Telomere-specific Fluorescence Research Articles

Predicting recurrence in pancreatic neuroendocrine tumours: role of ARX and alternative lengthening of telomeres (ALT).

While many pancreatic neuroendocrine tumours (PanNET) show indolent behaviour, predicting the biological behaviour of small nonfunctional PanNETs remains a challenge. Nonfunctional PanNETs with an epigenome and transcriptome that resemble isletalpha cells (ARX-positive) are more aggressive than neoplasms that resemble islet beta cells (PDX1-positive). In this study, we explore the ability of immunohistochemistry for ARX and PDX1 and telomere-specific fluorescence in situ hybridisation (FISH) for alternative lengthening of telomeres (ALT) to predict recurrence. Two hundred fifty-six patients with PanNETs were identified, and immunohistochemistry for ARX and PDX1 was performed. Positive staining was defined as strong nuclear staining in >5% of tumour cells. FISH for ALT was performed in a subset of cases. ARX reactivity correlated with worse disease-free survival (DFS) (P = 0.011), while there was no correlation between PDX1 reactivity and DFS (P = 0.52). ALT-positive tumours (n = 63, 31.8%) showed a significantly lower DFS (P < 0.0001) than ALT-negative tumours (n = 135, 68.2%). ARX reactivity correlated with ALT positivity (P < 0.0001). Among nonfunctional tumours, recurrence was noted in 18.5% (30/162) of ARX-positive tumours and 7.5% (5/67) of ARX-negative tumours. Among WHO grade 1 and 2 PanNETs with ≤2 cm tumour size, 14% (6/43) of ARX-positive tumours recurred compared to 0 of 33 ARX-negative tumours and 33.3% (3/9) ALT-positive tumours showed recurrence versus 4.4% (2/45) ALT-negative tumours. Immunohistochemistry for ARX and ALT FISH status may aid in distinguishing biologically indolent cases from aggressive small low-grade PanNETs, and help to identify patients who may preferentially benefit from surgical intervention.

Alternative Lengthening of Telomeres (ALT) and Telomerase Reverse Transcriptase Promoter Methylation in Recurrent Adult and Primary Pediatric Pituitary Neuroendocrine Tumors.

Neoplastic cells acquire the ability to proliferate endlessly by maintaining telomeres via telomerase, or alternative lengthening of telomeres (ALT). The role of telomere maintenance in pituitary neuroendocrine tumors (PitNETs) has yet to be thoroughly investigated. We analyzed surgical samples of 24 adult recurrent PitNETs (including onset and relapses for 14 of them) and 12 pediatric primary PitNETs. The presence of ALT was assessed using telomere-specific fluorescence in situ hybridization, methylation of telomerase reverse transcriptase promoter (TERTp) by methylation-specific PCR, and ATRX expression by immunohistochemistry. Among the adult recurrent PitNETs, we identified 3/24 (12.5%) ALT-positive cases. ALT was present from the onset and maintained in subsequent relapses, suggesting that this mechanism occurs early in tumorigenesis and is stable during progression. ATRX loss was only seen in one ALT-positive case. Noteworthy, ALT was observed in 3 out of 5 aggressive PitNETs, including two aggressive corticotroph tumors, eventually leading to patient's death. ALT-negative tumors (87.5%) were classified according to their low (29.2%), medium (50%), and high (8.3%) telomere fluorescence intensity, with no significant differences emerging in their molecular, clinical, or pathological characteristics. TERTp methylation was found in 6/24 cases (25%), with a total concordance in methylation status between onset and recurrences, suggesting that this mechanism remains stable throughout disease progression. TERTp methylation did not influence telomere length. In the pediatric cohort of PitNETs, TERTp methylation was also observed in 4/12 cases (33.3%), but no case of ALT activation was observed. In conclusion, ALT is triggered at onset and maintained during tumor progression in a subset of adult PitNETs, suggesting that it could be used for clinical purposes, as a potential predictor of aggressive behavior.

CBIO-08. ENDOGENOUS DNA DOUBLE STRAND BREAKS ACTIVATE HETEROGENOUS DNA DAMAGE SIGNALING IN IDH1/2 MUTANT GLIOMAS

Abstract Isocitrate dehydrogenase 1/2 (IDH1/2) mutations are common in astrocytic glioma and are frequently coupled with TP53 and ATRX mutations. Collectively, these alterations cause genomic instability leading to high basal DNA double strand breaks (DSBs). Understanding how IDH/TP53/ATRX mutant cells process endogenous DSBs may help exploit inhibitors of DNA damage response (DDR) for the treatment of patients with IDH mutant gliomas. Through systematic effort to uncover the mechanisms involved in repair of endogenous DSBs in IDH1/2 mutant GBMs, we have discovered that high basal phosphorylated DNA-PK (p-DNA-PK) was characteristic of an IDH1/TP53/ATRX mutant GBM164 patient derived xenograft (PDX) but not in another IDH1 mutant GBM196 PDX. Immunofluorescence (IF) studies in patient specimen from which GBM164 was derived showed that p-DNA-PK co-localized with g-H2AX, 53BP1 or H4K20me2 (but not p-RPA) the known surrogates of DSBs. In contrast, p-DNA-PK was absent in the patient specimen from which GBM196 was derived, which otherwise had equally intense g-H2AX immunostaining colocalized with p-RPA. An independent IF study involving 11 IDH1 wild-type (WT) and 11 IDH1 mutant GBM patient samples, the p-DNA-PK was observed in 3 (27%) of 11 IDH1 mutant samples while IDH1 WT tumors were negative for p-DNA-PK. A telomere specific fluorescence in situ hybridization (Tel-FISH) confirmed elevated alternative lengthening of telomere (ALT) activity in GBM196 (but not in GBM164) indicative of HR proficiency. Consistently, HR related genes, including BRCA1 and MRE11A, were found upregulated in ALT-positive GBM196 as compared to those in GBM164. Interestingly, ALT+ GBM196 cells were highly vulnerable to inhibitors of ATM and ATR pathways. In conclusion, IDH1/TP53/ATRX mutant gliomas can be subdivided into HR-mediated ALT-positive group, which repairs the endogenous DSBs by HR (e.g. GBM196) and an ALT-negative/p-DNA-PK group, which repairs DSBs by c-NHEJ (e.g. GBM164) and this subdivision can be developed as a prescient biomarker of sensitivity to DDR inhibitors.

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.

Alternative Lengthening of Telomeres Phenotype Predicts Progression Risk in Noninsulinomas in a Chinese Cohort

Introduction: Recent studies have suggested that alternative lengthening of telomeres (ALT) is associated with metastasis and poor survival in pancreatic neuroendocrine tumors (PanNETs). This study evaluated whether this association is applicable to Chinese patients as well as the potential somatic mutations associated with ALT. Methods: We assessed the prevalence of ALT by performing telomere-specific fluorescence in situ hybridization and analyzed DAXX/ATRX expression using immunohistochemistry in 112 Chinese patients with PanNETs to evaluate the association between ALT and clinical outcomes. A subset of the noninsulinoma samples (28/60) was subjected to Sanger sequencing and targeted sequencing. Results: The ALT-positive phenotype was identified in 23.2% (26/112) of the samples. The clinicopathologic factors significantly associated with progression in the noninsulinoma (n = 60) cohort were the female sex (p = 0.006), Ki-67 index (p < 0.001), World Health Organization grade (p = 0.031), and ALT positivity (p = 0.013). Patients with ALT-positive PanNETs had significantly shorter progression-free survival than those with ALT-negative PanNETs in the entire cohort (p < 0.001), noninsulinoma subgroup (p = 0.01), and G2 subgroup (p = 0.001). ALT-positive samples frequently harbored somatic mutations in DAXX, ATRX, MEN1, SETBP1, PRKDC, and GNAS. Conclusions: We confirmed that ALT positivity is an effective risk predictor, especially in the noninsulinoma and G2 subgroups. ALT is also related to somatic mutations in MEN1, SETBP1, PRKDC, and GNAS, in addition to DAXX and ATRX.

Clinicopathological and molecular characterization of chromophobe hepatocellular carcinoma.

Chromophobe hepatocellular carcinoma (HCC) is a newly included subtype of HCC in the 5th edition of the WHO classification with distinctive histological features (chromophobic cytoplasm with anaplastic nuclei and pseudocyst formation) and is strongly associated with the alternative lengthening of telomeres (ALT) phenotype. However, the clinicopathologic characterization and molecular features of chromophobe HCC are unknown. To comprehensively characterize chromophobe HCC, whole exome sequencing, copy number variation, and transcriptomic analyses were performed in 224 surgically resected HCC cases. Additionally, telomere-specific fluorescence in situ hybridization was used to assess ALT. These genomic profiles and ALT status were compared with clinicopathological features among subtypes of HCC, particularly chromophobe HCC and conventional HCC. Chromophobe HCC was observed in 10.3% (23/224) cases and, compared to conventional HCC, was more frequent in females (P=.023). The overall and recurrence-free survival outcomes were similar between patients with chromophobe HCC and conventional HCC. However, chromophobe HCC displayed significantly more upregulated genes involving cell cycle progression and DNA repair. Additionally, ALT was significantly enriched in chromophobe HCC (87%; 20/23) compared to conventional HCC (2.2%, 4/178; P<.001). Somatic mutations in ALT-associated genes, including ATRX, SMARCAL1, FANCG, FANCM, SP100, TSPYL5, and RAD52 were more frequent in chromophobe HCC (30.4%, 7/23 cases) compared to conventional HCC (11.8%, 21/178 cases; P=.024). Chromophobe HCC is a unique subtype of HCC with a prevalence of ~10%. Compared to conventional HCC, chromophobe HCC is associated with female predominance and ALT, although overall and recurrence-free outcomes are similar to conventional HCC.

Non-functional pancreatic neuroendocrine tumours: ATRX/DAXX and alternative lengthening of telomeres (ALT) are prognostically independent from ARX/PDX1 expression and tumour size

ObjectiveRecent studies have found aristaless-related homeobox gene (ARX)/pancreatic and duodenal homeobox 1 (PDX1), alpha-thalassemia/mental retardation X-linked (ATRX)/death domain-associated protein (DAXX) and alternative lengthening of telomeres (ALT) to be promising prognostic...

Telomere lengths differ significantly between small-cell neuroendocrine prostate carcinoma and adenocarcinoma of the prostate

Small-cell neuroendocrine carcinoma (SCNC) of the prostate is an aggressive subtype with frequent TP53 mutation and RB1 inactivation; however, the molecular phenotype remains an area of investigation. Here, we compared telomere lengths in prostatic SCNC and usual-type prostatic adenocarcinoma (AdCa). We studied 32 cases of prostatic SCNC (including 11 cases with concurrent AdCa) and 347 cases of usual-type AdCa on tissue microarrays. Telomere lengths in tumor cells were qualitatively compared with those in normal cells using a telomere-specific fluorescence in situ hybridization assay. ERG, PTEN, and TP53 status were assessed in a proportion of cases using genetically validated immunohistochemistry protocols. Clinicopathological and molecular characteristics of cases were compared between the telomere groups using the chi-square test.A significantly higher proportion of prostatic SCNC cases (50%, 16/32) showed normal/long telomeres compared with AdCa cases (11%, 39/347; P<0.0001). In 82% (9/11) of cases with concurrent SCNC and AdCa, the paired components were concordant for telomere length status. Among AdCa cases, the proportion of cases with normal/long telomeres significantly increased with increasing tumor grade group (P=0.01) and pathologic stage (P=0.02). Cases with normal/long telomeres were more likely to be ERG positive (P=0.04) and to have TP53 missense mutation (P=0.01) than cases with short telomeres.Normal or long telomere lengths are significantly more common in prostatic SCNC than in AdCa and are similar between concurrent SCNC and AdCa tumors, supporting a common origin. Among AdCa cases, longer telomere lengths are significantly associated with high-risk pathologic and molecular features.

Telomere length alterations and ATRX/DAXX loss in pituitary adenomas.

Telomeres are nucleoprotein complexes located at the termini of eukaryotic chromosomes that prevent exonucleolytic degradation and end-to-end chromosomal fusions. Cancers often have critically shortened, dysfunctional telomeres contributing to genomic instability. Telomere shortening has been reported in a wide range of precancerous lesions and invasive carcinomas. However, the role of telomere alterations, including the presence of alternative lengthening of telomeres (ALT), has not been studied in pituitary adenomas. Telomere length and the presence of ALT were assessed directly at the single cell level using a telomere-specific fluorescence in situ hybridization assay in tissue microarrays. Tumors were characterized as either ALT-positive or having short, normal, or long telomere lengths and then these categories were compared with clinicopathological characteristics. ATRX and DAXX expression was studied through immunohistochemistry. We characterized a discovery set of 106 pituitary adenomas including both functional and nonfunctional subsets (88 primary, 18 recurrent). Telomere lengths were estimated and we observed 64 (59.4%) cases with short, 39 (36.8%) cases with normal, and 0 (0%) cases with long telomeres. We did not observe significant differences in the clinicopathological characteristics of the group with abnormally shortened telomeres compared to the group with normal telomeres. However, three pituitary adenomas were identified as ALT-positive of which two were recurrent tumors. Two of these three ALT-positive cases had alterations in either of the chromatin remodeling proteins, ATRX and DAXX, which are routinely altered in other ALT-positive tumor subtypes. In a second cohort of 32 recurrent pituitary adenomas from 22 patients, we found that the tumors from 36% of patients (n = 8) were ALT-positive. This study demonstrates that short telomere lengths are prevalent in pituitary adenomas and that ALT-positive pituitary adenomas are enriched in recurrent disease.

Analysis of Telomere Lengths in p53 Signatures and Incidental Serous Tubal Intraepithelial Carcinomas Without Concurrent Ovarian Cancer.

Telomere alterations represent one of the major molecular changes in the development of human cancer. We have previously reported that telomere lengths in most serous tubal intraepithelial carcinomas (STIC) are shorter than they are in ovarian high-grade serous carcinomas (HGSC) or in normal-appearing fallopian tube epithelium from the same patients. However, it remains critical to determine if similar telomere alterations occur in TP53-mutated but histologically unremarkable "p53 signature" lesions, as well as incidental STICs without concurrent HGSC. In this study, we quantitatively measured telomere lengths by performing telomere-specific fluorescence in situ hybridization in conjunction with p53 immunolabeling in 15 p53 signatures and 30 incidental STICs without concurrent HGSC. We compared these new results with our previous data in paired STICs and concurrent HGSCs. We found that most p53 signatures (80%) and incidental STICs without HGSC (77%) exhibited significant telomere shortening compared with adjacent normal-appearing fallopian tube epithelium (P<0.01). Interestingly, however, p53 signatures and incidental STICs without HGSC displayed longer telomeres and less cell-to-cell telomere length heterogeneity than STICs associated with HGSC (P<0.001). These findings indicate that telomere shortening occurs in p53 signatures, the earliest precancer lesion. Moreover, incidental STICs without concurrent HGSC are indeed similar to p53 signatures as they have less telomere shortening and less cell-to-cell telomere length heterogeneity than STICs associated with HGSC.

Telomere elongation via alternative lengthening of telomeres (ALT) and telomerase activation in primary metastatic medulloblastoma of childhood.

Elongation of telomeres is necessary for tumor cell immortalization and senescence escape; neoplastic cells use to alternative pathways to elongate telomeres: telomerase reactivation or a telomerase-independent mechanism termed alternative lengthening of telomeres (ALT). Telomerase and ALT pathway has been explored in adult and pediatric gliomas and medulloblastomas (MDBs); however, these mechanisms were not previously investigated in MDBs metastatic at the onset. Therefore, we analyzed the activation of telomerase and ALT pathway in a homogenous cohort of 43 pediatric metastatic medulloblastomas, to investigate whether telomere elongation could play a role in the biology of metastatic MDB. We evaluated telomeres length via telomere-specific fluorescence in situ hybridization (Telo-FISH); we assessed nuclear expression of ATRX by immunohistochemistry (IHC). H3F3A and TERT promoter mutations were analyzed by pyrosequencing, while UTSS methylation status was analyzed via methylation-specific-PCR (MS-PCR). H3F3A mutations were absent in all MDBs, 30% of samples showed ATRX nuclear loss, 18.2% of cases were characterized by TERT promoter mutations, while 60.9% harboured TERT promoter hyper-methylation in the UTSS region. Elongation of telomeres was found in 42.8% of cases. Metastatic MDBs control telomere elongation via telomerase activation (10.7%), induced by TERT promoter mutations in association with UTSS hyper-methylation, and ALT mechanism (32.1%), triggered by ATRX inactivation. Among non-metastatic MDBs, only 5.9% (1/17) showed ATRX nuclear loss with activation of ALT. Our metastatic cases frequently activate ALT pathway, suggesting that it is a common process for senescence escape in primary metastatic medulloblastomas. Furthermore, the activation of mechanisms for telomere elongation is not restricted to certain molecular subgroups in this high-risk group of MDBs.

Performance of DAXX Immunohistochemistry as a Screen for DAXX Mutations in Pancreatic Neuroendocrine Tumors.

DAXX immunohistochemistry (IHC) is often used as a surrogate for sequencing. We aimed to elucidate the sensitivity of IHC for DAXX mutation. All pancreatic neuroendocrine tumors (PanNETs) with DAXX mutations detected by sequencing and a subset of DAXX wild-type PanNETs were analyzed for DAXX expression by IHC. Of 154 PanNETs with MSK-IMPACT testing, 36 (30%) harbored DAXX mutations. DAXX mutations were associated with TSC2 mutations (46% vs 10%, P < 0.0001), tended to co-occur with MEN1 mutations (63% vs 49%, P = 0.11), and tended to be mutually exclusive with ATRX mutations (11% vs 25%, P = 0.053). Of 27 available DAXX mutant PanNETs, 23 lost DAXX expression (85.2%). All 4 DAXX mutants with retained expression harbored DAXX mutations within the SUMO-interacting motif of the last exon. Telomere-specific fluorescence in situ hybridization demonstrated alternative lengthening of telomeres in all 4 cases. Of 20 PanNETs with wild-type DAXX, 19 retained DAXX IHC expression (95%). The sensitivity and specificity of IHC for DAXX mutation are 85% and 95%, respectively. Last exon DAXX mutant PanNETs often show alternative lengthening of telomeres despite retained DAXX expression, likely due to escape of nonmediated decay.

Unbalanced X;9 translocation in an infertile male with de novo duplication Xp22.31p22.33.

Male carriers of an X-autosome translocation are generally infertile, regardless of the position of the breakpoint on the X chromosome while the pathogenicity of Xp22.3 subtelomeric duplications is under debate. To shed light into this controversy, we present a rare case, of an azoospermic male with no other significant clinical findings, in whom classical cytogenetics revealed additional unbalanced chromosomal material, at the telomere of the long arm of one homolog of chromosome 9. In peripheral blood specimens of the index case and his parents, we performed GBanding, Inverted-DAPI Banding, AgNOR staining, Telomere specific Fluorescence in Situ Hybridization (FISH), Molecular karyotyping by Multi-color FISH, whole genome SNP microarrays, sub-telomeric MLPA, and transcription analysis of the expression of KAL1 gene by RT-PCR. Multi-color FISH revealed an unbalanced translocation involving the short arm of chromosome X. SNP microarray analysis combined to classical cytogenetics and MLPA demonstrated a de novo 8.796Mb duplication of Xp22.31-p22.33. Compared to three control specimens, the patient presented significantly elevated expression levels of KAL1 mRNA in peripheral blood, suggesting transcriptional functionality of the duplicated segment. The duplicated segment contains the pseudo-autosomal region PAR1 and more than 30 genes including SHOX, ARSE, STS, KAL1, and FAM9A and is not listed as polymorphic. Our data advocate that duplications of the Xp22.3 region may not be associated with a clinical consequence.

Loss of ATRX/DAXX expression and alternative lengthening of telomeres in uterine leiomyomas.

Uterine leiomyomas (ULs) are the most common gynecologic tumors and affect 3 of every 4 women by the age of 50 years. The majority of ULs are classified as conventional tumors, whereas 10% represent various histopathological subtypes with features that mimic malignancy. These subtypes include cellular and mitotically active ULs and ULs with bizarre nuclei. Uterine leiomyosarcoma (ULMS), the malignant counterpart of UL, is an aggressive cancer with poor overall survival. The early diagnosis and preoperative differentiation of ULMS from UL are often challenging because their symptoms and morphology resemble one another. Recent studies have shown frequent loss of alpha-thalassemia/mental retardation syndrome X-linked (ATRX) or death domain-associated protein (DAXX) expression in ULMS, and this is often associated with an alternative lengthening of telomeres (ALT) phenotype. To investigate ATRX and DAXX expression and the presence of ALT in UL subtypes, immunohistochemical and telomere-specific fluorescence in situ hybridization analyses were performed. The study material consisted of 142 formalin-fixed, paraffin-embedded tissue samples representing various UL subtypes and 64 conventional ULs. A loss of ATRX or DAXX and/or ALT was detected in 6.3% of the histopathological UL subtype samples (9 of 142). Two patients whose ULs showed either ATRX loss or ALT were later diagnosed with a pulmonary smooth muscle tumor. Pulmonary tumors displayed molecular alterations found in the corresponding uterine tumors, which indicated metastasis to the lungs. All conventional ULs displayed normal ATRX, DAXX, and telomeres. These results highlight the differences between conventional and histopathologically atypical ULs and indicate that some UL subtype tumors may harbor long-term malignant potential.

Abstract 5383: Tumor evolution of uterine leiomyosarcoma from a benign leiomyoma precursor

Abstract Uterine leiomyosarcoma (ULMS) is a highly aggressive smooth muscle tumor with poorly understood pathogenesis. Although ULMS is believed to develop as a de novo tumor, it has been suggested that in some cases uterine leiomyoma (UL) could serve as a precursor lesion for the pathogenesis. ULs are extremely common tumors affecting up to 70% of women. Whereas 90% of ULs are classified as conventional ULs, 10% of the tumors represent subtype histopathologies, some of which have features associated with malignancy. These subtypes include tumors with bizarre nuclei and increased cellularity. In this study, we investigated the genetic and molecular background of one ULMS showing simultaneously mixed histopathologies of UL with bizarre nuclei and cellular UL. The study material consisted of four formalin-fixed paraffin-embedded samples having distinct areas of ULMS, UL with bizarre nuclei, and cellular UL. Fallopian tube sample was used as a normal control. Libraries for exome sequencing were prepared with KAPA Library Preparation Kit and subjected to exome capture with NimbleGen SeqCap EZ System (Roche). Paired-end sequencing was performed with HiSeq2500 (Illumina). SNP genotyping data was produced with Infinium Omni2.5-8 Kit (Illumina). Immunohistochemistry was performed to proteins commonly displaying aberrant expression in UL and/or ULMS. Telomere-specific fluorescence in situ hybridization was conducted to detect alternatively lengthened telomeres (ALT), a feature often associated with ULMS pathogenesis. Exome sequencing data and protein expression analyses revealed that all tumor compartments were negative for UL driver alterations, including MED12 mutations, HMGA2 overexpression and biallelic FH inactivation. Data from exome sequencing indicated that ULMS shared the largest number of variants with UL with bizarre nuclei, including the pathogenic splice site mutation c.994-1G&amp;gt;A in tumor protein 53 (TP53). SNP genotyping data showed identical and large chromosomal aberrations in 6q, 9p, 10q and 19q in ULMS and UL with bizarre nuclei. No such aberrations were shared between ULMS and cellular UL. All tumor compartments shared changes in chromosomes 1q, 2p, 12q, 13q, 17q and 17p. ULMS showed prominent ALT, whereas UL with bizarre nuclei had a weaker ALT phenotype. Cellular UL had normal telomere lengths. In conclusion, these results suggest that the genome, including telomeres, of UL with bizarre nuclei resembles that of ULMS. Large genomic events have probably occurred in UL with bizarre nuclei during tumor evolution, leading to unstable chromosomes, which in turn favored the development of ULMS. Also, all tumor compartments displayed shared genetic variation, suggesting a common evolutionary origin. Understanding the pathogenesis of ULMS and the malignant potential of ULs is clinically highly relevant, as it may improve the diagnosis, prevent malignant transformation and enable the design of new treatments. Citation Format: Netta Mäkinen, Terhi Ahvenainen, Pernilla von Nandelstadh, Ralf Bützow, Pia Vahteristo. Tumor evolution of uterine leiomyosarcoma from a benign leiomyoma precursor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5383.

Current or recent smoking is associated with more variable telomere length in prostate stromal cells and prostate cancer cells.

Current and recent smoking have been associated with a greater risk of prostate cancer recurrence and mortality, though the underlying mechanism is unknown. To determine if telomere shortening, which has been associated with poor outcomes, may be a potential underlying mechanism, we prospectively evaluated the association between smoking status and telomere length in 567 participants in the Health Professionals Follow-up Study, who were surgically treated for prostate cancer. Using tissue microarrays (TMA), we measured telomere length in cancer and benign tissue, specifically stromal cells in the same TMA spot using a telomere-specific fluorescence in situ hybridization assay. Smoking status was collected via questionnaire 2-years before diagnosis. Adjusting for age, pathologic stage and grade, the median and standard deviation of the per-cell telomere signals were determined for each man for stromal cells and cancer cells by smoking categories. In sub-analyses, we restricted to men without major co-morbidities diagnosed before prostate cancer. Overall, there were no associations between smoking status and telomere length or variability in stromal cells or cancer cells. However, among men without comorbidities, current smokers and former smokers who quit <10 years ago had the most variable telomere length in stromal cells (29.3% more variable than never smokers; P-trend = 0.0005) and in cancer cells (27.7% more variable than never smokers; P-trend = 0.05). Among men without comorbidities, mean telomere length did not differ by smoking status in stromal cells or cancer cells. Telomere variability in prostate cells may be one mechanism through which smoking influences poor prostate cancer outcomes.

Telomere length variation in tumor cells and cancer-associated fibroblasts: potential biomarker for hepatocellular carcinoma.

The role of telomere dysfunction and aberrant telomerase activities in hepatocellular carcinoma (HCC) has been overlooked for many years. This study aimed to delineate the variation and prognostic value of telomere length in HCC. Telomere‐specific fluorescence in situ hybridization (FISH) and qPCR were used to evaluate telomere length in HCC cell lines, tumor tissues, and isolated non‐tumor cells within the tumor. Significant telomere attrition was found in tumor cells and cancer‐associated fibroblasts (CAFs) compared to their normal counterparts, but not in intratumor leukocytes or bile duct epithelial cells. Clinical relevance and prognostic value of telomere length were investigated on tissue microarrays of 257 surgically treated HCC patients. Reduced intensity of telomere signals in tumor cells or CAFs correlated with larger tumor size and the presence of vascular invasion (p < 0.05). Shortened telomeres in tumor cells or CAFs associated with reduced survival and increased recurrence, and were identified as independent prognosticators for HCC patients (p < 0.05). These findings were validated in an independent HCC cohort of 371 HCC patients from The Cancer Genome Atlas (TCGA) database, confirming telomere attrition and its prognostic value in HCC. We also showed that telomerase reverse transcriptase promoter (TERTp) mutation correlated with telomere shortening in HCC. Telomere variation in tumor cells and non‐tumor cells within the tumor microenvironment of HCC was a valuable prognostic biomarker for this fatal malignancy. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

1413P - ATRX-associated alternative lengthening of telomere (ALT) may be correlated with chemotherapy response in leiomyosarcoma (LMS)

Response to systemic chemotherapy in LMS varies and no predictive biomarker exists. ALT, a common mechanism to maintain telomere in sarcoma, is associated with altered DNA repair mechanism. Loss of a chromatin modifier &agr;-thalassemia/mental retardation syndrome X-linked (ATRX) has been implicated as one of the factors leading to ALT. We aimed to determine the relationship between ALT, ATRX and chemotherapy response in patients with advanced LMS. Histology-proven advanced LMS patients (pts) treated with systemic chemotherapy in National Taiwan University Hospital from January 1995 to December 2014 were selected. Patient demographic data and clinical outcomes including tumor response, progression-free survival and overall survival were recorded. We used formalin-fixed paraffin-embed tumor samples for ALT and ATRX status assessment by telomere-specific fluorescence in situ hybridization and immunohistochemistry, respectively. 30 pts with advanced LMS treated with systemic chemotherapy were identified. Table 1 summarizes the clinicopathological information. 21 pts received anthracycline-based first line chemotherapy. 20 pts (67%) were ALT-positive (ALT+). 10 pts (33%) were ATRX-negative (ATRX-), among which all but 1 were also ALT+. ALT positivity was not correlated with chemotherapy response (odds ratio = 1.25, p = 0.7844). In the 9 ALT + /ATRX- patients, a lower odds, albeit non-significant, of response to chemotherapy was noted compared to non ALT + /ATRX- patients (11.3% vs 42.9%, odds ratio = 0.17, 95% CI = 0.01-1.58, p = 0.11). PFS was significantly shorter in ALT + / ATRX- patients (median 2.0 mos vs 7.7 mos, p < 0.001, hazard ratio 6.5, 95% CI 2.4-17.7). No significant difference in OS was noted (median 11.7 mos vs 23.9 mon, p = 0.314) between ALT + /ATRX- and non-ALT + /ATRX- pts.Tabled 1Patient demographics and clinicopathological featuresn = 30 (%)Age, median (range)51 (28-67)Sex- Male2 (7)- Female28 (93)Tumor Origin- Uterus22 (73)- Retroperitoneum/intra-abdomen3 (10)- Others5 (17)Metastatic site- Lung25 (83)- Liver14 (47)- Bone7 (23)- Intra-abdomen (excluding liver)7 (23)Cell Morphology- Spindle19 (63)- Epithelioid/pleomorphic11 (37)FNCLCC Grade- Grade 1/212 (40)- Grade 318 (60)ALT FISH- Positive20 (67)- Negative10 (33)ATRX Expression- Present20 (67)- Loss10 (33)1st-line Chemotherapy- Anthracycline-based21 (70)- Nonanthracycline-based9 (30) Open table in a new tab ALT+ with ATRX- may predict poorer response to chemotherapy in advanced LMS.

High-resolution telomere fluorescence in situ hybridization reveals intriguing anomalies in germ cell tumors

Testicular germ cell tumor (TGCT) is the most common malignancy of young men. Most patients are completely cured, which distinguishes these from most other malignancies. Orchiectomy specimens (n=76) were evaluated using high-resolution (single-cell discriminative) telomere-specific fluorescence in situ hybridization (FISH) with simultaneous Oct4 immunofluorescence to describe telomere length phenotype in TGCT neoplastic cells. For the first time, the TGCT precursor lesion, germ cell neoplasia in situ (GCNIS) is also evaluated in depth. The intensity of the signals from cancerous cells was compared to the same patient's reference cells-namely, healthy germ cells (defined as "medium" length) and interstitial/somatic cells (defined as "short" telomere length). We observed short telomeres in most GCNIS and pure seminomas (P=.006 and P=.0005, respectively). In contrast, nonseminomas displayed longer telomeres. Lesion-specific telomere lengths were documented in mixed tumor cases. Embryonal carcinoma (EC) demonstrated the longest telomeres. A fraction of EC displays the telomerase-independent alternative lengthening of telomeres (ALT) phenotype (24% of cases). Loss of ATRX or DAXX nuclear expression was strongly associated with ALT; however, nuclear expression of both proteins was retained in half of ALT-positive ECs. The particular distribution of telomere lengths among TGCT and GCNIS precursors implicate telomeres anomalies in pathogenesis. These results may advise management decisions as well.

Exome Sequencing of Uterine Leiomyosarcomas Identifies Frequent Mutations in TP53, ATRX, and MED12.

Uterine leiomyosarcomas (ULMSs) are aggressive smooth muscle tumors associated with poor clinical outcome. Despite previous cytogenetic and molecular studies, their molecular background has remained elusive. To examine somatic variation in ULMS, we performed exome sequencing on 19 tumors. Altogether, 43 genes were mutated in at least two ULMSs. Most frequently mutated genes included tumor protein P53 (TP53; 6/19; 33%), alpha thalassemia/mental retardation syndrome X-linked (ATRX; 5/19; 26%), and mediator complex subunit 12 (MED12; 4/19; 21%). Unlike ATRX mutations, both TP53 and MED12 alterations have repeatedly been associated with ULMSs. All the observed ATRX alterations were either nonsense or frameshift mutations. ATRX protein levels were reliably analyzed by immunohistochemistry in altogether 44 ULMSs, and the majority of tumors (23/44; 52%) showed clearly reduced expression. Loss of ATRX expression has been associated with alternative lengthening of telomeres (ALT), and thus the telomere length was analyzed with telomere-specific fluorescence in situ hybridization. The ALT phenotype was confirmed in all ULMSs showing diminished ATRX expression. Exome data also revealed one nonsense mutation in death-domain associated protein (DAXX), another gene previously associated with ALT, and the tumor showed ALT positivity. In conclusion, exome sequencing revealed that TP53, ATRX, and MED12 are frequently mutated in ULMSs. ALT phenotype was commonly seen in tumors, indicating that ATR inhibitors, which were recently suggested as possible new drugs for ATRX-deficient tumors, could provide a potential novel therapeutic option for ULMS.