Molly Yancovitz

Intra- and Inter-Tumor Heterogeneity of BRAFV600EMutations in Primary and Metastatic Melanoma

The rationale for using small molecule inhibitors of oncogenic proteins as cancer therapies depends, at least in part, on the assumption that metastatic tumors are primarily clonal with respect to mutant oncogene. With the emergence of BRAFV600E as a therapeutic target, we investigated intra- and inter-tumor heterogeneity in melanoma using detection of the BRAFV600E mutation as a marker of clonality. BRAF mutant-specific PCR (MS-PCR) and conventional sequencing were performed on 112 tumors from 73 patients, including patients with matched primary and metastatic specimens (n = 18). Nineteen patients had tissues available from multiple metastatic sites. Mutations were detected in 36/112 (32%) melanomas using conventional sequencing, and 85/112 (76%) using MS-PCR. The better sensitivity of the MS-PCR to detect the mutant BRAFV600E allele was not due to the presence of contaminating normal tissue, suggesting that the tumor was comprised of subclones of differing BRAF genotypes. To determine if tumor subclones were present in individual primary melanomas, we performed laser microdissection and mutation detection via sequencing and BRAFV600E-specific SNaPshot analysis in 9 cases. Six of these cases demonstrated differing proportions of BRAFV600Eand BRAFwild-type cells in distinct microdissected regions within individual tumors. Additional analyses of multiple metastatic samples from individual patients using the highly sensitive MS-PCR without microdissection revealed that 5/19 (26%) patients had metastases that were discordant for the BRAFV600E mutation. In conclusion, we used highly sensitive BRAF mutation detection methods and observed substantial evidence for heterogeneity of the BRAFV600E mutation within individual melanoma tumor specimens, and among multiple specimens from individual patients. Given the varied clinical responses of patients to BRAF inhibitor therapy, these data suggest that additional studies to determine possible associations between clinical outcomes and intra- and inter-tumor heterogeneity could prove fruitful.

Providing hepatitis B vaccination to injection drug users: referral to health clinics vs on-site vaccination at a syringe exchange program.

Injection drug users (IDUs) are at very high risk for infection with hepatitis B virus (HBV) through multiperson use of injection equipment and through unprotected sexual contact. Although a safe and efficacious vaccine exists for hepatitis B, there are multiple problems in vaccinating IDUs in the United States, including (1) discrimination against drug users by health care providers, (2) the need to reach IDUs before they are exposed to HBV, (3) paying for the vaccinations, and (4) difficulties in completing the 3-injection vaccination series. We compared 2 methods for delivering free hepatitis B vaccination to IDUs: (1) referral by research staff to local health care providers and (2) on-site vaccination at a syringe exchange program.

Role of radiologic imaging at the time of initial diagnosis of stage T1b‐T3b melanoma

In patients with T1b‐T3b cutaneous melanoma the utility of radiologic imaging at the time of diagnosis is unclear. Whether initial imaging led to a change in stage or treatment plan was investigated.

Utility of Lesion Diameter in the Clinical Diagnosis of Cutaneous Melanoma

OBJECTIVE To determine the utility of the current diameter criterion of larger than 6 mm of the ABCDE acronym for the early diagnosis of cutaneous melanoma. DESIGN Cohort study. SETTING Dermatology hospital-based clinics and community practice offices. Patients A total of 1323 patients undergoing skin biopsies of 1657 pigmented lesions suggestive of melanoma. MAIN OUTCOME MEASURE The maximum lesion dimension (diameter) of each skin lesion was calculated before biopsy using a novel computerized skin imaging system. RESULTS Of 1657 biopsied lesions, 853 (51.5%) were 6 mm or smaller in diameter. Invasive melanomas were diagnosed in 13 of 853 lesions (1.5%) that were 6 mm or smaller in diameter and in 41 of 804 lesions (5.1%) that were larger than 6 mm in diameter. In situ melanomas were diagnosed in 22 of 853 lesions (2.6%) that were 6 mm or smaller in diameter and in 62 of 804 lesions (7.7%) that were larger than 6 mm in diameter. Conclusion The diameter guideline of larger than 6 mm provides a useful parameter for physicians and should continue to be used in combination with the A, B, C, and E criteria previously established in the selection of atypical lesions for skin biopsy.

Clinical relevance of neutral endopeptidase (NEP/CD10) in melanoma.

BackgroundOverexpression of Neutral Endopeptidase (NEP) has been reported in metastatic carcinomas, implicating NEP in tumor progression and suggesting a role for NEP inhibitors in its treatment. We investigated the role of NEP expression in the clinical progression of cutaneous melanoma.MethodsWe screened 7 melanoma cell lines for NEP protein expression. NEP-specific siRNA was transfected into the lines to examine the role of gene transcription in NEP expression. Immunohistochemistry was done for 93 specimens and correlated with clinicopathologic parameters. Thirty-seven metastatic melanoma specimens were examined for NEP transcript expression using Affymetrix GeneChips. In a subset of 25 specimens for which both transcript and protein expression was available, expression ratios were used to identify genes that co-express with NEP in GeneChip analysis.ResultsNEP was overexpressed in 4/7 human melanoma cell lines, and siRNA knock-down of NEP transcripts led to downregulation of its protein expression. NEP protein overexpression was significantly more common in metastatic versus primary tumors (P = 0.002). Twelve of 37 (32%) metastatic tumors had increased NEP transcript expression, and an association was observed between NEP transcript upregulation and protein overexpression (P < 0.0001). Thirty-eight genes were found to significantly co-express with NEP (p < 0.005). Thirty-three genes positively correlated with NEP, including genes involved in the MAP kinase pathway, antigen processing and presentation, apoptosis, and WNT signaling pathway, and 5 genes negatively correlated with NEP, including genes of focal adhesion and the notch signaling pathways.ConclusionNEP overexpression, which seems to be largely driven by increased transcription, is rare in primary melanoma and occurs late in melanoma progression. Functional studies are needed to better understand the mechanisms of NEP regulation in melanoma.

Nucleofection is a highly effective gene transfer technique for human melanoma cell lines

Abstract:  Despite the increasing use of gene transfer strategies in the study of cellular and molecular biology, melanoma cells have remained difficult to transfect in a safe, efficient, and reproducible manner. In the present study, we report the successful use of nucleofector technology to transfect human melanoma cell lines. This technology uses an empirically derived combination of cell line‐specific solutions and nucleofector programmes to electroporate nucleic acid substrates directly into the cell nucleus. Using a colorimetric β‐galactosidase assay, we optimized nucleofection parameters for 13 melanoma cell lines, leading to maximum transfection efficiency and cell survival. The combinations of cell solutions NHEM or T and nucleofector programmes A‐24 or U‐20 produced the best results. We compared nucleofection with two commercially available lipid‐based gene transfer systems, effectene and lipofectamine 2000 using a green fluorescent protein reporter vector. Nucleofection demonstrated a 3‐ to 40‐fold improvement in transfection efficiency when compared with the lipid‐based counterparts. Nucleofection was also superior in transfecting small‐interfering RNA (siRNA) as determined by Western blot analysis. Lastly, we applied nucleofection to the simultaneous transfection of a p53‐dependent luciferase plasmid and p53‐siRNA. Experiments using dual transfection showed knockdown of p53 expression and silencing of the reporter plasmid. In conclusion, nucleofection is highly effective for the transfer of nucleic acid substrates, singly or in combination, into human melanoma cell lines.