High frequency of BRAFV600E mutation in acquired nevi and small congenital nevi, but low frequency of mutation in medium-sized congenital nevi.

To investigate whether the frequency of the BRAF(V600E) (V-raf murine sarcoma virus oncogene homolog B1) mutation in melanocytic nevi is associated with sun exposure patterns, we examined 120 acquired melanocytic nevi excised from various anatomic sites, including glabrous skin, as well as 62 congenital nevi. We used a new mutation detection system based on the shifted termination assay, called Mutector, which was able to detect only 5% of heterozygous mutant cells within the samples. We detected the mutation in 105/120 (87.5%) acquired nevi and 43/62 (69.4%) congenital nevi. Notably, we found the mutation in 35/43 (81.4%) acquired nevi excised from glabrous skin and genitalia. These results strongly suggest that UV light is not necessarily required for the acquisition of the BRAF(V600E) mutation, and suggest that non-mutagenic effects of UV light to melanocytes may be more important in the nevogenesis. Additionally, we showed heterogeneous distribution of BRAF-mutated cells within the lesions of small congenital nevi by a combination of laser microdissection and direct sequencing. Finally, we found low frequency of BRAF(V600E) mutation (6/20, 30.0%) in medium-sized congenital nevi. Most of these nevi with wild-type BRAF had neroblastoma ras viral oncogene homolog mutations (9/14, 64.3%), suggesting different pathogenesis of medium-sized congenital nevi from acquired nevi and small congenital nevi.

X-Linked inhibitor of apoptosis protein expression level in colorectal cancer is regulated by hepatocyte growth factor/C-met pathway via Akt signaling.

PURPOSE: The inhibitor of the apoptosis protein (IAP) family members, such as the X-linked IAP (XIAP), survivin, and livin, are essential for cell survival and antiapoptosis in colorectal cancer cells. We hypothesized that the hepatocyte growth factor (HGF) activation in colorectal cancer via c-Met receptor regulates IAP proteins through Akt signaling. EXPERIMENTAL DESIGN: The level of IAPs and C-Met mRNA expression was assessed using a quantitative real-time reverse transcriptase-PCR (RT-PCR) assay on colorectal normal mucosa (n = 13), adenomas (n = 6), and colorectal cancer tumors (n = 50). The role of HGF/C-Met pathway through Akt and XIAP was investigated by small interfering RNA (siRNA) and quantitative RT-PCR analysis of colorectal cancer lines. RESULTS: Of the IAPs, only XIAP showed significant correlation to tumor development and progression. XIAP mRNA level in primary colorectal cancer was significantly higher than that in colorectal normal mucosa (P = 0.01); liver metastases was significantly higher than primary colorectal cancer tumors (P = 0.04); and primary colorectal cancer N1/N2 cases were significantly higher than N0 cases (P = 0.008). HGF stimulation of colorectal cancer lines enhanced XIAP mRNA expression but not other IAPs. Activation of XIAP expression by HGF was inhibited by siRNA targeting Akt1 and Akt2. CONCLUSIONS: Activation of C-MET enhances XIAP through the Akt pathway. XIAP up-regulation was shown to be correlated to colorectal cancer tumor progression. The Akt-XIAP pathway may be a potential molecular target for regulating colorectal cancer progression.

Constitutive activation of the mitogen-activated protein kinase signaling pathway in acral melanomas.

One of the most attractive clinical targets for melanoma is the mitogen-activated protein kinase (MAPK) signaling pathway. In this study, we examined MAPK signaling activation in a total of 28 acral melanoma samples, consisting of 13 primary tumors and 15 metastases. In line with the previous reports, NRAS/BRAF mutations were rare; only one metastatic tumor had an NRAS E61R mutation, and one primary tumor and two metastases harbored BRAF V599E mutations. Western blot analyses, however, revealed phosphorylated extracellular signal-regulated kinase (ERK)1/2 proteins in 11 of 14 (78.5%) of the acral melanoma tumors. Furthermore, fluorescence in situ hybridization analyses revealed the prominent amplification of the cyclin D1 (CCND1) gene, which is an important down-stream effecter of the MAPK pathway, in 5 of 21 (23.8%) tumors examined. Interestingly, two of three tumors that were negative for phosphorylated ERK proteins according to western blot harbored CCND1 amplifications, suggesting that the increased gene dosage of CCND1 may exert effects similar to phosphorylated ERK proteins in cell growth. We conclude that, despite the low frequency of BRAF/NRAS mutations, the MAPK signaling pathway is constitutively activated in the majority of acral melanomas. This provides a rational basis to include acral melanomas into the clinical trials with MAPK inhibitors.

Epigenetic inactivation of ID4 in colorectal carcinomas correlates with poor differentiation and unfavorable prognosis.

PURPOSE: ID4 gene is a member of the inhibitor of DNA binding (ID) family proteins that inhibit DNA binding of basic helix-loop-helix transcription factors. The epigenetic inactivation of ID4 gene on colorectal cancer (CRC) development and its clinical significance was assessed. EXPERIMENTAL DESIGN: In CRC cell lines, ID4 methylation status of the promoter region was assessed by methylation-specific PCR and bisulfite sequencing. The mRNA expression level was assessed by quantitative real-time reverse transcription-PCR. The methylation status of 9 normal epithelia, 13 adenomas, 92 primary CRCs, and 26 liver metastases was assessed by methylation-specific PCR. ID4 protein expression was assessed by immunohistochemistry analysis of tissue specimen. RESULTS: CRC cell lines were shown to be hypermethylated, and mRNA expression was suppressed and could be restored by 5-aza-cytidine treatment. In clinical specimens from normal epithelia, adenomas, primary CRCs, and liver metastases, the frequency of ID4 hypermethylation was 0 of 9 (0%), 0 of 13 (0%), 49 of 92 (53%), and 19 of 26 (73%), respectively, with a significant elevation according to CRC pathological progression. Methylation status of primary CRCs significantly correlated with histopathological tumor grade (P = 0.028). Immunohistochemistry analysis showed ID4 expression of normal colon epithelia, adenomas, and unmethylated primary CRCs but not hypermethylated CRC specimens. Among 76 American Joint Committee on Cancer stage I to IV patients who had undergone curative surgical resection, overall survival was significantly poorer in patients with hypermethylated ID4 bearing tumors (P = 0.0066). CONCLUSIONS: ID4 gene is a potential tumor suppressor gene for which methylation status may play an important role in the CRC progression.

Allelic imbalance of APAF-1 locus at 12q23 is related to progression of colorectal carcinoma.

APAF-1 gene, located at chromosome locus 12q23, is a key factor in the mitochondrial apoptotic pathway downstream of p53, and is a potential tumor suppressor gene. We hypothesized that APAF-1 gene dysfunction due to allelic imbalance (AI) contributes to the development and progression of colorectal carcinoma (CRC). AI at APAF-1 locus and microsatellite instability (MIN) in CRCs and adenomas were assessed by multiple microsatellite markers. The frequency of AI significantly increased with tumor progression; 0 of 33 (0%) adenomas, 14 of 49 (29%) primary CRCs, and 18 of 34 (53%) liver metastases had AI. A total of 12 metastases were matched with corresponding primary CRCs; in 11 of 12 (92%) pairs, the metastasis had same AI status as the corresponding primary tumor. APAF-1 mRNA transcription level was significantly decreased with AI in liver metastases (P=0.009). Promoter hypermethylation was found in three of 35 (9%) primary CRCs and one of 15 (7%) liver metastases by methylation-specific PCR but was not correlated with AI. MIN was observed in 11 of 49 (23%) primary CRCs and was a favorable prognostic factor. Our results suggest that APAF-1 gene haploinsufficiency caused by AI increases with tumor progression, and relates to hepatic metastasis.

Prognostic significance of molecular upstaging of paraffin-embedded sentinel lymph nodes in melanoma patients.

PURPOSE: Detection of micrometastases in sentinel lymph nodes (SLNs) is important for accurate staging and prognosis in melanoma patients. However, a significant number of patients with histopathology-negative SLNs subsequently develop recurrent disease. We hypothesized that a quantitative realtime reverse transcriptase polymerase chain reaction (qRT) assay using multiple specific mRNA markers could detect occult metastasis in paraffin-embedded (PE) SLNs to upstage and predict disease outcome. PATIENTS AND METHODS: qRT was performed on retrospectively collected PE SLNs from 215 clinically node-negative patients who underwent lymphatic mapping and sentinel lymphadenectomy for melanoma and were followed up for at least 8 years. PE SLNs (n = 308) from these patients were sectioned and assessed by qRT for mRNA of four melanoma-associated genes: MART-1 (antigen recognized by T cells-1), MAGE-A3 (melanoma antigen gene-A3 family), GalNAc-T (beta1-->4-N-acetylgalactosaminyl-transferase), and Pax3 (paired-box homeotic gene transcription factor 3). RESULTS: Fifty-three (25%) patients had histopathology-positive SLNs by hemotoxylin and eosin and/or immunohistochemistry. Of the 162 patients with histopathology-negative SLNs, 48 (30%) had nodes that expressed at least one of the four qRT markers, and these 48 patients also had a significantly increased risk of disease recurrence by a Cox proportional hazards model analysis (P <.0001; risk ratio, 7.48; 95% CI, 3.70 to 15.15). The presence of > or = one marker in histopathology-negative SLNs was also a significant independent prognostic factor by multivariate analysis for overall survival (P =.0002; risk ratio, 11.42; 95% CI, 3.17 to 41.1). CONCLUSION: Molecular upstaging of PE histopathology-negative SLNs by multiple-marker qRT assay is a significant independent prognostic factor for long-term disease recurrence and overall survival of patients with early-stage melanoma.

Detection of mitochondrial DNA alterations in plasma of malignant melanoma patients.

Genetic changes in mitochondrial DNA (mtDNA) have been detected in a variety of pathologic conditions including cancer. We hypothesized that malignant melanoma has genetic alterations in the displacement loop (D-loop) region and that these mtDNA alterations can be detected in blood as a circulating DNA melanoma marker. D-loop region from 20 melanoma cell lines, 12 metastatic melanoma specimens, and corresponding lymphocytes and plasma samples were sequenced using the CEQ 8000 XL Genetic Analysis System (Beckman Coulter). Nine of 20 (45%) melanoma cell lines and 5 of 12 (42%) melanoma specimens contained somatic mutations in the D-loop region of mtDNA. DNA alterations in the polycytosine tract (C-tract) of D-loop were detected in 6 of 20 (30%) cell lines and 2 of 12 (17%) specimens. Two of five paired plasma samples (40%) contained the same mutations as did melanoma specimens. In a comparison of lymphocytes and plasma of melanoma patients, 9 of 44 paired plasma samples (20%) contained at least one mutation compared to corresponding lymphocytes. Somatic mutations in the D-loop region of tumor and paired plasma did not correlate with the clinicopathological characteristics. However, circulating mtDNA alterations were more frequent in advanced disease. Studies indicate that circulating mtDNA mutations in the plasma of melanoma patients can be detected.

Allelic imbalance on 12q22-23 in serum circulating DNA of melanoma patients predicts disease outcome.

Allelic imbalance (AI) encompassing the apoptotic protease-activating factor 1 (APAF-1) locus (12q22-23) is found frequently in metastatic melanoma. Circulating DNA with AI on 12q22-23 in serum was evaluated as a surrogate marker to predict biochemotherapy (BC) treatment response in melanoma patients. Sera were collected from 49 American Joint Committee on Cancer stage IV melanoma patients treated with BC. Serum AI of the 12q22-23 region was demonstrated to be present before and/or after BC. BC responders showed a significantly lower frequency of AI (5 of 24, 21%) compared with nonresponders (11 of 20, 55%; Fisher's exact test, P < 0.029). Serum AI on 12q22-23 was associated with worse prognosis (log-rank test, P < 0.046). These findings indicate that serial serum genetic analysis of tumor-related AI on 12q22-23 may have clinical use in predicting tumor response to therapy.

CCL21 chemokine regulates chemokine receptor CCR7 bearing malignant melanoma cells.

PURPOSE: The chemokine CC-ligand 21/secondary lymphoid tissue chemokine (CCL21/SLC) regulates the homing of naïve T cells and dendritic cells that express CC-chemokine receptor 7 (CCR7) from distant sites to lymphoid tissue such as lymph nodes. We hypothesized that CCL21/SLC regulates the migration of CCR7-bearing melanoma cells from a primary lesion to regional tumor-draining lymph nodes. EXPERIMENTAL DESIGN: Quantitative real-time reverse transcriptase-PCR (qRT) assay and immunohistochemistry (IHC) were used to assess the level of CCR7 expression in melanoma cell lines and in primary and metastatic melanoma tumors. Cell migration assay using melanoma cell lines was performed under the induction of CCL21/SLC. The CCL21/SLC expression level in tumor-draining sentinel lymph nodes (SLNs) was assessed by both qRT assay and IHC. RESULTS: Melanoma cell lines and tumors demonstrated heterogeneous expression of CCR7 mRNA by qRT assay. There was strong functional correlation between CCR7 mRNA expression and cell migration induced by CCL21/SLC. IHC evidence of CCR7 expression in primary melanomas significantly (P = 0.02) correlated with Breslow thickness. Assessment of SLN from 55 melanoma patients by qRT assay demonstrated that CCL21/SLC mRNA expression level was significantly (P = 0.008) higher in pathologically melanoma-negative SLNs than in melanoma-positive SLNs. CONCLUSIONS: This report demonstrates a potential mechanism for recruitment and homing of CCR7(+) metastatic melanoma cells to tumor-draining lymph nodes, which express CCL21/SLC. The study also suggests that lymph nodes bearing metastasis may suppress CCL21/SLC production.

Incidence of BRAF oncogene mutation and clinical relevance for primary cutaneous melanomas.

PURPOSE: The purpose of the study was to clarify the incidence of B-raf oncogene (BRAF) mutations in primary cutaneous melanomas, their relation to tumor progression, and effect on disease outcome. Somatic mutations of BRAF kinase, a component of the Ras-mitogen-activated protein/extracellular signal-regulated kinase kinase-mitogen-activated protein kinase pathway, are frequently reported (>65%) in nevi and malignant melanomas. EXPERIMENTAL DESIGN: We assessed BRAF mutation frequency in exons 11 and 15 in primary (n = 59) and metastatic (n = 68) melanomas. Direct sequencing of PCR products was performed on DNA isolated and purified from microdissected tumors. RESULTS: Eighteen mutations (31%) at exon 15 were detected in primary melanoma with a significantly (P = 0.001) higher frequency in patients < 60 years old. Incidence of BRAF mutation did not correlate with Breslow thickness. Presence of BRAF mutation of primary tumors did not effect overall disease-free survival. BRAF mutation frequency in metastatic lesions was 57% and significantly (P = 0.0024) higher than primary melanomas. CONCLUSIONS: The study suggests that BRAF mutation may be acquired during development of metastasis but is not a significant factor for primary tumor development and disease outcome.

Allelic imbalance of 12q22-23 associated with APAF-1 locus correlates with poor disease outcome in cutaneous melanoma.

Cutaneous melanoma is a highly aggressive tumor that is relatively resistant to chemotherapy and radiotherapy. This resistance may be in part due to inhibition of apoptosis. Apoptotic protease activating factor-1(APAF-1), a candidate tumor suppressor gene, mediates p53-induced apoptosis, and its loss promotes oncogenic transformation. To determine whether loss of the APAF-1 locus influences tumor progression, we assessed loss of heterozygosity microsatellites on the APAF-1 locus (12q22-23) in 62 primary and 112 metastatic melanomas. We discovered that frequency of allelic imbalance was significantly higher in metastatic tumors (n = 36 of 98; 37%) than in primary melanomas (n = 10 of 54; 19%; P = 0.02). In metastatic melanomas, APAF-1 loss significantly correlated with a worse prognosis (P < 0.05) in the patients, and its loss during melanoma tumor progression suggests that APAF-1 is a tumor suppressor gene. Furthermore, loss of heterozygosity was frequent in the 12q22-23 chromosome region centromeric to the APAF-1 locus suggesting that other tumor-related genes may be present in the 12q22-23 region. In summary, the study demonstrates that allelic imbalance in the 12q22-23 region is a genomic surrogate of poor disease outcome for cutaneous melanoma patients.