Core D: Molecular Diagnostics

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The Core, Aims I/I I, III are involved in the accrual of patients with paraffin-embedded (PE) SLNs from the individual multicenter sites. To date, we have received 1,143 patients who have entered into the trial that were qualified for RT-PCR randomization of which 29 were ineligible. Of these 274 were PCR+ (26%) and 794 were PCR- (74%). Forty patients were ineligible and 21 are still pending results. The PE tissue sections prepared as documented in the SOPs were sent from different sites in the USA or around the world to the core and been processed for PCR analysis. The studies have been running accordingly as planned. All centers are receiving results in a timely manner. Overall, there have been no major problems in the study workflow. Discussions have been carried out with each center to improve processing and shipping of tissue specimens. For centers which have not complied with our guidelines nor have poor quality specimens, were dropped from the PCR randomized study. For Aim IV, we are collecting PE and/or IHC positive SLN blocks from JWCI and from the MLST-I trial centers. The blocks have been sectioned for immunohistochemistry (IHC) and RNA isolation. The sections are being used to assess new melanoma IHC and RT-PCR biomarkers in Project II. For Aim V, we have been isolating and quantifying RNA from SLN, non-SLN and metastatic melanoma tissues. The RNA is isolated and cryopreserved for Project II. Over 200 tissue specimens have been processed. These specimens have served as a valuable resource for development and validation of new mRNA markers. In addition, we have received over 250 primary and metastatic tumor specimens from the Sydney Melanoma Unit for RNA analysis. The blocks have been sent to us, cut into sections, processed and sent back to them. These specimens have been important for studies we do not have particular types of melanomas or metastasis to specific sites. Aim VI, we have continued to improve the SOPs and streamline operations. We have expanded the ItemTracker program for all our specimen banking. Microsoft SQL Server serves as the database management engine, which enables ItemTracker to input and store millions of records. The system's intuitive visually-driven user interface incorporates functionality for barcodes, label making, data filtering, data fields customization, visual representation of storage layouts, and much more. All specimens are barcoded prior to freezing. We have a virtual specimen tracker in each freezer to monitor storage. In Aim VII, we have accrued 121 primary melanomas. Centers have provided tissue blocks whereby the Core cuts the tissues for IHC, and DNA/RNA analysis. The blocks are returned back to the center within a month. Specific subtypes of melanoma are being collected. These specimens are being used in Project II. DMA analysis and IHC have been performed on the samples and have shown good quality results. Studies such as the CCR9 receptor mRNA analysis was performed on these specimens. This was recently published in Clin Cancer Res and received a special editorial review. The primary tumor tissue was also used for assessment of methylated tumor-related DMA markers. The core performs laser microdissection using the Veritas Arcturus system equipped with both UV and laser light. For Aim VII, serum has been collected from the patients entered into the MSLT-II trial. The serum has been processed for molecular/genetic analysis. Bloods are being collected for molecular studies from selected MLST sites. The blood is received and processed under a specific molecular SOP and then cryostored. We have 1,836 bleeds from the multicenter sites. The bloods' serum is processed and will be used for DMA studies in Project II. Aim VIII will not be performed any longer as the need for this is not a major priority any longer due to funds available.