Stimulation of the mouse hindlimb via the sciatic nerve was performed for a 4-h period to investigate acute muscle gene activation in a model of muscle phenotype conversion. Initial force production (1.6 +/- 0.1 g/g body wt) declined 45% within 10 min and was maintained for the remainder of the experiment. Force returned to initial levels upon study completion. An immediate-early growth response was present in the extensor digitorum longus (EDL) muscle (FOS, JUN, activating transcription factor 3, and musculoaponeurotic fibrosarcoma oncogene) with a similar but attenuated pattern in the soleus muscle. Transcript profiles showed decreased fast fiber-specific mRNA (myosin heavy chains 2A and 2B, fast troponins T(3) and I, alpha-tropomyosin, muscle creatine kinase, and parvalbumin) and increased slow transcripts (myosin heavy chain-1beta/slow, troponin C slow, and tropomyosin 3y) in the EDL versus soleus muscles. Histological analysis of the EDL revealed glycogen depletion without inflammatory cell infiltration in stimulated versus control muscles, whereas ultrastructural analysis showed no evidence of myofiber damage after stimulation. Multiple fiber type-specific transcription factors (tea domain family member 1, nuclear factor of activated T cells 1, peroxisome proliferator-activated receptor-gamma coactivator-1alpha and -beta, circadian locomotor output cycles kaput, and hypoxia-inducible factor-1alpha) increased in the EDL along with transcription factors characteristic of embryogenesis (Kruppel-like factor 4; SRY box containing 17; transcription factor 15; PBX/knotted 1 homeobox 1; and embryonic lethal, abnormal vision). No established in vivo satellite cell markers or genes activated in our parallel experiments of satellite cell proliferation in vitro (cyclins A(2), B(2), C, and E(1) and MyoD) were differentially increased in the stimulated muscles. These results indicated that the molecular onset of fast to slow phenotype conversion occurred in the EDL within 4 h of stimulation without injury or satellite cell recruitment. This conversion was associated with the expression of phenotype-specific transcription factors from resident fiber myonuclei, including the activation of nascent developmental transcriptional programs.

21161 Background: This multi-clinical laboratory validation assessed the accuracy, reproducibility and robustness of the Pathwork™ Tissue of Origin Test. We focused on tumor specimens that are poorly differentiated to undifferentiated (G3-G4; high grade) because this type of specimen can consume a disproportionate amount of physician time and diagnostic resources. A subset of this type of specimen ultimately will be categorized as cancer of unknown primary (CUP), which comprises 4–5% of all malignancies. The Test uses proprietary analytics and a companion genomic microarray (Pathchip™) to compare expression levels of poorly differentiated to undifferentiated specimens with 15 types of cancer. Test development involved over 2000 specimens from 14 labs, representing 15 cancers and sixty morphologies. Methods: The studies addressed operability limits, specimen stability, potential interferences and reproducibility, as well as validation in molecular diagnostics and commercial laboratories. In all, over 700 frozen tumor specimens and microarrays were analyzed. Results: Clinical validation data demonstrated that the percent agreement of the Tissue of Origin Test was 88% across all tissue types. Between-lab concordance was 95%. If the quantitative result (Similarity Score) exceeded the recommended threshold, the probability that the indicated tissue is present was 95% across all tissue types. If the Similarity Score was less than 5, the probability that the indicated tissue is absent was 98% across all tissues types. Conclusions: The Pathwork™ Tissue of Origin Test, which is under development, is accurate and highly reproducible across laboratories in this study. The test gave consistent results for different tissue handling and extraction methods and has the potential to be an effective aid in the diagnosis of cancer patients presenting with poorly differentiated and undifferentiated tumors. No significant financial relationships to disclose.