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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 ATRX KO cells. Telomerase activity was subsequently crippled in these LAPC-4 ATRX KO cells by introducing mutations in the TERC locus, the essential RNA component of telomerase. These LAPC-4 ATRX KO TERC mut 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.

Purpose: To assess the impact of OCT signal strength (SS) and artifact on retinal nerve fiber layer (RNFL) measurement reliability and to understand whether glaucoma severity modifies this relationship. Design: Retrospective, longitudinal cohort study. Participants: Two thousand nine hundred ninety-two OCT scans from 474 eyes of 241 patients with glaucoma or glaucoma suspect status. Methods: We extracted mean RNFL thickness and SS and manually graded scans for artifact. To analyze the effect of SS and artifact on OCT reliability, we (1) created a multilevel linear model using measured RNFL thickness values and demographic and clinical data to estimate the true (predicted) RNFL thickness, (2) calculated model residuals (DRNFL) as our reliability measure, and (3) created a second multilevel linear model with splines and interaction terms that modeled overall and quadrant specific reliability (DRNFL) as the outcome, using SS and artifact as predictors. Main Outcome Measures: Impact of SS and artifact on DRNFL. Results: For SS between 10 and 3, the impact of decreases in SS on OCT reliability is modest (e0.67 to e1.25 DRNFL per 1-point decrease in SS; P < 0.05). But at less than 3, changes in SS have a large impact on reliability (e15.70 to e16.34 DRNFL per 1-point decrease in SS; P < 0.05). At SS between 10 and 3, decreases in SS tend to have a larger impact on reliability in eyes with severe glaucoma (e1.25 per 1-point decrease in SS; P < 0.05) compared with eyes with mild or moderate glaucoma (e0.67 to e0.75 per 1-point decrease in SS; P < 0.05). The presence of artifact has a significant impact on OCT reliability independent of the effects of SS (e4.76 DRNFL; P < 0.05). Artifact affects reliability solely in the quadrant in which it occurs, with artifact in one quadrant showing no impact on DRNFL in the opposite quadrant (P > 0.05). Conclusions: Signal strength decreases down to 3 have relatively mild impacts on OCT reliability. At less than 3, the impact of further decreases in SS on reliability are substantial. The effect of SS on reliability is greater in severe glaucoma. Artifacts result in a decrease in reliability independent of the effect of SS. We propose evidence-based guidelines to guide physicians on whether to trust the results of an OCT scan.

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