Stephan Ariyan

The pectoralis major myocutaneous flap. A versatile flap for reconstruction in the head and neck.

A compound flap is described that utilizes skin from the anterior chest on a narrow segment of pectoralis major muscle, with its underlying axial neurovascular bundle. This flap has been used successfully to reconstruct large defects in 4 consecutive patients. Our experience with this flap suggests that it may be more versatile than the deltopectoral flap.

Expression Profiling Reveals Novel Pathways in the Transformation of Melanocytes to Melanomas

Affymetrix and spotted oligonucleotide microarrays were used to assess global differential gene expression comparing normal human melanocytes with six independent melanoma cell strains from advanced lesions. The data, validated at the protein level for selected genes, confirmed the overexpression in melanoma cells relative to normal melanocytes of several genes in the growth factor/receptor family that confer growth advantage and metastasis. In addition, novel pathways and patterns of associated expression in melanoma cells not reported before emerged, including the following: (a) activation of the NOTCH pathway; (b) increased Twist expression and altered expression of additional transcriptional regulators implicated in embryonic development and epidermal/mesenchymal transition; (c) coordinated activation of cancer/testis antigens; (d) coordinated down-regulation of several immune modulation genes, in particular in the IFN pathways; (e) down-regulation of several genes implicated in membrane trafficking events; and (f) down-regulation of growth suppressors, such as the Prader-Willi gene NECDIN, whose function was confirmed by overexpression of ectopic Flag-necdin. Validation of differential expression using melanoma tissue microarrays showed that reduced ubiquitin COOH-terminal esterase L1 in primary melanoma is associated with worse outcome and that increased expression of the basic helix-loop-helix protein Twist is associated with worse outcome. Some differentially expressed genes reside on chromosomal regions displaying common loss or gain in melanomas or are known to be regulated by CpG promoter methylation. These results provide a comprehensive view of changes in advanced melanoma relative to normal melanocytes and reveal new targets that can be used in assessing prognosis, staging, and therapy of melanoma patients.

PLX4032, a selective BRAFV600E kinase inhibitor, activates the ERK pathway and enhances cell migration and proliferation of BRAFWT melanoma cells

BRAFV600E/K is a frequent mutationally active tumor‐specific kinase in melanomas that is currently targeted for therapy by the specific inhibitor PLX4032. Our studies with melanoma tumor cells that are BRAFV600E/K and BRAFWT showed that, paradoxically, while PLX4032 inhibited ERK1/2 in the highly sensitive BRAFV600E/K, it activated the pathway in the resistant BRAFWT cells, via RAF1 activation, regardless of the status of mutations in NRAS or PTEN. The persistently active ERK1/2 triggered downstream effectors in BRAFWT melanoma cells and induced changes in the expression of a wide‐spectrum of genes associated with cell cycle control. Furthermore, PLX4032 increased the rate of proliferation of growth factor‐dependent NRAS Q61L mutant primary melanoma cells, reduced cell adherence and increased mobility of cells from advanced lesions. The results suggest that the drug can confer an advantage to BRAFWT primary and metastatic tumor cells in vivo and provide markers for monitoring clinical responses.

Incidence of the V600K mutation among melanoma patients with BRAF mutations, and potential therapeutic response to the specific BRAF inhibitor PLX4032

Activating mutations in BRAF kinase are common in melanomas. Clinical trials with PLX4032, the mutant-BRAF inhibitor, show promising preliminary results in patients selected for the presence of V600E mutation. However, activating V600K mutation is the other most common mutation, yet patients with this variant are currently excluded from the PLX4032 trials. Here we present evidence that a patient bearing the BRAF V600K mutation responded remarkably to PLX4032, suggesting that clinical trials should include all patients with activating BRAF V600E/K mutations.

Further experiences with the pectoralis major myocutaneous flap for the immediate repair of defects from excisions of head and neck cancers.

In our experience with 14 pectoralis major myocutaneous flaps they have been very reliable for reconstructions in a variety of clinical situations--especially in the repair of defects from extensive cancer resections in the head and neck area, including orbital exenterations. The flaps can be transferred immediately, without a delay.

Genome-wide screen of promoter methylation identifies novel markers in melanoma

DNA methylation is an important component of epigenetic modifications, which influences the transcriptional machinery aberrant in many human diseases. In this study we present the first genome-wide integrative analysis of promoter methylation and gene expression for the identification of methylation markers in melanoma. Genome-wide promoter methylation and gene expression of eight early-passage human melanoma cell strains were compared with newborn and adult melanocytes. We used linear mixed effect models (LME) in combination with a series of filters based on the localization of promoter methylation relative to the transcription start site, overall promoter CpG content, and differential gene expression to discover DNA methylation markers. This approach identified 76 markers, of which 68 were hyper- and eight hypomethylated (LME, P < 0.05). Promoter methylation and differential gene expression of five markers (COL1A2, NPM2, HSPB6, DDIT4L, MT1G) were validated by sequencing of bisulfite-modified DNA and real-time reverse transcriptase PCR, respectively. Importantly, the incidence of promoter methylation of the validated markers increased moderately in early and significantly in advanced-stage melanomas, using early-passage cell strains and snap-frozen tissues (n = 18 and n = 24, respectively) compared with normal melanocytes and nevi (n = 11 and n = 9, respectively). Our approach allows robust identification of methylation markers that can be applied to other studies involving genome-wide promoter methylation. In conclusion, this study represents the first unbiased systematic effort to determine methylation markers in melanoma and revealed several novel genes regulated by promoter methylation that were not described in cancer cells before.

Exome sequencing identifies recurrent mutations in NF1 and RASopathy genes in sun-exposed melanomas

We report on whole-exome sequencing (WES) of 213 melanomas. Our analysis established NF1, encoding a negative regulator of RAS, as the third most frequently mutated gene in melanoma, after BRAF and NRAS. Inactivating NF1 mutations were present in 46% of melanomas expressing wild-type BRAF and RAS, occurred in older patients and showed a distinct pattern of co-mutation with other RASopathy genes, particularly RASA2. Functional studies showed that NF1 suppression led to increased RAS activation in most, but not all, melanoma cases. In addition, loss of NF1 did not predict sensitivity to MEK or ERK inhibitors. The rebound pathway, as seen by the induction of phosphorylated MEK, occurred in cells both sensitive and resistant to the studied drugs. We conclude that NF1 is a key tumor suppressor lost in melanomas, and that concurrent RASopathy gene mutations may enhance its role in melanomagenesis.

Prospective Multi-Institutional Study of Reverse Transcriptase Polymerase Chain Reaction for Molecular Staging of Melanoma

PURPOSE To evaluate the prognostic significance of molecular staging using reverse transcriptase polymerase chain reaction (RT-PCR) in detecting occult melanoma cells in sentinel lymph nodes (SLNs) and circulating bloodstream. PATIENTS AND METHODS In this multicenter study, eligibility criteria included patient age 18 to 71 years, invasive melanoma > or = 1.0 mm Breslow thickness, and no clinical evidence of metastasis. SLN biopsy and wide excision of the primary tumor were performed. SLNs were examined by serial-section histopathology and S-100 immunohistochemistry. A portion of each SLN was frozen for RT-PCR. In addition, RT-PCR was performed on peripheral-blood mononuclear cells (PBMCs). RT-PCR analysis was performed using four markers: tyrosinase, MART1, MAGE3, and GP-100. Disease-free survival (DFS), distant-DFS (DDFS), and overall survival (OS) were analyzed. RESULTS A total of 1,446 patients with histologically negative SLNs underwent RT-PCR analysis. At a median follow-up of 30 months, there was no difference in DFS, DDFS, or OS between the RT-PCR-positive (n = 620) and RT-PCR-negative (n = 826) patients. Analysis of PBMC from 820 patients revealed significant differences in DFS and DDFS, but not OS, for patients with detection of more than one RT-PCR marker in peripheral blood. CONCLUSION In this large, prospective, multi-institutional study, RT-PCR analysis on SLNs and PBMCs provides no additional prognostic information beyond standard histopathologic analysis of SLNs. Detection of more than one marker in PBMC is associated with a worse prognosis. RT-PCR remains investigational and should not be used to direct adjuvant therapy at this time.

Follow-up recommendations for patients with American Joint Committee on Cancer Stages I-III malignant melanoma.

Guidelines for follow‐up of melanoma patients are not established. In 1987, a follow‐up protocol was instituted at the Yale Melanoma Unit to improve upon the detection of disease recurrence in patients with American Joint Committee on Cancer Stage I–III cutaneous melanoma. The follow‐up protocol consists of a patient education program and a surveillance schedule based on stage of disease.

Gender-Related Differences in Outcome for Melanoma Patients

Objective:To better understand the factors associated with the well-established gender difference in survival for patients with melanoma. Summary Background Data:Gender is an important factor in patients with cutaneous melanoma. Male patients have a worse outcome when compared with females. The reasons for this difference are poorly understood. Methods:This prospective multi-institutional study included patients aged 18 to 70 years with melanomas ≥1.0 mm Breslow thickness. Wide excision and sentinel lymph node (SLN) biopsy was performed in all patients. Clinicopathologic factors, including gender, were assessed and correlated with disease-free survival (DFS), distant disease-free survival (DDFS), and overall survival (OS). Results:A total of 3324 patients were included in the covariate analyses; 1829 patients had follow-up data available and were included in the survival analyses. Median follow-up was 30 months. On univariate analysis, men (n = 1906) were more likely than women to be older than 60 years (P < 0.0001), have thicker melanomas (P < 0.0001), have primary tumor regression (P = 0.0054), ulceration (P < 0.0001), and axial primary tumor location (P < 0.0001). On multivariate analysis, age (P = 0.0002), thickness (P < 0.0001), ulceration (P = 0.015), and location (P < 0.0001) remained significant in the model. There was no difference in the rate of SLN metastasis between men and women (P = 0.37) on multivariate analysis. When factors affecting survival were considered, the prognosis was worse for men as validated by lower DFS (P = 0.0005), DDFS (P < 0.0001), and OS (P < 0.0001). Conclusions:Male gender is associated with a greater incidence of unfavorable primary tumor characteristics without an increased risk for nodal metastasis. Nonetheless, gender is an independent factor affecting survival.