Histone H3 trimethylation on lysine 27 immunostaining pattern in DICER1-associated tumors.

Background: DICER1-associated tumors are heterogeneous and affect several organs. DICER1-associated primary intracranial sarcoma is associated with histone H3 trimethylation on lysine 27 (H3K27me3) loss in nucleus by immunohistochemistry. Methods: We explored the H3K27me3 immunostaining pattern in other DICER1-associated tumors. Twelve tumors from eleven patients with confirmed DICER1 mutations (sporadic and germline) data from a pancancer next-generation sequencing panel, and four tumors of pleuropulmonary blastoma (PPB) were retrieved from our database and stained with anti-H3K27me3 antibody. Results: The H3K27me3 expression in the nucleus showed heterogeneous mosaic loss in neoplastic Sertoli cell components in three of the five cases of moderately to poorly differentiated Sertoli-Leydig cell tumors. Among two tumors of DICER1-associated primary intracranial sarcoma, one showed complete loss of H3K27me3 in all neoplastic cells, whereas the other showed mosaic loss in the sarcomatous spindle cells. One DICER1-associated tumor with epithelial and mesenchymal differentiation, including pulmonary blastoma and PPB, showed mosaic loss of glandular epithelial and mesenchymal components. Four cases of type II PPB and a single case of type III PPB showed a similar mosaic loss of H3K27me3 staining restricted to large spindle cell components. All other components in all tumors-including Leydig cells; the areas of epithelial, cartilaginous, and rhabdomyomatous differentiation; and all cells of the remaining three cases (one papillary thyroid carcinoma and two cases of PPB type I)-demonstrated retained H3K27me3 staining. Conclusions: H3K27me3 expression is not universally lost in DICER1-associated tumors and thus is not predictive of DICER1 mutation status. The mosaic regional loss of H3K27me3 immunostaining is consistent in PPB type II and III, which can be a helpful diagnostic marker for these tumors and suggests a similarity to DICER1-associated intracranial sarcoma.

Comparative Clinicopathologic and Genomic Analysis of Hepatocellular Neoplasm, Not Otherwise Specified, and Hepatoblastoma.

Accurate diagnosis and treatment of hepatocellular neoplasm, not otherwise specified (HCN-NOS), poses significant challenges. Our study aimed to investigate the clinicopathologic and genomic similarities and differences between HCN-NOS and hepatoblastoma (HB) to guide diagnostic and treatment strategies. The clinicopathologic characteristics of 16 patients with HCN-NOS and 23 patients with HB were compared. Molecular studies, including the OncoKids DNA- and RNA-based next-generation sequencing panel, chromosomal microarray, and targeted Sanger sequencing analyses of CTNNB1 and TERT promoters, were employed. We found that patients with HCN-NOS were older (P < .001) and more frequently classified as high risk (P < .01), yet they showed no significant differences in alpha fetoprotein levels or survival outcomes compared with those with HB. HCN-NOS and HB had a comparable frequency of sequence variants, with CTNNB1 mutations being predominant in both groups. Notably, TERT promoter mutations (37.5%) and rare clinically significant variants (BRAF, NRAS, and KMT2D) were exclusive to HCN-NOS. HCN-NOS demonstrated a higher prevalence of gains in 1q, encompassing the MDM4 locus (17/17 vs 11/24; P < .001), as well as loss/loss of heterozygosity (LOH) of 1p (11/17 vs 6/24; P < .05) and chromosome 11 (7/17 vs 1/24; P < .01) when compared with HB. Furthermore, the recurrent loss/LOH of chromosomes 3, 4p, 9, 15q, and Y was only observed in HCN-NOS. However, no significant differences were noted in gains of chromosomes 2, 8, and 20, or loss/LOH of 4q and 11p between the 2 groups. Notably, no clinically significant gene fusions were detected in either group. In conclusion, our study reveals that HCN-NOS exhibits high-risk clinicopathologic features and greater structural complexity compared with HB. However, patients with HCN-NOS exhibit comparable alpha fetoprotein levels at diagnosis, CTNNB1 mutation rates, and survival outcomes when subjected to aggressive treatment, as compared with those with HB. These findings have the potential to enhance diagnostic accuracy and inform more effective treatments for HCN-NOS.

Pediatric Sertoli-Leydig Cell Tumors of the Ovary: An Integrated Study of Clinicopathological Features, Pan-cancer-Targeted Next-generation Sequencing and Chromosomal Microarray Analysis From a Single Institution.

Sertoli-Leydig cell tumors (SLCTs) are currently classified into 3 molecular subtypes: DICER1 -mutant (younger patient age), FOXL2 -mutant, and DICER1/FOXL2 -wildtype. However, it is not clear whether all pediatric SLCTs are DICER1 -mutant molecular subtypes and whether other molecular genetic aberrations besides DICER1 are involved in the pathogenesis and prognosis of these tumors. We studied comprehensive data for 8 cases of pediatric SLCTs, including clinicopathological features, pan-cancer-targeted next-generation sequencing/OncoKids panel, and chromosomal microarray analysis, to further analyze the correlation among clinicopathological features, molecular genetic aberrations, and prognosis. The ages of the patients ranged from 4 to 16 years (median, 14 y). Seven cases were moderately differentiated, and one was poorly differentiated with heterologous mesenchymal elements. Two cases had heterologous epithelium or retiform elements. Follow-up was available for all 8 patients (median, 49.5 mo). Seven patients were alive without evidence of recurrence or metastasis, and only case 5 developed metastases (synchronous bilateral pulmonary tumors with rhabdomyosarcomatous differentiation). All 8 tumors were found to harbor somatic hotspot DICER1 mutations, and 5 patients carried germline DICER1 mutations (2 of them had the phenotype of DICER1 syndrome). Together with recent studies, the DICER1 mutation frequency is 100% in pediatric SLCTs (n=27, age≤16 y). Copy number alterations were detected in 3 tumors; the only recurrent copy number alterations was the gain of whole chromosome 6 in case 5 and case 8. This is the first report describing clinicopathological features and molecular alterations in pediatric SLCTs. Our results demonstrate that all pediatric SLCTs belong to the DICER1 -mutant molecular subtype, highlighting that somatic hotspot DICER1 mutation detection has high sensitivity (100%) for the auxiliary diagnosis of pediatric SLCTs (age ≤16 y). Some pediatric SLCTs harbor molecular genetic aberrations other than DICER1 mutation, and their significance needs further study.

Lysophosphatidic acid receptor 1 antagonist (EPGN2154) causes regression of NASH in preclinical NASH models.

BACKGROUND: NASH causes a tremendous health care burden in the United States. A glucagon-like peptide-1 agonist, semaglutide (Sema), treatment resulted in hepatic steatosis reduction in clinical trials of NASH. Lysophosphatidic acid receptor 1 antagonists are known to have antifibrotic effects in several organs. We tested Sema and a novel lysophosphatidic acid receptor 1 antagonist, EPGN2154, individually and in combination to evaluate their efficacy for NASH remission in preclinical models. METHODS: In the present study, we used (1) C57Bl6/J wild-type mice fed on a high-fat, high-carbohydrate (HFHC) diet for 16 weeks and (2) leptin-deficient mice (ob/ob) fed on an Amylin liver NASH diet for 16 weeks. After 16 weeks, the mice were randomly distributed in equal numbers in (1) no-drug, (2) EPGN2154, (3) Sema, and (4) EPGN2154+Sema treatment groups for 8 additional weeks at a dosage of 10 mg/kg body weight for EPGN2154 (oral gavage, 5 days a week) and 6.17 µg/kg body weight of Sema (subcutaneous injection every alternate day, 3 days a week). RESULTS: In the wild-type-high-fat, high-carbohydrate model, we observed the most body weight loss in the EPGN2154+Sema combination group compared to the other treatment groups. All groups led to a significant reduction in alanine transaminase levels when compared to high-fat, high-carbohydrate-fed wild type. However, no significant difference in alanine transaminase levels was observed among the treatment groups. In the ob/ob mice study, Sema did not cause body weight loss. Moreover, the EPGN2154 and the combination groups had a lower NAFLD Activity Score and incidence of advanced-stage hepatic fibrosis than the Sema group. CONCLUSIONS: EPGN2154 demonstrated a hepato-protective effect independent of body weight loss in preclinical NASH models.

Current Utilization of Electron Microscopy in the Pediatric Pathology Setting: A Survey by the SPP Practice Committee.

BACKGROUND: Electron microscopy (EM), once an important component in diagnosing pediatric diseases, has experienced a decline in its use. To assess the impact of this, pediatric pathology practices were surveyed regarding EM services. METHODS: The Society of Pediatric Pathology Practice Committee surveyed 113 society members from 74 hospitals. Settings included 36 academic tertiary, 32 free-standing children's, and 6 community hospitals. RESULTS: Over 60% maintained in-house EM services and had more than 2 pathologists interpreting EM while reporting a shortage of EM technologists. Freestanding children's hospitals had the most specimens (100-200 per year) and more diverse specimen types. Hospitals with fewer than 50 yearly specimens often used reference laboratories. Seventeen had terminated all in-house EM services. Challenges included decreasing caseloads due to alternative diagnostic methods, high operating costs, and shortages of EM technologists and EM-proficient pathologists. Kidney, liver, cilia, heart, and muscle biopsies most often required EM. Lung/bronchoalveolar lavage, tumor, skin, gastrointestinal, nerve, platelet, and autopsy samples less commonly needed EM. CONCLUSIONS: The survey revealed challenges in maintaining EM services but demonstrated its sustained value in pediatric pathology. Pediatric pathologists may need to address the centralization of services and training to preserve EM diagnostic proficiency among pathologists who perform ultrastructural interpretations.

Chromosome 6p amplification detected in blood cell-free DNA in advanced intraocular retinoblastoma.

BACKGROUND: In patients with retinoblastoma, gains of chromosome 6p have been associated with less differentiated tumors. In cell-free DNA from the aqueous humor (AH), 6p gain has been associated with an increased risk of enucleation. While the identification of somatic copy number alterations (SCNAs) via the AH has been well established, these alterations are not routinely identified in the blood due to low tumor fraction. MATERIALS AND METHODS: SCNAs were considered positive at 20% deflection from the baseline. Somatic RB1 pathogenic variants were identified with targeted sequencing using a panel including all RB1 exons. RESULTS: A 24-month-old patient presented with unilateral retinoblastoma (Group D/AJCC Stage cT2B) and was treated with primary enucleation. In the peripheral blood, a heterozygous mutation (c.3920T>A) in the APC gene was reported. Genomic analysis of the tumor and AH revealed two novel somatic RB1 mutations (c.1589_1590del and c.2330dupC). Both also demonstrated highly recurrent RB-related SCNAs. Chromosome 6p gain was detected in the blood with an amplitude suggesting approximately 12% tumor fraction. At a follow-up of 24 months, there has been no evidence of metastatic disease. CONCLUSIONS: To our knowledge, this is the first time an SCNA has been detected in the blood of an RB patient, suggesting in some advanced eyes there may be a high enough tumor fraction to detect these alterations (>5% needed). It remains unclear whether 6p gain or increased tumor fraction in the blood is indicative of increased risk of metastatic disease or new primary cancer; studies to address this are ongoing.

In vivo 3D profiling of site-specific human cancer cell morphotypes in zebrafish.

Tissue microenvironments affect the functional states of cancer cells, but determining these influences in vivo has remained a challenge. We present a quantitative high-resolution imaging assay of single cancer cells in zebrafish xenografts to probe functional adaptation to variable cell-extrinsic cues and molecular interventions. Using cell morphology as a surrogate readout of cell functional states, we examine environmental influences on the morphotype distribution of Ewing Sarcoma, a pediatric cancer associated with the oncogene EWSR1-FLI1 and whose plasticity is thought to determine disease outcome through non-genomic mechanisms. Computer vision analysis reveals systematic shifts in the distribution of 3D morphotypes as a function of cell type and seeding site, as well as tissue-specific cellular organizations that recapitulate those observed in human tumors. Reduced expression of the EWSR1-FLI1 protein product causes a shift to more protrusive cells and decreased tissue specificity of the morphotype distribution. Overall, this work establishes a framework for a statistically robust study of cancer cell plasticity in diverse tissue microenvironments.

Loss of FOCAD, operating via the SKI messenger RNA surveillance pathway, causes a pediatric syndrome with liver cirrhosis.

Cirrhosis is usually a late-onset and life-threatening disease characterized by fibrotic scarring and inflammation that disrupts liver architecture and function. While it is typically the result of alcoholism or hepatitis viral infection in adults, its etiology in infants is much less understood. In this study, we report 14 children from ten unrelated families presenting with a syndromic form of pediatric liver cirrhosis. By genome/exome sequencing, we found recessive variants in FOCAD segregating with the disease. Zebrafish lacking focad phenocopied the human disease, revealing a signature of altered messenger RNA (mRNA) degradation processes in the liver. Using patient's primary cells and CRISPR-Cas9-mediated inactivation in human hepatic cell lines, we found that FOCAD deficiency compromises the SKI mRNA surveillance pathway by reducing the levels of the RNA helicase SKIC2 and its cofactor SKIC3. FOCAD knockout hepatocytes exhibited lowered albumin expression and signs of persistent injury accompanied by CCL2 overproduction. Our results reveal the importance of FOCAD in maintaining liver homeostasis and disclose a possible therapeutic intervention point via inhibition of the CCL2/CCR2 signaling axis.

Dysregulated heparan sulfate proteoglycan metabolism promotes Ewing sarcoma tumor growth.

The Ewing sarcoma family of tumors is a group of malignant small round blue cell tumors (SRBCTs) that affect children, adolescents, and young adults. The tumors are characterized by reciprocal chromosomal translocations that generate chimeric fusion oncogenes, the most common of which is EWSR1-FLI1. Survival is extremely poor for patients with metastatic or relapsed disease, and no molecularly targeted therapy for this disease currently exists. The absence of a reliable genetic animal model of Ewing sarcoma has impaired investigation of tumor cell/microenvironmental interactions in vivo. We have developed a new genetic model of Ewing sarcoma based on Cre-inducible expression of human EWSR1-FLI1 in wild-type zebrafish, which causes rapid onset of SRBCTs at high penetrance. The tumors express canonical EWSR1-FLI1 target genes and stain for known Ewing sarcoma markers including CD99. Growth of tumors is associated with activation of the MAPK/ERK pathway, which we link to dysregulated extracellular matrix metabolism in general and heparan sulfate proteoglycan catabolism in particular. Targeting heparan sulfate proteoglycans with the specific heparan sulfate antagonist Surfen reduces ERK1/2 signaling and decreases tumorigenicity of Ewing sarcoma cells in vitro and in vivo. These results highlight the important role of the extracellular matrix in Ewing sarcoma tumor growth and the potential of agents targeting proteoglycan metabolism as novel therapies for this disease.

Large Mesenteric Gaucheroma Responds to Substrate Reduction Therapy: A New Management of Gaucheromas.

Gaucheromas, which are pseudotumors consisting of a cluster of Gaucher cells, are rare complications in Gaucher's disease (GD) and reported in patients treated with enzyme replacement therapy (ERT). Gaucheromas commonly develop in the lymph nodes in the mesenteric and mediastinal regions and can cause serious complications including protein-losing enteropathy. A large mesenteric Gaucheroma showed a significant reduction in size after initiation of substrate reduction therapy (SRT) with eliglustat in an adult patient with GD type 3. Combination therapy with ERT and SRT should be considered to prevent Gaucheromas in patients with GD.

PLAG1 Immunohistochemical Staining Is a Surrogate Marker for PLAG1 Fusions in Lipoblastomas.

BACKGROUND: The hallmark of lipoblastoma is a PLAG1 fusion. PLAG1 protein overexpression has been reported in sporadic PLAG1-rearranged lipoblastomas. METHODS: We evaluated the utility of PLAG1 immunohistochemical staining (IHC) in 34 pediatric lipomatous tumors, correlating the results with histology and conventional cytogenetics, FISH and/or next generation sequencing (NGS) results. RESULTS: The study included 24 lipoblastomas, divided into 2 groups designated as "Lipoblastoma 1" with both lipoblastoma histology and PLAG1 rearrangement (n = 16) and "Lipoblastoma 2" with lipoblastoma histology but without PLAG1 cytogenetic rearrangement (n = 8), and 10 lipomas with neither lipoblastoma histology nor a PLAG1 rearrangement. Using the presence of a fusion as the "gold standard" for diagnosing lipoblastoma (Lipoblastoma 1), the sensitivity of PLAG1 IHC was 94%. Using histologic features alone (Lipoblastoma 1 + 2), the sensitivity was 96%. Specificity, as defined by the ability to distinguish lipoma from lipoblastoma, was 100%, as there were no false positives in the lipoma group. CONCLUSIONS: Cytogenetics/molecular testing is expensive and may not be ideal for detecting PLAG1 fusions because PLAG1 fusions are often cytogenetically cryptic and NGS panels may not include all partner genes. PLAG1 IHC is an inexpensive surrogate marker of PLAG1 fusions and may be useful in distinguishing lipoblastomas from lipomas.

Kaposiform hemangioendothelioma of the bone in children and adolescents.

Kaposiform hemangioendothelioma (KHE) is a rare, locally aggressive vascular tumor that mainly occurs during infancy or early childhood. Approximately 70% of cases are complicated by Kasabach-Merritt phenomenon. Although osseous extension of the primary lesion is relatively common, primary bone involvement by KHE is rare. Given the paucity of literature on primary KHE of the bone, we report a case series of primary KHE of the bone treated at our institution and describe the clinical presentation, radiologic and pathologic findings, management and outcomes.

Primary Adrenal Malignant Rhabdoid Tumor in a 14-Year-Old Female: A Case Report and Literature Review.

Malignant rhabdoid tumor (MRT) is a rare, SWItch/sucrose nonfermentable-related matrix-associated actin-dependent regulator of chromatin subfamily B member 1 (SMARCB1)-deficient, aggressive tumor, occurring predominantly in children below 3 years of age. Primary adrenal MRT is extremely rare, with only 3 cases reported in the literature. A previously healthy 14-year-old female presented with left upper quadrant/epigastric abdominal pain. Imaging studies revealed an 8.0 × 8.0 × 6.5 cm, heterogeneous, partially enhancing mass along the superior margin of the left kidney encasing the adrenal gland. Surgical resection of the tumor revealed a hypercellular heterogeneous neoplasm arising from the adrenal gland. It was composed predominantly of primitive small round blue cells with focal true rosettes and areas of vague glandular epithelial differentiation and chondroid differentiation. Classic rhabdoid-type cytoplasmic inclusions were focally present. Mitoses, tumor necrosis, and hemorrhage were readily seen. Tumor cells showed complete loss of SMARCB1 (INI1) nuclear staining, demonstrated strong, and diffuse positivity for glypican 3, patchy positivity for CD99, cytokeratin, Sal-like protein 4, Lin-28 homolog A, epithelial membrane antigen, and S100. Molecular studies revealed biallelic frameshift mutations in the SMARCB1 gene (c.673delG and c.683dupT) without pathogenic copy number aberrations. The histologic, immunohistochemical, and molecular findings support a diagnosis of MRT. The unusual age, location, and mutations of this case expand the clinicopathologic and molecular spectrum of MRT.

Mycophenolate Mofetil Hepatotoxicity Associated With Mitochondrial Abnormality in Liver Transplant Recipients and Mice.

OBJECTIVES: Mycophenolate mofetil (MMF) is a widely used immunosuppressive agent. MMF hepatotoxicity has been reported in non-transplant and renal transplant patients with minimal histologic description. This is the first study describing detailed histology and ultrastructure of MMF hepatotoxicity. METHODS: Four liver-transplant recipients (Cases 1-4) were suspected to have MMF hepatotoxicity. Cases 1-3 (two females and one male; 4-17 years) had multiple biopsies for liver function test (LFT) abnormalities. Case 4 (female; 16 years) had a surveillance biopsy. Electron-microscopic examination (EM) was requested on Cases 1-3 for unexplained, persistent LFT elevation and histologic abnormalities despite therapy and Case 4 for unexplained histologic abnormalities despite a stable clinical course. To confirm the pathologic changes in the human allografts, livers from MMF-treated and untreated mice were also reviewed. RESULTS: While the allograft biopsies showed nonspecific histologic changes, EM revealed unequivocal mitochondrial abnormalities similar to those seen in primary and secondary mitochondrial disorders. In Cases 1 and 2, LFTs improved after stopping and reducing MMF, respectively. In Case 3, pre- and post-MMF treatment biopsies were performed and only the post-MMF biopsy demonstrated mitochondrial abnormalities. Mitochondrial abnormality in Case 4 was subclinical. The mouse study confirmed that MMF caused various stress changes in the mitochondria; number of mitochondria/cell (mean ± standard deviation; untreated group: 58.25 ±â€Š8.426; MMF-treated group: 76.37 ±â€Š18.66), number of lipid droplets/cell (untreated: 0.9691 ±â€Š1.150; MMF-treated: 3.649 ±â€Š4.143) and sizes of mitochondria (µm, untreated: 0.8550 ±â€Š0.3409; MMF-treated: 0.9598 ±â€Š0.5312) were significantly increased in hepatocytes in the MMF-treated mice compared with the untreated mice (P < 0.0001). CONCLUSIONS: Although MMF is safe for the majority of patients, MMF can cause mitochondrial stress, which may trigger more severe mitochondrial abnormalities in a small subset. MMF hepatotoxicity should be considered for MMF-treated patients with unexplained, persistent LFT abnormalities and nonspecific histologic findings. EM should be requested for these cases.

Hepatic Ago2 Regulates PPARα for Oxidative Metabolism Linked to Glycemic Control in Obesity and Post Bariatric Surgery.

Argonaute 2 (Ago2) is the main component of the RNA-induced silencing complex. We recently showed that liver-specific Ago2-deficiency in mice (L-Ago2 knockout [KO] mice) enhances mitochondrial oxidation and alleviates obesity-associated pathophysiology. However, the precise mechanisms behind the role of hepatic Ago2 in regulating the mitochondrial oxidation associated with glucose metabolism are still unclear. Here, we show that hepatic Ago2 regulates the function of peroxisome proliferator-activated receptor α (PPARα) for oxidative metabolism. In both genetically and diet-induced severe obese conditions, L-Ago2 KO mice developed obesity and hepatic steatosis but exhibited improved glucose metabolism accompanied by lowered expression levels of pathologic microRNAs (miRNAs), including miR-802, miR-103/107, and miR-152, and enhanced expression of PPARα and its target genes regulating oxidative metabolism in the liver. We then investigated the role of hepatic Ago2 in the outcomes of vertical sleeve gastrectomy (VSG) in which PPARα plays a crucial role in a drastic transcription reprogram associated with improved glycemia post VSG. Whereas VSG reduced body weight and improved fatty liver in wild-type mice, these effects were not observed in hepatic Ago2-deficient mice. Conversely, glucose metabolism was improved in a hepatic Ago2-dependent manner post VSG. Treating Ago2-deficient primary hepatocytes with WY-14643, a PPARα agonist, showed that Ago2-deficiency enhances sensitivity to WY-14643 and increases expression of PPARα target genes and mitochondrial oxidation. Our findings suggest that hepatic Ago2 function is intrinsically associated with PPARα that links Ago2-mediated RNA silencing with mitochondrial functions for oxidation and obesity-associated pathophysiology.

Bronchiectasis and Bronchiolectasis With Severe Herniating Pattern Associated With STAT1 Gain-of-Function Mutation: Detailed Clinicopathological Findings.

STAT1 gain-of-function (GOF) mutations are associated with a rare autosomal dominant immunodeficiency disorder with main clinical manifestations including chronic mucocutaneous candidiasis (CMC) and bronchiectasis. In addition, these patients show higher incidences of cerebral and extracerebral aneurysm, malignancies and various autoimmune conditions compared to the general population. Although previous publications have reported clinical findings in patients with STAT1 GOF mutation, they did not include histopathologic features. Herein, we describe the first case with detailed histologic findings in the lung of a 5-year-old patient with a de novo STAT1 GOF mutation, who presented with CMC and bronchiectasis. The biopsy showed severe bronchiolectasis with extensive airway dilatation and occasional disruptions. Peribronchiolar inflammation was not always present and evident mainly in areas of airway disruption; inflammation may have not been a main driver of the airway damage in this case. The airway dilatation often showed an interesting herniating pattern, possibly implying a connective tissue etiology. This case also demonstrates the diagnostic utility of whole exome sequencing as STAT1 GOF mutations are not detected by routine workup. The definitive diagnosis will lead to more specific treatments and increased surveillance for serious conditions, such as cerebral aneurysms and malignancies.

PD-L1 expression in angiomatoid fibrous histiocytoma.

Angiomatoid fibrous histiocytoma (AFH) is a rare tumor of intermediate malignancy. Treatment options for unresectable and/or metastatic tumors are very limited. Immunotherapy with PD-1/PD-L1 inhibitors may be worth exploring. The aim of this study was to evaluate the expression of PD-L1 in AFHs. PD-L1 expression was assessed on 36 AFHs from 36 pediatric patients by immunohistochemical staining of PD-L1 (clone 22C3). Positivity was defined as membranous expression in ≥ 1% of either tumor or immune cells. The correlations between PD-L1 expression and clinicopathologic features were assessed. Two patients had lymph node metastasis. All patients underwent surgical resection; three of them also had systemic chemotherapy. Three patients had recurrence after initial resection; all patients were alive with a median follow-up of 2.5 years. Overall, twenty-two (61%) tumors were positively stained for PD-L1 and positivity was seen on both tumor and immune cells in eighteen of the 22 tumors. A positive correlation was found between tumor cell PD-L1 expression and CD8+ T-cell infiltration. There were no statistically significant differences between the status of PD-L1 expression and the clinicopathological features assessed. PD-L1 expression was identified in 61% of AFHs with a predominantly adaptive pattern. Our findings provide a rationale for future studies evaluating the potential of checkpoint immunotherapy for patients with unresectable and/or metastatic tumor.

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.

Phox2b Immunohistochemical Staining in Detecting Enteric Neural Crest Cells in Hirschsprung Disease.

BACKGROUND: It can be challenging to recognize undifferentiated/immature ganglion cells, especially single forms. Ganglion cells and glia are derived from enteric neural crest cells (ENCCs), a group of autonomic nervous system (ANS)-lineage neural crest progenitors that PHOX2B regulates. Phox2b is an excellent marker for neoplastic and non-neoplastic ANS cells (eg, peripheral neuroblastic tumors [pNTs]). We hypothesized that Phox2b immunohistochemical staining (IHC) would also be useful for detecting ENCCs. METHODS: Hematoxylin and eosin, calretinin IHC, and Phox2b IHC were reviewed on 21 pull-through specimens and on a cohort of 12 rectal biopsies. RESULTS: Phox2b IHC demonstrated nuclear positivity in all of the ganglion cells across the different phases of differentiation without background staining. The Phox2b result correlated with the morphological findings, calretinin IHC results, and diagnoses based on the routine diagnostic method. The intensity was uniformly strong in the undifferentiated/immature forms and became variable in the mature forms; this pattern was similar to that seen in pNTs. CONCLUSION: Phox2b IHC was highly sensitive and specific for detecting ganglion cells. It worked especially well for immature ganglion cells, seen in premature neonates, and scattered single forms in transition zones. In basic research settings, Phox2b can be a useful marker for early differentiation of ENCCs.