The overall studies have been to develop and monitor the molecular prognosis and upstaging of tumordraininglymph nodes and primary melanoma tumors. In addition, the other major program is development ofDNA prognostic biomarkers for primary melanomas and serum.
Aims I and II: It is clear that a successful sentinel node biopsy (SNB) requires a collaborative effortbetween the nuclear medicine physician, the surgeon and the pathologist, and a failure in any of these areasmay result in an unsatisfactory outcome.
The aim of the study was to investigate a cohort of patients with falsenegative (FN) sentinel node (SNs) to identify deficiencies that may have caused the FN result. A FN SN wasdefined as a patient who had a negative SNB result but subsequently developed their first recurrence withinthe biopsied nodal field. In a major collaborative study with the Sydney Melanoma Unit (SMU, Australia); weinvestigated 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 thelymphoseintigraphy, surgical data and histopathology, and assessing nodal tissue using multimarker real-timequantitative reverse transcriptase polymerase chain reaction (qRT), and antimony concentrationmeasurements (as a marker of 'true' SN status) using inductively coupled plasma mass spectroscopy. TheqRT studies were performed on archival paraffin-embedded (PE) tissues from SMU. A multimarker real-timeqRT assay with 5 mRNA markers (MAGE 3, MART-1, GalNAc-T, PAX-3 and TRP-2) was used as is for thecurrent 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; 8were 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 afterreview of all modalities in 4 patients . SNs from all 4 patients had antimony levels indicative of a SN. Of theSNs evaluable by qRT, 1 was qRT(+) and 7 SNs from 2 patients were qRT(-).10 SNs (14%) from 9 of the 33patients (27%) were found to have evidence of metastatic melanoma on histopathologic review and/or IHCanalysis. Of the 8 evaluable by qRT, 5 of these SNs were qRT(+) and 3 were qRT(-). 2 of the qRT(-) SNs withhistopathologic/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 candetect 'occult' metastatic melanoma in SNs that were identified as negative by histopathology. These studiesconfer 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(-) thatrecurred as well as those that had SLN(+) with complete lymph node dissection (CLND) that had negativeNSLN. 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 retrievingthese lymph node blocks to perform qRT.
Aims III :
This Aim i s focused on developing genomic prognostic biomarkers in primary tumors and blood.We have focused on developing epigenetic biomarkers examining methylation of gene promoter regions ofCpG islands. Several types of assays have been developed such as real time PCR used on a real-timethermocycler, 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 absolutequantitative analysis of genomic DMA from PE tissues and blood. We have been able to develop theseassays. 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 31and 17. Along with these markers we have assessed multiple tumor-related genes such as GATA4, GATAbinding 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, Wntinhibitory factor-1.The CpG island methylator phenotype (CIMP) may be associated with development of malignancythrough coordinated inactivation of tumor-suppressor and tumor-related genes (TRGs) and methylation ofmultiple non-coding, methylated-in-tumor (MINT) loci. These epigenetic changes create a distinct CIMPpattern that has been linked to recurrence and survival in gastrointestinal cancers. Because epigeneticinactivation of TRGs also has been implicated in development and progression of malignant melanoma, wehypothesized the existence of a clinically significant CIMP in cutaneous melanoma. We examined themethylation status of the CpG island promoter region of TRGs related to melanoma pathophysiology and apanel of MINT loci (MINT-1, 2, 3, 12, 17, 25, and 31) in primary and metastatic tumors of different clinicalstages. We showed an increase in the extent of methylation of the TRGs WIF-1, TFPI-2, RASSF1A, andSOCS-1 with advancing clinical tumor stage. Furthermore, we find a significant positive association betweenthe methylation status of MINT-17, MINT-31, and TRGs. These findings demonstrate the significance of aCIMP pattern that is associated with advancing clinical stage of malignant melanoma, and may be used toidentify primary melanomas that have a high risk of metastasis or recurrence. The study has been submittedfor publication. This is the first major finding on epigenetic changes in primary melanomas related to tumorprogression. The studies will be further expanded to examine the clinical utility of the CIMP as a prognosticgenotype 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 assaysto assess non-coding region DNA in the serum.
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