Molecular restoration of archived transcriptional profiles by complementary-template reverse-transcription (CT-RT)

Gene expression profiling of formalin-fixed and paraffin-embedded (FFPE) specimens, banked from completed clinical trials and routine clinical care, has the potential to yield valuable information implicating and linking genes with clinical parameters. In order to prepare high-quality cDNA from highly fragmented FFPE-RNA, previously precluded from high-throughput analyses, we have designed a novel strategy based on the nucleic acid restoration of incomplete cDNA sequences prior to T7 in vitro transcription (IVT) amplification. We describe this strategy as complementary-template reverse-transcription (CT-RT) because short single-stranded T7-oligo-dT24-VN-DNA sequences, obtained from FFPE-RNA, are used as primers for the RT of complementary RNA templates contained in a sense-RNA library. We validated our assay by determining the correlation between expression profiles of a matched 10-year-old frozen and FFPE breast cancer sample. We show that T7 IVT-amplification of cDNA transcripts restored by CT-RT is a specific and reliable process that allows recovery of transcriptional features undetectable by direct T7 IVT-amplification of FFPE-RNA. Furthermore, CT-RT restored 35–41% of the transcripts from archived breast and cervical specimens when compared to matched frozen tissue; and profiles included tissue-specific transcripts. Our results indicate that CT-RT allows microarray profiling of severely degraded RNA that could not be analyzed by previous methods

Sialoblastoma- long-term follow-up and remission for a rare salivary malignancy

Sialoblastoma is a rare salivary neoplasm which presents either congenitally or during early infancy. It was originally considered a benign neoplasm, however a number of reported cases have documented locoregional recurrence and distant metastases. Currently, there is no consensus on the appropriate treatment for this neoplasm. We report on long term follow-up of a patient with metastatic sialoblastoma, and a brief discussion of the possible treatment modalities currently being considered

In Vivo Fluorescence Immunohistochemistry:Localization of Fluorescently Labeled Cetuximab in Squamous Cell Carcinomas

Anti-EGFR (epidermal growth factor receptor) antibody based treatment strategies have been successfully implemented in head and neck squamous cell carcinoma (HNSCC). Unfortunately, predicting an accurate and reliable therapeutic response remains a challenge on a per-patient basis. Although significant efforts have been invested in understanding EGFR-mediated changes in cell signaling related to treatment efficacy, the delivery and histological localization in (peri-) tumoral compartments of antibody-based therapeutics in human tumors is poorly understood nor ever made visible. In this first in-human study of a systemically administered near-infrared (NIR) fluorescently labeled therapeutic antibody, cetuximab-IRDye800CW (2.5 mg/m(2), 25 mg/m(2), and 62.5 mg/m(2)), we show that by optical molecular imaging (i.e. denominated as In vivo Fluorescence Immunohistochemistry) we were able to evaluate localization of fluorescently labeled cetuximab. Clearly, optical molecular imaging with fluorescently labeled antibodies correlating morphological (peri-) tumoral characteristics to levels of antibody delivery, may improve treatment paradigms based on understanding true tumoral antibody delivery

Photoimmunotherapy of residual disease after incomplete surgical resection in head and neck cancer models

Antibody-based photodynamic therapy, or photoimmunotherapy (PIT), is a novel, targeted cancer therapy, which can serve as both a diagnostic and a therapeutic agent. The primary objective of this study was to evaluate the capacity of panitumumab-IRDye700DX (Pan-IR700) to eliminate microscopic tumor remnants in the postsurgical setting, which was accomplished using novel in vitro and in vivo models of residual disease after incomplete resection. - Additionally, PIT was evaluated in fresh human-derived cancer tissue. To - determine a threshold for cellular regrowth after PIT, an in vitro assay was performed using a range of cells representing microscopic disease quantities. Long-term growth inhibition was induced after treatment of 5 x 10(3) and 1 x 10(4) cells at 6 J. A novel in vivo mouse model of subtotal tumor resection was used to assess the effectiveness of Pan-IR700 mediated PIT to eliminate residual disease and inhibit recurrence in the post-surgical wound bed. Mice receiving surgical treatment plus adjuvant PIT showed a threefold and fourfold reduction in tumor regrowth at 30 days post PIT in the 50% and 90% subtotal resection groups, respectively (as measured by bioluminescence imaging), demonstrating a significant (P <0.001) reduction in tumor regrowth. To determine the translatability of epidermal growth factor receptor (EGFR)-targeted PIT, SCCHN human tissues (n = 12) were treated with Pan-IR700. A significant reduction (P <0.001) in ATP levels was observed after treatment with Pan-IR700 and 100 J cm(-2) (48% +/- 5%) and 150 J cm(-2) (49% +/- 7%) when compared to baseline. Targeting EGFR with Pan-IR700 has robust potential to provide a tumor-specific mechanism for eliminating residual disease in the surgical setting, thereby increasing therapeutic efficacy, prolonging progression-free survival, and decreasing morbidity