Abstract Telomeres, the terminal-most structures of chromosomes, are critical to maintain the integrity of chromosomal DNA. As tumor cells bypass normal cell cycle controls and expand their proliferative capacity, accelerated telomere erosion necessitates the activation of a telomere maintenance mechanism. This mechanism can be telomerase-dependent or -independent. Telomerase-independent maintenance is termed alternative lengthening of telomeres, or ALT, and may represent an amalgamation of several recombination-associated pathways. ALT likely uses other telomeres or extrachromosomal telomeric DNA as a template for recombination-based addition of telomere repeats to a shortened chromosome. While most human tumors use telomerase to maintain telomere length, a subset uses ALT. Osteosarcomas are more commonly in this category, as almost 60% demonstrate ALT characteristics. Previous studies to analyze mechanisms of telomere maintenance have shown that while some tumors are ALT or telomerase-dependent, a small number demonstrates mixed Results: both a heterogeneous telomere length consistent with ALT and telomerase activity, incompatible with ALT, or vice versa (reviewed in Henson and Reddel, 2010). These results suggest the idea of a mixed population of cells within some tumors that uses both telomerase-dependent and -independent telomere maintenance mechanisms; however, this has not yet been confirmed. Our studies have used a human osteosarcoma tissue microarray to demonstrate both ALT and telomerase-dependent cells within the same tumor. Tumor sections were stained for PML, TRF2 and telomerase, followed by analysis with the Nuance Multispectral Imaging System. Some tumors (4/6) displayed robust staining and colocalization of PML and TRF2, indicative of ALT-associated PML bodies (APBs), a marker of ALT cells. Some of these tumors (3/4) also contained cells staining strongly for telomerase that were exclusive of ALT-specific colocalization. These two types of cells within this subset of tumors were distinct from one another. To confirm these initial findings, five frozen human osteosarcoma samples were evaluated to correlate in situ staining results with classical methods of gross telomere classification, characterization of telomere length via telomere restriction fragment (TRF) Southern blot and identification of telomerase activity via the telomere repeat amplification protocol (TRAP). Two tumors displayed ALT characteristics only, exhibiting heterogeneous telomere lengths with TRF Southern blots and negative findings with TRAP; one tumor displayed positive TRAP results and more homogeneous telomere lengths associated with telomerase activity. Of the remaining two tumors, one was characterized by telomerase-associated homogenous telomere length and one was characterized by telomerase-independent heterogeneous telomere length by TRF Southern blots; both tumor lysates displayed variable and weak telomerase activity with TRAP. These data suggest that cells within these tumors use more than one mechanism of telomere maintenance. Tumor sections from these five osteosarcomas are now being analyzed using our in situ immunohistochemistry methods. Given recent interest in telomerase-targeted cancer therapies, the coexistence of both telomere maintenance mechanisms within a single tumor would have significant implications for understanding the mechanisms of telomere maintenance and for developing precise therapies to target ALT mechanisms of telomere elongation. Henson, J. D. and Reddel, R. R. Assaying and investigating Alternative Lengthening of Telomeres activity in human cells and cancers. FEBS Lett., 584: 3800–3811, 2010. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B57.