosa that are visible along the surface of the colon (Ross and Pawlina, 2016, page 594).

The recto-anal junction can be seen by looking at the epithelium, which shifts from a simple columnar epithelium of the rectum to eventually a stratified squamous epithelium. The anus is characterized by a stratified squamous epithelium that undergoes a gradual transition to skin. This type of epithelium is important for protection from the outside environment.

The anal canal has three zones based on the epithelial lining (Ross and Pawlina,

2016, page 599). The colorectal zone is the superior one-third of the anal canal and has simple columnar epithelium (Ross and Pawlina, 2016, page 599). The anal transitional

18 zone (ATZ) is the middle one-third of the anal canal and may have a mix of simple columnar epithelium, stratified columnar epithelium, and stratified squamous epithelium (Ross and Pawlina, 2016, page 599). The squamous zone is the inferior one- third of the anal canal and has non-keratinized stratified squamous epithelium (Ross and

Pawlina, 2016, page 599). At the end is a mucocutaneous junction, which transitions from a mucous membrane to skin.

Gross Anatomy of the Large Intestine and Anal Canal

The large intestine is important for the absorption of water and electrolytes and for the storage of feces. The large intestine is composed of the cecum, appendix, ascending colon, transverse colon, descending colon, sigmoid colon, rectum, and anal canal (Moore, 2015). The first part of the large intestine is the cecum. The ileum protrudes into the cecum at the ileal orifice (Moore, 2015). At this orifice there are two folds called ileocecal folds that prevent contents in the cecum from going back into the ileum. The cecum has an appendage known as the vermiform appendix (Moore, 2015).

Following the cecum is the beginning of the colon, which has four parts: ascending, transverse, descending, and sigmoid (Moore, 2015). The ascending colon rises up from the cecum on the right side of the abdomen. The ascending colon turns into the transverse colon at the right colic (hepatic) flexure (Moore, 2015).

The transverse colon is intraperitoneal and is held in place by the gastrocolic ligament, which extends from the greater curvature of the stomach to the transverse

19 colon. The transverse colon turns at the left colic (splenic) flexure into the descending colon, which descends down the left side of the abdomen (Moore, 2015).

The sigmoid colon is an S-shaped segment distal to the descending colon

(Moore, 2015). The sigmoid colon is continuous with the rectum. The rectum has no haustra and the taeniae coli end at the junction of the sigmoid and rectum (Chiva and

Magrina, 2019, Ch 2 pages 3-49). The rectum transitions into the anal canal, which has vertical folds known as anal columns. The anal canal is surrounded by the internal anal sphincter, which is involuntary smooth muscle, and the external anal sphincter and levator ani muscles, which are voluntary skeletal muscles (Chiva and Magrina, 2019, Ch

2 pages 3-49). There is an 80-degree anorectal flexure that contracts the puborectalis muscle (part of the levator ani m.), which forms a sling around the anus. This muscular sling is important for fecal continence. The anal canal has the dentate line, which indicates the superior two thirds of the anal canal originating from the hindgut from the inferior one third of the anal canal originating from the proctodeum.

The large intestine has other characteristics that make it distinct from the small intestine. Pouches of the large intestine are called haustra (Moore, 2015). Small fat- filled pouches found on the large intestine are termed epiploic or omental appendices

(Moore, 2015). There are three layers of longitudinal smooth muscle called taenia coli that begin at the appendix and end at the rectum (Drake et al., 2015, page 320).

Innervation of the Large Intestine and Anal Canal

The intestines have extrinsic innervation and intrinsic innervation (Furness et al.,

2015, page 252). Extrinsic innervation comes from the parasympathetic fibers, sympathetic fibers, and sensory nerves. The parasympathetic nervous system stimulates the intestines, while the sympathetic nervous system inhibits motility of the intestines.

Intrinsic innervation comes from the ganglia and nerves of the enteric nervous system, and well as glial cells (Nagy and Goldstein, 2017, page 95). The intrinsic innervation includes the submucosal (Meissner’s) plexus in the submucosal layer and the myenteric

(Auerbach’s) plexus in between the inner circular and outer longitudinal layers of the muscularis externa. The myenteric plexus is present throughout the gastrointestinal tract, but the submucosal plexus is not present in the esophagus (Nagy and Goldstein,

2017, page 95).

The sympathetic chain is found at the superior thoracic ganglion in the neck and descends down to the ganglion impar next to the coccyx (Moore, 2015). The cell bodies of preganglionic sympathetic fibers are in the lateral horns of the spinal cord, which are present only at thoracolumbar regions from T1 to L2 (Moore, 2015). Sympathetic presynaptic fibers will travel from the cell bodies in the lateral horns, through the ventral rootlets, ventral root, spinal nerve, and white rami communicants to get onto the sympathetic chain. The presynaptic sympathetic fibers can take multiple paths, including the following: 1) ascending the sympathetic chain and synapsing with the

21 postsynaptic sympathetic fibers; 2) entering and synapsing instantly; 3) descending the sympathetic chain and synapsing with postsynaptic sympathetic fibers; and 4) exiting the chain without synapsing at all. The last path is the one taken for sympathetic fibers going to organs in the abdomen (Moore, 2015).

The splanchnic nerves supply sympathetic fibers to the organs of the abdomen

(Moore, 2015). Splanchnic nerves do not synapse in the sympathetic chain, but instead leave the chain without synapsing. The greater splanchnic nerve exits the spinal cord from levels T5 to T9, the lesser splanchnic nerve exits at T10 and T11, and the least splanchnic nerve exits at T12. Below the level of T12, the lumbar splanchnic nerves supply sympathetic innervation (Moore, 2015).

The greater splanchnic nerves run to the celiac ganglion and synapse with postganglionic sympathetic fibers. These fibers will go to innervate the foregut structures. The lesser splanchnic nerves run to the superior mesenteric ganglion and synapse with postganglionic sympathetic fibers. These fibers will go on to innervate the midgut structures. The lesser splanchnic nerves may also go on to the aorticorenal ganglion, synapse with postganglionic sympathetic fibers, and innervate the kidneys and ureter. The least splanchnic nerves go on to the aorticorenal ganglion and synapse with postganglionic sympathetic fibers. These fibers will go to innervate the kidneys. The lumbar splanchnic nerves supply the hindgut structures (Moore, 2015).

The parasympathetic fibers are from cranial nerve X, the vagus nerve, and from pelvic splanchnic nerves (S2-S4) (Moore, 2015). The presynaptic parasympathetic fibers

22 arise in either the brainstem or in the sacral region, travel to the target organs, and synapse in or near the wall of the target organ. The transition point from supply of the vagus nerve to pelvic splanchnic nerves is at the left colic flexure (Moore, 2015).

The extrinsic innervation regulates the actions of the intrinsic nervous system

(Kapur, 2006). The submucosal and myenteric plexuses have both sympathetic and parasympathetic fibers. The submucosal and myenteric plexuses insert into various layers of the gastrointestinal tract to innervate those layers (Kapur, 2006).

Nerves in both plexuses are a mixture of intrinsic and extrinsic fibers and specialized enteric glial cells (Kapur, 2006).

2.2: Abnormal Anatomy of the Large Intestine and Anal Canal in Hirschsprung Disease

In Hirschsprung disease there is failure of the neural crest cells to migrate to the distal colon and potentially regions proximal to the distal colon as well. These areas where neural crest cells do not migrate to do not form the ganglia of the enteric nervous system, which includes the submucosal and myenteric plexuses. Without these plexuses in the submucosal and muscularis externa layers, respectively, peristalsis cannot occur and the aganglionic segment remains contracted. The intrinsic nerves and ganglia of the gut are needed to relax the smooth muscle and push the fecal matter forward (Kapur, 2006).

In Hirschsprung disease, the ganglion cells of the submucosal plexus and myenteric plexus are absent. The nerves in both of the plexuses are also hypertrophic

(Das and Mohanty, 2017). Hypertrophic nerves are defined as greater than or equal to

40 microns in greatest diameter (Kapur, 2006). Non-hypertrophic submucosal nerves have diameters less than 40 microns (Kapur, 2006).

Chapter 3: Purpose of Study

This study has three objectives:

Objective 1: Determine the distribution of submucosal nerve diameters in the submucosal plexus based on age in patients without HSCR.

Ho: There is no statistically significant difference between the submucosal nerve diameters in patients without HSCR across ages.

Ha: There is a statistically significant difference between the submucosal nerve diameters in patients without HSCR across ages.

We expect that as the non-HSCR patients increase in age, the submucosal nerve diameters will also increase.

Objective 2: Compare the average submucosal nerve diameters in patients with HSCR who have not had a prior pull-through procedure and patients without HSCR across all age groups.

Ho: There is no statistically significant difference between the submucosal nerve diameters in patients with and without HSCR across all ages.

Ha: There is a statistically significant difference between the submucosal nerve diameters in patients with and without HSCR across all ages.

25 We expect that the HSCR cases will have hypertrophic nerves within the first year of life. We expect the non-HSCR cases will have non-hypertrophic nerves within the first year, but that the nerve diameters will increase with increasing age.

Objective 3: Determine an appropriate nerve cutoff that would be supportive of a

HSCR diagnosis.

Ho: There is no difference in the nerve cutoff points that are supportive of a

HSCR diagnosis.

Ha: There is a certain nerve cutoff point for supportive of a diagnosis of HSCR which has a high specificity while maintaining sensitivity.

We expect that a nerve cutoff supportive of a HSCR diagnosis will be close to 40 microns, as this is the current cutoff point used clinically and cited in the literature.

Chapter 4: Methods

4.1 Study approval and gathering of the cases

This study was approved by the institutional review board at Nationwide

Children’s Hospital (IRB17-00503). The pathology specimen database (CoPath) at

Nationwide Children’s Hospital was searched from January 1, 2017 to December 31,

2018 to identify all suction rectal biopsies and full-thickness rectal biopsies. Clinical information was gathered by searching the electronic medical record (EPIC). Clinical data for each patient included the indication for a biopsy, if the patient had a prior pull- through procedure, and which prior pull-through procedure was performed. The information was entered in a Microsoft Excel spreadsheet.

There were a total of 309 cases from 2017-2018 (Figure 2). Biopsies were included if the patient had not had a prior pull-through procedure and the biopsy was from native rectum (179 patients). Of the included cases, 28 biopsies were from patients with HSCR, and 151 biopsies were from patients without HSCR. There were 130 cases excluded. Ninety-nine cases were excluded because they were not native rectal biopsies, meaning that they had previously had a resection procedure. Non-rectal biopsies were excluded, including two cases of small bowel biopsies and three cases

27 with proximal colon biopsies. Three cases were excluded because they lacked the submucosa layer. Thus, the submucosal nerves could not be measured for these cases.

Seven cases were excluded because they were determined to be inadequate, due to the presence of stratified squamous epithelium, indicative of being taken in the anal canal.

Seven cases were considered to be indeterminant for a diagnosis of HSCR. Four rectal biopsies were excluded because they were taken for intraoperative frozen section evaluation at the time of resection and were not diagnostic biopsies. Five cases were excluded because H&E slides from the pathology archived files were not available for review.

Figure 2. Inclusion and exclusion criteria. From 2017-2018, 309 cases were identified,

130 cases were excluded, and 179 cases were included for the study. Of the included cases, there were 28 HSCR cases and 151 non-HSCR cases.

For each case, the presence or absence of ganglion cells was recorded. If there were no ganglion cells present, the case was recorded in a Microsoft Excel spreadsheet as positive for Hirschsprung disease (1=HSCR). If ganglion cells were present, the case was recorded as not having Hirschsprung disease (0=non-HSCR).

4.2 Measuring the submucosal nerve diameters

A retrospective review of the H&E slides was performed for all patients included in the study. Select slides from each subject were viewed under a Zeiss microscope at

10X and 40X magnifications. The slides for each case were chosen based on the appearance of the stained sections on the slides. For cases with numerous slides, the first and last few slides where typically not viewed due to the lack of tissue and staining.

Slides with adequate pieces of specimen and staining were viewed. The submucosal nerves were measured at 40X magnification using a micrometer in the lens of the microscope. The nerves were measured along the shortest axis of the nerve. The area with the largest width of the nerve was recorded. The submucosal nerve diameter and the corresponding slide numbers were recorded. Submucosal nerves with ganglion cells were not measured, as ganglion cells may distort the appearance of nerves and make them appear larger. The submucosal nerve with the largest diameter measurement for each patient was documented into a Microsoft Excel spreadsheet by recording the

29 number of marks on the micrometer and multiplying by a conversion factor of 2.5 to convert into micrometers. All nerve measurements were measured by Sarah Beach and confirmed by pathologist Miriam Conces, M.D.

4.3 Comparing non-HSCR and HSCR nerve diameters across ages

Using our data, pivot tables were created in Microsoft Excel to identify the number of non-HSCR and HSCR cases for each age (Table 1) and the average nerve diameter with standard deviation for the non-HSCR and HSCR cases for that age (Table

4). The average nerve diameters with standard deviations, maximum nerve diameter, and minimum nerve diameter were also determined for each age group using the pivot tables (Tables 2 and 3).

The 28 HSCR cases were then categorized as false negatives and true positives.

The false negative cases had been diagnosed with HSCR but had nerve diameters less than 40 micrometers. The true positive cases had been diagnosed with HSCR and had nerve diameters greater than or equal to 40 micrometers. For each age group we found the number of false negative and true positive cases (Table 6).

The non-HSCR cases were categorized as true negatives or false positives. The true negative cases were not diagnosed with HSCR and had nerve diameters less than 40 micrometers. The false positive cases were not diagnosed with HSCR but had nerve

30 diameters that were greater than or equal to 40 micrometers. For each age group we found the number of true negative and false positive cases (Table 7).

Comparing submucosal nerve diameter between HSCR and non-HSCR rectal biopsies, the sensitivity and specificity of a diameter of 40 microns or greater was determined for each age group. A Fisher’s exact test was used to determine the p-values for each age group. The values were recorded into the Microsoft Excel spreadsheet. A p- value less than or equal to 0.05 was considered significant.

4.4 Determining an optimal nerve cutoff point supportive of HSCR

We wanted to determine a value for average submucosal nerve diameter that would best support a diagnosis of HSCR. To do so, we generated a receiver operating characteristic (ROC) curve to determine the cutoff point showing the greatest sensitivity while maintaining specificity. We included all cases that were less than 1 year old, as we found hypertrophic submucosal nerves were significantly associated with HSCR for ages less than 1 year old. For each nerve diameter we determined the true positive rate

(sensitivity) and false positive rate (1-specificity). We looked only at the cases that were less than 1 year old, which we found to be statistically significant.

The true positive rate (sensitivity) was found using the formula: True positive rate = number of true positive cases / (number of true positive cases + number of false negative cases)

31 The specificity was found using the formula: Specificity = number of true negative cases / (number of true negative cases + number of false positive cases)

The false positive rate (1-specificity) was found using the formula: 1 - the value we found for specificity

Chapter 5: Results

5.1 Distribution of nerve diameter in non-HSCR patients

We first wanted to see the distribution of the average submucosal nerve diameters across age groups in patients without HSCR (Figure 3). There were 151 non-

HSCR cases total from ages less than 1 month to greater than 10 years (Table 1). Overall, submucosal nerve diameter ranged from 17.5 micrometers to 101.5 micrometers in biopsies from patients without HSCR (Table 2). The average nerve diameter across all ages was 40.7 ± 16.3 micrometers (Table 2). There was an increase in average submucosal nerve diameter as the patient age increased (Figure 3). In patients less than

1 year of age, the average submucosal nerve diameter was 34.1 ± 11.6 micrometers

(Table 2). Hypertrophic submucosal nerves were identified more frequently in biopsies from patients greater than 1 year of age, with average submucosal nerve diameters being larger than 40 microns for ages 1 year and older (Table 2).

Table 1. Number of non-HSCR and HSCR cases across ages.

Number of Patients Non-HSCR HSCR Age All Ages 151 28 < 1 month 24 17 1 month 26 1 2-5 months 35 2 6-12 months 7 4 < 1 year 91 24 1 year 9 0 2-5 years 20 1 6-10 years 15 2 >10 years 16 1

Table 2. Non-HSCR average nerve diameter with standard deviation, maximum nerve diameter, and minimum nerve diameter for age groups.

Non-HSCR Non-HSCR Non-HSCR Age Average Nerve Maximum Nerve Minimum Nerve Diameter with Diameter Diameter Standard Deviation All Ages 40.7 ± 16.3 101.5 17.5 < 1 month 34.6 ± 12.9 66.5 17.5 1 month 34.1 ± 11.9 66.5 17.5 2-5 months 33.6 ± 11.5 73.5 17.5 6-12 months 36.5 ± 7 49 28 < 1 year 34.1 ± 11.6 73.5 17.5 1 year 44.7 ± 14.4 77 31.5 2-5 years 50.2 ± 15.9 80.5 17.5 6-10 years 49 ± 16 70 21 >10 years 56.7 ± 20.9 101.5 31.5

Figure 3. Average submucosal nerve diameter in non-HSCR patients. The overall average nerve diameter increases with increasing patient age in non-HSCR patients.

5.2 Comparison between nerve diameters of non-HSCR and HSCR patients

Next, we wanted to compare the distribution of average submucosal nerve diameter in non-HSCR and HSCR patients (Figure 4). In biopsies from patients with HSCR, the submucosal nerve diameter ranged from 14 to 98 micrometers, and the average nerve diameter across all ages was 60.4 ± 18.1 micrometers (Table 3). The error bars in

Figure 4 represent the standard deviation, which signifies if the nerve diameters for each age group are close together (cluster around the average) or spread apart (farther from the average). A low standard deviation suggests that the nerve diameters of

35 patients that were close to the average and a high standard deviation suggests that the nerve diameters of patients were not clustered around the average. Figure 5 represents the numeral results in images, with a non-HSCR case less than 1 month old without hypertrophic nerves measuring 29 microns, and a HSCR case 5 months old with large, hypertrophic nerves measuring 98 microns.

Table 3. HSCR average nerve diameter with standard deviation, maximum nerve diameter, and minimum nerve diameter for age groups.

HSCR HSCR HSCR Age Average Nerve Maximum Nerve Minimum Nerve Diameter with Diameter Diameter Standard Deviation All Ages 60.4 ± 18.1 98 14 < 1 month 64.6 ± 14.2 84 35 1 month 45.5 45.5 45.5 2-5 months 66.5 ± 44.5 98 35 6-12 months 58.6 ± 11.6 70 45.5 < 1 year 63 ± 16.2 98 35 1 year - - - 2-5 years 14 14 14 6-10 years 56 ± 19.8 70 42 >10 years 52.5 52.5 52.5

Table 4. Average submucosal nerve diameters in micrometers for non-HSCR and HSCR patients across ages with standard deviations.

Age Non-HSCR Average Submucosal HSCR Average Submucosal Nerve Nerve Diameter (micrometers) with Diameter (micrometers) with Standard Deviation Standard Deviation All ages 40.7 ± 16.3 60.4 ± 18.1 < 1 month 34.6 ± 12.9 64.6 ± 14.2 1 month 34.1 ± 11.9 45.5 2-5 months 33.6 ± 11.5 66.5 ± 44.5 6-12 months 36.5 ± 7 58.6 ± 11.6 <1 year 34.1 ± 11.6 63 ± 16.2 1 year 44.7 ± 14.4 - 2-5 years 50.2 ± 15.9 14 6-10 years 49 ± 16 56 ± 19.8 >10 years 56.7 ± 20.9 52.5

Figure 4. Comparison of average submucosal nerve diameters in HSCR and non-HSCR patients across ages. The error bars represent the standard deviations for each age group. Average nerve diameter increased with patient age in non-HSCR patients (blue).

Average nerve diameters were significantly larger in patients with HSCR (red) at ages less than 1 year.

20x 20x

Figure 5. Comparison of submucosal nerve diameters in non-HSCR and HSCR patients. A)

Rectal biopsy from a less than 1-month-old non-HSCR case with a submucosal nerve measuring 29 microns. B) Rectal biopsy from a 5-month-old HSCR case with a submucosal nerve measuring 98 microns, which is hypertrophic.

Across all ages, hypertrophic submucosal nerves (≥ 40 microns) were significantly associated with HSCR [HSCR = 25/28 (89%) vs non-HSCR = 59/151 (39.1%), p=<0.0001]

(Table 5). When evaluating by age, hypertrophic submucosal nerves were significantly associated with HSCR for ages less than 1 year (p<0.0001) (Table 5). As shown in Figure

4, the average nerve diameter in biopsies of HSCR (red bars) cases is greater compared to the non-HSCR cases (blue bars) for ages less than 1 year old. The average nerve diameters were not significantly different between HSCR and non-HSCR patients ages 1 and older (p=1) (Table 5). As shown in Figure 4, the HSCR and non-HSCR have similar nerve diameters at ages 6 and older.

Table 5. Hypertrophic nerves in non-HSCR and HSCR biopsies by age, sensitivities and specificities for HSCR, and p-values across all ages.

Age Non-HSCR HSCR Sensitivity Specificity p-value ≥ 40 µm ≥ 40 µm for HSCR for HSCR All 59/151 25/28 89.29% 60.93% <0.0001 <1 month 6/24 16/17 94.12% 75.00% <0.0001 1 month 4/26 1/1 100.00% 84.62% 0.1852 2-5 months 8/35 1/2 50.00% 77.14% 0.4324 6-12 months 2/7 4/4 100.00% 71.43% 0.0278 <1 year 19/91 22/24 91.67% 79.12% <0.0001 1 year 4/9 0/0 - - 1 ≥ 1 year 40/60 3/4 75.00% 33.33% 1

There were 28 biopsies from patients with HSCR (Table 1). The majority of patients with HSCR were less than 1 year of age. There were 24 HSCR cases less than 1 year old, and 4 HSCR cases that were greater than 1 year old (Table 1). Of the 28 HSCR cases, 25 showed hypertrophic submucosal nerves (25/28=89.3%) and 3 showed no hypertrophic submucosal nerves (3/28 = 10.7%) (Table 6).

40 Table 6. HSCR patients separated as the number of false negative and true positive cases across ages.

Hirschsprung Disease (n=28) <40 휇푚 in diameter >/= 40 휇푚 in diameter Age (False Negative) (True Positive) All Ages 3 25 < 1 month 1 16 1 month 0 1 2-5 months 1 1 6-11 months 0 4 < 1 year 2 22 1 year 0 0 ≥ 1 year 1 3 2-5 years 1 0 6-10 years 0 2 >10 years 0 1

Out of the 151 non-HSCR cases, nearly 40% of cases showed hypertrophic submucosal nerves. Overall, 92 biopsies showed no hypertrophic nerves (92/151 =

60.9%) and 59 showed hypertrophic nerves (59/151 = 39.1%) (Table 7). Hypertrophic nerves were more commonly seen in older children, especially in patients greater than 1 year of age. For ages less than 1 year, 72 biopsies showed no hypertrophic nerves

(72/92 = 78.3%) and 19 showed hypertrophic nerves (19/59=32.2%) (Table 7). For ages greater than or equal to 1 year of age, 20 cases showed no hypertrophic nerves (20/92 =

21.7%) and 40 biopsies showed hypertrophic nerves (40/59 = 67.8%) (Table 7).

41 Table 7. Non-HSCR patients separated as the number of false negative and true positive cases across ages.

Non-Hirschsprung (n=151) <40 휇푚 in diameter >/= 40 휇푚 in diameter Age (True Negative) (False Positive) All Ages 92 59 <1 month 18 6 1 month 22 4 2-5 months 27 8 6-10 months 5 1 11-12 months 0 1 >12 months 20 39 6-12 months 5 2 <1 year 72 19 1 year 5 4 ≥ 1 year 20 40 2-5 years 6 14 6-10 years 5 10 >10 years 4 12

The sensitivity, specificity, and p-values were determined for each age group

(Table 5). Over all ages, the sensitivity of hypertrophic submucosal nerves for HSCR is

89.29% and the specificity is 60.93%. The p-value was statistically significant (p<0.0001)

(Table 5). The sensitivity and specificity for cases less than 1 year of age is 91.67% and

79.12%, respectively. The cases less than 1 year of age were statistically significant

(p<0.0001) (Table 5). The sensitivity and specificity for cases greater than or equal to one year of age is 75.00% and 33.33%, respectively. The cases greater than or equal to 1 year of age were not statistically significant (p=1) (Table 5). The highest sensitivity

(100%) and specificity (84.62%) values were for 1 month of age, but the p-value did not show statistical significance (p = 0.1852) (Table 5). Although the groupings for ages in

42 months did not all have p-values that were significant, overall the p-value for ages less than 1 year was statistically significant (p<0.0001) (Table 5).

5.3 Determining optimal cutoff of nerve diameter in HSCR patients

For the ROC curve there were 24 HSCR cases and 92 non-HSCR cases that were less than 1 year old, and thus a total of 116 cases. The true positive rate was on the y- axis and the false positive rate was on the x-axis (Figure 6). Based on the ROC curve, a nerve diameter of 45 micrometers is located at the coordinate points (0.1739, 0.875) and shows a true positive rate of 87.5% and a false positive rate of 17.4% (Figure 6). For our study, a cutoff point greater than or equal to 45 microns is supportive of HSCR as this value has a high sensitivity while maintaining specificity. This cutoff point shows greater specificity compared to 40 microns at the coordinate points (0.2174, 0.9167), which shows a sensitivity of 91.67%, a specificity of 78.26%, and a p value <0.0001. The nerve cutoff of 40 microns has a higher sensitivity, but a smaller sensitivity (true positive rate), and does not maintain the sensitivity. By obtaining a high sensitivity and maintaining specificity, 45 microns is a good approximate cutoff point for a nerve diameter suggestive of HSCR for patients less than 1 year of age.

Figure 6. Receiver operating characteristic curve. ROC curve with the true positive rate

(sensitivity) on the y-axis and the false positive rate (1-specificity) on the x-axis. A nerve diameter of 45 microns has a false positive rate of 0.1739 (17.4%), a specificity of 0.826

(82.6%), and a true positive rate of 0.875 (87.5%). A nerve diameter of 40 microns has a false positive rate of 0.2174 (21.7%), a specificity of 0.783 (78.3%), and a true positive rate of 0.9167 (91.7%).

Chapter 6: Discussion

In the literature, there have been few studies that have shown the distribution of nerve diameter in non-HSCR patients. A study by Kapur and Kennedy in 2013 looked at submucosal nerve diameter in autopsies of 9 non-HSCR patients, all less than 1 year old.

They found that 3 of the 9 non-HSCR patients had hypertrophic nerves, suggesting that non-HSCR patients less than 1 year of age may also present with hypertrophic nerves.

These results question if nerve diameter is useful at all. Another study by Coe and colleagues in 2012 looked at 5 non-HSCR cases with familial adenomatous polyposis who underwent colectomies due to the presence of numerous adenomas. The submucosal nerves in these 5 cases were measured, and they found that there were hypertrophic submucosal nerves in the two oldest patients that were 13.5 and 16 years old. This suggested that nerve diameter increased with increasing age in non-HSCR patients. In 2016, Kapur then added to his previous autopsy study, including patients who were older than 1 year. He found that nerve diameter increases with increasing age

(Kapur, 2016). Our study was able to expand on and support the current knowledge, providing a larger subset of data from non-HSCR patients to see submucosal nerve diameter distribution across ages, from less than 1 month, to greater than 1 year.

45 We first sought to determine the distribution of submucosal nerve diameter across ages in cases without HSCR to then compare these nerve measurements to cases with HSCR. In patients without HSCR, the submucosal nerve diameter increases with increasing age. Once establishing this knowledge, we could then compare the HSCR nerve diameters to the non-HSCR nerve diameters across ages.

We found that average submucosal nerve diameters in HSCR cases are significantly larger than non-HSCR nerve diameters at ages less than one year of age.

Based on the receiver operating characteristic curve, a measurement of 45 microns or larger may be supportive of a HSCR diagnosis when the patient is less than 1 year of age.

However, if the patient is older than 1 year and shows aganglionosis, the submucosal nerve measurement should not be applied, as the nerves of non-HSCR cases have similar nerve diameter values to those of HSCR cases at one year of age and beyond.

Based on the literature, hypertrophic nerves are defined as being 40 microns or larger (Sangkhathat et al., 2002; Monforte-Munoz, 1998). This nerve measurement has been applied clinically for aiding in the diagnosis of HSCR. Most cases of HSCR are diagnosed within the first year of life. However, pathologists were unsure if the nerves were still considered to be hypertrophic if nerve measurements were 40 microns or larger for patients ages one and older. Based on the results of our study, a submucosal nerve measurement of 45 microns or larger should be supportive of a HSCR diagnosis if the patient is less than 1 year of age.

46 One limitation of this study would be the lack of HSCR cases older than 1 year of age, as many HSCR cases are diagnosed within the first year. Another limitation was that this study only looked at rectal biopsies taken from 2017 to 2018, although this study did have more cases than other studies in the literature as Nationwide Children’s

Hospital is a referral center for HSCR. A third limitation was that only one pathologist confirmed the slides. The results could have been different if other pathologists were included as the method of measuring the nerves may slightly different from person to person.

In the future, more HSCR cases can be collected over a larger time span so that average submucosal nerve measurements from these patients, especially at age 1 year and older, can be collected. We could study the impact of body mass index (BMI) on nerve diameter, as a larger BMI may increase nerve diameters. It would also be interesting to see the proportion of HSCR cases with genetic abnormalities, such as

Down Syndrome, as approximately ten percent of HSCR patients also have Down

Syndrome (Szylberg and Marszalek, 2014).

References

Bjørn, N., Rasmussen, L., Qvist, N., Detlefsen, S., Bremholm, M.B. (2018, October) Full-

thickness rectal biopsy in children suspicious for Hirschsprung’s Disease is safe and

yields a low number of insufficient biopsies. Journal of Pediatric Surgery 53, no. 10:

1942–44.

Carlson, B. (2018) Human Embryology and Developmental Biology. 6th ed. Elsevier

Chiva, L.M. & Magrina, J. (2019) Abdominal and pelvic anatomy. Principles of Gynecologic

Oncology Surgery, 3–49. Philadelphia, PA: Elsevier

Coe, A., Collins, M.H., Lawal, T., Louden, E., Levitt, M.A., Peña, A. (2012, February)

Reoperation for Hirschsprung Disease: pathology of the resected problematic distal pull-

through. Pediatric and Developmental Pathology: The Official Journal of the Society for

Pediatric Pathology and the Paediatric Pathology Society 15, no. 1: 30–38.

Das, K. & Mohanty, S. (2017, August) Hirschsprung Disease - current diagnosis and

management. Indian Journal of Pediatrics 84, no. 8: 618–23.

De La Torre, L. & Langer, C. (2010, May) Transanal endorectal pull-through for Hirschsprung

Disease: technique, controversies, pearls, pitfalls, and an organized approach to the

management of postoperative obstructive symptoms. Seminars in Pediatric Surgery 19,

no. 2: 96–106.

48 Drake, R., Vogl, A.W., Mitchell, A., Gray, H. (2015) Gray’s Anatomy for Students. 3rd edition.

Philadelphia, PA: Elsevier Health Sciences

Duhamel, B. (1960, February) A new operation for the treatment of Hirschsprung’s Disease.

Archives of Disease in Childhood 35, no. 179: 38–39.

Furness, J.B., Poole, D.P., Cho, H., Callaghan, B.P., Rivera, L.R. (2015) The innervation of the

gastrointestinal tract. Yamada’ s Textbook of Gastroenterology, 239–58. John Wiley &

Sons, Ltd.

Guinard-Samuel, V., Bonnard, A., De Lagausie, P., Philippe-Chomette, P., Alberti, C., El

Ghoneimi, A., Peuchmaur, M., Berrebi-Binczak, D. (2009, October) Calretinin

immunohistochemistry: a simple and efficient tool to diagnose Hirschsprung Disease.

Modern Pathology: An Official Journal of the United States and Canadian Academy of

Pathology, Inc 22, no. 10: 1379–84.

Heuckeroth, R.O. (2018, March) Hirschsprung Disease — integrating basic science and clinical

medicine to improve outcomes. Nature Reviews Gastroenterology & Hepatology 15, no.

3: 152–67.

Kapur, R.P. (2016, December) Histology of the transition zone in Hirschsprung Disease. The

American Journal of Surgical Pathology 40, no. 12: 1637–46.

Kapur, R.P. (2006, July) Can we stop looking? Immunohistochemistry and the diagnosis of

Hirschsprung Disease. American Journal of Clinical Pathology 126, no. 1: 9–12.

49 Kapir, R.P. (2016, October) Submucosal nerve diameter of greater than 40 µm is not a valid

diagnostic index of transition zone pull-through. Journal of Pediatric Surgery 51, no. 10:

1585–91.

Kapur, R.P. & Kennedy, A.J. (2013, August) Histopathologic delineation of the transition zone

in short-segment Hirschsprung Disease. Pediatric and Developmental Pathology: The

Official Journal of the Society for Pediatric Pathology and the Paediatric Pathology

Society 16, no. 4: 252–66.

Langer, J. (2013, June) Hirschsprung Disease. Current Opinion in Pediatrics 25, no. 3: 368–74.

Lowe, J., Anderson, P., Anderson, S.. Steven & Lowe’s Human Histology. (2019) 5th ed.

Elsevier

Lukac, M., Antunović, S.S., Vujović, D., Petronić, I., Nikolić, D., Radlović, V., Krstajić, T., Krstić,

Z. (2016, March) Effectiveness of various surgical methods in treatment of

Hirschsprung’s Disease in children. Vojnosanitetski Pregled 73, no. 3: 246–50.

Monforte-Muñoz, H., Gonzalez-Gomez, I., Rowland, J.M., Landing, B.H. (1998, August)

Increased submucosal nerve trunk caliber in aganglionosis: a ‘positive’ and objective

finding in suction biopsies and segmental resections in Hirschsprung’s Disease. Archives

of Pathology & Laboratory Medicine 122, no. 8: 721–25.

Moore, K.L. (2015) Essential Clinical Anatomy. 5th edition. Philadelphia, PA: Lippincott

Williams & Wilkins

50 Muise, E.D., Hardee, S., Morotti, R.A., Cowles, R.A. (2016, March) A comparison of suction

and full-thickness rectal biopsy in children. Journal of Surgical Research 201, no. 1: 149–

55.

Nagy, N. & Goldstein, A.M. (2017, June 1) Enteric nervous system development: a crest cell’s

journey from neural tube to colon. Seminars in Cell & Developmental Biology,

Development of the digestive organs, 66: 94–106.

Qualman, S.J., Jaffe, R., Bove, K.E., Monforte-Muñoz, H. (1999, November 1) Diagnosis of

Hirschsprung Disease using the rectal biopsy: multi-institutional survey. Pediatric and

Developmental Pathology 2, no. 6: 588–96.

Ross, M.H. & Pawlina, W. (2016) Histology: A Text and Atlas, with Correlated Cell and

Molecular Biology. 7th ed. Philadelphia, PA: Lippincott Williams & Wilkins

Sadler, T.W. (2014) Langman’s Medical Embryology. 13th ed. Lippincott Williams & Wilkins

Sangkhathat, S., Tadtayathikom, K., Patrapinyokul, S., Mitarnun, W. (2002, February)

Congenital megacolon: diagnosis using size of sub-mucosal nerve trunk as a criteria.

Journal of the Medical Association of Thailand = Chotmaihet Thangphaet 85, no. 2: 250–

55.

Schoenwolf, G.C., Bleyl, S.B., Brauer, P.R., Francis-West, P.H. (2015) Larsen’s Human

Embryology. Fifth.

Smith, C., Ambartsumyan, L., Kapur, R.P. (2020, February) Surgery, surgical pathology, and

postoperative management of patients with Hirschsprung Disease. Pediatric and

51 Developmental Pathology: The Official Journal of the Society for Pediatric Pathology and

the Paediatric Pathology Society 23, no. 1: 23–39.

Szylberg, L. & Marszałek, A. (2014) Diagnosis of Hirschsprung’s Disease with particular

emphasis on histopathology: a systematic review of current literature. Przeglad

Gastroenterologiczny 9, no. 5: 264–69.

Wester, T. & Granström, A.L. (2017, October) Hirschsprung Disease-bowel function beyond

childhood. Seminars in Pediatric Surgery 26, no. 5: 322–27.

Xi, M. & Lui, F. (2019) Neuroanatomy, Neural Crest. StatPearls. Treasure Island (FL):

StatPearls Publishing

Xu, Z., Zhao, Z., Wang, L., An, Q., Tao, W. (2009, December 5) A new modification of transanal

swenson pull-through procedure for Hirschsprung’s Disease. Chinese Medical Journal

121, no. 23: 2420–23.