Hirschsprung disease.

Hirschsprung disease (HSCR) is a rare congenital intestinal disease that occurs in 1 in 5,000 live births. HSCR is characterized by the absence of ganglion cells in the myenteric and submucosal plexuses of the intestine. Most patients present during the neonatal period with the first meconium passage delayed beyond 24 h, abdominal distension and vomiting. Syndromes associated with HSCR include trisomy 21, Mowat-Wilson syndrome, congenital central hypoventilation syndrome, Shah-Waardenburg syndrome and cartilage-hair hypoplasia. Multiple putative genes are involved in familial and isolated HSCR, of which the most common are the RET proto-oncogene and EDNRB. Diagnosis consists of visualization of a transition zone on contrast enema and confirmation via rectal biopsy. HSCR is typically managed by surgical removal of the aganglionic bowel and reconstruction of the intestinal tract by connecting the normally innervated bowel down to the anus while preserving normal sphincter function. Several procedures, namely Swenson, Soave and Duhamel procedures, can be undertaken and may include a laparoscopically assisted approach. Short-term and long-term comorbidities include persistent obstructive symptoms, enterocolitis and soiling. Continued research and innovation to better understand disease mechanisms holds promise for developing novel techniques for diagnosis and therapy, and improving outcomes in patients.

Computer-Based Natural Language Search Applied to the Electronic Medical Record for Tonsil Triage.

OBJECTIVES: To determine significant histologic findings in tonsils and categorize clinical settings in which they occur to identify cases benefiting from histopathologic examination using a computer-based natural language search (NLS) applied to the electronic medical record. METHODS: The pathology database was queried for tonsillectomy cases accessioned between 2002 and 2018. Tonsils with microscopic examination were reviewed, and indication for examination and diagnoses were tallied. Clinical risk of malignancy was correlated with findings. A NLS was used to interrogate preoperative clinical records of the same group of patients. The search identified cases at risk of significant histologic findings and was implemented as part of standard practice. RESULTS: Of the 18,733 bilateral tonsillectomies identified in the pathology database, 494 were palatine tonsils that underwent microscopic examination, 134 had indications concerning for malignancy, and 14 had significant findings on histologic examination. When the NLS was applied to the medical record of the same group, 223 cases were identified as having risk of malignancy, including all flagged by surgeons and pathologists and 89 additional cases. Clinical implementation resulted in identification of all cases benefiting from examination. CONCLUSIONS: A NLS applied to the electronic medical record to select tonsils for examination was superior to relying on surgeons and pathologists.

Spatial profiling of the placental chorioamniotic membranes reveals upregulation of immune checkpoint proteins during Group B Streptococcus infection in a nonhuman primate model.

Background: Preterm birth is a leading cause of neonatal mortality, which is often complicated by intrauterine infection and inflammation. We have established a nonhuman primate model of Group B Streptococcus (GBS, Streptococcus agalactiae) infection-associated preterm birth. Immune checkpoints are modulators of the immune response by activating or suppressing leukocyte function and are understudied in preterm birth. The objective of this study was to spatially profile changes in immune protein expression at the maternal-fetal interface during a GBS infection with a focus on immune checkpoints. Methods: Twelve nonhuman primates (pigtail macaques, Macaca nemestrina) received a choriodecidual inoculation of either: 1) 1-5 X 108 colony forming units (CFU) of hyperhemolytic/hypervirulent GBS (GBSΔcovR, N=4); 2) an isogenic/nonpigmented strain (GBS ΔcovRΔcylE, N=4); or, 3) saline (N=4). A Cesarean section was performed at preterm labor or 3 days after GBS infection or 7 days after saline inoculation. Nanostring GeoMx® Digital Spatial Profiling technology was used to segment protein expression within the amnion, chorion, and maternal decidua at the inoculation site using an immuno-oncology panel targeting 56 immunoproteins enriched in stimulatory and inhibitory immune checkpoint proteins or their protein ligands. Statistical analysis included R studio, Kruskal-Wallis, Pearson and Spearman tests. Results: Both inhibitory and stimulatory immune checkpoint proteins were significantly upregulated within the chorioamniotic membranes and decidua (VISTA, LAG3, PD-1, CD40, GITR), as well as their ligands (PD-L1, PD-L2, CD40L; all p<0.05). Immunostaining for VISTA revealed positive (VISTA+) cells, predominantly in the chorion and decidua. There were strong correlations between VISTA and amniotic fluid concentrations of IL-1ß, IL-6, IL-8, and TNF-α (all p<0.05), as well as maternal placental histopathology scores (p<0.05). Conclusion: Differential regulation of multiple immune checkpoint proteins in the decidua at the site of a GBS infection indicates a major perturbation in immunologic homeostasis that could benefit the host by restricting immune-driven pathologies or the pathogen by limiting immune surveillance. Protein expression of VISTA, an inhibitory immune checkpoint, was upregulated in the chorion and decidua after GBS infection. Investigating the impact of innate immune cell expression of inhibitory immune checkpoints may reveal new insights into placental host-pathogen interactions at the maternal-fetal interface.

Rectal suction biopsy versus incisional rectal biopsy in the diagnosis of Hirschsprung disease.

BACKGROUND: Hirschsprung disease is one of the most common congenital anomalies that affect colorectal function. Rectal biopsy demonstrating the absence of ganglion cells in the affected bowel is the gold standard for diagnosis. Suction and incisional rectal biopsies are appropriate methods for obtaining diagnostic tissue. The goal of this study is to determine if any differences in adequacy exist between suction and incisional rectal biopsies at our institution. METHODS: We conducted a retrospective review of suction and incisional rectal biopsies for inadequacy per procedure at a tertiary pediatric hospital. Each procedure for rectal biopsy was also evaluated by a number of biopsies per procedure. We used a two-sample test of proportions to compare the inadequacy of suction vs. incisional biopsies. RESULTS: 133 rectal suction biopsy procedures (227 biopsies) and 125 incisional biopsy procedures (140 biopsies) were analyzed. In patients 6 months of age and older, the percentage of inadequate procedures was substantially higher in the suction biopsy group (24.1% vs 0.9%, p < 0.01). CONCLUSIONS: A substantially higher proportion of inadequacy was found in the suction rectal biopsy group compared to the incisional cohort among the older patient cohort, suggesting incisional biopsies should be strongly considered as the primary rectal biopsy method in patients older than 6 months.

Recurrent Gingival Lesions in a Pediatric Patient.

We present the case of a 13-year-old girl who developed numerous gingival masses that recurred after two prior resections. Following the initial resection as a child, she reported that there was a period of resolution for several years before recurrence as a teenager. After the second resection, the masses recurred after 4 months. The lesions obscured the majority of her dentition and interfered with speech, eating, and oral hygiene. The patient underwent staged resection of the masses, and the wounds were allowed to heal by secondary intention. The histopathologic findings of the specimens were consistent with a diagnosis of peripheral ossifying fibroma, which is unusual as these are generally solitary lesions. We believe that this case brings attention to an underrecognized and atypical presentation of peripheral ossifying fibroma, and it should be considered in the differential diagnosis of multicentric gingival masses.

Intestinal Pathology in Patients With Pathogenic ACTG2-Variant Visceral Myopathy: 16 Patients From 12 Families and Review of the Literature.

BACKGROUND: Dominant gamma-smooth muscle actin gene (ACTG2) variants cause clinically diverse forms of visceral myopathy. Many patients undergo intestinal resection or biopsy before identification of their genetic defect. The pathology of ACTG2-variant visceral myopathy has not been evaluated systematically. METHODS: Glass slides, ultrastructural images, molecular genetic reports, and clinical records from 16 patients with pathogenic (15) or likely pathogenic (1) ACTG2 variants were reviewed and compared with surgical specimens from controls (no evidence of a primary myopathy or pseudo-obstruction due to Hirschsprung disease) and published descriptions. RESULTS: The variable clinical manifestations in our cohort matched those in the literature. Only non-specific light and electron microscopic findings observed in non-myopathic controls were encountered in 13 of 16 patients. The remaining 3 patients harbored hyalinized cytoplasmic inclusions in smooth muscle cells and 1 of them had polyglucosan bodies in the muscularis propria. CONCLUSIONS: Apart from hyalinized inclusions, which were only observed in 3/16 patients, intestinal pathology in the majority of patients with ACTG2 variants is not indicative of an underlying visceral myopathy. Molecular testing should be considered even when no diagnostic intestinal pathology is identified.

Profiling PI3K-AKT-MTOR variants in focal brain malformations reveals new insights for diagnostic care.

Focal malformations of cortical development including focal cortical dysplasia, hemimegalencephaly and megalencephaly, are a spectrum of neurodevelopmental disorders associated with brain overgrowth, cellular and architectural dysplasia, intractable epilepsy, autism and intellectual disability. Importantly, focal cortical dysplasia is the most common cause of focal intractable paediatric epilepsy. Gain and loss of function variants in the PI3K-AKT-MTOR pathway have been identified in this spectrum, with variable levels of mosaicism and tissue distribution. In this study, we performed deep molecular profiling of common PI3K-AKT-MTOR pathway variants in surgically resected tissues using droplet digital polymerase chain reaction (ddPCR), combined with analysis of key phenotype data. A total of 159 samples, including 124 brain tissue samples, were collected from 58 children with focal malformations of cortical development. We designed an ultra-sensitive and highly targeted molecular diagnostic panel using ddPCR for six mutational hotspots in three PI3K-AKT-MTOR pathway genes, namely PIK3CA (p.E542K, p.E545K, p.H1047R), AKT3 (p.E17K) and MTOR (p.S2215F, p.S2215Y). We quantified the level of mosaicism across all samples and correlated genotypes with key clinical, neuroimaging and histopathological data. Pathogenic variants were identified in 17 individuals, with an overall molecular solve rate of 29.31%. Variant allele fractions ranged from 0.14 to 22.67% across all mutation-positive samples. Our data show that pathogenic MTOR variants are mostly associated with focal cortical dysplasia, whereas pathogenic PIK3CA variants are more frequent in hemimegalencephaly. Further, the presence of one of these hotspot mutations correlated with earlier onset of epilepsy. However, levels of mosaicism did not correlate with the severity of the cortical malformation by neuroimaging or histopathology. Importantly, we could not identify these mutational hotspots in other types of surgically resected epileptic lesions (e.g. polymicrogyria or mesial temporal sclerosis) suggesting that PI3K-AKT-MTOR mutations are specifically causal in the focal cortical dysplasia-hemimegalencephaly spectrum. Finally, our data suggest that ultra-sensitive molecular profiling of the most common PI3K-AKT-MTOR mutations by targeted sequencing droplet digital polymerase chain reaction is an effective molecular approach for these disorders with a good diagnostic yield when paired with neuroimaging and histopathology.

Amniotic fluid interleukin 6 and interleukin 8 are superior predictors of fetal lung injury compared with maternal or fetal plasma cytokines or placental histopathology in a nonhuman primate model.

BACKGROUND: Intra-amniotic infection or inflammation is common in early preterm birth and associated with substantial neonatal lung morbidity owing to fetal exposure to proinflammatory cytokines and infectious organisms. Amniotic fluid interleukin 8, a proinflammatory cytokine, was previously correlated with the development of neonatal bronchopulmonary dysplasia, but whether amniotic fluid cytokines or placental pathology more accurately predicts neonatal lung pathology and morbidity is unknown. We have used a pregnant nonhuman primate model of group B Streptococcus infection to study the pathogenesis of intra-amniotic infection, bacterial invasion of the amniotic cavity and fetus, and microbial-host interactions. In this nonhuman primate model, we have studied the pathogenesis of group B Streptococcus strains with differing potential for virulence, which has resulted in a spectrum of intra-amniotic infection and fetal lung injury that affords the opportunity to study the inflammatory predictors of fetal lung pathology and injury. OBJECTIVE: This study aimed to determine whether fetal lung injury is best predicted by placental histopathology or the cytokine response in amniotic fluid or maternal plasma. STUDY DESIGN: Chronically catheterized pregnant monkeys (Macaca nemestrina, pigtail macaque) at 116 to 125 days gestation (term at 172 days) received a choriodecidual inoculation of saline (n=5), weakly hemolytic group B Streptococcus strain (n=5, low virulence), or hyperhemolytic group B Streptococcus strain (n=5, high virulence). Adverse pregnancy outcomes were defined as either preterm labor, microbial invasion of the amniotic cavity, or development of the fetal inflammatory response syndrome. Amniotic fluid and maternal and fetal plasma samples were collected after inoculation, and proinflammatory cytokines (tumor necrosis factor alpha, interleukin beta, interleukin 6, interleukin 8) were measured by a multiplex assay. Cesarean delivery was performed at the time of preterm labor or within 1 week of inoculation. Fetal necropsy was performed at the time of delivery. Placental pathology was scored in a blinded fashion by a pediatric pathologist, and fetal lung injury was determined by a semiquantitative score from histopathology evaluating inflammatory infiltrate, necrosis, tissue thickening, or collapse scored by a veterinary pathologist. RESULTS: The principal findings in our study are as follows: (1) adverse pregnancy outcomes occurred more frequently in animals receiving hyperhemolytic group B Streptococcus (80% with preterm labor, 80% with fetal inflammatory response syndrome) than in animals receiving weakly hemolytic group B Streptococcus (40% with preterm labor, 20% with fetal inflammatory response syndrome) and in controls (0% preterm labor, 0% fetal inflammatory response syndrome); (2) despite differences in the rate of adverse pregnancy outcomes and fetal inflammatory response syndrome, fetal lung injury scores were similar between animals receiving the weakly hemolytic group B Streptococcus strains and animals receiving the hyperhemolytic group B Streptococcus strains; (3) fetal lung injury score was significantly correlated with peak amniotic fluid cytokines interleukin 6 and interleukin 8 but not tumor necrosis factor alpha or interleukin 1 beta; and (4) fetal lung scores were poorly correlated with maternal and fetal plasma cytokine levels and placental pathology. CONCLUSION: Amniotic fluid interleukin 6 and interleukin 8 levels were superior predictors of fetal lung injury than placental histopathology or maternal plasma cytokines. This evidence supports a role for amniocentesis in the prediction of neonatal lung morbidity owing to intra-amniotic infection, which cannot be provided by cytokine analysis of maternal plasma or placental histopathology.

Congenital Myenteric Hypoganglionosis.

Congenital myenteric hypoganglionosis is a rare developmental disorder characterized clinically by severe and persistent neonatal intestinal pseudoobstruction. The diagnosis is established by the prevalence of small myenteric ganglia composed of closely spaced ganglion cells with sparse surrounding neuropil. In practice, the diagnosis entails familiarity with the normal appearance of myenteric ganglia in young infants and the ability to confidently recognize significant deviations in ganglion size and morphology. We review clinical, histologic, and immunohistochemical findings from 12 patients with congenital myenteric hypoganglionosis in comparison with similar data from age-matched controls and clearly delineate the diagnostic features of the condition. Practical guidelines are provided to assist surgical pathologists, who are likely to encounter this condition only infrequently. The diagnosis typically requires full-thickness intestinal biopsy as the abnormality is confined to the myenteric plexus in many patients. Immunohistochemistry for Hu C/D may be used to confirm hypoganglionosis. Reduced staining for calretinin and NeuN implicates a selective deficiency of intrinsic primary afferent neurons in this disease.

Advances and Pitfalls in the Diagnosis of Hirschsprung Disease.

Surgical pathology for Hirschsprung disease (HSCR) occasionally is difficult, especially for those who encounter the disorder infrequently. This article reviews pathologic features of HSCR, considers various specimens the pathologist is required to evaluate, and discusses useful ancillary tests. Potential diagnostic pitfalls are highlighted, and helpful hints are provided to successfully navigate challenging situations. Finally, the article looks forward to new ancillary tests on the horizon and future topics for HSCR research.

ADAR1 mediated regulation of neural crest derived melanocytes and Schwann cell development.

The neural crest gives rise to numerous cell types, dysfunction of which contributes to many disorders. Here, we report that adenosine deaminase acting on RNA (ADAR1), responsible for adenosine-to-inosine editing of RNA, is required for regulating the development of two neural crest derivatives: melanocytes and Schwann cells. Neural crest specific conditional deletion of Adar1 in mice leads to global depigmentation and absence of myelin from peripheral nerves, resulting from alterations in melanocyte survival and differentiation of Schwann cells, respectively. Upregulation of interferon stimulated genes precedes these defects, which are associated with the triggering of a signature resembling response to injury in peripheral nerves. Simultaneous extinction of MDA5, a key sensor of unedited RNA, rescues both melanocytes and myelin defects in vitro, suggesting that ADAR1 safeguards neural crest derivatives from aberrant MDA5-mediated interferon production. We thus extend the landscape of ADAR1 function to the fields of neural crest development and disease.

Diagnosis of Hirschsprung Disease.

Diagnosis or exclusion of Hirschsprung disease (HSCR) is a frequent exercise in any pediatric hospital. Although HSCR may present at different ages and with varied clinical findings, the most common presentation is a neonate with severe constipation or signs of intestinal obstruction. A variety of diagnostic tests including contrast enema and anorectal manometry may be used as diagnostic screens, but diagnosis ultimately rests upon histopathological evaluation of a rectal biopsy. For the experienced pathologist, conventional hematoxylin-and-eosin-stained sections often suffice to exclude HSCR or establish the diagnosis. However, ancillary diagnostic tests such as acetylcholinesterase histochemistry or calretinin immunohistochemistry are complementary and extremely helpful in some cases. In this Perspectives article, we review the clinical and pathological features of HSCR, highlight those that are found in most patients, and discuss how to address particularly challenging aspects of the diagnostic workup.

Are We Underdiagnosing Hirschsprung Disease?

Hirschsprung disease (HSCR) is conventionally defined as aganglionosis of the distal rectum and a variable length of proximal contiguous bowel with a transition zone of ganglionic, but neuroanatomically abnormal, bowel located immediately upstream. Recent improvement in our understanding of the pathology and genetics of HSCR and relevant animal models indicates highly variable expressivity. The spectrum of intestinal neuropathology includes patients with very short-segment aganglionosis, limited to the distal 1 to 2 cm of the rectum, and possibly patients with no true aganglionic segment, but nonphysiological transition zone pathology in their distal rectums. The presence or absence of submucosal ganglion cells in a rectal biopsy is not sufficient to exclude these patients, in whom submucosal nerve hypertrophy and/or abnormal cholinergic mucosal innervation may be the only diagnostic clues. In addition, diagnosis or exclusion of HSCR by rectal biopsy now relies in part on mucosal patterns of calretinin immunohistochemistry, with less emphasis on submucosal tissue adequacy and assessment of cholinergic innervation. These recent trends in the surgical pathology approach to rectal biopsies may miss patients at the phenotypically milder end of the malformation spectrum, with profound implications for subsequent management, prognosis, and genetic counseling.

Surgery, Surgical Pathology, and Postoperative Management of Patients With Hirschsprung Disease.

Endorectal pullthrough surgery is integral in the treatment of patients with Hirschsprung disease. Several different surgical procedures exist, which share as common goals to excise the aganglionic segment and upstream transition zone and attach ganglionic bowel just proximal to the anal canal. The operation requires collaboration between surgeon and pathologist to localize ganglionic bowel and prevent retention of transition zone. Intraoperative frozen sections are extremely important, first to establish that ganglion cells are present and subsequently to exclude features of transition zone (partial circumferential aganglionosis, myenteric hypoganglionosis, and submucosal nerve hypertrophy) at the proximal surgical (anastomotic) margin. Postoperative histopathological analysis of resection specimens should be tailored to document distal aganglionosis, document the length of the aganglionic segment and its proximity to the anastomotic margin, and confirm that transition zone has been resected completely. Adherence to the recommendations described in this review will reduce the likelihood of transition zone pullthrough and should decrease the incidence of persistent postoperative obstructive symptoms.

Postoperative Pullthrough Obstruction in Hirschsprung Disease: Etiologies and Diagnosis.

Some patients continue to have obstructive symptoms and/or incontinence after pullthrough surgery for Hirschsprung disease. Incontinence can be due to injury to the anal sphincter and/or dentate line, abnormal colonic motility (nonretentive), or a chronic large stool burden (retentive). A diagnostic algorithm based on clinical and pathological evaluations can be applied to distinguish potential etiologies for obstructive symptoms, which segregate into anatomic (mechanical or histopathological) or physiologic subgroups. Valuable clinical information may be obtained by anorectal examination under anesthesia, radiographic studies, and anorectal or colonic manometry. In addition, histopathological review of a patient's original resection specimen(s) as well as postoperative biopsies of the neorectum usually are an important component of the diagnostic workup. Goals for the surgical pathologist are to exclude incomplete resection of the aganglionic segment or transition zone and to identify other neuromuscular pathology that might explain the patient's dysmotility. Diagnoses established from a combination of clinical and pathological data dramatically alter management strategies. In rare instances, reoperative pullthrough surgery is required, in which case the pathologist must be aware of histopathological features specific to redo pullthrough resection specimens.

Multimodality and multiterritory vascular imaging in a toddler with acquired, complex, and unusual aortic arch anatomy.

A 20-month-old otherwise well child with no cardiac history presented for evaluation of a recent onset cardiac murmur. His initial examination revealed significant right carotid bruit along with a transthoracic echocardiogram (ECHO) concerning for severe transverse arch hypoplasia with unusual appearance and left ventricular hypertrophy with preserved function. This report demonstrates the utility of various modalities including echo, CT, and MRI to assess vascular structures in different vascular territories in a challenging patient who underwent a satisfactory arch repair with unexpected pathology findings.

Guidelines for synoptic reporting of surgery and pathology in Hirschsprung disease.

BACKGROUND/PURPOSE: Synoptic, or standardized, reporting of surgery and pathology reports has been widely adopted in surgical oncology. Patients with Hirschsprung disease may experience morbidity related to surgical factors or underlying pathology and often undergo multiple operations. Our aim is to improve the postoperative outcome and care of patients with Hirschsprung disease by proposing a standardized set of data that should be included in every surgery and pathology report. METHODS: Members of the American Pediatric Surgical Association Hirschsprung Disease Interest Group and experts in pediatric pathology of Hirschsprung disease participated in group discussions, performed literature review and arrived at expert consensus guidelines for surgery and pathology reporting. RESULTS: The importance of accurate operative and pathologic reports and the implications of inadequate documentation in patients with Hirschsprung disease are discussed and guidelines for standardizing these reports are provided. CONCLUSIONS: Adherence to the principles of reporting for operations and surgical pathology may improve outcomes for Hirschsprung disease patients and will facilitate identification of correlations among morphology, function, genetics and outcomes, which are required to improve the overall management of these patients. LEVEL OF EVIDENCE: V.

Infra-anastomotic Innervation of Residual Aganglionic Distal Rectum After Pull-through Surgery for Hirschsprung Disease.

BACKGROUND: Descending neurons are important for intestinal reflex activities, including the recto-anal inhibitory reflex involved in normal defecation. Pull-through surgery for Hirschsprung disease results in the anastomosis of ganglionic bowel to native aganglionic rectum just superior to the internal anal sphincter, which potentially could allow for physiologically significant infra-anastomotic innervation. METHODS: The density and distribution of intramuscular neuronal nitric oxide synthase (nNOS)- and mucosal calretinin-immunoreactive nerves were evaluated proximal and distal to the anastomosis in redo resection specimens after pull-through surgery for Hirschsprung disease. The findings were compared with data collected from cadaveric controls with no history of dysmotility and the distal aganglionic segments of primary rectal resections from patients with Hirschsprung disease. RESULTS: Native aganglionic rectum of Hirschsprung patients lacks the normal lush intramuscular nNOS- and mucosal calretinin-immunoreactive nerves present in normal bowel. In post-pull-through resection specimens obtained more than 7 months after pull-through surgery, nNOS- and calretinin-positive innervation is at least partially restored for variable distances up to 10 to 12 mm inferior to the anastomosis, respectively. CONCLUSIONS: Innervation of infra-anastomotic muscularis propria and mucosa in the aganglionic distal rectum occurs to a variable degree after pull-through surgery for Hirschsprung disease and may contribute to individual differences in postoperative obstructive symptoms. Strategies to enhance infra-anastomotic innervation may improve clinical outcome.

Atypical Hepatic Mesenchymal Hamartoma: Histologic Appearance, Immunophenotype, and Molecular Findings.

Hepatic mesenchymal hamartoma is a rare benign neoplasm principally encountered in young children. Its origin is unknown. We report an unusual hepatic mesenchymal hamartoma in a 7-month-old girl, including histopathologic findings, immunophenotype, and karyotype. Chromosomal microarray analysis of tumoral tissue and circulating lymphocytes found 4 copies of a segment at 1q44 and fluorescence in situ hybridization indicated tandem triplication, ascribed to expansion of a paternal tandem duplication. This genetic abnormality may have played a role in pathogenesis.