An exploration in pitfalls in interpreting SDHB immunohistochemistry.

AIMS: Mutations and epimutations in genes encoding the succinate dehydrogenase complex (SDHx) are associated with multiple tumour types in which identification of SDH-deficiency has significant management implications. Immunohistochemistry (IHC) for the succinate dehydrogenase B (SDHB) subunit can help to detect SDH-deficiency, which manifests as complete loss of staining in tumour cells. However, a subset of SDH-deficient tumours can show aberrant cytoplasmic SDHB-IHC staining patterns and be misinterpreted as 'retained', a diagnostic pitfall complicating interpretation. Herein, we characterise in detail aberrant SDHB-IHC staining patterns in SDH-deficient tumours. METHODS AND RESULTS: We identified 23 tumours from patients with known germline SDHx and/or molecularly confirmed SDHx pathogenic/likely-pathogenic variants in their tumour. Of these, eight (35%) showed significant SDHB-IHC staining: one SDHA-, one SDHB-, three SDHC- and three SDHD-mutated cases. In all eight cases, closer inspection revealed differences in intensity and intracellular distribution of SDHB-IHC staining in tumour cells compared to adjacent non-neoplastic cells: non-neoplastic cells showed intense cytoplasmic coarse granular staining; tumour cells in seven of eight cases showed weak to focally strong, cytoplasmic blush to fine granular staining, in > 80% of cells. The remaining case in the initial block showed variably strong non-granular cytoplasmic staining with globular perinuclear accentuation throughout, only subtly distinct from the staining pattern of non-neoplastic cells. SDHB-IHC performed on two additional blocks in this latter case revealed significant intratumoral heterogeneity, including convincing areas of complete loss. CONCLUSIONS: When evaluating SDHB-IHC, care should be taken to distinguish true retained expression from aberrant cytoplasmic expression, which may be difficult to appreciate. Sometimes this may require additional molecular testing.

The utility of high-risk human papillomavirus in situ hybridization in cytology cell block material from cystic head and neck lesions.

BACKGROUND: Human papillomavirus-related oropharyngeal squamous cell carcinoma (HPV-OPSCC) presents frequently as metastasis in a neck lymph node that may be cystic or necrotic. Fine-needle aspiration (FNA) biopsies are often first-line diagnostic procedures. p16 immunohistochemistry (IHC) is a surrogate marker for high-risk HPV (hrHPV) infection but can be challenging to interpret. This study evaluated the use of hrHPV in situ hybridization (ISH) in cytology cell blocks of cystic neck lesions. METHODS: Twenty-four FNA cases with cell blocks and surgical correlates were evaluated. p16 IHC and hrHPV ISH were assessed on cell blocks (C-p16 and C-hrHPV ISH), and hrHPV ISH on surgical samples (S-hrHPV ISH). All results were classified as negative, positive, or equivocal. RESULTS: Two cases were excluded because of insufficient tissue on recut. On the basis of C-hrHPV ISH cases, 12 were positive, 5 were negative, and 5 were equivocal. All 12 positive C-hrHPV ISH cases had concordant S-hrHPV ISH with no false positives. Of the 5 negative C-hrHPV ISH cases, 4 had concordant S-hrHPV ISH, and 1 had a discordant S-hrHPV ISH. Of the 5 equivocal C-hrHPV ISH cases, S-hrHPV ISH were both positive and negative. Fourteen cases were equivocal by C-p16; 9 cases were reliably classified by C-hrHPV ISH (5 positive, 4 negative; 64%). CONCLUSIONS: C-hrHPV ISH can be reliably used, especially when positive. A negative or equivocal interpretation of C-hrHPV ISH may warrant repeat testing. Compared to C-p16, C-hrHPV ISH is more frequently diagnostic and could be helpful for HPV-OSCC diagnosis and management.

Inhibin-positive hepatic carcinoma: proposal for a solid-tubulocystic variant of intrahepatic cholangiocarcinoma.

Inhibin-positive hepatic carcinoma is a rare primary liver neoplasm that resembles sex cord-stromal tumor and thyroid follicular tumors. The term "cholangioblastic variant of intrahepatic cholangiocarcinoma" has been proposed. This study describes the clinicopathologic, immunophenotypic, and molecular features of a small series (n = 6) of this rare tumor. Albumin in situ hybridization (ISH) and capture-based next-generation sequencing (NGS) were also performed. All tumors occurred in young women (mean age 32.5 years, range 19-44 years) as a solitary large mass (mean 15.8 cm, range 6.9-23.5 cm). All tumors showed a highly distinctive morphology with sheets and large nests of tumor cells alternating with tubular and cystic areas imparting a sex cord-like or thyroid follicle-like morphology. Cytologic atypia was mild, and mitotic activity was low. All cases were positive for inhibin, as well as pancytokeratin, CK7, CK19, and albumin ISH. Synaptophysin and chromogranin showed focal or patchy staining, whereas INSM1 was negative. Markers for hepatocellular differentiation, thyroid origin, and sex cord-stromal tumor were negative. There were no recurrent genomic changes based on capture-based NGS of ∼500 cancer genes. Recurrence and/or metastasis was seen in three (50%) cases (follow-up time range for all cases: 5 months to 2 years). In conclusion, this series describes the distinctive morphology, immunophenotypic features, and diffuse albumin staining in six cases of a rare inhibin-positive primary liver carcinoma that runs an aggressive course similar to intrahepatic cholangiocarcinoma. Genomic changes typical of cholangiocarcinoma or hepatocellular carcinoma were not identified, and there were no recurrent genetic abnormalities. We propose the term "solid-tubulocystic variant of intrahepatic cholangiocarcinoma" to reflect the spectrum of morphologic patterns observed in this tumor.

High-grade neuroepithelial tumor with BCOR exon 15 internal tandem duplication-a comprehensive clinical, radiographic, pathologic, and genomic analysis.

High-grade neuroepithelial tumor with BCOR exon 15 internal tandem duplication (HGNET BCOR ex15 ITD) is a recently proposed tumor entity of the central nervous system (CNS) with a distinct methylation profile and characteristic genetic alteration. The complete spectrum of histologic features, accompanying genetic alterations, clinical outcomes, and optimal treatment for this new tumor entity are largely unknown. Here, we performed a comprehensive assessment of 10 new cases of HGNET BCOR ex15 ITD. The tumors mostly occurred in young children and were located in the cerebral or cerebellar hemispheres. On imaging all tumors were large, well-circumscribed, heterogeneous masses with variable enhancement and reduced diffusion. They were histologically characterized by predominantly solid growth, glioma-like fibrillarity, perivascular pseudorosettes, and palisading necrosis, but absence of microvascular proliferation. They demonstrated sparse to absent GFAP expression, no synaptophysin expression, variable OLIG2 and NeuN positivity, and diffuse strong BCOR nuclear positivity. While BCOR exon 15 internal tandem duplication was the solitary pathogenic alteration identified in six cases, four cases contained additional alterations including CDKN2A/B homozygous deletion, TERT amplification or promoter hotspot mutation, and damaging mutations in TP53, BCORL1, EP300, SMARCA2 and STAG2. While the limited clinical follow-up in prior reports had indicated a uniformly dismal prognosis for this tumor entity, this cohort includes multiple long-term survivors. Our study further supports inclusion of HGNET BCOR ex15 ITD as a distinct CNS tumor entity and expands the known clinicopathologic, radiographic, and genetic features.

Identification of high-risk human papillomavirus and Rb/E2F pathway genomic alterations in mutually exclusive subsets of colorectal neuroendocrine carcinoma.

Colorectal neuroendocrine carcinomas, both small cell and large cell types, are highly aggressive tumors with poor prognosis compared with colorectal adenocarcinoma. The molecular drivers of neuroendocrine carcinoma are best defined in small cell lung cancer, which shows near-universal genomic alterations in TP53 and RB1. The genetics of colorectal neuroendocrine carcinoma remain poorly understood; recent studies demonstrated infrequent RB1 alterations and genetics closely resembling colorectal adenocarcinoma. To better define the molecular pathogenesis of colorectal neuroendocrine carcinoma, we performed capture-based next-generation sequencing on 25 cases and evaluated for expression of p53, Rb, p16, and high-risk human papillomavirus (HR-HPV) subtypes using immunohistochemistry, in situ hybridization, and polymerase chain reaction. Rb/E2F pathway dysregulation was identified in nearly all cases (23/25, 92%) and occurred via three distinct mechanisms. First, RB1 genomic alteration was present in 56% (14/25) of cases and was accompanied by Rb protein loss, high p16 expression, and absence of HR-HPV; these cases also had frequent genomic alterations in TP53, the PI3K/Ras and Wnt pathways, as well as in DNA repair genes, with 4/14 cases being hypermutated. Second, 16% (4/25) of cases, all left-sided, had TP53 alteration without RB1 alteration; half of these harbored high-level amplifications in CCNE1 and MYC or MYCN and arose in patients with ulcerative colitis. Finally, 28% (7/25) of cases, all rectal or anal, lacked genomic alterations in RB1 or TP53 but were positive for HR-HPV. Our data demonstrate that Rb/E2F pathway dysregulation is essential in the pathogenesis of colorectal neuroendocrine carcinoma, akin to neuroendocrine carcinomas in other anatomic sites. Moreover, colorectal neuroendocrine carcinomas stratify into three distinct molecular subgroups, which can be differentiated based on Rb protein and HR-HPV status. HR-HPV infection represents a distinct mechanism for Rb and p53 inactivation in cases lacking genomic alterations in either gene. Differential treatment strategies for hypermutated and HPV-driven cases could improve patient outcomes.

Discordant Findings on 18F-NaF and 99mTc-HDP Bone Scans in a Patient With ATTR Cardiac Amyloidosis.

Cardiac amyloidosis is an important cause of restrictive cardiomyopathy and congestive heart failure. Bone scintigraphy with [Tc]hydroxymethylene diphosphonate (Tc-HDP) and [F]sodium fluoride (F-NaF) have been investigated in the noninvasive diagnosis of transthyretin (ATTR)-related cardiac amyloidosis. We present a case of a 76-year-old man with metastatic prostate cancer who underwent Tc-HDP bone scintigraphy with an incidental finding of diffuse left ventricular abnormal uptake suggesting ATTR cardiac amyloidosis. Review of previous imaging showed a negative F-NaF PET/CT study. This example suggests that the mechanism of uptake of Tc-HDP and F-NaF may differ in patients with ATTR cardiac amyloidosis.

Intrapancreatic accessory spleen: utilization of fine needle aspiration for diagnosis of a potential mimic of a pancreatic neoplasm.

Accessory spleen (AS) is not a rare occurrence, and with the second most common site being the tail of the pancreas, intrapancreatic AS (IPAS) can easily mimic a pancreatic neoplasm. Together with radiologic imaging findings, endoscopic ultrasound-guided fine needle aspiration (FNA) can be used to assist in the diagnosis, preventing potentially unnecessary surgical procedures. The most common cytologic findings that have been described in the literature include a heterogenous population of small lymphocytes along with traversing small vessels. Immunohistochemical staining for CD8 has also been documented as a useful tool to support the diagnosis as it specifically highlights the endothelial cells of the splenic sinus. Here, we report two additional cases of IPAS diagnosed by FNA and discuss the potential pitfalls in diagnosis of this entity.

Silent intralesional microhemorrhage as a risk factor for brain arteriovenous malformation rupture.

BACKGROUND AND PURPOSE: We investigated whether brain arteriovenous malformation silent intralesional microhemorrhage, that is, asymptomatic bleeding in the nidal compartment, might serve as a marker for increased risk of symptomatic intracranial hemorrhage (ICH). We evaluated 2 markers to assess the occurrence of silent intralesional microhemorrhage: neuroradiological assessment of evidence of old hemorrhage-imaging evidence of bleeding before the outcome events-and hemosiderin positivity in hematoxylin and eosin-stained paraffin block sections. METHODS: We identified cases from our brain arteriovenous malformation database with recorded neuroradiological data or available surgical paraffin blocks. Using 2 end points, index ICH or new ICH after diagnosis (censored at treatment, loss to follow-up, or death), we performed logistic or Cox regression to assess evidence of old hemorrhage and hemosiderin positivity adjusting for age, sex, deep-only venous drainage, maximal brain arteriovenous malformation size, deep location, and associated arterial aneurysms. RESULTS: Evidence of old hemorrhage was present in 6.5% (n=975) of patients and highly predictive of index ICH (P<0.001; OR, 3.97; 95% CI, 2.1-7.5) adjusting for other risk factors. In a multivariable model (n=643), evidence of old hemorrhage was an independent predictor of new ICH (hazard ratio, 3.53; 95% CI, 1.35-9.23; P=0.010). Hemosiderin positivity was found in 36.2% (29.6% in unruptured; 47.8% in ruptured; P=0.04) and associated with index ICH in univariate (OR, 2.18; 95% CI, 1.03-4.61; P=0.042; n=127) and multivariable models (OR, 3.64; 95% CI, 1.11-12.00; P=0.034; n=79). CONCLUSIONS: The prevalence of silent intralesional microhemorrhage is high and there is evidence for an association with both index and subsequent ICH. Further development of means to detect silent intralesional microhemorrhage during brain arteriovenous malformation evaluation may present an opportunity to improve risk stratification, especially for unruptured brain arteriovenous malformations.

Intestinal barrier failure during experimental necrotizing enterocolitis: protective effect of EGF treatment.

Necrotizing enterocolitis (NEC) is the most common intestinal disease of premature infants. Although increased mucosal permeability and altered epithelial structure have been associated with many intestinal disorders, the role of intestinal barrier function in NEC pathogenesis is currently unknown. We investigated the structural and functional changes of the intestinal barrier in a rat model of NEC. In addition, the effect of EGF treatment on intestinal barrier function was evaluated. Premature rats were divided into three groups: dam fed (DF), formula fed (NEC), or fed with formula supplemented with 500 ng/ml EGF (NEC + EGF); all groups were exposed to asphyxia/cold stress to develop NEC. Intestinal permeability, goblet cell density, mucin production, and composition of tight junction (TJ) proteins were evaluated in the terminal ileum, the site of NEC injury, and compared with the proximal jejunum, which was unaffected by NEC. Animals with NEC had significantly increased intestinal paracellular permeability compared with DF pups. Ileal goblet cell morphology, mucin production, and TJ composition were altered in animals with NEC. EGF treatment significantly decreased intestinal paracellular permeability, increased goblet cell density and mucin production, and normalized expression of two major TJ proteins, occludin and claudin-3, in the ileum. In conclusion, experimental NEC is associated with disruption of the intestinal barrier. EGF treatment maintains intestinal integrity at the site of injury by accelerating goblet cell maturation and mucin production and normalizing expression of TJ proteins, leading to improved intestinal barrier function.

Bile acids induce ileal damage during experimental necrotizing enterocolitis.

BACKGROUND & AIMS: Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency of premature infants. While the effect of bile acids (BAs) on intestinal mucosal injury is known, we investigated the contribution of BAs during the development of NEC in neonatal rats. METHODS: Premature rats were fed with cow's milk-based formula and subjected to asphyxia and cold stress to develop NEC. Jejunal and ileal luminal BAs, portal blood BAs, and messenger RNA and protein for the apical sodium-dependent bile acid transporter, the ileal bile acid binding protein, and the heteromeric organic solute transporter (Ostalpha/Ostbeta)were evaluated. RESULTS: Ileal luminal BAs levels were increased significantly during disease development and the removal of ileal BAs significantly decreased the incidence and severity of disease. Furthermore, when NEC was reduced via treatment with epidermal growth factor (EGF), BA levels were reduced significantly. Jejunal luminal BA levels were similar between animals with NEC and controls, but portal/ileal luminal BA ratios were decreased significantly in animals with NEC. The apical sodium-dependent bile acid transporter was up-regulated at the site of injury in animals with NEC and decreased after EGF treatment; however, the ileal bile acid binding protein was up-regulated only in the NEC and EGF group. Ostalpha/Ostbeta expression was low in all groups, and only slightly increased in the NEC group. CONCLUSIONS: These data strongly suggest that BAs play a role in the development of ileal damage in experimental NEC and that alterations in BA transport in the neonatal ileum may contribute to disease development.

Epidermal growth factor reduces hepatic sequelae in experimental necrotizing enterocolitis.

BACKGROUND AND AIM: Neonatal necrotizing enterocolitis (NEC) is the most common gastrointestinal disease of premature infants. We recently demonstrated that the gut/liver axis plays an important role in the pathophysiology of NEC through the release of inflammatory mediators into the intestinal lumen. We have also shown that supplementation of formula with epidermal growth factor (EGF) dramatically decreases ileal pathology associated with experimental NEC. In this study, we examined the effects of EGF on the liver portion of the gut/liver axis in the neonatal rat model of NEC. METHODS: Newborn rats were divided into three experimental groups, NEC, hand-fed with growth-factor free formula; NEC + EGF, hand-fed with formula supplemented with 500 ng/ml rat EGF; or DF, dam fed. All animals were exposed to asphyxia and cold stress twice daily for 4 days to develop NEC. RESULTS: EGF receptor expression was significantly (p

Reduction of experimental necrotizing enterocolitis with anti-TNF-alpha.

Necrotizing enterocolitis (NEC) is the most common gastrointestinal disease of premature infants. However, despite significant morbidity and mortality, the etiology and pathogenesis of NEC are poorly understood. Evidence suggests that ileal proinflammatory mediators such as IL-18 contribute to the pathology associated with this disease. In addition, we have previously shown that upregulation of TNF-alpha in the liver is correlated with ileal disease severity in a neonatal rat model of NEC. With the use of a neonatal rat model of NEC, we evaluated the incidence and severity of ileal damage along with the production of both hepatic and ileal proinflammatory cytokines in animals injected with (anti-TNF-alpha; n = 23) or without (NEC; n = 25) a monoclonal anti-TNF-alpha antibody. In addition, we assessed changes in apoptosis and ileal permeability in the NEC and anti-TNF-alpha groups. Ileal damage was significantly decreased, and the incidence of NEC was reduced from 80% to 17% in animals receiving anti-TNF-alpha. Hepatic TNF-alpha and hepatic and ileal IL-18 were significantly decreased in pups given anti-TNF-alpha compared with those sham injected. In addition, ileal luminal levels of both TNF-alpha and IL-18 were significantly decreased in the anti-TNF-alpha-injected group. Ileal paracellular permeability and the proapoptotic markers Bax and cleaved caspase-3 were significantly decreased in the anti-TNF-alpha group. These data show that hepatic TNF-alpha is an important component for the development of NEC in the neonatal rat model and suggest that anti-TNF-alpha could be used as a potential therapy for human NEC.