NAB2::STAT6 fusions and genome-wide DNA methylation profiling: Predictors of patient outcomes in meningeal solitary fibrous tumors.

Meningeal solitary fibrous tumors (SFT) are rare and have a high frequency of local recurrence and distant metastasis. In a cohort of 126 patients (57 female, 69 male; mean age at surgery 53.0 years) with pathologically confirmed meningeal SFTs with extended clinical follow-up (median 9.9 years; range 15 days-43 years), we performed extensive molecular characterization including genome-wide DNA methylation profiling (n = 80) and targeted TERT promoter mutation testing (n = 98). Associations were examined with NAB2::STAT6 fusion status (n = 101 cases; 51 = ex5-7::ex16-17, 26 = ex4::ex2-3; 12 = ex2-3::exANY/other and 12 = no fusion) and placed in the context of 2021 Central Nervous System (CNS) WHO grade. NAB2::STAT6 fusion breakpoints (fusion type) were significantly associated with metastasis-free survival (MFS) (p = 0.03) and, on multivariate analysis, disease-specific survival (DSS) when adjusting for CNS WHO grade (p = 0.03). DNA methylation profiling revealed three distinct clusters: Cluster 1 (n = 38), Cluster 2 (n = 22), and Cluster 3 (n = 20). Methylation clusters were significantly associated with fusion type (p < 0.001), with Cluster 2 harboring ex4::ex2-3 fusion in 16 (of 20; 80.0%), nearly all TERT promoter mutations (7 of 8; 87.5%), and predominantly an "SFT" histologic phenotype (15 of 22; 68.2%). Clusters 1 and 3 were less distinct, both dominated by tumors having ex5-7::ex16-17 fusion (respectively, 25 of 33; 75.8%, and 12 of 18; 66.7%) and with variable histological phenotypes. Methylation clusters were significantly associated with MFS (p = 0.027), but not overall survival (OS). In summary, NAB2::STAT6 fusion type was significantly associated with MFS and DSS, suggesting that tumors with an ex5::ex16-17 fusion may have inferior patient outcomes. Methylation clusters were significantly associated with fusion type, TERT promoter mutation status, histologic phenotype, and MFS.

Machine Learning-Supported Diagnosis of Small Blue Round Cell Sarcomas Using Targeted RNA Sequencing.

Small blue round cell sarcomas (SBRCSs) are a heterogeneous group of tumors with overlapping morphologic features but markedly varying prognosis. They are characterized by distinct chromosomal alterations, particularly rearrangements leading to gene fusions, whose detection currently represents the most reliable diagnostic marker. Ewing sarcomas are the most common SBRCSs, defined by gene fusions involving EWSR1 and transcription factors of the ETS family, and the most frequent non-EWSR1-rearranged SBRCSs harbor a CIC rearrangement. Unfortunately, currently the identification of CIC::DUX4 translocation events, the most common CIC rearrangement, is challenging. Here, we present a machine-learning approach to support SBRCS diagnosis that relies on gene expression profiles measured via targeted sequencing. The analyses on a curated cohort of 69 soft-tissue tumors showed markedly distinct expression patterns for SBRCS subgroups. A random forest classifier trained on Ewing sarcoma and CIC-rearranged cases predicted probabilities of being CIC-rearranged >0.9 for CIC-rearranged-like sarcomas and <0.6 for other SBRCSs. Testing on a retrospective cohort of 1335 routine diagnostic cases identified 15 candidate CIC-rearranged tumors with a probability >0.75, all of which were supported by expert histopathologic reassessment. Furthermore, the multigene random forest classifier appeared advantageous over using high ETV4 expression alone, previously proposed as a surrogate to identify CIC rearrangement. Taken together, the expression-based classifier can offer valuable support for SBRCS pathologic diagnosis.

Determinants Affecting the Clinical Implementation of a Molecularly Informed Molecular Tumor Board Recommendation: Experience from a Tertiary Cancer Center.

Molecular Tumor Boards (MTBs) converge state-of-the-art next-generation sequencing (NGS) methods with the expertise of an interdisciplinary team consisting of clinicians, pathologists, human geneticists, and molecular biologists to provide molecularly informed guidance in clinical decision making to the treating physician. In the present study, we particularly focused on elucidating the factors impacting on the clinical translation of MTB recommendations, utilizing data generated from gene panel mediated comprehensive genomic profiling (CGP) of 554 patients at the MTB of the Comprehensive Cancer Center Erlangen, Germany, during the years 2016 to 2020. A subgroup analysis of cases with available follow-up data (n = 332) revealed 139 cases with a molecularly informed MTB recommendation, which was successfully implemented in the clinic in 44 (31.7%) of these cases. Here, the molecularly matched treatment was applied in 45.4% (n = 20/44) of cases for ≥6 months and in 25% (n = 11/44) of cases for 12 months or longer (median time to treatment failure, TTF: 5 months, min: 1 month, max: 38 months, ongoing at data cut-off). In general, recommendations were preferentially implemented in the clinic when of high (i.e., tier 1) clinical evidence level. In particular, this was the case for MTB recommendations suggesting the application of PARP, PIK3CA, and IDH1/2 inhibitors. The main reason for non-compliance to the MTB recommendation was either the application of non-matched treatment modalities (n = 30)/stable disease (n = 7), or deteriorating patient condition (n = 22)/death of patient (n = 9). In summary, this study provides an insight into the factors affecting the clinical implementation of molecularly informed MTB recommendations, and careful considerations of these factors may guide future processes of clinical decision making.

DNA-methylation signature accurately differentiates pancreatic cancer from chronic pancreatitis in tissue and plasma.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy. Differentiation from chronic pancreatitis (CP) is currently inaccurate in about one-third of cases. Misdiagnoses in both directions, however, have severe consequences for patients. We set out to identify molecular markers for a clear distinction between PDAC and CP. DESIGN: Genome-wide variations of DNA-methylation, messenger RNA and microRNA level as well as combinations thereof were analysed in 345 tissue samples for marker identification. To improve diagnostic performance, we established a random-forest machine-learning approach. Results were validated on another 48 samples and further corroborated in 16 liquid biopsy samples. RESULTS: Machine-learning succeeded in defining markers to differentiate between patients with PDAC and CP, while low-dimensional embedding and cluster analysis failed to do so. DNA-methylation yielded the best diagnostic accuracy by far, dwarfing the importance of transcript levels. Identified changes were confirmed with data taken from public repositories and validated in independent sample sets. A signature of six DNA-methylation sites in a CpG-island of the protein kinase C beta type gene achieved a validated diagnostic accuracy of 100% in tissue and in circulating free DNA isolated from patient plasma. CONCLUSION: The success of machine-learning to identify an effective marker signature documents the power of this approach. The high diagnostic accuracy of discriminating PDAC from CP could have tremendous consequences for treatment success, once the result from still a limited number of liquid biopsy samples would be confirmed in a larger cohort of patients with suspected pancreatic cancer.

BiasCorrector: Fast and accurate correction of all types of experimental biases in quantitative DNA methylation data derived by different technologies.

Quantification of DNA methylation in neoplastic cells is crucial both from mechanistic and diagnostic perspectives. However, such measurements are prone to different experimental biases. Polymerase chain reaction (PCR) bias results in an unequal recovery of methylated and unmethylated alleles at the sample preparation step. Post-PCR biases get introduced additionally by the readout processes. Correcting the biases is more practicable than optimising experimental conditions, as demonstrated previously. However, utilisation of our earlier developed algorithm strongly necessitates automation. Here, we present two R packages: rBiasCorrection, the core algorithms to correct biases; and BiasCorrector, its web-based graphical user interface frontend. The software detects and analyses experimental biases in calibration DNA samples at a single base resolution by using cubic polynomial and hyperbolic regression. The correction coefficients from the best regression type are employed to compensate for the bias. Three common technologies-bisulphite pyrosequencing, next-generation sequencing and oligonucleotide microarrays-were used to comprehensively test BiasCorrector. We demonstrate the accuracy of BiasCorrector's performance and reveal technology-specific PCR- and post-PCR biases. BiasCorrector effectively eliminates biases regardless of their nature, locus, the number of interrogated methylation sites and the detection method, thus representing a user-friendly tool for producing accurate epigenetic results.

Lipomatous Solitary Fibrous Tumors Harbor Rare NAB2-STAT6 Fusion Variants and Show Up-Regulation of the Gene PPARG, Encoding for a Regulator of Adipocyte Differentiation.

Solitary fibrous tumors (SFTs) harbor activating NAB2-STAT6 gene fusions. Different variants of the NAB2-STAT6 gene fusion have been associated with distinct clinicopathologic features. Lipomatous SFTs are a morphologic variant of SFTs, characterized by a fat-forming tumor component. Our aim was to evaluate NAB2-STAT6 fusion variants and to further study the molecular genetic features in a cohort of lipomatous SFTs. A hybrid-capture-based next-generation sequencing panel was employed to detect NAB2-STAT6 gene fusions at the RNA level. In addition, the RNA expression levels of 507 genes were evaluated using this panel, and were compared with a control cohort of nonlipomatous SFTs. Notably, 5 of 11 (45%) of lipomatous SFTs in the current series harbored the uncommon NAB2 exon 4-STAT6 exon 4 gene fusion variant, which is observed in only 0.9% to 1.4% of nonlipomatous SFTs. Furthermore, lipomatous SFTs displayed significant differences in gene expression compared with their nonlipomatous counterparts, including up-regulation of the gene peroxisome proliferator activated receptor-γ (PPARG). Peroxisome proliferator activated receptor-γ is a nuclear receptor regulating adipocyte differentiation, providing a possible explanation for the fat-forming component in lipomatous SFTs. In summary, the current study provides a possible molecular genetic basis for the distinct morphologic features of lipomatous SFTs.

Gene Expression in Solitary Fibrous Tumors (SFTs) Correlates with Anatomic Localization and NAB2-STAT6 Gene Fusion Variants.

Solitary fibrous tumors (SFTs) harbor recurrent NAB2-STAT6 gene fusions, promoting constitutional up-regulation of oncogenic early growth response 1 (EGR1)-dependent gene expression. SFTs with the most common canonical NAB2 exon 4-STAT6 exon 2 fusion variant are often located in the thorax (pleuropulmonary) and are less cellular with abundant collagen. In contrast, SFTs with NAB2 exon 6-STAT6 exon 16/17 fusion variants typically display a cellular round to ovoid cell morphology and are often located in the deep soft tissue of the retroperitoneum and intra-abdominal pelvic region or in the meninges. Here, we employed next-generation sequencing-based gene expression profiling to identify significant differences in gene expression associated with anatomic localization and NAB2-STAT6 gene fusion variants. SFTs with the NAB2 exon 4-STAT6 exon 2 fusion variant showed a transcriptional signature enriched for genes involved in DNA binding, gene transcription, and nuclear localization, whereas SFTs with the NAB2 exon 6-STAT6 exon 16/17 fusion variants were enriched for genes involved in tyrosine kinase signaling, cell proliferation, and cytoplasmic localization. Specific transcription factor binding motifs were enriched among differentially expressed genes in SFTs with different fusion variants, implicating co-transcription factors in the modification of chimeric NGFI-A binding protein 2 (NAB2)-STAT6-dependent deregulation of EGR1-dependent gene expression. In summary, this study establishes a potential molecular biologic basis for clinicopathologic differences in SFTs with distinct NAB2-STAT6 gene fusion variants.

SST gene hypermethylation acts as a pan-cancer marker for pancreatic ductal adenocarcinoma and multiple other tumors: toward its use for blood-based diagnosis.

Aberrant DNA methylation is often involved in carcinogenesis. Our initial goal was to identify DNA methylation biomarkers associated with pancreatic cancer. A genomewide methylation study was performed on DNA from pancreatic ductal adenocarcinoma (PDAC) and endocrine pancreas tumors. Validation of DNA methylation patterns and concomitant alterations in expression of gene candidates was performed on patient samples and pancreatic cancer cell lines. Furthermore, validation was done on independent data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). Finally, droplet digital PCR was employed to detect DNA methylation marks in cell-free (cf) DNA isolated from plasma samples of PDAC patients and cancer-free blood donors. Hypermethylation of the SST gene (encoding somatostatin) and concomitant downregulation of its expression were discovered in PDAC and endocrine tumor tissues while not being present in chronic pancreatitis (inflamed) tissues and normal pancreas. Fittingly, treatment with a somatostatin agonist (octreotide) reduced cell proliferation and migration of pancreatic cancer cells. Diagnostic performance of SST methylation in a receiver operating characteristic curve analysis was 100% and 89% for tissue and plasma samples, respectively. A large body of TCGA and GEO data confirmed SST hypermethylation and downregulation in PDAC and showed a similar effect in a broad spectrum of other tumor entities. SST promoter methylation represents a sensitive and promising molecular, pan-cancer biomarker detectable in tumor tissue, and liquid biopsy samples.

Distinct genetic alterations and luminal molecular subtype in nested variant of urothelial carcinoma.

AIMS: Nested variant of urothelial carcinoma (NVUC) is rare, and only a few small series exist. Molecular characteristics and the classifying marker profile as well as therapeutic targets of this specific variant are mostly unknown. The aim of this study was to characterise NVUC at the molecular level in one of the largest cohorts to date. In addition, we applied an immunohistochemical marker panel in order to define the molecular subtype. METHODS AND RESULTS: Sixty NVUC cases were collected from different departments. TERT promoter mutation analysis was carried out in all samples using SNaPshot analysis. Targeted sequencing of 48 cancer-related genes by next-generation sequencing (NGS) analysis was performed in a subset of 26 cases. Immunohistochemical markers CD44, CK5, CK14, EGFR, p63, FOXA1, GATA3, CD24 and CK20 were used to elucidate the molecular subtype. A total of 62.5% of NVUC cases harboured a mutation of the TERT promoter. Additionally, TP53, JAK3 and CTNNB1 were among the most frequently mutated genes identified by NGS analysis. Subtyping revealed that all NVUC express luminal markers such as CD24, FOXA1, GATA3 and CK20. CONCLUSIONS: In summary, NVUC belong to the luminal molecular subtype. Moreover, a subset of NVUC seems to be characterised by mutations of the Wnt and inflammatory pathways, including JAK3 mutations, indicating a different biological background compared to conventional urothelial bladder cancer.

Nuclear NR4A3 Immunostaining Is a Specific and Sensitive Novel Marker for Acinic Cell Carcinoma of the Salivary Glands.

Recently, we discovered the recurrent genomic rearrangement [t(4;9)(q13;q31)] enabling upregulation of the transcription factor Nuclear Receptor Subfamily 4 Group A Member 3 (NR4A3) through enhancer hijacking as the oncogenic driver event in acinic cell carcinoma (AciCC) of the salivary glands. In the current study, we evaluated the usefulness of NR4A3 immunostaining and NR4A3 fluorescence in situ hybridization (FISH) in the differential diagnosis of AciCC, comparing a total of 64 AciCCs including 17% cases with high-grade transformation, 29 secretory (mammary analog) carcinomas (MASC), and 70 other salivary gland carcinomas. Nuclear NR4A3 immunostaining was a highly specific (100%) and sensitive (98%) marker for AciCC with only 1 negative case, whereas NR4A3 FISH was less sensitive (84%). None of the MASCs or other salivary gland carcinomas displayed any nuclear NR4A3 immunostaining. The recently described HTN3-MSANTD3 gene fusion was observed in 4 of 49 (8%) evaluable AciCCs, all with nuclear NR4A3 immunostaining. In summary, NR4A3 immunostaining is a highly specific and sensitive marker for AciCC, which may be especially valuable in cases with high-grade transformation and in "zymogen granule"-poor examples within the differential diagnostic spectrum of AciCC and MASC.

Enhancer hijacking activates oncogenic transcription factor NR4A3 in acinic cell carcinomas of the salivary glands.

The molecular pathogenesis of salivary gland acinic cell carcinoma (AciCC) is poorly understood. The secretory Ca-binding phosphoprotein (SCPP) gene cluster at 4q13 encodes structurally related phosphoproteins of which some are specifically expressed at high levels in the salivary glands and constitute major components of saliva. Here we report on recurrent rearrangements [t(4;9)(q13;q31)] in AciCC that translocate active enhancer regions from the SCPP gene cluster to the region upstream of Nuclear Receptor Subfamily 4 Group A Member 3 (NR4A3) at 9q31. We show that NR4A3 is specifically upregulated in AciCCs, and that active chromatin regions and gene expression signatures in AciCCs are highly correlated with the NR4A3 transcription factor binding motif. Overexpression of NR4A3 in mouse salivary gland cells increases expression of known NR4A3 target genes and has a stimulatory functional effect on cell proliferation. We conclude that NR4A3 is upregulated through enhancer hijacking and has important oncogenic functions in AciCC.

The impact of histopathology and NAB2-STAT6 fusion subtype in classification and grading of meningeal solitary fibrous tumor/hemangiopericytoma.

Meningeal solitary fibrous tumor (SFT)/hemangiopericytoma (HPC) is a rare tumor with propensity for recurrence and metastasis. Although multiple classification schemes have been proposed, optimal risk stratification remains unclear, and the prognostic impact of fusion status is uncertain. We compared the 2016 WHO CNS tumor grading scheme (CNS-G), a three-tier system based on histopathologic phenotype and mitotic count, to the 2013 WHO soft-tissue counterpart (ST-G), a two-tier system based on mitotic count alone, in a cohort of 133 patients [59 female, 74 male; mean age 54 years (range 20-87)] with meningeal SFT/HPC. Tumors were pathologically confirmed through review of the first tumor resection (n = 97), local recurrence (n = 35), or distant metastasis (n = 1). A STAT6 immunostain showed nuclear expression in 132 cases. NAB2-STAT6 fusion was detected in 99 of 111 successfully tested tumors (89%) including the single STAT6 immunonegative tumor. Tumors were classified by CNS-G as grade 1 (n = 43), 2 (n = 41), or 3 (n = 49), and by ST-G as SFT (n = 84) or malignant SFT (n = 49). Necrosis was present in 16 cases (12%). On follow-up, 42 patients had at least one subsequent recurrence or metastasis (7 metastasis only, 33 recurrence only, 2 patients had both). Twenty-nine patients died. On univariate analysis, necrosis (p = 0.002), CNS-G (p = 0.01), and ST-G (p = 0.004) were associated with recurrence-free (RFS) but not overall survival (OS). NAB2-STAT6 fusion type was not significantly associated with RFS or OS, but was associated with phenotype. A modified ST-G incorporating necrosis showed higher correlation with RFS (p = 0.0006) and remained significant (p = 0.02) when considering only the primary tumors. From our data, mitotic rate and necrosis appear to stratify this family of tumors most accurately and could be incorporated in a future grading scheme.

Long noncoding RNA HOTAIR is upregulated in an aggressive subgroup of gastrointestinal stromal tumors (GIST) and mediates the establishment of gene-specific DNA methylation patterns.

Aberrant alterations of DNA methylation are common events in oncogenesis. The origin of cancer-associated epigenetic defects is of interest for mechanistic understanding of malignant transformation and-in the long run-therapeutic modulation of DNA methylation in a locus-specific manner. Given the ability of certain long noncoding RNAs to operate as an interface between DNA and the epigenetic modification machinery which can interact with DNA methyltransferases, we hypothesized-considering HOTAIR as an example-that this transcript may contribute to gene specificity of DNA methylation. Using gastrointestinal stromal tumors (GISTs, n = 67) as a model, we confirmed upregulation of HOTAIR in tumors with high risk of recurrence and showed high abundance of the transcript in GIST cell lines. HOTAIR knockdown in GIST-T1 cells triggered transcriptional response of genes involved in the organization and disassembly of the extracellular matrix and, notably, induced global locus-specific alterations of DNA methylation patterns. Hypomethylation was induced at a total of 507 CpG sites, whereas 382 CpG dinucleotides underwent gain of methylation upon HOTAIR depletion. Importantly, orchestrated gain or loss of methylation at multiple individual CpG sites was shown for cancer-related DPP4, RASSF1, ALDH1A3, and other targets. Collectively, our data indicate that HOTAIR enables target specificity of DNA methylation in GIST and is capable of dual (hypo- and hypermethylation) regulation by a yet to be defined mechanism. The results further suggest the feasibility of manipulating DNA methylation in a targeted manner and are of interest in the context of epigenetic cancer therapy.

Intraductal Papillary Mucinous Neoplasms of Minor Salivary Glands With AKT1 p.Glu17Lys Mutation.

The spectrum of low-grade intraductal papillary proliferations of the salivary glands is heterogenous, and reproducible morphologic diagnostic criteria have not yet been established. Recognized types are sialadenoma papilliferum, inverted ductal papilloma, and intraductal papilloma, but some lesions have been possibly included in the morphologic spectrum of cystadenoma or low-grade intraductal carcinomas. We herein present detailed morphologic, immunophenotypic, and genotypic features of 3 minor salivary gland neoplasms affecting 2 men (aged 65 and 71 y) and 1 woman (aged 78 y). They ranged in size from 1 to 2.5 cm. All tumors showed atypical papillary intraductal growth that presented either as uninodular/unicystic lesions (intraductal papilloma-like; n=2) or as a discontinuous growth along the ductal system in a manner similar to pancreatic intraductal papillary mucinous neoplasm (n=1). Variable cytologic and architectural atypia was observed, ranging from bland intraductal papilloma-like features, to areas mimicking atypical ductal hyperplasia and low-grade ductal carcinoma in situ of the breast. Amplicon-based massive parallel sequencing revealed an identical AKT1 p.Glu17Lys mutation in all 3 cases, but absence of concurring mutations in other genes of the RAS or PI3K pathway. This small series represents the first genetic study on salivary intraductal papillary neoplasms. Our cases showed significant variation in the degree of cytologic and architectural atypia, which overlaps with intraductal papillomas at the one end and with low-grade intraductal carcinoma at the other end of the spectrum, suggesting a disease continuum. As the full biological and morphologic characteristics of these ductal papillary lesions remain to be defined, the noncommitted term "intraductal papillary neoplasms" might be more appropriate. Our novel genetic findings mirror similar activating mutations of AKT1 and other PI3K pathway members in intraductal papillary lesions of the breast and anogenital glands.

Low-grade Endometrioid Stromal Sarcoma of the Paratestis: A Novel Report With Molecular Confirmation of JAZF1/SUZ12 Translocation.

Tumors with Müllerian-like serous or mucinous phenotypes originating in the testis and its adnexa are rare neoplasms that have been increasingly recognized in recent years. Cystadenomas with or without ovarian-type stroma, borderline tumors, and adenocarcinomas are the main documented types. Although a handful cases of putative endometrioid adenocarcinomas have been reported, to our knowledge no case of endometrial stromal-type neoplasm has ever been reported in the literature. A 59-year-old man presented with a 2 cm left intrascrotal mass that was found on sonographic examination to arise from the epididymal tail with prominent vascularization. He was otherwise healthy without significant clinical history, endocrinopathy, or external hormone therapy. His testicular tumor markers (beta-HCG, AFP) were normal. Histologic examination of the resection showed a multinodular tumor closely associated with the epididymis and composed of monotonous rounded to ovoid cells with scanty cytoplasm and prominent spiral-like arterioles and capillaries. Mitotic activity was high. No other tumor component was seen. Immunohistochemistry revealed strong and diffuse expression of vimentin, CD10, estrogen receptor, and progesterone receptor. Molecular examination (performed on paraffin-embedded tumor tissue using a 517 gene fusion next-generation sequencing assay) showed a JAZF1/SUZ12 translocation, which was then confirmed by fluorescence in situ hybridization (FISH). These findings are consistent with a low-grade endometrioid stromal sarcoma originating in the paratestis. This report represents a novel addition to the growing spectrum of Müllerian-analog testicular adnexal neoplasms.

The expression of hematopoietic progenitor cell antigen CD34 is regulated by DNA methylation in a site-dependent manner in gastrointestinal stromal tumours.

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.

SMARCA4-deficient pulmonary adenocarcinoma: clinicopathological, immunohistochemical, and molecular characteristics of a novel aggressive neoplasm with a consistent TTF1neg/CK7pos/HepPar-1pos immunophenotype.

Alterations in SMARCA4, a member of the chromatin remodeling Switch Sucrose Non-Fermentable (SWI/SNF) complex, characterize a subset of non-small cell lung cancer (NSCLC), but detailed morphological and immunophenotypic description of this tumor type is lacking. We describe 20 NSCLC cases found on routine screening not to express SMARCA4 by immunohistochemistry (IHC). These tumors were stained for CK7, TTF1, SMARCA2, SMARCA4, SMARCB1, and HepPar-1 and analyzed for molecular alterations, using a 160 cancer-related gene panel including the full coding sequence of SMARCA4. Patients were eight females and 12 males aged 41 to 76 (median, 60). Of 18 tumors with detailed data, 14 presented with synchronous distant metastases (M1). Histological examination showed predominantly solid adenocarcinoma (n = 15), frankly rhabdoid (n = 3) and mucinous (n = 2) patterns. Except for the rhabdoid cases, all tumors showed at least focal unequivocal glands and lacked squamous differentiation, justifying a diagnosis of adenocarcinoma. IHC showed a distinctive uniform immunophenotype (CK7+/HepPar-1+/TTF1-) in 18/20 cases. Only 2/16 cases showed limited weak expression of neuroendocrine markers. EGFR mutations and EML4-ALK and ROS1 gene rearrangements were not found in any of the examined cases. Next-generation sequencing, using a 160 cancer-related gene panel, revealed concurrent SMARCA4 and TP53 mutations in nine of the 12 (75%) successfully tested cases. Our study highlights (1) the morphological diversity of SMARCA4-deficient lung adenocarcinoma, (2) the consistent absence of expression of TTF1 in the presence of expression of HepPar-1, (3) absence of EGFR driver mutations, and (4) frequent inactivating SMARCA4 mutations as underlying mechanism of the observed SMARCA4 protein loss. SMARCA4-deficient pulmonary adenocarcinoma is emerging as a distinctive, albeit phenotypically heterogeneous molecular subgroup of TTF1-negative NSCLC. Uniform HepPar-1 expression in this subset of NSCLC may represent a diagnostic pitfall and merits further studies to explore the mechanisms involved.

Epigenetic Regulation of CD133 in Gastrointestinal Stromal Tumors.

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.

Recurrent Somatic PDGFRB Mutations in Sporadic Infantile/Solitary Adult Myofibromas But Not in Angioleiomyomas and Myopericytomas.

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.