Faculty Bibliography

Brain metastasis (B-Met) from melanoma remains mostly incurable and the main cause of death from the disease. Early stage clinical trials and case studies show some promise for targeted therapies in the treatment of melanoma B-Met. However, the progression-free survival for currently available therapies, although significantly improved, is still very short. The development of new potent agents to eradicate melanoma B-Met relies on the elucidation of the molecular mechanisms that allow melanoma cells to reach and colonize the brain. The discovery of such mechanisms depends heavily on pre-clinical models that enable the testing of candidate factors and therapeutic agents in vivo. In this review we summarize the effects of available targeted therapies on melanoma B-Met in the clinic. We provide an overview of existing pre-clinical models to study the disease and discuss specific molecules and mechanisms reported to modulate different aspects of melanoma B-Met and finally, by integrating both clinical and basic data, we summarize both opportunities and challenges currently presented to researchers in the field.

The prognostic value of mitotic rate in melanoma is increasingly recognized, particularly in thin melanoma in which the presence or absence of a single mitosis/mm can change staging from T1a to T1b. Still, accurate mitotic rate calculation (mitoses/mm) on hematoxylin and eosin (H&E)-stained sections can be challenging. Antimonoclonal mitotic protein-2 (MPM-2) and antiphosphohistone-H3 (PHH3) are 2 antibodies reported to be more mitosis-specific than other markers of proliferation such as Ki-67. We used light microscopy and computer-assisted image analysis software to quantify MPM-2 and PHH3 staining in melanoma. We then compared mitotic rates by each method with conventional H&E-based mitotic rate for correlation with clinical outcomes. Our study included primary tissues from 190 nonconsecutive cutaneous melanoma patients who were prospectively enrolled at New York University Langone Medical Center with information on age, gender, and primary tumor characteristics. The mitotic rate was quantified manually by light microscopy of corresponding H&E-stained, MPM-2-stained, and PHH3-stained sections. Computer-assisted image analysis was then used to quantify immunolabeled mitoses on the previously examined PHH3 and MPM-2 slides. We then analyzed the association between mitotic rate and both progression-free and melanoma-specific survival. Univariate analysis of PHH3 found significant correlation between increased PHH3 mitotic rate and decreased progression-free survival (P=0.04). Computer-assisted image analysis enhanced the correlation of PHH3 mitotic rate with progression-free survival (P=0.02). Regardless of the detection method, neither MPM-2 nor PHH3 offered significant advantage over conventional H&E determination of mitotic rate.

T cells expressing antigen-specific T-cell receptors (TCRs) can mediate effective tumor regression, but they often also are accompanied by autoimmune responses. To determine the TCR affinity threshold defining the optimal balance between effective antitumor activity and autoimmunity in vivo, we used a unique self-antigen system comprising seven human melanoma gp100(209-217)-specific TCRs spanning physiological affinities (1-100 muM). We found that in vitro and in vivo T-cell responses are determined by TCR affinity, except in one case that was compensated by substantial CD8 involvement. Strikingly, we found that T-cell antitumor activity and autoimmunity are closely coupled but plateau at a defined TCR affinity of 10 microM, likely due to diminished contribution of TCR affinity to avidity above the threshold. Together, these results suggest that a relatively low-affinity threshold is necessary for the immune system to avoid self-damage, given the close relationship between antitumor activity and autoimmunity. The low threshold, in turn, indicates that adoptive T-cell therapy treatment strategies using in vitro-generated high-affinity TCRs do not necessarily improve efficacy.

The incidence of melanoma is increasing faster than that of any other cancer, and predicted to double every 10-20 years. Surgery can be curative in Stage I, II, or III disease, but 75% of patients with deep primary lesions develop extensive recurrence or distant metastases and have dismal prognosis. In fact, there is no curative treatment for stage IV melanoma. Although novel targeted therapies, such as BRAF inhibitors and anti-CTLA4 antibodies are showing promising results in melanoma clinical trials, resistance to these agents and patient relapse rapidly ensue. Therapeutic resistance has been commonly attributed to functional redundancy between intimately hardwired cellular pathways responsible for tumor cell maintenance and survival. In order to avoid redundancy, we propose to directly inhibit the transcription of multiple genes required for the establishment or maintenance of tumors. First, we analyzed the expression of Bromodomain (BrD)-containing proteins, a family of epigenetic readers that bind acetylated lysine. BrDs are present in histone acetyl transferases (i.e. CBP/p300, PCAF, GCN5) and transcriptional regulators (i.e. BET family members: BRD2, 3, and 4). mRNA expression arrays showed several BrD-containing genes as upregulated in primary and metastatic melanoma cell lines compared to normal melanocytes. Analysis of available expression profiles also revealed higher levels of BRD2 and BRD4 in melanoma tissues relative to nevi or normal skin (P<0.001). Furthermore, immunohistochemistry staining of a melanoma tissue microarray confirmed overexpression of BRD4 protein in primary (P<0.001) and metastatic tumors (P<0.001) compared to nevi. BRD4 knockdown using siRNA or shRNA suppressed the proliferation and colony formation capacity of several metastatic melanoma cell lines. Moreover, melanoma cells stably infected with shBRD4 carrying lentivirus displayed reduced tumor growth in vivo compared with their counterpart infected with a non-silencing control. To identify cellular pathways mo!

the molecular characterization of melanoma has ex- panded to include studies of microRNA (miRNA) ex- pression. As miR-16 has been utilized as a normalizer in serum-based miRNA studies in several cancers, we evaluated miR-16 expression as a potential reference for normalization of serum miRNA expression in melanoma patients. Methods: 143 primary cutaneous melanoma patients who presented to New York Uni- versity (NYU) Langone Medical Center for surgical resection of AJCC stage I-III disease were studied. In addition, sera samples from 60 control subjects were utilized including 22 healthy volunteers, 13 rheuma- toid arthritis patients, 20 non-melanoma cancer pa- tients (10 renal cell carcinoma and 10 bladder cancer), and 5 Atypical Mole Syndrome patients. The Kruskal- Wallis test (k = 6) or Wilcoxon test (k = 2) with Bon- ferroni correction was used for analyses of miR-16 expression in melanoma patients compared to various control groups, using raw Ct values directly. The Kruskal-Wallis test was used to compare miR-16 ex- pression across stages of melanoma. The equivalence test for independent samples was used to test the equivalence of miR-16 expression among different groups. Results: No significant differential expression of miR-16 was observed between melanoma patients and healthy volunteers (Wilcoxon test, p = 0.37). How- ever, miR-16 did show a significant difference in ex- pression as it related to stage of melanoma (p = 0.015). Additionally, the equivalence test was unable to con- firm equivalent expression of miR-16 in any melanoma versus control group pair. Conclusion: Our data in- dicate that miR-16 cannot be used as a universal normalizer in sera studies of melanoma patients