AIM1 and LINE-1 epigenetic aberrations in tumor and serum relate to melanoma progression and disease outcome.

Aberrations in the methylation status of noncoding genomic repeat DNA sequences and specific gene promoter region are important epigenetic events in melanoma progression. Promoter methylation status in long interspersed nucleotide element-1 (LINE-1) and absent in melanoma-1 (AIM1; 6q21) associated with melanoma progression and disease outcome was assessed. LINE-1 and AIM1 methylation status was assessed in paraffin-embedded archival tissue (PEAT; n = 133) and in melanoma patients' serum (n = 56). LINE-1 U-Index (hypomethylation) and AIM1 were analyzed in microdissected melanoma PEAT sections. The LINE-1 U-Index of melanoma (n = 100) was significantly higher than that of normal skin (n = 14) and nevi (n = 12; P = 0.0004). LINE-1 U-Index level was elevated with increasing American Joint Committee on Cancer (AJCC) stage (P<0.0001). AIM1 promoter hypermethylation was found in higher frequency (P = 0.005) in metastatic melanoma (65%) than in primary melanomas (38%). When analyzed, high LINE-1 U-Index and/or AIM1 methylation in melanomas were associated with disease-free survival (DFS) and overall survival (OS) in stage I/II patients (P = 0.017 and 0.027, respectively). In multivariate analysis, melanoma AIM1 methylation status was a significant prognostic factor of OS (P = 0.032). Furthermore, serum unmethylated LINE-1 was at higher levels in both stage III (n = 20) and stage IV (n = 36) patients compared with healthy donors (n = 14; P = 0.022). Circulating methylated AIM1 was detected in patients' serum and was predictive of OS in stage IV patients (P = 0.009). LINE-1 hypomethylation and AIM1 hypermethylation have prognostic utility in both melanoma patients' tumors and serum.

Peptide nucleic acid clamp PCR: a novel K-ras mutation detection assay for colorectal cancer micrometastases in lymph nodes.

Inaccurate staging of colorectal cancer (CRC) has been attributed to the failure to detect lymph node metastases by conventional pathology. We have previously reported the use of lymphatic mapping to accurately identify those lymph nodes most likely to harbor micrometastatic disease and permit focused pathologic examination. Mutation of K-ras allele at codons 12 or 13 occurs frequently in early stages of CRC development. The purpose of our study was to assess sentinel lymph nodes (SLN) for occult CRC micrometastases using a unique peptide nucleic acid (PNA) clamp PCR assay specific for K-ras mutations. Seventy-two paraffin-embedded primary CRC and paired SLN were evaluated by PNA clamp PCR for K-ras mutations. Thirty primary tumors (42%) were positive for K-ras mutations, and in 5 of these cases the SLN were positive for metastases by Hematoxylin and Eosin staining. PNA clamp PCR identified occult metastases in an additional 6 patients, upstaging 24% of K-ras positive primary CRCs (p = 0.014). No K-ras mutations were detected among the 20 noncancer lymph nodes assessed. This study demonstrates the utility, specificity and sensitivity of PNA clamp PCR assay in identifying occult micrometastases in the SLN of CRC patients by single-base mutation analysis.

Prediction of disease outcome in melanoma patients by molecular analysis of paraffin-embedded sentinel lymph nodes.

PURPOSE: A significant number of patients who develop recurrence after a histopathologically negative sentinel lymph node (SLN) biopsy will demonstrate occult metastases on re-evaluation of the SLNs with serial sectioning and immunohistochemistry. Reverse transcriptase polymerase chain reaction (RT-PCR) has been evaluated to improve disease staging and avoid false-negative findings in fresh or frozen-section SLNs. The purpose of this study was to develop a multimarker RT-PCR assay for assessing melanoma patients' archived paraffin-embedded (PE) SLNs. PATIENTS AND METHODS: Archived PE histopathologically positive (n = 37) and negative (n = 40) SLNs from patients with primary melanoma were analyzed using a semiquantitative multimarker RT-PCR assay. RESULTS: Marker expression in histopathologically positive and negative SLNs were as follows: 89%, 92%, 35%, and 43% (positive) and 40%, 33%, 5%, and 13% (negative) for tyrosinase, melanoma antigen recognized by T cells-1, tyrosinase-related protein-1, and tyrosinase-related protein-2, respectively. Twenty-five percent of histopathologically negative SLN patients were upstaged using at least two markers. Of these, 80% developed a recurrence. Furthermore, at a median follow-up of 55 months, patients with histopathologically negative SLNs who expressed zero or one marker had a significantly improved disease-free (P <.002) and overall (P <.03) survival versus those expressing two or more markers. CONCLUSION: These findings demonstrate the feasibility of a multimarker RT-PCR assay for evaluating archived PE SLNs. More significantly, identification of molecular risk factors can be detected in histopathologically negative SLNs for distinguishing early-stage melanoma patients with a worse prognosis.

Molecular lymphatic mapping of the sentinel lymph node.

Intraoperative lymphatic mapping to identify the sentinel lymph node (SLN) has significantly changed the management of regional lymph node basin of patients with various types of solid tumors such as melanoma and breast cancer. The procedure has improved the diagnosis of micrometastasis in the regional tumor-draining lymph nodes by providing a focused histopathological assessment of select lymph nodes most likely to harbor occult disease. Blue dye and/or radioisotopes are efficient mapping agents but the lack of accurate methods to quantify their presence and the potential for dissolution and decay, respectively, throughout time limit their role as reliable markers for identifying a sentinel node from additional secondary lymph nodes that may be either blue and/or radioactive to some degree. A consistently durable marker is needed that can be introduced during surgery and successfully quantitated among tumor-draining lymph nodes to permit a more accurate assessment of hierarchical organization. This may be of particular importance in retrospective analysis of archival tissues as there are no inherent markers to denote the SLN from successive echelon nodes. A procedure of molecular lymphatic mapping (MLM) was developed in a rat model to label the SLN preoperatively with rice gene DNA containing plasmid or linear rice DNA fragment (rDNA). The MLM efficiency was demonstrated by polymerase chain reaction (PCR) detection of the molecular marker in both frozen and paraffin-embedded SLN; 1.25 micro g of rDNA injected with blue dye could be reproducibly detected by PCR. The MLM procedure was validated in a rat breast tumor model with lymph node metastasis. The procedure was successful in permanently labeling and identifying by PCR both frozen and paraffin-embedded SLN. MLM in conjunction with a conventional mapping agent can be used as a valuable asset for molecular assessment of the SLN and retrospective analysis of paraffin-embedded specimens.

Overexpression of beta 1,4N-acetylgalactosaminyl- transferase mRNA as a molecular marker for various types of cancers.

OBJECTIVE: To determine GalNAcT mRNA expression in human carcinoma cell lines and primary tumor tissues. Assessment of the potential use of GalNAcT mRNA as a molecular marker for detection of metastatic cancer cells in the peripheral blood of patients with hepatocellular carcinoma. METHODS/RESULTS: We investigated GalNAcT mRNA expression in various human cancer cell lines and primary cancer tissues using RT-PCR assay for GalNAcT mRNA. The expression of GalNAcT mRNA was detected in 25 of 26 cancer cell lines tested and in the majority of primary tumors from different organs: 8 of 10 colon cancers, 9 of 9 breast cancers, 11 of 12 esophageal cancers, 14 of 14 gastric cancers, 4 of 18 pancreatic cancers, 6 of 12 biliary tract cancers, 17 of 18 hepatocellular carcinomas and 13 of 14 lung cancers. Semi-quantitative analysis with duplex RT-PCR showed that the amount of the GalNAcT mRNA was enhanced in cancer tissues as compared to the surrounding cancer-free tissues. Blood specimens of 5 of 14 patients with hepatocellular carcinoma were positive for GalNAcT mRNA, all of whom developed recurrent disease in less than 24 months. Peripheral blood samples of 30 normal subjects were negative for GalNAcT mRNA. CONCLUSION: Our results suggest that the RT-PCR assay for GalNAcT mRNA could be a potentially useful molecular marker for detecting cancer dissemination in blood circulation of patients with malignancy.