TERT Alterations Research Articles

Analysis of Even a Limited Number of Genes Indicates a Strong Inherited Component in Otherwise Typical Sporadic MDS

Analysis of Even a Limited Number of Genes Indicates a Strong Inherited Component in Otherwise Typical Sporadic MDS

The genetic landscape of anaplastic pleomorphic xanthoastrocytoma.

Pleomorphic xanthoastrocytoma (PXA) is an astrocytic neoplasm that is typically well circumscribed and can have a relatively favorable prognosis. Tumor progression to anaplastic PXA (WHO grade III), however, is associated with a more aggressive biologic behavior and worse prognosis. The factors that drive anaplastic progression are largely unknown. We performed comprehensive genomic profiling on a set of 23 PXAs from 19 patients, including 15 with anaplastic PXA. Four patients had tumor tissue from multiple recurrences, including two with anaplastic progression. We find that PXAs are genetically defined by the combination of CDKN2A biallelic inactivation and RAF alterations that were present in all 19 cases, most commonly as CDKN2A homozygous deletion and BRAF p.V600E mutation but also occasionally BRAF or RAF1 fusions or other rearrangements. The third most commonly altered gene in anaplastic PXA was TERT, with 47% (7/15) harboring TERT alterations, either gene amplification (n=2) or promoter hotspot mutation (n=5). In tumor pairs analyzed before and after anaplastic progression, two had increased copy number alterations and one had TERT promoter mutation at recurrence. Less commonly altered genes included TP53, BCOR, BCORL1, ARID1A, ATRX, PTEN, and BCL6. All PXA in this cohort were IDH and histone H3 wildtype, and did not contain alterations in EGFR. Genetic profiling performed on six regions from the same tumor identified intratumoral genomic heterogeneity, likely reflecting clonal evolution during tumor progression. Overall, anaplastic PXA is characterized by the combination of CDKN2A biallelic inactivation and oncogenic RAF kinase signaling as well as a relatively small number of additional genetic alterations, with the most common being TERT amplification or promoter mutation. These data define a distinct molecular profile for PXA and suggest additional genetic alterations, including TERT, may be associated with anaplastic progression.

The genetic landscape of gliomas arising after therapeutic radiation.

Radiotherapy improves survival for common childhood cancers such as medulloblastoma, leukemia, and germ cell tumors. Unfortunately, long-term survivors suffer sequelae that can include secondary neoplasia. Gliomas are common secondary neoplasms after cranial or craniospinal radiation, most often manifesting as high-grade astrocytomas with poor clinical outcomes. Here, we performed genetic profiling on a cohort of 12 gliomas arising after therapeutic radiation to determine their molecular pathogenesis and assess for differences in genomic signature compared to their spontaneous counterparts. We identified a high frequency of TP53 mutations, CDK4 amplification or CDKN2A homozygous deletion, and amplifications or rearrangements involving receptor tyrosine kinase and Ras-Raf-MAP kinase pathway genes including PDGFRA, MET, BRAF, and RRAS2. Notably, all tumors lacked alterations in IDH1, IDH2, H3F3A, HIST1H3B, HIST1H3C, TERT (including promoter region), and PTEN, which genetically define the major subtypes of diffuse gliomas in children and adults. All gliomas in this cohort had very low somatic mutation burden (less than three somatic single nucleotide variants or small indels per Mb). The ten high-grade gliomas demonstrated markedly aneuploid genomes, with significantly increased quantity of intrachromosomal copy number breakpoints and focal amplifications/homozygous deletions compared to spontaneous high-grade gliomas, likely as a result of DNA double-strand breaks induced by gamma radiation. Together, these findings demonstrate a distinct molecular pathogenesis of secondary gliomas arising after radiation therapy and identify a genomic signature that may aid in differentiating these tumors from their spontaneous counterparts.

DMD genomic deletions characterize a subset of progressive/higher-grade meningiomas with poor outcome.

Progressive meningiomas that have failed surgery and radiation have a poor prognosis and no standard therapy. While meningiomas are more common in females overall, progressive meningiomas are enriched in males. We performed a comprehensive molecular characterization of 169 meningiomas from 53 patients with progressive/high-grade tumors, including matched primary and recurrent samples. Exome sequencing in an initial cohort (n = 24) detected frequent alterations in genes residing on the X chromosome, with somatic intragenic deletions of the dystrophin-encoding and muscular dystrophy-associated DMD gene as the most common alteration (n = 5, 20.8%), along with alterations of other known X-linked cancer-related genes KDM6A (n =2, 8.3%), DDX3X, RBM10 and STAG2 (n = 1, 4.1% each). DMD inactivation (by genomic deletion or loss of protein expression) was ultimately detected in 17/53 progressive meningioma patients (32%). Importantly, patients with tumors harboring DMD inactivation had a shorter overall survival (OS) than their wild-type counterparts [5.1years (95% CI 1.3-9.0) vs. median not reached (95% CI 2.9-not reached, p = 0.006)]. Given the known poor prognostic association of TERT alterations in these tumors, we also assessed for these events, and found seven patients with TERT promoter mutations and three with TERT rearrangements in this cohort (n = 10, 18.8%), including a recurrent novel RETREG1-TERT rearrangement that was present in two patients. In a multivariate model, DMD inactivation (p = 0.033, HR = 2.6, 95% CI 1.0-6.6) and TERT alterations (p = 0.005, HR = 3.8, 95% CI 1.5-9.9) were mutually independent in predicting unfavorable outcomes. Thus, DMD alterations identify a subset of progressive/high-grade meningiomas with worse outcomes.

Comprehensive genomic profiling of brain tumors to provide targeted therapy options and diagnostic certainty for oligodendrogliomas.

2039 Background: Genomic profiling of gliomas is vital to ensure diagnostic accuracy, inform prognosis, and identify therapeutic options for primary and recurrent tumors. The integration of genomic biomarkers into brain tumor classification has advanced the development of molecularly stratified clinical trials and the need to characterize tumors by genomic signature. Methods: Comprehensive genomic profiling (CGP) was performed on FFPE material from 6304 consecutive cases of pediatric and adult brain tumors initially diagnosed by submitting institutions based on histology. We analyzed tumors via CGP in 395 cancer-associated genes (including IDH1/2) and for 1p/19q codeletion using a validated algorithm. Results: Of 6304 brain tumor samples, known IDH point mutations included 1182 IDH1 R132, 50 IDH2 R172, and 1 IDH2 R140 variants. In the IDH-mutant cohort, 1p/19q codeletion was detected in 72% (260/363) of histologically defined oligodendrogliomas (ODGs), 21% (17/82) of oligoastrocytomas (OAs), 6% (22/360) of glioma (NOS, not otherwise specified), 4% (2/50) of gliosarcomas, 3% (26/859) of astrocytomas (NOS), and 1% (32/3200) of glioblastomas. ODG with 1p/19q loss were enriched for TERT, CIC, and FUBP1 alterations, whereas 1p/19q intact tumors were enriched for TP53 and ATRX alterations. Actionable alterations in ODG included 8% (29/363) with high tumor mutational burden (potential immunotherapy responsiveness) and 15% (56/363) with PIK3CA mutation. Analysis of OA (mixed glioma) revealed genomic subtypes similar to well-defined gliomas including ODGs (IDH-mutant, 1p/19q loss, TERT, CIC, FUBP1), diffuse astrocytomas (IDH-mutant, TP53, ATRX), and high-grade gliomas (IDH-wild-type, EGFR, NF1). Conclusions: Using co-occurring IDH mutation and 1p/19q codeletion as the diagnostic signature of ODG, we show that as many as 25% may be misclassified on morphologic criteria alone. OAs exhibit genomic features of defined glioma subtypes, suggesting CGP may provide diagnostic clarity in this setting. This study highlights how CGP can improve diagnostic accuracy and provide additional treatment options for patients.

Association of clinicopathological features and prognosis of TERT alterations in phyllodes tumor of breast

Phyllodes tumor (PT) of the breast is a rare but clinically important fibroepithelial tumor with potential risks of recurrence and metastasis. Recent studies identified recurrent TERT promoter mutations in PTs. However, the clinical significance of this alteration has not been fully examined. Two hundred and seven PTs from two intuitions were included. All cases were subjected to immunohistochemical analysis for TERT expression. Analysis of TERT promoter mutations was further performed by Sanger sequencing targeting the hotspot mutation region on cases from one of the involved institutions. The expression of TERT was correlated with clinicopathologic features, mutation status and recurrence. There was an association of TERT expression and its promoter mutation. Both stromal TERT expression and its promoter mutation correlated with PT grading and older patient age. Recurrence free survival (RFS) of PT patients with high stromal TERT expression was shorter if the excision margin was positive. Our findings suggested a possible pathogenic role of TERT alteration in PT malignancy. Currently there is no consensus for re-excision for PT patients with positive surgical margin, particularly for low grade cases. Stromal TERT expression could be potentially useful to guide management patients with benign PTs.

Utility of next-generation sequencing in clinical decision making in hepatocellular carcinoma (HCC).

295 Background: HCC is a heterogeneous disease with diverse genomic alterations. The prior genomic studies have identified common alterations in TERT, P53, WNT pathways. However, most of these alterations are not targetable with current FDA approved targeted agents. In this study, we determine the clinical impact of targeted next generation sequencing in patients with advanced HCC. Methods: We retrospectively assessed all patients with gastrointestinal (GI) malignancies who have undergone next generation sequencing (NGS) between January 2013 and August 2017. The primary endpoint is to determine the frequency of clinically actionable mutations in HCC. Secondary endpoint is to identify number of patients eligible for current FDA approved targeted agents. Results: Of the 299 consecutive GI tumors sequenced, 29 cases were identified as HCC. Clinically actionable mutations were noted in 22 of 29 patients (pts) (76%). Most pts (52%) were found to harbor more than one potentially actionable genetic alterations (n = 15). The common pathways involved: P53 in 9 pts (22%), cell-cycle regulation in 7 pts (17.1%) and mitogen-activated protein kinase in 6 pts (14.6%). Other pathways involved were DNA repair in 4 pts (9.8%), WNT in 3 pts (7.3%), MYC in 3 pts (7.3%), NOTCH in 2 pts (4.9%), HNPCC in 2 pts (4.9%), APC in 2 pts (4.9%), phosphatidylinositol 3-kinase-AKT-mTOR in 1 patient (pt) (2.4%), BRINP in 1 pt (2.4%) and angiogenesis in 1 pt (2.4%). We noted 10 pts (34.5%) harbor alterations that could potentially be targeted with FDA approved treatments such as palbociclib (for CCND1/2 amplification), PARP inhibitors (for DNA repair defects), and immunotherapy (for MMR defects) on precision medicine clinical trials such as TAPUR or MATCH. Conclusions: Mutational profiling using a targeted NGS panel identified clinically actionable alterations in nearly 75% of advanced HCC patients. Almost one third of these patients were potential candidates for current FDA approved treatments. NGS and enrollment in clinical trials should be considered in all fit patients with HCC who had progression on current standard of care treatments.

Intratumoral heterogeneity and TERT promoter mutations in progressive/higher-grade meningiomas.

BackgroundRecent studies have reported mutations in the telomerase reverse transcriptase promoter (TERTp) in meningiomas. We sought to determine the frequency, clonality and clinical significance of telomere gene alterations in a cohort of patients with progressive/higher-grade meningiomas.MethodsWe characterized 64 temporally- and regionally-distinct specimens from 26 WHO grade III meningioma patients. On initial diagnoses, the meningiomas spanned all WHO grades (3 grade I, 13 grade II and 10 grade III). The tumor samples were screened for TERTp and ATRX/DAXX mutations, and TERT rearrangements. Additionally, TERTp was sequenced in a separate cohort of 19 patients with radiation-associated meningiomas. We examined the impact of mutational status on patients’ progression and overall survival.ResultsSomatic TERTp mutations were detected in six patients (6/26 = 23%). Regional intratumoral heterogeneity in TERTp mutation status was noted. In 4 patients, TERTp mutations were detected in recurrent specimens but not in the available specimens of the first surgery. Additionally, a TERT gene fusion (LPCAT1-TERT) was found in one sample. In contrary, none of the investigated samples harbored an ATRX or DAXX mutation. In the cohort of radiation-induced meningiomas, TERTp mutation was detected in two patients (10.5%). Importantly, we found that patients with emergence of TERTp mutations had a substantially shorter OS than their TERTp wild-type counterparts (2.7 years, 95% CI 0.9 – 4.5 years versus 10.8 years, 95% CI 7.8 -12.8 years, p=0.003).ConclusionsIn progressive/higher-grade meningiomas,TERTp mutations are associated with poor survival, supporting a model in which selection of this alteration is a harbinger of aggressive tumor development. In addition, we observe spatial intratumoral heterogeneity of TERTp mutation status, consistent with this model of late emergence in tumor evolution. Thus, early detection of TERTp mutations may define patients with more aggressive meningiomas. Stratification for TERT alterations should be adopted in future clinical trials of progressive/higher-grade meningiomas.

Genomic Analysis of Pigmented Epithelioid Melanocytomas Reveals Recurrent Alterations in PRKAR1A, and PRKCA Genes.

Pigmented epithelioid melanocytoma (PEM) is a rare cutaneous melanocytic tumor first described as epithelioid blue nevus in patients with the Carney Complex (CC). PEM was among the first established examples of an intermediate class of melanocytic tumors, including atypical Spitz tumors, with frequent metastasis to lymph nodes but only rare extranodal spread. Sporadic and CC-associated PEM are essentially histologically indistinguishable. A subset of PEM shows loss of cytoplasmic expression of the protein kinase A regulatory subunit alpha (PRKAR1A), a tumor suppressor gene mutated in 70% of families with CC. However, molecular studies of such tumors have been limited. Therefore, we used next-generation sequencing to assess 480 cancer-related genes and performed PrkaR1α immunohistochemistry on 13 cases morphologically consistent with PEM. Six cases demonstrated loss of PrkaR1α expression by immunohistochemistry. Three cases were "combined" PEM arising in association with a common nevus. These lesions harbored PRKAR1A genetic alterations in addition to BRAF mutations. Three "pure" PEM, not associated with a common nevus, showed no evidence of PRKAR1A genetic alterations despite loss of PrkaR1α expression. Two of these PEM demonstrated MAP2K1 in frame deletions. PrkaR1α protein expression was preserved in 7 cases. Two of these lesions revealed fusions of the gene encoding the protein kinase C alpha isoform (PRKCA) to 2 distinct partners (ATP2B4-PRKCA and RNF13-PRKCA). Two lesions may represent misdiagnosed "blue nevus with epithelioid features" as they demonstrated GNAQ hotspot mutations. A conceivable explanation, but one we do not favor is that rare PEM are caused by GNAQ mutations. No genetic aberrations were detected in 3 lesions. None of our 13 cases demonstrated TERT alterations or significant chromosomal copy number changes. These results further validate the concept of PEM as a distinctive intermediate/borderline melanocytic tumor, and also illustrate its molecular heterogeneity.

Prognostic Value of TERT Alterations, Mutational and Copy Number Alterations Burden in Urothelial Carcinoma

Point mutations in the TERT gene promoter occur at high frequency in multiple cancers, including urothelial carcinoma (UC). However, the relationship between TERT promoter mutations and UC patient outcomes is unclear due to conflicting reports in the literature. In this study, we examined the association of TERT alterations, tumor mutational burden per megabase (Mb), and copy number alteration (CNA) burden with clinical parameters and their prognostic value in a cohort of 398 urothelial tumors. The majority of TERT mutations were located at two promoter region hotspots (chromosome 5, 1 295 228 C>T and 1 295 250 C>T). TERT alterations were more frequently present in bladder tumors than in upper tract tumors (73% vs 53%; p=0.001). ARID1A, PIK3CA, RB1, ERCC2, ERBB2, TSC1, CDKN1A, CDKN2A, CDKN2B, and PTPRD alterations showed significant co-occurrence with TERT alterations (all p<0.0025). TERT alterations and the mutational burden/Mb were independently associated with overall survival (hazard ratio[HR] 2.31, 95% confidence interval [CI] 1.46–3.65; p<0.001; and HR 0.96, 95% CI 0.93–0.99; p=0.002), disease-specific survival (HR 2.23, 95% CI 1.41–3.53; p<0.001; and HR 0.96, 95% CI 0.93–0.99; p=0.002), and metastasis-free survival (HR 1.63, 95% CI 1.05–2.53; p=0.029; and HR 0.98, 95% CI 0.96–1.00; p=0.063) in multivariate models. Patient summaryThe majority of TERT gene mutations that we detected in urothelial carcinoma are located at two promoter hotspots. Urothelial tumors with TERT alterations had worse prognosis compared to tumors without TERT alterations, whereas tumors with a higher mutational burden had more favorable outcome compared to tumors with low mutational burden.

Identification of somatic TERT promoter mutations in familial nonmedullary thyroid carcinomas.

The genes causing familial nonmedullary thyroid carcinoma (FNMTC) identified to date are only involved in a small fraction of the families. Recently, somatic mutations in TERT promoter region and in EIF1AX gene were reported in thyroid tumours of undefined familial status. The aim of this study was to investigate the role of TERT and EIF1AX mutations in familial thyroid tumours. The promoter region of TERT was sequenced in leucocyte DNA of the probands from 75 FNMTC families. In thyroid tumours from 54 familial cases, we assessed somatic TERT promoter, RAS and BRAF hotspot mutations, and the whole EIF1AX gene. No potentially pathogenic germline variants were identified in TERT in the 75 FNMTC families' probands. In the 54 carcinomas, we identified five cases (9%) with hotspot somatic TERT promoter mutations. BRAF mutations were found in 41% of the tumours. All TERT-positive samples were also positive for BRAF p.Val600Glu, and this co-occurrence was found to be statistically significant (P=.008). RAS mutations were detected in four tumours wild-type for TERT (7%). Evaluation of tumour mutation data together with the patients' clinicopathological features revealed a significant correlation between TERT plus BRAF mutations and advanced tumour stage (T4) (P=.020). No mutations were identified in EIF1AX. The results of this study suggest that TERT promoter and EIF1AX mutations are not frequently involved in FNMTC aetiology. However, we show for the first time that TERT alterations are associated with familial thyroid tumour progression. Our data also suggest that TERT mutations are more often found in concomitance with BRAF mutations in advanced stages of FNMTC.

Activity of RX-3117, an oral antimetabolite nucleoside, in subjects with metastatic bladder cancer resistant to gemcitabine: Preliminary results of a phase Ia/IIb study.

4544 Background: RX-3117 is an oral small molecule antimetabolite, cyclopentyl pyrimidyl nucleoside that is activated by uridine cytidine kinase 2. RX-3117 has shown efficacy in xenograft models of gemcitabine resistant pancreatic, bladder and colorectal cancer. Preliminary data from an analysis of a phase 1b/2a clinical study of RX3117 in metastatic bladder cancer is described. Methods: This phase 1b/2a study (NCT02030067) was designed to evaluate safety, tolerability and efficacy following treatment with 700 mg administered orally once-daily for 5 consecutive days with 2 days off per week for 3 weeks with 1 week off in each 4 week cycle in a 2-stage design. Eligible subjects (aged ≥ 18 years) were those with relapsed/refractory metastatic bladder cancer with any number of prior therapies. Prior therapy with platinum-based chemotherapy was required. The primary endpoint was to assess the efficacy and safety of RX-3117 in metastatic bladder cancer, with secondary aims of evaluating PFS and CBR. Results: With 9 subjects enrolled, median age was 66 years, ECOG PS was 0-1. All subjects had received gemcitabine/cisplatin in the perioperative or metastatic setting, and 4 subjects had received 3 or more prior therapies. The most frequent related adverse events were anemia, mild-moderate fatigue, vomiting and diarrhea. No dose limiting toxicities were observed. PFS and CBR will be presented at the meeting, as 5 subjects continue to receive therapy at the time of this submission. One subject continues on treatment at 139 days with persistent stable disease. Molecular profiling of his bladder tumor showed alterations in ARID1A, FBXW7, FGFR3, NF1, and TERT. The patient previously responded to an FGFR3 inhibitor but progressed after 9 months, with ctDNA assessments showing incurrence of TP53 alteration. Clinical benefit with RX-3117 was achieved in spite of incurrence of this alteration. Conclusions: RX-3117 demonstrated an excellent safety profile, and prolonged stable disease was seen in 1 subject who failed prior cisplatin/gemcitabine and FGFR3 inhibition. Activity persisted despite development a putative resistance alteration detected by ctDNA. Clinical trial information: NCT02030067.

Histological subtypes of hepatocellular carcinoma are related to gene mutations and molecular tumour classification

Our increasing understanding of hepatocellular carcinoma (HCC) biology holds promise for personalized care, however its translation into clinical practice requires a precise knowledge of its relationship to tumour phenotype. We aimed at investigating molecular-phenotypic correlations in a large series of HCC. To this purpose, 343 surgically resected HCC samples were investigated by pathological review, immunohistochemistry, gene expression profiling and sequencing. CTNNB1 (40%) and TP53 (21%) mutations were mutually exclusive and defined two major groups of HCC characterized by distinct phenotypes. CTNNB1 mutated tumours were large (p=0.002), well-differentiated (p<0.001), cholestatic (p<0.001), with microtrabecular (p<0.001) and pseudoglandular (p<0.001) patterns and without inflammatory infiltrates (p<0.001). TP53 mutated tumours were poorly differentiated (p<0.001) with a compact pattern (p=0.02), multinucleated (p=0.01) and pleomorphic (p=0.02) cells and frequent vascular invasion (p=0.02). World Health Organization (WHO) classification of histological subtypes were also strongly related to molecular features. The scirrhous subtype was associated with TSC1/TSC2 mutations (p=0.005), epithelial-to-mesenchymal transition and a progenitor expression profile. The steatohepatitic subtype showed frequent IL-6/JAK/STAT activation without CTNNB1, TERT and TP53 pathway alterations (p=0.01). Pathological review identified a novel subtype, designated as "macrotrabecular-massive" associated with poor survival (p<0.001), high alpha-fetoprotein serum level (p=0.02), vascular invasion (p<0.001), TP53 mutations (p<0.001) and FGF19 amplifications (p=0.02), features also validated in The Cancer Genome Atlas (TCGA) data. Finally, integration of HCC pathological characteristics with its transcriptomic classification showed phenotypically distinct tumour subclasses closely related to G1-G6 subgroups. HCC phenotypes are tightly associated with gene mutations and transcriptomic classification. These findings may help in translating our knowledge of HCC biology into clinical practice. Lay summary: HCC is a very heterogenous tumour, both at the pathological and molecular levels. We show here that HCC phenotype is tightly associated to its molecular alterations and underlying oncogenic pathways.

Genetic and Epigenetic Alterations of TERT Are Associated with Inferior Outcome in Adolescent and Young Adult Patients with Melanoma

Progression of melanoma to distant sites in adolescents and young adults (AYAs) is not reliably predicted by clinicopathologic criteria. TERT promoter mutations when combined with BRAF/NRAS mutations correlate with adverse outcome in adult melanoma. To determine the prognostic value of TERT alterations in AYA melanoma, we investigated the association of TERT promoter mutations, as well as promoter methylation, an epigenetic alteration also linked to TERT upregulation, with TERT mRNA expression and outcome using a well-characterized cohort of 27 patients with melanoma (ages 8–25, mean 20). TERT mRNA expression levels were significantly higher in tumors harboring TERT promoter mutation and/or hypermethylation than those without either aberration (P = 0.046). TERT promoter mutations alone did not predict adverse outcomes (P = 0.50), but the presence of TERT promoter methylation, alone or concurrent with promoter mutations, correlated with reduced recurrence-free survival (P = 0.001). These data suggest that genetic and epigenetic alterations of TERT are associated with TERT upregulation and may predict clinical outcomes in AYA melanoma. A more exhaustive understanding of the different molecular mechanisms leading to increased TERT expression may guide development of prognostic assays to stratify AYA melanoma patients according to clinical risk.

Accelerated cellular senescence as underlying mechanism for functionally impaired bone marrow-derived progenitor cells in ischemic heart disease

Background and aimsBone marrow (BM)-derived progenitor cells are functionally impaired in patients with ischemic heart disease (IHD), thereby hampering the outcome of autologous stem cell therapy. In search for underlying mechanisms for this BM dysfunction, accelerated cellular senescence was explored. MethodsWe analysed telomere length of BM-derived mononuclear cells (MNC) by MMqPCR in patients with coronary artery disease (n = 12), ischemic heart failure (HF; n = 9), non-ischemic HF (n = 7) and controls (n = 10), and related it to their myeloid differentiation capacity. Expressions of senescence-associated genes p53, p21Cip1 and p16lnk4A; and telomere maintenance genes TERT, TRF1/2, Sirt1 in BM-MNC were evaluated using qPCR. Pro-inflammatory cytokine levels (TNFα, IFNy, IL-6) in BM were measured by MSD. ResultsBM-MNC telomere length was shortened in patients with IHD, irrespective of associated cardiomyopathy, and shortened further with increasing angiographic lesions. This telomere shortening was associated with reduced myeloid differentiation capacity of BM-MNC, suggesting accelerated senescence as underlying cause for progenitor cell dysfunction in IHD. Both p16lnk4A and p21Cip1 were activated in IHD and inversely related to myeloid differentiation capacity of BM-MNC; hence, the BM-MNC functional impairment worsens with increasing senescence. While BM-MNC telomere attrition was not related with alterations in TERT, TRF1/2 and Sirt1 expression, IFNy levels were associated with p21Cip1/p16lnk4A upregulation, suggesting a link between inflammation and cellular senescence. Still, the trigger for telomere shortening in IHD needs to be elucidated. ConclusionsAccelerated replicative senescence is associated with a functional impairment of BM-derived progenitor cells in IHD and could be targeted to improve efficacy of stem cell therapy.

Comprehensive genomic sequencing of urothelial tumors to identify rare driver genomic alterations in SMARCB1 in a subset of patients.

385 Background: Multiple genomic alterations leading to overlapping dysregulation of cell cycle, kinase and phosphotidyl \inositol-3-OHkinase signaling and chromatin remodeling are the hallmark of urothelial carcinoma (UC). ARID1A and SMARCA2 loss are frequently altered leading to SWI/SNF nucleosome-remodeling complex dysregulation in urothelial cancers. Mutations affecting components of the SWI/SNF complex lead to aggressive de-differentiated neoplasms. SMARCB1 loss has been implicated in malignant rhabdoid tumors, epithelioid sarcoma and renal medullary carcinoma. Upper tract UC patients with SMARCB1 loss may represent a novel, rare subset. Methods: Tissue from UC patients (n=1099) was assayed by hybrid-capture based comprehensive genomic profiling (CGP) in the course of clinical care to evaluate genomic alterations (GA: base substitutions, indels, amplifications, copy number alterations, fusions/rearrangements) and targeted therapy opportunities; 315 genes frequently altered in cancer were assayed. One patient had an advanced poorly differentiated neoplasm and received conventional chemotherapy. Results: CGP identified 10 (1%) patients with SMARCB1 alterations and different patterns overall in GAs. Three patients (1 kidney, 1 ureter, and 1 bladder) had bi-allelic loss of SMARCB1, and 7 others had a loss of function alteration with loss of heterozygosity. These patients with SMARCB1 bi-allelic loss had few or no additional alterations, notably no alterations in TP53, CDKN2A/B, or TERT, which are observed in greater than 50% of urothelial tumors, or RB1 alterations observed in ~22% of UC. The patient treated with cisplatin based chemotherapy achieved a pathologic CR. Conclusions: Genomic profiles of tumors from patients with UC revealed a rare population of patients with alterations impacting chromatin regulation and tumor suppression, specifically through loss of SMARCB1. Tumors with SMARCB1 loss have a very low burden of genetic alterations and SMARCB1 loss or deletion may be the sole driving mutation. Conventional chemotherapy may be effective. Inhibitors of EZH2, HDAC and CDK4 may be potential treatment strategies to target this subset of tumors.

Distribution of TERT promoter mutations in primary and metastatic melanomas in Austrian patients.

TERT promoter mutations were detected at high frequencies in several cancer types including melanoma. Previous reports showed that these recurrent mutations increase TERT gene expression and the use of TERT mutation status as prognostic factor was suggested. Here we screen a panel of 115 melanoma tumor samples from Austrian patients to evaluate the prevalence and distribution of TERT promoter mutations. The association with clinical and tumor characteristics and the effect on overall survival was analyzed. Genomic DNA from formalin-fixed paraffin-embedded tumor samples was isolated followed by PCR amplification, Sanger sequencing and statistical analysis. We identified TERT promoter mutations in 63 of 115 (54.8%) tumor samples. No statistical significant difference in mutation frequency between primary (22/40 [55%]) and metastatic lesions (41/75 [54.7%]) was detected. BRAF-/NRAS-mutated tumors showed a higher frequency of TERT mutations (pT OR 2.24, 95% CI 0.56-9.02, p=0.3) (met OR 2.74, 95% CI 0.98-7.66, p=0.05). In primary melanoma, the presence of alterations in TERT was associated with the carrier status of a common single-nucleotide polymorphism rs2853669 (OR 4.55, CI 1.18-17.52, p=0.03). In this patient cohort, TERT promoter mutations were not associated with clinical characteristics such as the presence of ulceration or Breslow thickness or showed an effect on overall survival. Alterations in the TERT promoter region are one of the most frequent mutations in melanoma. Based on this analysis and preliminary evidence, prospective studies will be needed to evaluate the reliability of TERT promoter mutations as prognostic factors in melanoma.

Abstract 2430: Neuroblastoma: Telomere elongation is responsible for aggressive behavior

Abstract Neuroblastoma (NB) is a highly malignant pediatric tumor of the sympathetic nervous system that commonly displays low overall mutation frequency. We searched for new structural rearrangements in a series of 275 NBs of all stages using high density SNP microarrays. Exome sequencing was also performed on 15 tumor/constitutional DNA pairs and 25 additional tumors, mainly high-risk NBs, using paired-end sequencing on Illumina instrument. Normalized coverage data were used to generate array comparative genomic hybridization (CGH)-like profiles. Structural rearrangements leading to gain of TERT gene were observed in 15 of 275 neuroblastomas as judged by SNP microarray. 12 out of these 15 tumors with TERT rearrangement belong to 11q-deleted subgroup of NB patients that marks tumors with a poor prognosis. These rearrangements occurred only in high-risk NBs in an almost mutually exclusive fashion with MYCN amplifications, which is a known genetic event in this tumor type. Furthermore, in the small series of NBs analyzed by exome sequencing, we detect extensive structural rearrangements of chromosome 2, 5 and 7 leading to amplification of MYCN, TERT and CDK6 respectively in one tumor. The exome sequencing approach detected at average 14 (range 2-77) somatic protein-changing alterations per tumor. Combining structural and mutational data from the exome sequencing show recurrent alterations in ATRX gene. In our NB cohort we see alterations of ATRX in four non MYCN-amplified tumors: two focal deletions, one nonsense mutation and one deleterious missense mutation. ATRX inactivation results in alternative lengthening of telomeres (ALT). This means that high-risk neuroblastomas cancer cells can preserve their telomeres by ALT or by activation of telomerase reverse transcriptase (encoded by TERT). Alterations in TERT and ATRX were mutually exclusive, which is in agreement with the independent activation by these genes of telomere lengthening. Moreover, three cases with chromothripsis of chromosome 5 with rearrangements affecting the TERT were identified through SNP microarrays. These results identify TERT as at least one of the target genes activated by chromothripsis of chromosome 5. This remodeling of the genomic context likely abrogates transcriptional silencing of TERT in high-risk neuroblastoma and places telomerase activation in the centre of transformation in a large fraction of these tumors. This study identifies recurrent TERT and ATRX rearrangements and telomere lengthening as an important mechanism characterizing high-risk tumors and it supports the pharmacological inhibition of these targets to improve the outcome for this patient group. Citation Format: Niloufar Javanmardi, Susanne Fransson, Rose-Marie Sjöberg, Anna Djos, Per Kogner, Tommy Martinsson. Neuroblastoma: Telomere elongation is responsible for aggressive behavior. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2430.

Massively parallel sequencing of phyllodes tumours of the breast reveals actionable mutations, and TERT promoter hotspot mutations and TERT gene amplification as likely drivers of progression.

Phyllodes tumours (PTs) are breast fibroepithelial lesions that are graded based on histological criteria as benign, borderline or malignant. PTs may recur locally. Borderline PTs and malignant PTs may metastasize to distant sites. Breast fibroepithelial lesions, including PTs and fibroadenomas, are characterized by recurrent MED12 exon 2 somatic mutations. We sought to define the repertoire of somatic genetic alterations in PTs and whether these may assist in the differential diagnosis of these lesions. We collected 100 fibroadenomas, 40 benign PTs, 14 borderline PTs and 22 malignant PTs; six, six and 13 benign, borderline and malignant PTs, respectively, and their matched normal tissue, were subjected to targeted massively parallel sequencing (MPS) using the MSK-IMPACT sequencing assay. Recurrent MED12 mutations were found in 56% of PTs; in addition, mutations affecting cancer genes (eg TP53, RB1, SETD2 and EGFR) were exclusively detected in borderline and malignant PTs. We found a novel recurrent clonal hotspot mutation in the TERT promoter (-124 C>T) in 52% and TERT gene amplification in 4% of PTs. Laser capture microdissection revealed that these mutations were restricted to the mesenchymal component of PTs. Sequencing analysis of the entire cohort revealed that the frequency of TERT alterations increased from benign (18%) to borderline (57%) and to malignant PTs (68%; p < 0.01), and TERT alterations were associated with increased levels of TERT mRNA (p < 0.001). No TERT alterations were observed in fibroadenomas. An analysis of TERT promoter sequencing and gene amplification distinguished PTs from fibroadenomas with a sensitivity and a positive predictive value of 100% (CI 95.38-100%) and 100% (CI 85.86-100%), respectively, and a sensitivity and a negative predictive value of 39% (CI 28.65-51.36%) and 68% (CI 60.21-75.78%), respectively. Our results suggest that TERT alterations may drive the progression of PTs, and may assist in the differential diagnosis between PTs and fibroadenomas. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Telomerase activation by genomic rearrangements in high-risk neuroblastoma.

Neuroblastoma is a malignant paediatric tumour of the sympathetic nervous system. Roughly half of these tumours regress spontaneously or are cured by limited therapy. By contrast, high-risk neuroblastomas have an unfavourable clinical course despite intensive multimodal treatment, and their molecular basis has remained largely elusive. Here we have performed whole-genome sequencing of 56 neuroblastomas (high-risk, n = 39; low-risk, n = 17) and discovered recurrent genomic rearrangements affecting a chromosomal region at 5p15.33 proximal of the telomerase reverse transcriptase gene (TERT). These rearrangements occurred only in high-risk neuroblastomas (12/39, 31%) in a mutually exclusive fashion with MYCN amplifications and ATRX mutations, which are known genetic events in this tumour type. In an extended case series (n = 217), TERT rearrangements defined a subgroup of high-risk tumours with particularly poor outcome. Despite a large structural diversity of these rearrangements, they all induced massive transcriptional upregulation of TERT. In the remaining high-risk tumours, TERT expression was also elevated in MYCN-amplified tumours, whereas alternative lengthening of telomeres was present in neuroblastomas without TERT or MYCN alterations, suggesting that telomere lengthening represents a central mechanism defining this subtype. The 5p15.33 rearrangements juxtapose the TERT coding sequence to strong enhancer elements, resulting in massive chromatin remodelling and DNA methylation of the affected region. Supporting a functional role of TERT, neuroblastoma cell lines bearing rearrangements or amplified MYCN exhibited both upregulated TERT expression and enzymatic telomerase activity. In summary, our findings show that remodelling of the genomic context abrogates transcriptional silencing of TERT in high-risk neuroblastoma and places telomerase activation in the centre of transformation in a large fraction of these tumours.