Chromogenic detection of telomere lengths in situ aids the identification of precancerous lesions in the prostate.

BACKGROUND: Telomeres are terminal chromosomal elements that are essential for the maintenance of genomic integrity. The measurement of telomere content provides useful diagnostic and prognostic information, and fluorescent methods have been developed for this purpose. However, fluorescent-based tissue assays are cumbersome for investigators to undertake, both in research and clinical settings. METHODS: A robust chromogenic in situ hybridization (CISH) approach was developed to visualize and quantify telomere content at single cell resolution in human prostate tissues, both frozen and formalin-fixed, paraffin-embedded (FFPE). RESULTS: This new assay (telomere chromogenic in situ hybridization ["Telo-CISH"]) produces permanently stained slides that are viewable with a standard light microscope, thus avoiding the need for specialized equipment and storage. The assay is compatible with standard immunohistochemistry, thereby allowing simultaneous assessment of histomorphology, identification of specific cell types, and assessment of telomere status. In addition, Telo-CISH eliminates the problem of autofluorescent interference that frequently occurs with fluorescent-based methods. Using this new assay, we demonstrate successful application of Telo-CISH to help identify precancerous lesions in the prostate by the presence of markedly short telomeres specifically in the luminal epithelial cells. CONCLUSIONS: In summary, with fewer restrictions on the types of tissues that can be tested, and increased histologic information provided, the advantages presented by this novel chromogenic assay should extend the applicability of tissue-based telomere length assessment in research and clinical settings.

Chromogenic detection of telomere lengths in situ aids the identification of precancerous lesions in the prostate.

Telomeres are terminal chromosomal elements that are essential for the maintenance of genomic integrity. The measurement of telomere content provides useful diagnostic and prognostic information, and fluorescent methods have been developed for this purpose. However, fluorescent-based tissue assays are cumbersome for investigators to undertake, both in research and clinical settings. Here, a robust chromogenic in situ hybridization (CISH) approach was developed to visualize and quantify telomere content at single cell resolution in human prostate tissues, both frozen and formalin-fixed, paraffin-embedded (FFPE). This new assay ("Telo-CISH") produces permanently stained slides that are viewable with a standard light microscope, thus avoiding the need for specialized equipment and storage. The assay is compatible with standard immunohistochemistry, thereby allowing simultaneous assessment of histomorphology, identification of specific cell types, and assessment of telomere status. In addition, Telo-CISH eliminates the problem of autofluorescent interference that frequently occurs with fluorescent-based methods. Using this new assay, we demonstrate successful application of Telo-CISH to help identify precancerous lesions in the prostate by the presence of markedly short telomeres specifically in the luminal epithelial cells. In summary, with fewer restrictions on the types of tissues that can be tested, and increased histologic information provided, the advantages presented by this novel chromogenic assay should extend the applicability of tissue-based telomere length assessment in research and clinical settings.

ATRX-Deficient High-Grade Glioma Cells Exhibit Increased Sensitivity to RTK and PDGFR Inhibitors.

High-grade glioma, including anaplastic astrocytoma and glioblastoma (GBM) patients, have a poor prognosis due to the lack of effective treatments. Therefore, the development of new therapeutic strategies to treat these gliomas is urgently required. Given that high-grade gliomas frequently harbor mutations in the SNF2 family chromatin remodeler ATRX, we performed a screen to identify FDA-approved drugs that are toxic to ATRX-deficient cells. Our findings reveal that multi-targeted receptor tyrosine kinase (RTK) and platelet-derived growth factor receptor (PDGFR) inhibitors cause higher cellular toxicity in high-grade glioma ATRX-deficient cells. Furthermore, we demonstrate that a combinatorial treatment of RTKi with temozolomide (TMZ)-the current standard of care treatment for GBM patients-causes pronounced toxicity in ATRX-deficient high-grade glioma cells. Our findings suggest that combinatorial treatments with TMZ and RTKi may increase the therapeutic window of opportunity in patients who suffer high-grade gliomas with ATRX mutations. Thus, we recommend incorporating the ATRX status into the analyses of clinical trials with RTKi and PDGFRi.

SMARCAL1 loss and alternative lengthening of telomeres (ALT) are enriched in giant cell glioblastoma.

Subsets of high-grade gliomas, including glioblastoma (GBM), are known to utilize the alternative lengthening of telomeres (ALT) pathway for telomere length maintenance. However, the telomere maintenance profile of one subtype of GBM-giant cell GBM-has not been extensively studied. Here, we investigated the prevalence of ALT, as well as ATRX and SMARCAL1 protein loss, in a cohort of classic giant cell GBM and GBM with giant cell features. To determine the presence of ALT, a telomere-specific fluorescence in situ hybridization assay was performed on 15 cases of classic giant cell GBM, 28 additional GBMs found to have giant cell features, and 1 anaplastic astrocytoma with giant cell features. ATRX, SMARCAL1, and IDH1 protein status were assessed in a proportion of cases by immunohistochemistry and were compared to clinical-pathologic and molecular characteristics. In the overall cohort of 44 cases, 19 (43%) showed evidence of ALT. Intriguingly, of the ALT-positive cases, only 9 (47.4%) displayed loss of the ALT suppressor ATRX by immunohistochemistry. Since inactivating mutations in SMARCAL1 have been identified in ATRX wild-type ALT-positive gliomas, we developed an immunohistochemistry assay for SMARCAL1 protein expression using genetically validated controls. Of the 19 ALT-positive cases, 6 (31.5%) showed loss or mis-localization of SMARCAL1 by immunohistochemistry. Of these cases, four retained ATRX protein expression, while two cases also displayed ATRX loss. Additionally, we assessed five cases from which multiple temporal samples were available and ALT status was concordant between both tumor biopsies. In summary, we have identified a subset of giant cell GBM that utilize the ALT telomere maintenance mechanism. Importantly, in addition to ATRX loss, ALT-positive tumors harboring SMARCAL1 alterations are prevalent in giant cell GBM.

Pervasive promoter hypermethylation of silenced TERT alleles in human cancers.

BACKGROUND: In cancers, maintenance of telomeres often occurs through activation of the catalytic subunit of telomerase, encoded by TERT. Yet, most cancers show only modest levels of TERT gene expression, even in the context of activating hotspot promoter mutations (C228T and C250T). The role of epigenetic mechanisms, including DNA methylation, in regulating TERT gene expression in cancer cells is as yet not fully understood. METHODS: Here, we have carried out the most comprehensive characterization to date of TERT promoter methylation using ultra-deep bisulfite sequencing spanning the CpG island surrounding the core TERT promoter in 96 different human cell lines, including primary, immortalized and cancer cell types, as well as in control and reference samples. RESULTS: In general, we observed that immortalized and cancer cell lines were hypermethylated in a region upstream of the recurrent C228T and C250T TERT promoter mutations, while non-malignant primary cells were comparatively hypomethylated in this region. However, at the allele-level, we generally found that hypermethylation of promoter sequences in cancer cells is associated with repressed expression, and the remaining unmethylated alleles marked with open chromatin are largely responsible for the observed TERT expression in cancer cells. CONCLUSIONS: Our findings suggest that hypermethylation of the TERT promoter alleles signals transcriptional repression of those alleles, leading to attenuation of TERT activation in cancer cells. This type of fine tuning of TERT expression may account for the modest activation of TERT expression in most cancers.

Pancreatic acinar cell carcinomas and mixed acinar-neuroendocrine carcinomas are more clinically aggressive than grade 1 pancreatic neuroendocrine tumours.

Acinar cell carcinomas (ACCs) and mixed acinar-neuroendocrine carcinomas (MAcNECs) of the pancreas are extremely rare carcinomas with a significant component with acinar differentiation. To date, the clinicopathological behaviours of these neoplasms remain unclear. In this study, we evaluated the histopathological and molecular characteristics of 20 ACCs and 13 MAcNECs and compared them to a cohort of 269 well-differentiated pancreatic neuroendocrine tumours (PanNETs). Compared to PanNETs, both ACCs and MAcNECs had an advanced pT classification (p<0.001), as well as more prevalent lymphovascular and perineural invasion (p=0.002) and lymph node and distant metastases (p<0.001). Patients with MAcNECs had worse overall (p<0.001) and recurrence-free survival (p<0.001) than those with PanNETs, but no significant difference with those with ACCs. Subgroup analyses revealed that patients with ACCs and MAcNECs had significantly worse recurrence-free survival than those with grade 1 PanNET (p<0.001), and patients with MAcNECs also had worse overall survival than those with grade 1 and 2 PanNETs (p<0.001, and p=0.001). ACCs presented more commonly with intraductal growth (p=0.014) than MAcNECs, while MAcNECs more often had lymph node metastasis (p=0.012) than ACCs. The telomere maintenance mechanism Alternative Lengthening of Telomeres (ALT) was assessed by telomere-specific FISH, and ALT was detected in 1 of 20 ACCs and in three of the 13 MAcNECs. Patients with MAcNECs and ACCs had worse survival and more aggressive behaviour than those with grade 1 PanNETs; thus, the clinicopathological behaviour of MAcNECs resembles ACCs rather than PanNETs. Combined neuroendocrine and acinar cell immunohistochemical markers are helpful for differentiating these different tumour types.

Functional Loss of ATRX and TERC Activates Alternative Lengthening of Telomeres (ALT) in LAPC4 Prostate Cancer Cells.

A key hallmark of cancer, unlimited replication, requires cancer cells to evade both replicative senescence and potentially lethal chromosomal instability induced by telomere dysfunction. The majority of cancers overcome these critical barriers by upregulating telomerase, a telomere-specific reverse transcriptase. However, a subset of cancers maintains telomere lengths by the telomerase-independent Alternative Lengthening of Telomeres (ALT) pathway. The presence of ALT is strongly associated with recurrent cancer-specific somatic inactivating mutations in the ATRX-DAXX chromatin-remodeling complex. Here, we generate an ALT-positive adenocarcinoma cell line following functional inactivation of ATRX and telomerase in a telomerase-positive adenocarcinoma cell line. Inactivating mutations in ATRX were introduced using CRISPR-cas9 nickase into two prostate cancer cell lines, LAPC-4 (derived from a lymph node metastasis) and CWR22Rv1 (sourced from a xenograft established from a primary prostate cancer). In LAPC-4, but not CWR22Rv1, abolishing ATRX was sufficient to induce multiple ALT-associated hallmarks, including the presence of ALT-associated promyelocytic leukemia bodies (APB), extrachromosomal telomere C-circles, and dramatic telomere length heterogeneity. However, telomerase activity was still present in these ATRXKO cells. Telomerase activity was subsequently crippled in these LAPC-4 ATRXKO cells by introducing mutations in the TERC locus, the essential RNA component of telomerase. These LAPC-4 ATRXKO TERCmut cells continued to proliferate long-term and retained ALT-associated hallmarks, thereby demonstrating their reliance on the ALT mechanism for telomere maintenance. IMPLICATIONS: These prostate cancer cell line models provide a unique system to explore the distinct molecular alterations that occur upon induction of ALT, and may be useful tools to screen for ALT-specific therapies.

Telomere alterations in neurofibromatosis type 1-associated solid tumors.

The presence of Alternative lengthening of telomeres (ALT) and/or ATRX loss, as well as the role of other telomere abnormalities, have not been formally studied across the spectrum of NF1-associated solid tumors. Utilizing a telomere-specific FISH assay, we classified tumors as either ALT-positive or having long (without ALT), short, or normal telomere lengths. A total of 426 tumors from 256 NF1 patients were evaluated, as well as 99 MPNST tumor samples that were sporadic or of unknown NF1 status. In the NF1-glioma dataset, ALT was present in the majority of high-grade gliomas: 14 (of 23; 60%) in contrast to only 9 (of 47; 19%) low-grade gliomas (p = 0.0009). In the subset of ALT-negative glioma cases, telomere lengths were estimated and we observed 17 (57%) cases with normal, 12 (40%) cases with abnormally long, and only 1 (3%) case with short telomeres. In the NF1-associated malignant nerve sheath tumor (NF1-MPNST) set (n = 75), ALT was present in 9 (12%). In the subset of ALT-negative NF1-MPNST cases, telomeres were short in 9 (38%), normal in 14 (58%) and long in 1 (3%). In the glioma set, overall survival was significantly decreased for patients with ALT-positive tumors (p < 0.0001). In the NF1-MPNST group, overall survival was superior for patients with tumors with short telomeres (p = 0.003). ALT occurs in a subset of NF1-associated solid tumors and is usually restricted to malignant subsets. In contrast, alterations in telomere lengths are more prevalent than ALT.

Alternative lengthening of telomeres, ATRX loss and H3-K27M mutations in histologically defined pilocytic astrocytoma with anaplasia.

Anaplasia may be identified in a subset of tumors with a presumed pilocytic astrocytoma (PA) component or piloid features, which may be associated with aggressive behavior, but the biologic basis of this change remains unclear. Fifty-seven resections from 36 patients (23 M, 13 F, mean age 32 years, range 3-75) were included. A clinical diagnosis of NF1 was present in 8 (22%). Alternative lengthening of telomeres (ALT) was assessed by telomere-specific FISH and/or CISH. A combination of immunohistochemistry, DNA sequencing and FISH were used to study BRAF, ATRX, CDKN2A/p16, mutant IDH1 p.R132H and H3-K27M proteins. ALT was present in 25 (69%) cases and ATRX loss in 20 (57%), mostly in the expected association of ALT+/ATRX- (20/24, 83%) or ALT-/ATRX+ (11/11, 100%). BRAF duplication was present in 8 (of 26) (31%). H3-K27M was present in 5 of 32 (16%) cases, all with concurrent ATRX loss and ALT. ALT was also present in 9 (of 11) cases in the benign PA precursor, 7 of which also had ATRX loss in both the precursor and the anaplastic tumor. In a single pediatric case, ALT and ATRX loss developed in the anaplastic component only, and in another adult case, ALT was present in the PA-A component only, but ATRX was not tested. Features associated with worse prognosis included subtotal resection, adult vs. pediatric, presence of a PA precursor preceding a diagnosis of anaplasia, necrosis, presence of ALT and ATRX expression loss. ALT and ATRX loss, as well as alterations involving the MAPK pathway, are frequent in PA with anaplasia at the time of development of anaplasia or in their precursors. Additionally, a small subset of PA with anaplasia have H3-K27M mutations. These findings further support the concept that PA with anaplasia is a neoplasm with heterogeneous genetic features and alterations typical of both PA and diffuse gliomas.

ATRX loss induces multiple hallmarks of the alternative lengthening of telomeres (ALT) phenotype in human glioma cell lines in a cell line-specific manner.

Cancers must maintain their telomeres at lengths sufficient for cell survival. In several cancer subtypes, a recombination-like mechanism termed alternative lengthening of telomeres (ALT), is frequently used for telomere length maintenance. Cancers utilizing ALT often have lost functional ATRX, a chromatin remodeling protein, through mutation or deletion, thereby strongly implicating ATRX as an ALT suppressor. Herein, we have generated functional ATRX knockouts in four telomerase-positive, ALT-negative human glioma cell lines: MOG-G-UVW, SF188, U-251 and UW479. After loss of ATRX, two of the four cell lines (U-251 and UW479) show multiple characteristics of ALT-positive cells, including ultrabright telomeric DNA foci, ALT-associated PML bodies, and c-circles. However, telomerase activity and overall telomere length heterogeneity are unaffected after ATRX loss, regardless of cellular context. The two cell lines that showed ALT hallmarks after complete ATRX loss also did so upon ATRX depletion via shRNA-mediated knockdown. These results suggest that other genomic or epigenetic events, in addition to ATRX loss, are necessary for the induction of ALT in human cancer.

Subependymal giant cell astrocytoma-like astrocytoma: a neoplasm with a distinct phenotype and frequent neurofibromatosis type-1-association.

Neurofibromatosis type-1 is a familial genetic syndrome associated with a predisposition to develop peripheral and central nervous system neoplasms. We have previously reported on a subset of gliomas developing in these patients with morphologic features resembling subependymal giant cell astrocytoma, but the molecular features of these tumors remain undefined. A total of 14 tumors were studied and all available slides were reviewed. Immunohistochemical stains and telomere-specific FISH were performed on all cases. In addition, next-generation sequencing was performed on 11 cases using a platform targeting 644 cancer-related genes. The average age at diagnosis was 28 years (range: 4-60, 9F/5M). All tumors involved the supratentorial compartment. Tumors were predominantly low grade (n = 12), with two high-grade tumors, and displayed consistent expression of glial markers. Next-generation sequencing demonstrated inactivating NF1 mutations in 10 (of 11) cases. Concurrent TSC2 and RPTOR mutations were present in two cases (1 sporadic and 1 neurofibromatosis type-1-associated). Interestingly, alternative lengthening of telomeres was present in 4 (of 14) (29%) cases. However, an ATRX mutation associated with aberrant nuclear ATRX expression was identified in only one (of four) cases with alternative lenghtening of telomeres. Gene variants in the DNA helicase RECQL4 (n = 2) and components of the Fanconi anemia complementation group (FANCD2, FANCF, FANCG) (n = 1) were identified in two alternative lenghtening of telomere-positive/ATRX-intact cases. Other variants involved genes related to NOTCH signaling, DNA maintenance/repair pathways, and epigenetic modulators. There were no mutations identified in DAXX, PTEN, PIK3C genes, TP53, H3F3A, HIST1H3B, or in canonical hotspots of IDH1, IDH2, or BRAF. A subset of subependymal giant cell astrocytoma-like astrocytomas are alternative lenghtening of telomere-positive and occur in the absence of ATRX alterations, thereby suggesting mutations in other DNA repair/maintenance genes may also facilitate alternative lenghtening of telomeres. These findings suggest that subependymal giant cell astrocytoma-like astrocytoma represents a biologically distinct group that merits further investigation.

The genomic landscape of TERT promoter wildtype-IDH wildtype glioblastoma.

The majority of glioblastomas can be classified into molecular subgroups based on mutations in the TERT promoter (TERTp) and isocitrate dehydrogenase 1 or 2 (IDH). These molecular subgroups utilize distinct genetic mechanisms of telomere maintenance, either TERTp mutation leading to telomerase activation or ATRX-mutation leading to an alternative lengthening of telomeres phenotype (ALT). However, about 20% of glioblastomas lack alterations in TERTp and IDH. These tumors, designated TERTpWT-IDHWT glioblastomas, do not have well-established genetic biomarkers or defined mechanisms of telomere maintenance. Here we report the genetic landscape of TERTpWT-IDHWT glioblastoma and identify SMARCAL1 inactivating mutations as a novel genetic mechanism of ALT. Furthermore, we identify a novel mechanism of telomerase activation in glioblastomas that occurs via chromosomal rearrangements upstream of TERT. Collectively, our findings define novel molecular subgroups of glioblastoma, including a telomerase-positive subgroup driven by TERT-structural rearrangements (IDHWT-TERTSV), and an ALT-positive subgroup (IDHWT-ALT) with mutations in ATRX or SMARCAL1.

Mutated Chromatin Regulatory Factors as Tumor Drivers in Cancer.

Genes encoding proteins that regulate chromatin structure and DNA modifications [i.e., chromatin regulatory factors (CRF)] and genes encoding histone proteins harbor recurrent mutations in most human cancers. These mutations lead to modifications in tumor chromatin and DNA structure and an altered epigenetic state that contribute to tumorigenesis. Mutated CRFs have now been identified in most types of cancer and are increasingly regarded as novel therapeutic targets. In this review, we discuss DNA alterations in CRFs and how these influence tumor chromatin structure and function, which in turn leads to tumorigenesis. We also discuss the clinical implications and review concepts of targeted treatments for these mutations. Continued research on CRF mutations will be critical for our future understanding of cancer biology and the development and implementation of novel cancer therapies. Cancer Res; 77(2); 227-33. ©2017 AACR.

Alternative Lengthening of Telomeres in Primary Pancreatic Neuroendocrine Tumors Is Associated with Aggressive Clinical Behavior and Poor Survival.

Purpose: Alternative lengthening of telomeres (ALT), a telomerase-independent telomere maintenance mechanism, is strongly associated with ATRX and DAXX alterations and occurs frequently in pancreatic neuroendocrine tumors (PanNET).Experimental Design: In a Korean cohort of 269 surgically resected primary PanNETs and 19 sporadic microadenomas, ALT status and nuclear ATRX and DAXX protein expression were assessed and compared with clinicopathologic factors.Results: In PanNETs, ALT or loss of ATRX/DAXX nuclear expression was observed in 20.8% and 19.3%, respectively, whereas microadenomas were not altered. ALT-positive PanNETs displayed a significantly higher grade, size, and pT classification (all, P < 0.001). ALT also strongly correlated with lymphovascular (P < 0.001) and perineural invasion (P = 0.001) and the presence of lymph node (P < 0.001) and distant metastases (P = 0.002). Furthermore, patients with ALT-positive primary PanNETs had a shorter recurrence-free survival [HR = 3.38; 95% confidence interval (CI), 1.83-6.27; P < 0.001]. Interestingly, when limiting to patients with distant metastases, those with ALT-positive primary tumors had significantly better overall survival (HR = 0.23; 95% CI, 0.08-0.68; P = 0.008). Similarly, tumors with loss of ATRX/DAXX expression were significantly associated with ALT (P < 0.001), aggressive clinical behavior, and reduced recurrence-free survival (P < 0.001). However, similar to ALT, when limiting to patients with distant metastases, loss of ATRX/DAXX expression was associated with better overall survival (P = 0.003).Conclusions: Both primary ALT-positive and ATRX/DAXX-negative PanNETs are independently associated with aggressive clinicopathologic behavior and displayed reduced recurrence-free survival. In contrast, ALT activation and loss of ATRX/DAXX are both associated with better overall survival in patients with metastases. Therefore, these biomarkers may be used as prognostic markers depending on the context of the disease. Clin Cancer Res; 23(6); 1598-606. ©2016 AACR.

ATRX loss promotes tumor growth and impairs nonhomologous end joining DNA repair in glioma.

Recent work in human glioblastoma (GBM) has documented recurrent mutations in the histone chaperone protein ATRX. We developed an animal model of ATRX-deficient GBM and showed that loss of ATRX reduces median survival and increases genetic instability. Further, analysis of genome-wide data for human gliomas showed that ATRX mutation is associated with increased mutation rate at the single-nucleotide variant (SNV) level. In mouse tumors, ATRX deficiency impairs nonhomologous end joining and increases sensitivity to DNA-damaging agents that induce double-stranded DNA breaks. We propose that ATRX loss results in a genetically unstable tumor, which is more aggressive when left untreated but is more responsive to double-stranded DNA-damaging agents, resulting in improved overall survival.