Russell S. Berman

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.

Immunization of Malignant Melanoma Patients with Full-Length NY-ESO-1 Protein Using TLR7 Agonist Imiquimod as Vaccine Adjuvant

T cell-mediated immunity to microbes and to cancer can be enhanced by the activation of dendritic cells (DCs) via TLRs. In this study, we evaluated the safety and feasibility of topical imiquimod, a TLR7 agonist, in a series of vaccinations against the cancer/testis Ag NY-ESO-1 in patients with malignant melanoma. Recombinant, full-length NY-ESO-1 protein was administered intradermally into imiquimod preconditioned sites followed by additional topical applications of imiquimod. The regimen was very well tolerated with only mild and transient local reactions and constitutional symptoms. Secondarily, we examined the systemic immune response induced by the imiquimod/NY-ESO-1 combination, and show that it elicited both humoral and cellular responses in a significant fraction of patients. Skin biopsies were assessed for imiquimod’s in situ immunomodulatory effects. Compared with untreated skin, topical imiquimod induced dermal mononuclear cell infiltrates in all patients composed primarily of T cells, monocytes, macrophages, myeloid DCs, NK cells, and, to a lesser extent, plasmacytoid DCs. DC activation was evident. This study demonstrates the feasibility and excellent safety profile of a topically applied TLR7 agonist used as a vaccine adjuvant in cancer patients. Imiquimod’s adjuvant effects require further evaluation and likely need optimization of parameters such as formulation, dose, and timing relative to Ag exposure for maximal immunogenicity.

Immune profile and mitotic index of metastatic melanoma lesions enhance clinical staging in predicting patient survival

Although remission rates for metastatic melanoma are generally very poor, some patients can survive for prolonged periods following metastasis. We used gene expression profiling, mitotic index (MI), and quantification of tumor infiltrating leukocytes (TILs) and CD3+ cells in metastatic lesions to search for a molecular basis for this observation and to develop improved methods for predicting patient survival. We identified a group of 266 genes associated with postrecurrence survival. Genes positively associated with survival were predominantly immune response related (e.g., ICOS, CD3d, ZAP70, TRAT1, TARP, GZMK, LCK, CD2, CXCL13, CCL19, CCR7, VCAM1) while genes negatively associated with survival were cell proliferation related (e.g., PDE4D, CDK2, GREF1, NUSAP1, SPC24). Furthermore, any of the 4 parameters (prevalidated gene expression signature, TILs, CD3, and in particular MI) improved the ability of Tumor, Node, Metastasis (TNM) staging to predict postrecurrence survival; MI was the most significant contributor (HR = 2.13, P = 0.0008). An immune response gene expression signature and presence of TILs and CD3+ cells signify immune surveillance as a mechanism for prolonged survival in these patients and indicate improved patient subcategorization beyond current TNM staging.

Melanoma MicroRNA Signature Predicts Post-Recurrence Survival

Purpose: To identify a melanoma microRNA (miRNA) expression signature that is predictive of outcome and then evaluate its potential to improve risk stratification when added to the standard-of-care staging criteria. Experimental Design: Total RNA was extracted from 59 formalin-fixed paraffin-embedded melanoma metastases and hybridized to miRNA arrays containing 911 probes. We then correlated miRNA expression with post-recurrence survival and other clinicopathologic criteria. Results: We identified a signature of 18 miRNAs whose overexpression was significantly correlated with longer survival, defined as more than 18 months post-recurrence survival. Subsequent cross-validation showed that a small subset of these miRNAs can predict post-recurrence survival in metastatic melanoma with an estimated accuracy of 80.2% (95% confidence interval, 79.8−80.6%). In contrast to standard-of-care staging criteria, a six-miRNA signature significantly stratified stage III patients into “better” and “worse” prognostic categories, and a multivariate Cox regression analysis revealed the signature to be an independent predictor of survival. Furthermore, we showed that most miRNAs from the signature also showed differential expression between patients with better and worse prognoses in the corresponding paired primary melanoma. Conclusions: MiRNA signatures have potential as clinically relevant biomarkers of prognosis in metastatic melanoma. Our data suggest that molecularly based models of risk assessment can improve the standard staging criteria and support the incorporation of miRNAs into such models. Clin Cancer Res; 16(5); 1577–86

Increased apoptosis in metastatic human colonic adenocarcinomas

Recent work suggests that apoptosis is disrupted during the progression of many solid tumors. Isogenic metastatic colon adenocarcinoma cells displayed significantly higher levels of staurosporine-induced apoptosis compared to their nonmetastatic counterparts in vitro. In addition, analysis of 15 matched primary tumors and liver metastases demonstrated that the levels of apoptosis were significantly higher in the metastases, and this increased cell death was associated with significantly lower levels of Bcl-2 protein expression. Our data demonstrate that the molecular events associated with acquisition of the metastatic phenotype sensitize colon cancer cells to some pro-apoptotic stimuli.

Meta-analysis of sentinel lymph node positivity in thin melanoma (≤1 mm)

Despite the lack of an established survival benefit of sentinel lymph node (SLN) biopsy, this technique has been increasingly applied in the staging of thin (≤1 mm) melanoma patients, without clear evidence to support this recommendation. The authors performed a meta‐analysis to estimate the risk, potential predictors, and outcome of SLN positivity in this group of patients.

PTEN Expression in Melanoma: Relationship with Patient Survival, Bcl-2 Expression, and Proliferation

Purpose: Inactivation of the tumor suppressor gene, phosphatase and tensin homologue (PTEN), is a major alteration in preclinical melanoma models. We investigated the clinical relevance of PTEN expression in the primary melanoma patients with extended follow-up. Experimental Design: We correlated PTEN expression with clinicopathologic variables and outcome in 127 primary melanomas (median follow-up, 12.8 years). We evaluated the associations between PTEN expression and proliferation and resistance to apoptosis (assessed by Ki-67 and Bcl-2, respectively). We also examined the effect of a favorable phenotype, defined as retained PTEN, low proliferative index, and low expression of Bcl-2 on disease-free survival and overall survival. Results: Altered PTEN, Bcl-2, and Ki-67 expressions were observed in 55 of 127 (43.3%), 61 of 127 (48%), and 43 of 114 (37.7%) of cases, respectively. Decreased PTEN expression correlated significantly with the ulceration (P = 0.01). Rates of disease-free survival and overall survival in patients with favorable phenotype were 72% and 74% at 5 years versus 64% and 64% in patients with an unfavorable phenotype. At 10 years, the rates of disease-free survival and overall survival were 72% and 68% for patients with a favorable phenotype but declined to 60% and 55% in patients with an unfavorable phenotype. However, relationships between both PTEN and Bcl2 and patient survival were not significant as well as the associations between PTEN and Bcl-2 or Ki-67. Conclusions: Our data suggest that altered PTEN expression is common in primary melanomas and is associated with aggressive tumor behavior. However, PTEN alone provided limited prognostic value. Our findings show the need to examine molecular alterations identified in preclinical studies using an adequately large cohort of patients with extended follow-up to better assess the magnitude of their clinical relevance.

Serum microRNAs as biomarkers for recurrence in melanoma

BackgroundIdentification of melanoma patients at high risk for recurrence and monitoring for recurrence are critical for informed management decisions. We hypothesized that serum microRNAs (miRNAs) could provide prognostic information at the time of diagnosis unaccounted for by the current staging system and could be useful in detecting recurrence after resection.MethodsWe screened 355 miRNAs in sera from 80 melanoma patients at primary diagnosis (discovery cohort) using a unique quantitative reverse transcription-PCR (qRT-PCR) panel. Cox proportional hazard models and Kaplan-Meier recurrence-free survival (RFS) curves were used to identify a miRNA signature with prognostic potential adjusting for stage. We then tested the miRNA signature in an independent cohort of 50 primary melanoma patients (validation cohort). Logistic regression analysis was performed to determine if the miRNA signature can determine risk of recurrence in both cohorts. Selected miRNAs were measured longitudinally in subsets of patients pre-/post-operatively and pre-/post-recurrence.ResultsA signature of 5 miRNAs successfully classified melanoma patients into high and low recurrence risk groups with significant separation of RFS in both discovery and validation cohorts (p = 0.0036, p = 0.0093, respectively). Significant separation of RFS was maintained when a logistic model containing the same signature set was used to predict recurrence risk in both discovery and validation cohorts (p < 0.0001, p = 0.033, respectively). Longitudinal expression of 4 miRNAs in a subset of patients was dynamic, suggesting miRNAs can be associated with tumor burden.ConclusionOur data demonstrate that serum miRNAs can improve accuracy in identifying primary melanoma patients with high recurrence risk and in monitoring melanoma tumor burden over time.

Can professionalism be taught? Encouraging evidence

BACKGROUND Teaching and assessing the Accreditation Council for Graduate Medical Education (ACGME) competencies of Professionalism and Communication have proven to be a challenge for surgical residency training programs. This study used innovative pedagogic approaches and tools in teaching these two competencies. The purpose of this study was to determine whether the learners actually are assimilating and using the concepts and values communicated through this curriculum. METHODS A six-station Objective Structured Clinical Examination (OSCE) was designed using standardized patients to create varying Professionalism and Communication scenarios. The surgical resident learners were evaluated using these OSCEs as a baseline. The faculty then facilitated a specially designed curriculum consisting of six interactive sessions focusing on information gathering, rapport building, patient education, delivering bad news, responding to emotion, and interdisciplinary respect. At the conclusion of this curriculum, the surgical resident learners took the same six-station OSCE to determine if their professionalism and communication skills had improved. RESULTS The surgical resident learners were rated by the standardized patients according to a strict task checklist of criteria at both the precurricular and postcurricular OSCEs. Improvement in the competencies of Professionalism and Communication did achieve statistical significance (P = .029 and P = .011, respectively). CONCLUSIONS This study suggests that the Communication and Professionalism ACGME competencies can be taught to surgical resident learners through a carefully crafted curriculum. Furthermore, these newly learned competencies can affect surgical resident interactions with their patients positively.

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.