Project 2: Molecular Approaches for Assessing Metastasis and Disease Relapse

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The overall studies have been to develop and monitor the molecular prognosis and upstaging of tumordraining lymph nodes and primary melanoma tumors. In addition, the other major program is development of DNA prognostic biomarkers for primary melanomas and serum. Aims I and II: It is clear that a successful sentinel node biopsy (SNB) requires a collaborative effort between the nuclear medicine physician, the surgeon and the pathologist, and a failure in any of these areas may result in an unsatisfactory outcome. The aim of the study was to investigate a cohort of patients with false negative (FN) sentinel node (SNs) to identify deficiencies that may have caused the FN result. A FN SN was defined as a patient who had a negative SNB result but subsequently developed their first recurrence within the biopsied nodal field. In a major collaborative study with the Sydney Melanoma Unit (SMU, Australia); we investigated a cohort of melanoma patients with FN SN biopsies to identify possible reasons for the FN result. Seventy-four SNs from 33 patients found to have had a FN SNB were analyzed by reviewing the lymphoseintigraphy, surgical data and histopathology, and assessing nodal tissue using multimarker real-time quantitative reverse transcriptase polymerase chain reaction (qRT), and antimony concentration measurements (as a marker of "true" SN status) using inductively coupled plasma mass spectroscopy. The qRT studies were performed on archival paraffin-embedded (PE) tissues from SMU. A multimarker real-time qRT assay with 5 mRNA markers (MAGE 3, MART-1, GalNAc-T, PAX-3 and TRP-2) was used as is for the current MLST-II study. Specimens were blinded to the qRT assay performer and clinician. Nine SNs (12%) from 9 patients (27%) were found to have evidence of melanoma on histopathologic review. 12 SNs (16%) from 10 patients (30%) were found to be qRT(+). Four of these 12 SNs were positive on histopathology; 8 were negative. Four patients (12%) were upstaged by qRT. Sixteen patients had their SNB histology, lymphoseintigraphy and surgical data reviewed. Identifiable causes of the FN SNBs were not found after review of all modalities in 4 patients . SNs from all 4 patients had antimony levels indicative of a SN. Of the SNs evaluable by qRT, 1 was qRT(+) and 7 SNs from 2 patients were qRT(-).10 SNs (14%) from 9 of the 33 patients (27%) were found to have evidence of metastatic melanoma on histopathologic review and/or IHC analysis. Of the 8 evaluable by qRT, 5 of these SNs were qRT(+) and 3 were qRT(-). 2 of the qRT(-) SNs with histopathologic/IHC evidence of melanoma had tiny deposits (0.1mm and 0.15mm in maximum dimension). A FN SN can occur because of deficiencies in nuclear medicine, surgery or histopathology. qRT can detect "occult" metastatic melanoma in SNs that were identified as negative by histopathology. These studies confer that qRT upstaging on PE tissue sections can be of clinical utility (Ann Surg, in press, see ref. 10). Currently, we are tracking MLST-I patients from the multicenter site in which patients had SLN(-) that recurred as well as those that had SLN(+) with complete lymph node dissection (CLND) that had negative NSLN. The objective will be to determine if our qRT multimarkers can upstage these different patient cohorts. Tracking of these patients specimens have been started with JWCI and SMU. This year, we will be retrieving these lymph node blocks to perform qRT. Aims III: This Aim is focused on developing genomic prognostic biomarkers in primary tumors and blood. We have focused on developing epigenetic biomarkers examining methylation of gene promoter regions of CpG islands. Several types of assays have been developed such as real time PCR used on a real-time thermocycler, absolute quantitative allele methylation assay (AQAMA) also used on a real-time thermocycler, and Sequnom (MALDI-TOF) mass spectrometry. The approach with the latter two assays is absolute quantitative analysis of genomic DMA from PE tissues and blood. We have been able to develop these assays. In the PE analysis we have assessed 7 MINT markers which are methylated tumor-related loci (non-coding) in PE primary and metastatic tissues. We have demonstrated the prognostic utility of MINT 31 and 17. Along with these markers we have assessed multiple tumor-related genes such as GATA4, GATA binding protein 4; RARbeta2, retinoic acid receptor-beta 2, RASSF1A, Ras association domain family 1A; SOCS-1, suppressor of cytokine signaling-1; TFPI-2, tissue factor pathway inhibitor-2; and WIF-1, Wnt inhibitory factor-1. The CpG island methylator phenotype (CIMP) may be associated with development of malignancy through coordinated inactivation of tumor-suppressor and tumor-related genes (TRGs) and methylation of multiple non-coding, methylated-in-tumor (MINT) loci. These epigenetic changes create a distinct CIMP pattern that has been linked to recurrence and survival in gastrointestinal cancers. Because epigenetic inactivation of TRGs also has been implicated in development and progression of malignant melanoma, we hypothesized the existence of a clinically significant CIMP in cutaneous melanoma. We examined the methylation status of the CpG island promoter region of TRGs related to melanoma pathophysiology and a panel of MINT loci (MINT-1, 2, 3, 12, 17, 25, and 31) in primary and metastatic tumors of different clinical stages. We showed an increase in the extent of methylation of the TRGs WIF-1, TFPI-2, RASSF1A, and SOCS-1 with advancing clinical tumor stage. Furthermore, we find a significant positive association between the methylation status of MINT-17, MINT-31, and TRGs. These findings demonstrate the significance of a CIMP pattern that is associated with advancing clinical stage of malignant melanoma, and may be used to identify primary melanomas that have a high risk of metastasis or recurrence. The study has been submitted for publication. This is the first major finding on epigenetic changes in primary melanomas related to tumor progression. The studies will be further expanded to examine the clinical utility of the CIMP as a prognostic genotype of primary tumors. Studies on circulating DNA are being performed using methylated TRGs and non-coding genomic loci. Methods to improved DNA isolation serum and bisulfite are being revised. We are focusing developing assays to assess non-coding region DNA in the serum.