Second-Generation Neuroendocrine Immunohistochemical Markers: Reflections from Clinical Implementation

When analyzing tumors by histopathology, endocrine pathologists have traditionally been restricted to a few key immunohistochemical markers related to secretory vesicles in order to pinpoint neuroendocrine differentiation—most notably Chromogranin A (CGA) and Synaptophysin (SYP). Although proven of great clinical utility, these markers sometimes exhibit tissue-specific patterns depending on tumor origin, and non-neuroendocrine tumors might sometimes display focal expression. Moreover, CGA and SYP might be partially or totally absent in highly proliferative neuroendocrine carcinomas, making the diagnosis particularly challenging on small biopsies of metastatic lesions with unknown location of the primary tumor. The advent of second-generation neuroendocrine markers ISL LIM Homeobox 1 (ISL1), INSM Transcriptional Repressor 1 (INSM1) and Secretagogin (SECG) have expanded the pathology toolbox considerably, constituting markers that often retain expression even in poorly differentiated neuroendocrine carcinomas. As non-neuroendocrine tumors seldom express these antigens, the specificity of ISL1, INSM1 and SECG make them welcome additions to clinical practice. In this commentary, recent advances of this field as well as initial clinical experiences from a tertiary neuroendocrine center are discussed.

Phaeochromocytomas overexpress insulin transcript and produce insulin

Introduction: Phaeochromocytomas are tumours originating in the medulla of the adrenal gland. They produce catecholamines and some tumours also produce ectopic hormones. Production of insulin has not been reported. However, two types of glucose imbalances occur in pheaochromocytoma patients, hyperglycaemia and hypoglycaemic attacks. Therefore, we tested whether insulin was expressed in phaeochromocytomas. Methods: We measured the expression of insulin using immunohistochemistry. The expression of insulin transcript (INS) and a hybrid read-through transcript between exons from insulin and insulin-like growth factor 2 (INS-IGF2) was determined by qRT-PCR in formalin-fixed and paraffin-embedded tissue from 20 phaeochromocytomas. The expression of INS and INS-IGF2 transcripts was also analysed in 182 phaeochromocytomas and paragangliomas using publicly available datasets in The Cancer Genome Atlas (TCGA) Database. Results: Of 20 phaeochromocytomas, 16 stained positive for insulin. The distribution of positive cells was mostly scattered, with some focal expression indicating clonal expansion. 19 tumours expressed high levels of INS and INS-IGF2 transcripts. The expression of the two transcripts correlated positively. In the TCGA dataset, phaeochromocytoma express higher levels of INS and INS-IGF2 transcripts compared to normal non-tumour adrenal gland tissue (p=0.01). Thus, expression of INS and INS-IGF2 seems to be a general phenomenon in phaeochromocytoma. Conclusion: Most phaeochromocytomas contain cells that express the INS and INS-IGF2 transcripts. As most tumours also express polypeptides immunoreactive to monoclonal anti-insulin antibodies, expression of the transcripts are likely functionally relevant. Clinically this may relate to both hyperglycaemia and hypoglycaemic attacks seen in patients with a phaeochromocytoma.

RHEB/mTOR hyperactivity causes cortical malformations and epileptic seizures through increased axonal connectivity

Hyperactivation of the mammalian target of rapamycin (mTOR) pathway can cause malformation of cortical development (MCD) with associated epilepsy and intellectual disability (ID) through a yet unknown mechanism. Here, we made use of the recently identified dominant-active mutation in Ras Homolog Enriched in Brain 1 (RHEB), RHEBp.P37L, to gain insight in the mechanism underlying the epilepsy caused by hyperactivation of the mTOR pathway. Focal expression of RHEBp.P37L in mouse somatosensory cortex (SScx) results in an MCD-like phenotype, with increased mTOR signaling, ectopic localization of neurons, and reliable generalized seizures. We show that in this model, the mTOR-dependent seizures are caused by enhanced axonal connectivity, causing hyperexcitability of distally connected neurons. Indeed, blocking axonal vesicle release from the RHEBp.P37L neurons alone completely stopped the seizures and normalized the hyperexcitability of the distally connected neurons. These results provide new evidence of the extent of anatomical and physiological abnormalities caused by mTOR hyperactivity, beyond local malformations, which can lead to generalized epilepsy.

CD133 Expression in Placenta Chorioangioma Presenting as a Giant Asymptomatic Mass

Background: Placental chorioangioma is the most common benign non-trophoblastic neoplasm of the placenta. Its clinical relevance lies in the size of the tumor since larger masses cause pregnancy complications, including an unfavorable neonatal outcome. Case presentation: We report the case of a 34-year-old second gravida and nullipara at the 35th week of gestation, admitted to the gynecological department for antibiotic-resistant fever. The cardiotocography performed during hospitalization showed an abnormal fetal pattern. A 2250 g newborn was delivered by cesarean section. No complications were observed during childbirth and postpartum was insignificant. On gross inspection a white fleshy intraparenchymal mass blooming on the maternal surface was noted; routinely stained sections revealed features consistent with chorioangioma with vascular channels lined by inconspicuous endothelial cells immunoreactive for CD31 and CD133. Focal expression of CD133 was also observed in placental villi. Discussion: CD133 expression indicated the presence of stem cells in chorioangioma, suggesting their possible role in the development of mesenchymal lesions including chorioangioma.

Identification of a novel 4-gene diagnostic model for atrial fibrillation risk based on integrated analysis across independent data sets

Background: Atrial fibrillation (AF) is the most common persistent arrhythmia and an important factor leading to cardiovascular morbidity and mortality. Several key genes and diagnostic markers have been discovered with the development of advanced modern molecular biology techniques, but the etiology and pathogenesis of AF remained unknown. Methods: In this study, three chip-seq data sets and a RNA-seq data set were integrated as a comprehensive network for pathway analysis of the biological functions of related genes in AF, hoping to provide a better understanding on the etiology and pathogenesis of AF. Results: Differential co-expression analysis identified 360 genes with specific expression in AF, and functional enrichment analysis further revealed that these genes were significantly correlated with focal expression (p <0.01), autophagy (p <0.01), and thyroid cancer. In addition, Af-specific protein-protein interaction (PPI) networks were constructed based on AF-specific expression genes. Network topology analysis identified PLEKHA7, YWHAQ, PPP1CB, WDR1, AKT1, IGF1R, CANX, MAPK1, SRPK2 and SRSF10 genes as hub genes of the networks, and they were considered as potential biomarkers of AF because they were found to participate in the development of AF through Oocyte meiosis and focal expression. Finally, a diagnostic model for AF established with support vector machine (SVM), demonstrated excellent predictive performance in internal and external data sets (AUC>0.9) and in different platform data sets (mean AUC>0.75). Conclusion: Finally, a diagnostic model for AF established, thus showing its potential in the early identification and prediction of AF.

Novel t(1;8)(p31.3;q21.3) NFIA-RUNX1T1 Translocation in an Infant Erythroblastic Sarcoma

Objectives Pure erythroid leukemia (PEL) is exceptionally rare in the pediatric setting. Four pediatric PEL cases with t(1;16)(p31;q24) NFIA-CBFA2T3 were reported previously. We present a case of an infant with PEL presenting with erythroblastic sarcoma and harboring a novel t(1;8)(p31.3;q21.3) NFIA-RUNX1T1 fusion detected by RNA sequencing and conventional karyotype. Methods Bone marrow (BM) and abdominal mass biopsies from the patient were evaluated with extensive immunohistochemical, flow cytometric, cytogenetic, and molecular studies. Results The patient was a female infant who presented between 2 and 5 months of age with cytopenias and an enlarging abdominal mass. Blasts in the BM and abdominal mass expressed CD71 and CD117 with focal expression of CD43, E-cadherin, epithelial membrane antigen, and hemoglobin A. They were negative for additional myeloid, lymphoid, and nonhematolymphoid markers. These findings were most consistent with PEL and erythroblastic sarcoma. RNA sequencing revealed the novel NFIA-RUNX1T1 fusion. Conclusions Along with the previously reported PELs with NFIA-CBFA2T3 fusions, we describe a subset of PELs that occur in children, that frequently display extramedullary disease, and that harbor rearrangements of NFIA with core binding factor genes. We hypothesize that, together, these cases represent a rare but distinct clinicopathologic group of pediatric PELs with recurrent genetic abnormality.

Abnormal p16 Expression and Prognostic Significance in Chinese Esophageal Squamous Cell Carcinoma

Background: The purpose of this study was to analysis p16 expression status and evaluate whether abnormal p16 expression was associated with prognosis in a large-scale Chinese esophageal squamous cell carcinoma (ESCC) patients. Methods: We retrospectively evaluated p16 expression status of 525 ESCC samples using immunohistochemistry. Associations between abnormal p16 expression and survival were analyzed. Results: P16 negative, focal expression and overexpression were found in 87.6%, 6.9% and 5.5% of ESCC patients. No significant association was observed between abnormal p16 expression and age, sex, tumor site and location, differentiation, vessel and nerve invasion, T stage and lymph node metastasis. In all patients, the survival of p16 focal expression group tended to be better compared with negative group (disease free survival/DFS P=0.040 and overall survival/OS P=0.052) and overexpression group (DFS P=0.201 and OS P=0.258), and there was no survival difference between negative group and overexpression group. The multivariate analysis for OS and DFS only found clinical stage was a significantly independent prognostic factor (P<0.001). When patients were divided into I-II stage (n=290) and III-IVa stage (n=235), the survival of focal expression group was better compared with negative group (DFS P=0.015 and OS P=0.019), tended to be better compared with overexpression group (DFS P=0.405 and OS P=0.432) in I-II stage ESCC, which was not found in III-IVa stage ESCC.Conclusion: P16 overexpression or negative tend to be associated with unfavorable outcomes, especially in I-II stage ESCC. Our study will help to identify a subgroup of ESCC patients with excellent prognosis after surgical therapy.

RHEB/mTOR-hyperactivity causing cortical malformations drives seizures through increased axonal connectivity

ABSTRACTDominant-active mutations in Ras Homolog Enriched in Brain 1 (RHEB), such as the recently identified RHEBp.P37L mutation, can cause malformations of cortical development (MCD) with associated epilepsy and intellectual disability through a yet unknown mechanism. We found that focal expression of RHEBp.P37L in mouse somatosensory cortex results in an MCD-like phenotype, with increased mammalian target of rapamycin (mTOR) signaling, ectopic localization of neurons and generalized seizures. In addition, the RHEBp.P37L expressing neurons showed increased axonal length and branching. By temporally controlling RHEBp.P37L expression, we found that the cortical malformation by itself was neither necessary nor sufficient to generate seizures. Rather, seizures were contingent on persistent mTOR activation and enhanced axonal connectivity of RHEBp.P37L expressing neurons, causing hyperexcitability of distally connected neurons. These results provide new evidence of the extent of anatomical and physiological abnormalities caused by mTOR hyperactivity, beyond local malformations, that can lead to generalized epilepsy.

PROGNOSTIC MARKERS IN IMMUNOHISTOCHEMICAL DIAGNOSIS OF METASTASES OF COLORECTAL PHENOTYPE CARCINOMA OF UNKNOWN PRIMARY

Most metastases of cancers of unknown primary (80% -85%) are known as having unfavourable prognostic variants due to their large extant and low susceptibility to the therapy. Studies of prognostic immunohistochemical markers will provide an opportunity to understand the biological features of the rapid dissemination of individual colorectal phenotypes. In recent decades, p53 and Ki-67, the most popular antibodies have been investigated as prognostic factors for colorectal cancer. The p53 protein is a regulatory protein that is at the crossroads of cell division and cell death. The goal of this study is to explore the expression features of prognostic immunohistochemical markers Ki-67, p53, β-catenin, AMACR (p503s), HER2-new in carcinomas of unknown primary of the colorectal phenotype compared with primary tumours of the colon, to improve diagnostic algorithms. Materials and methods. A study of biopsy material taken from 37 patients aged 28 to 81 years (mean 58.46 ± 12.28; median 58), group 1, with metastases without primary localization was carried out; the immunohistochemical investigation revealed adenocarcinomas of the colorectal phenotype CK20 + / CDX2 + / CK7; the investigation of postoperative material in 41 patients aged 27 to 76 years (mean 60.56 ± 12.81; median 64), group 2, revealed primary colon tumour. Results. Positive focal expression of p53 was found in the colorectal cancers of the proximal sections (located to the right), it is they that more often have microsatellite instability, compared to distal carcinomas. Amplification of the Her-2-new gene (expression at the level of 2+ and 3+) was found in 35% (13 of 37) of metastasis of colorectal cancers, and in almost 15% (6 of 41) of primary colon adenocarcinomas that suggests the appropriateness in applying targeted therapy.Conclusions. Nuclear translocation of β-catenin, positive “focal” expression of p53 and amplification of the Her-2-new gene are statistically significantly more common in colorectal phenotype metastases compared with primary colon adenocarcinomas (all р> 0.05).