Search

Abstract Methods We examined gene expression profiles of tumor cells from 29 untreated patients with lung cancer (10 adenocarcinomas (AC), 10 squamous cell carcinomas (SCC), and 9 small cell lung cancer (SCLC)) in comparison to 5 samples of normal lung tissue (NT). The European and American methodological quality guidelines for microarray experiments were followed, including the stipulated use of laser capture microdissection for separation and purification of the lung cancer tumor cells from surrounding tissue. Results Based on differentially expressed genes, different lung cancer samples could be distinguished from each other and from normal lung tissue using hierarchical clustering. Comparing AC, SCC and SCLC with NT, we found 205, 335 and 404 genes, respectively, that were at least 2-fold differentially expressed (estimated false discovery rate: < 2.6%). Different lung cancer subtypes had distinct molecular phenotypes, which also reflected their biological characteristics. Differentially expressed genes in human lung tumors which may be of relevance in the respective lung cancer subtypes were corroborated by quantitative real-time PCR. Genetic programming (GP) was performed to construct a classifier for distinguishing between AC, SCC, SCLC, and NT. Forty genes, that could be used to correctly classify the tumor or NT samples, have been identified. In addition, all samples from an independent test set of 13 further tumors (AC or SCC) were also correctly classified. Conclusion The data from this research identified potential candidate genes which could be used as the basis for the development of diagnostic tools and lung tumor type-specific targeted therapies.

Eosinophilic esophagitis is an important differential diagnosis in the presence of dysphagia or bolus obstruction of the esophagus. Delayed diagnosis of eosinophilic esophagitis can lead to strictures of the esophagus.We report on a young patient who presented with initially unclear retrosternal symptoms to our department. The diagnosis of eosinophilic esophagitis, complicated by an intramural abscess of the esophagus, was established. After spontaneous drainage of the abscess, antibiotic therapy and subsequent remission induction of eosinophilic esophagitis with orodispersible budesonide resulted in a good therapeutic outcome., Competing Interests: Die Autorinnen/Autoren geben an, dass kein Interessenkonflikt besteht., (Thieme. All rights reserved.)

The anatomic site-dependent expression of hematopoietic progenitor cell antigen CD34 is a feature of gastrointestinal stromal tumours (GISTs). The basis for the differential CD34 expression is only incompletely understood. This study aimed at understanding the regulation of CD34 in GISTs and clarification of its site-dependent expression. Two sample sets of primary GISTs were interrogated including 52 fresh-frozen and 134 paraffin-embedded and formalin-fixed specimens. DNA methylation analysis was performed by HumanMethylation450 BeadChip array in three cell lines derived from gastric and intestinal GISTs, and differentially methylated CpG sites were established upstream of CD34. The methylation degree was further quantified by pyrosequencing, and inverse correlation with CD34 mRNA and protein abundance was revealed. The gene's expression could be activated upon induction of DNA hypomethylation with 5-aza-2'-deoxycytidine in GIST-T1 cells. In patient samples, a strong inverse correlation of DNA methylation degree with immunohistochemically evaluated CD34 expression was documented. Both CD34 expression and DNA methylation levels were specific to the tumours' anatomic location and mutation status. A constant decrease in methylation levels was observed ranging from almost 100% hypermethylation in intestinal GISTs from duodenum to hypomethylation in rectum. CD34 was heavily methylated in gastric PDGFRA-mutant GISTs in comparison to hypomethylated KIT-mutant counterparts. Next to CD34 hypermethylation, miR-665 was predicted and experimentally confirmed to target CD34 mRNA in GIST-T1 cells. Our results suggest that CD34 expression in GISTs may undergo a complex control by DNA methylation and miR-665. Differential methylation and expression of CD34 in GISTs along the gastrointestinal tract axis and in tumours that harbour different gain-of-function mutations suggest the origin from different cell populations in the gastrointestinal tract., (© 2017 UICC.)

Objectives: This study ascertained the regulation of the stem cell marker CD133 and its potential applicability for prognostication of gastrointestinal stromal tumors (GISTs)., Methods: A total of 95 resected GISTs were included in the study. CD133 protein expression was assessed immunohistochemically on tissue microarrays. Methylation percentage was quantified by pyrosequencing. Gene expression in cell lines GIST48b and GIST882 upon treatment with DNA demethylation agent 5-aza-2'-deoxycytidine was analyzed by quantitative polymerase chain reaction., Results: The expression of hypermethylated CD133 could be reactivated in the GIST cell line upon hypomethylation with the drug. Similarly, in patient material, CD133 methylation percentage correlated inversely with the protein expression and reflected tumor size with hypermethylation in small (<2 cm) tumors and virtually no methylation in large (>10 cm) GISTs. The gene's methylation percentage and expression level were clearly specific to anatomic sites and distinct driver mutations. KIT -mutant gastric GISTs exhibited significantly lower methylation degrees and concomitant high CD133 protein abundance compared with KIT -mutant GISTs from the small intestine. CD133 hypermethylation was documented in PDGFRA -mutant gastric GISTs along with low CD133 expression compared with KIT -mutant gastric GISTs. High CD133 expression was a prognosticator of shorter disease-free survival in all patients. In a subgroup of KIT -mutant gastric GISTs, low CD133 methylation degree was correlated with a shorter disease-free survival., Conclusions: Our results strongly suggest epigenetic regulation of CD133 expression by promoter methylation in GISTs. Pending further validation studies, high abundance of the protein can serve as a marker for malignant GISTs., (© American Society for Clinical Pathology, 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com)

To more fully characterize the clinical and pathologic spectrum of a recently described tumor entity of the sinonasal tract characterized by loss of nuclear expression of SMARCB1 (INI1), we analyzed 39 SMARCB1-deficient sinonasal carcinomas collected from multiple medical centers. The tumors affected 23 males and 16 females with an age range of 19 to 89 years (median, 52). All patients presented with locally advanced disease (T3, n=5; T4, n=27) involving the sinuses (mainly ethmoid) with variable involvement of the nasal cavity. Thirty patients received surgery and/or radiochemotherapy with curative intent. At last follow-up, 56% of patients died of disease 0 to 102 months after diagnosis (median, 15), 2 were alive with disease, and 1 died of an unrelated cause. Only 9 patients (30%) were alive without disease at last follow-up (range, 11 to 115 mo; median, 26). The original diagnosis of retrospectively identified cases was most often sinonasal undifferentiated carcinoma (n=14) and nonkeratinizing/basaloid squamous cell carcinoma (n=5). Histologically, most tumors displayed either a predominantly basaloid (61%) or plasmacytoid/rhabdoid morphology (36%). The plasmacytoid/rhabdoid form consisted of sheets of tumor cells with abundant, eccentrically placed eosinophilic cytoplasm, whereas similar cells were typically rare and singly distributed in the basaloid variant. Glandular differentiation was seen in a few tumors. None of the cases showed squamous differentiation or surface dysplasia. By immunohistochemistry, the tumors were positive for pancytokeratin (97%), CK5 (64%), p63 (55%), and CK7 (48%); and they were negative for NUT (0%). Epstein-Barr virus and high-risk human papillomavirus was not detected by in situ hybridization. Immunohistochemical loss of SMARCB1 (INI1) expression was confirmed for all 39 tumors. Investigation of other proteins in the SWI/SNF complex revealed co-loss of SMARCA2 in 4 cases, but none were SMARCA4 deficient or ARID1A deficient. Of 27 tumors with SMARCB1 fluorescence in situ hybridization analysis, 14 showed homozygous (biallelic) deletions and 7 showed heterozygous (monoallelic) deletions. SMARCB1-deficient sinonasal carcinoma represents an emerging poorly differentiated/undifferentiated sinonasal carcinoma that (1) cannot be better classified as another specific tumor type, (2) has consistent histopathologic findings (albeit with some variability) with varying proportions of plasmacytoid/rhabdoid cells, and (3) demonstrates an aggressive clinical course. This entity should be considered in any difficult-to-classify sinonasal carcinoma, as correct diagnosis will be mandatory for optimizing therapy and for further delineation of this likely underdiagnosed disease.

Pulmonary Langerhans cell histiocytosis (PLCH) is a rare, smoking-related histiocytic disorder with variable clinical symptoms. Like in other non-pulmonary Langerhans cell proliferations, PLCH has recently been shown to harbour BRAF V600E mutations in a significant subset of cases, thus challenging the concept of PLCH being a reactive disorder. Here, we analysed 38 formalin-fixed and paraffin-embedded PLCH nodules of nine patients for BRAF mutation using two different molecular methods. Using pyrosequencing and allele-specific quantitative PCR (AS-PCR), BRAF V600E mutations were found in 16/38 (42%) and 31/37 (84%) nodules, respectively. Analysing different nodules of the same patients with pyrosequencing 3/6 patients showed a concordant BRAF mutation status. When allele-specific quantitative PCR was used, condordant results were found in 5/6 patients. Our findings clearly indicate that (a) the sensitivity of the method used is crucial in analysing BRAF mutation status, (b) AS-PCR is more sensitive in detecting BRAF V600E mutations than pyrosequencing,, ((c) BRAF mutation is frequent and might play a key role in the pathogenesis of PLCH and (d) PLCH is a true neoplastic disease.)

Infantile myofibroma (MF) is an uncommon benign myofibroblastic tumor of infancy and childhood. Solitary adult MF shares similar features with infantile MF. The lesions occur in 3 clinicopathologic settings: solitary, multicentric, and generalized and can be either sporadic or familial. Traditionally, infantile MF has been included in the spectrum of infantile hemangiopericytoma. The recent World Health Organization classification listed MF, angioleiomyoma, and myopericytoma under the general heading of perivascular tumors in the sense of a morphologic spectrum of perivascular myoid cell neoplasms. Although activating germline PDGFRB mutations have recently been linked to familial infantile MF, the molecular pathogenesis of sporadic infantile and adult solitary MF remained unclear. In this study, we analyzed 25 solitary MFs without evidence of familial disease (9 infantile and 16 adult MFs) to address the question whether somatic PDGFRB mutations might be responsible for the sporadic form of the disease. Given the presumed histogenetic link of MF to myopericytoma and angioleiomyoma, we additionally analyzed a control group of 6 myopericytomas and 9 angioleiomyomas for PDGFRB mutations. We detected PDGFRB mutations in 6/8 (75%) analyzable infantile and in 11/16 (69%) adult MFs but in none of the angioleiomyomas or myopericytomas. In 2 infantile MFs, additional sequencing of the germline confirmed the somatic nature of PDGFRB mutations. To our knowledge, this is the first study reporting apparently somatic recurrent PDGFRB mutations as molecular driver events in the majority of sporadic infantile and adult solitary MFs. Our results suggest molecular distinctness of MF as compared with angioleiomyoma/myopericytoma. Investigation of more cases including those with atypical and worrisome features, as well as other mimickers in the heterogenous morphologic spectrum of MF, is mandatory for validating the potential diagnostic value of PDGFRB mutation testing as a possible surrogate in difficult-to-classify lesions.

Neuroendocrine carcinomas (NECs) of the colorectum are rare but highly aggressive neoplasms. These tumors show some shared genetic alterations with colorectal adenocarcinomas, and most of them have adjacent glandular adenoma or adenocarcinoma components. However, genetic data on colorectal NECs still are sparse and insufficient for definite conclusions regarding their molecular origin. Based on morphological characterization, panel and whole-exome sequencing, we here present results from an in-depth analysis of a collection of 15 colorectal NECs with glandular components, 10 of which by definition were mixed adenoneuroendocrine carcinomas (MANECs). Among shared genetic alterations of both tumor components, we most frequently found TP53, KRAS and APC mutations that also had highest allele frequencies. Mutations exclusive to glandular or neuroendocrine components outnumbered shared mutations but occurred at lower allele frequencies. Our findings not only provide additional evidence for a common clonal origin of colorectal NECs and adjacent glandular tumor components, but strongly suggest their development through the classical adenoma-carcinoma sequence. Moreover, our data imply early separation of glandular and neuroendocrine components during malignant transformation with subsequent independent mutational evolution.

Objectives: Loss-of-function mutations in TP53 and CDKN2A have been found at varying frequencies in gastrointestinal stromal tumors (GISTs), while no mutations of RB1 have been reported to date. The aim of the current study was to determine the mutation frequency of TP53, RB1, and CDKN2A in GISTs., Methods: A cohort of 83 primary untreated GISTs was analyzed for mutations in TP53, RB1, and CDKN2A by massive parallel sequencing. Tumors with mutations in TP53 and RB1 were analyzed by fluorescence in situ hybridization for the corresponding gene loci., Results: Two GISTs harbored inactivating mutations in RB1, and two other GISTs displayed inactivating mutations in TP53 All four tumors were KIT mutant high-risk tumors with highly cellular sarcomatous histomorphology and variable combinations of plump spindle cells to epithelioid highly atypical cells and high mitotic activity. Three of these patients developed recurrent or metastatic disease, while the fourth patient showed tumor rupture intraoperatively. The combined overall frequency of TP53 and RB1 mutations was 13% considering high-risk or malignant GISTs., Conclusions: TP53 and RB1 mutations seem to be restricted to high-risk/malignant GISTs and occur at an equal although relatively low frequency., (© American Society for Clinical Pathology, 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Gastrointestinal stromal tumors (GISTs) have distinct gene expression patterns according to localization, genotype and aggressiveness. DNA methylation at CpG dinucleotides is an important mechanism for regulation of gene expression. We performed targeted DNA methylation analysis of 1.505 CpG loci in 807 cancer-related genes in a cohort of 76 GISTs, combined with genome-wide mRNA expression analysis in 22 GISTs, to identify signatures associated with clinicopathological parameters and prognosis. Principal component analysis revealed distinct DNA methylation patterns associated with anatomical localization, genotype, mitotic counts and clinical follow-up. Methylation of a single CpG dinucleotide in the non-CpG island promoter of SPP1 was significantly correlated with shorter disease-free survival. Hypomethylation of this CpG was an independent prognostic parameter in a multivariate analysis compared to anatomical localization, genotype, tumor size and mitotic counts in a cohort of 141 GISTs with clinical follow-up. The epigenetic regulation of SPP1 was confirmed in vitro, and the functional impact of SPP1 protein on tumorigenesis-related signaling pathways was demonstrated. In summary, SPP1 promoter methylation is a novel and independent prognostic parameter in GISTs, and might be helpful in estimating the aggressiveness of GISTs from the intermediate-risk category., (© 2014 UICC.)

Sinonasal hemangiopericytoma (SN-HPC) is an uncommon, site-specific, low-grade mesenchymal neoplasm of probable perivascular myoid cell origin. In contrast to solitary fibrous tumors of soft tissue and sinonasal tract origin, SN-HPCs were recently shown to lack recurrent NAB2-STAT6 fusion variants. Other molecular alterations known to occur in some of soft tissue perivascular myoid cell neoplasms were also absent in SN-HPC; thus, the molecular pathogenesis of SN-HPCs remained unknown. Guided by whole-genome sequencing combined with RNA sequencing of an index case, we analyzed a total of six SN-HPCs for mutations within the amino-terminal region of the gene CTNNB1 (cadherin-associated protein), β 1, 88 kDa, encoding β-catenin. All six cases showed missense mutations, with amino acid substitutions clustering at positions 33 to 45, corresponding to the recognition site of the β-catenin destruction complex. Similar CTNNB1 mutations have been described in a variety of epithelial and mesenchymal neoplasms. These mutations prevent β-catenin phosphorylation and proteasomal degradation but promote its nuclear accumulation and subsequent increased transcription of Wingless-related integration site target genes. Consistent with these molecular findings, β-catenin IHC showed consistent diffuse and strong nuclear staining of the tumor cells in all six SN-HPCs. Our results highlight, for the first time, CTNNB1 mutations as the likely initiating molecular events driving SN-HPC tumorigenesis, which places SN-HPC among the growing family of β-catenin-driven mesenchymal neoplasms., (Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Aims: Sinonasal haemangiopericytoma (SN-HPC) is a rare sinonasal mesenchymal neoplasm of perivascular myoid cell origin. Solitary fibrous tumour (SFT) occurs only very rarely in the sinonasal tract. SFT and soft tissue HPC have been considered a single entity. Recently, recurrent gene fusions involving NAB2-STAT6 resulting in differential expression of STAT6 were characterized as central molecular events in SFT. However, no data exist for NAB2-STAT6 status or STAT6 expression in SN-HPC., Methods and Results: We examined six SN-HPCs and two sinonasal SFTs by immunohistochemistry and RT-PCR for NAB2-STAT6 fusions. SN-HPC affected three females and three males (mean age: 72 years). They expressed smooth muscle actin, lacked strong CD34 reactivity and were negative for nuclear STAT6 expression. RT-PCR analysis confirmed the absence of NAB2-STAT6 fusions in all cases. Conversely, both sinonasal SFTs (in males aged 39 and 52 years) displayed classical features of pleuropulmonary and soft-tissue SFTs (uniformly CD34-positive with strong nuclear expression of STAT6). RT-PCR revealed NAB2-STAT6 fusions in both cases., Conclusions: These findings confirm the molecular and phenotypical distinctness of these two entities. While SN-HPC is a site-specific sinonasal neoplasm of as yet unknown molecular pathogenesis, sinonasal SFTs show phenotypical and molecular identity to their pleural/extrapleural counterparts., (© 2014 John Wiley & Sons Ltd.)