Purpose: Multiple BRAF inhibitor resistance mechanisms have been described, however, their relative frequency, clinical correlates, and effect on subsequent therapy have not been assessed in patients with metastatic melanoma.Experimental Design: Fifty-nine BRAF V600 -mutant melanoma metastases from patients treated with dabrafenib or vemurafenib were analyzed. The genetic profile of resistance mechanisms and tumor signaling pathway activity was correlated with clinicopathologic features and therapeutic outcomes.Results: Resistance mechanisms were identified in 58% progressing tumors and BRAF alterations were common. Gene expression analysis revealed that mitogen-activated protein kinase (MAPK) activity remained inhibited in 21% of resistant tumors, and the outcomes of patients with these tumors were poor. Resistance mechanisms also occurred in pretreatment biopsies and heterogeneity of resistance mechanisms occurred within patients and within tumors. There were no responses to subsequent targeted therapy, even when a progressing tumor had a resistance mechanism predicted to be responsive.Conclusions: Selecting sequential drugs based on the molecular characteristics of a single progressing biopsy is unlikely to provide improved responses, and first-line therapies targeting multiple pathways will be required.

Purpose: To evaluate the effects of treatment with the potent mutant BRAF inhibitors GSK2118436 or vemurafenib (PLX4720) on immune responses to metastatic melanoma in tissues taken before and after treatment.Experimental Design: Thirty-seven tumor biopsies were collected from 15 patients with unresectable American Joint Committee on Cancer stage III or IV melanoma immediately before and approximately 7 days after the commencement of BRAF inhibitor treatment and at the time of tumor progression. Immunohistochemical staining was carried out on the biopsies using specific antibodies for CD8, CD4, CD20, CD1a, and Granzyme B.

Purpose: Certain clinicopathologic features correlate with BRAF mutation status in melanoma including younger age and primary subtype. This study sought to determine the BRAF mutation status by age-decade and whether BRAF-mutant genotypes correlated with clinicopathologic features and outcome in patients with metastatic melanoma.Methods: A prospectively assembled cohort of Australian patients were followed from diagnosis of metastatic melanoma (N ¼ 308). Clinicopathologic variables were correlated with BRAF mutational status, genotype, and survival.Results: Forty-six percent of patients had a BRAF mutation; 73% V600E, 19% V600K, and 8% other genotypes. An inverse relationship existed between BRAF mutation prevalence and age-decade (P < 0.001). All patients <30 years and only 25% 70 years had BRAF-mutant melanoma. Amongst BRAF-mutant melanoma, the frequency of non-V600E genotypes (including V600K) increased with increasing age. Non-V600E genotypes comprised <20% in patients <50 years and >40% in those 70 years. A higher degree of cumulative sun-induced damage correlated with V600K but not V600E melanoma (P ¼ 0.002). The diseasefree interval from diagnosis of primary melanoma to first distant metastasis was shorter for patients with V600K compared with V600E melanoma (17.4 vs. 39.2 months, P ¼ 0.048), with no difference in survival thereafter. In patients BRAF tested at diagnosis of metastatic melanoma, one year survival from diagnosis of metastasis was significantly longer for patients with BRAF-mutant melanoma treated with an inhibitor (83%), than those not treated with an inhibitor (29%, P < 0.001), or patients with BRAF wild-type melanoma (37%, P < 0.001).Conclusion: Different genotypes exist within BRAF-mutant metastatic melanoma, representing biologically and clinically discrete subtypes, suggesting distinct etiology and behavior.

One-third of BRAF-mutant metastatic melanoma patients treated with combined BRAF and MEK inhibition progress within 6 months. Treatment options for these patients remain limited. Here we analyse 20 BRAF V600 -mutant melanoma metastases derived from 10 patients treated with the combination of dabrafenib and trametinib for resistance mechanisms and genetic correlates of response. Resistance mechanisms are identified in 9/11 progressing tumours and MAPK reactivation occurred in 9/10 tumours, commonly via BRAF amplification and mutations activating NRAS and MEK2. Our data confirming that MEK2 C125S , but not the synonymous MEK1 C121S protein, confers resistance to combination therapy highlight the functional differences between these kinases and the preponderance of MEK2 mutations in combination therapy-resistant melanomas. Exome sequencing did not identify additional progression-specific resistance candidates. Nevertheless, most melanomas carried additional oncogenic mutations at baseline (for example, RAC1 and AKT3) that activate the MAPK and PI3K pathways and are thus predicted to diminish response to MAPK inhibitors.

Disruption of PD-L1/cytotoxic T-cell PD-1 signaling by immune checkpoint inhibitors improves survival in cancer patients. This study sought to identify changes in tumoral PD-L1 expression and tumor-associated immune cell flux with anti-PD-1 therapies in patients with melanoma, particularly early during treatment, and correlate them with treatment response. Forty-six tumor biopsies from 23 patients with unresectable AJCC stage III/IV melanoma receiving pembrolizumab/nivolumab were analyzed. Biopsies were collected prior to (PRE, = 21), within 2 months of commencing treatment (EDT, = 20) and on disease progression after previous response (PROG, = 5). Thirteen patients responded (defined as CR, PR, or durable SD by RECIST/irRC criteria), and 10 did not respond. PRE intratumoral and peritumoral PD-1 T-cell densities were sevenfold ( = 0.006) and fivefold higher ( = 0.011), respectively, in responders compared with nonresponders and correlated with degree of radiologic tumor response ( = -0.729, = 0.001 and = -0.725, = 0.001, respectively). PRE PD-L1 expression on tumor and macrophages was not significantly different between the patient groups, but tumoral PD-L1 and macrophage PD-L1 expression was higher in the EDT of responders versus nonresponders ( = 0.025 and = 0.033). Responder EDT biopsies (compared with PRE) also showed significant increases in intratumoral CD8 lymphocytes ( = 0.046) and intratumoral CD68 macrophages ( = 0.046). Higher PRE PD-1 T cells in responders suggest active suppression of an engaged immune system that is disinhibited by anti-PD-1 therapies. Furthermore, immunoprofiling of EDT biopsies for increased PD-L1 expression and immune cell infiltration showed greater predictive utility than PRE biopsies and may allow better selection of patients most likely to benefit from anti-PD-1 therapies and warrants further evaluation. .

Merkel cell carcinoma (MCC) is a rare aggressive cutaneous neuroendocrine malignancy that frequently metastasizes to the regional lymphatic basin. Pathological assessment of regional lymph nodes with sentinel lymph node biopsy (SLNB) in patients without clinical involvement has permitted more accurate staging and more appropriate management. Nonetheless, concerns have been raised regarding the accuracy of this technique and its prognostic implications. We conducted a review of previously published data analysing the positive and false negative rates of SLNB in MCC. A search of the Medline and Embase databases to April 2015 identified 36 published studies between 1997 and 2015 comprising 692 patients. With the addition of 29 patients treated at our own institution, we conducted an analysis of 721 patients. Among this cumulative cohort, SLNBs were performed from 736 regional sites with 29·6% recorded as positive. Regional metastasis occurred in 45 cases following a negative SLNB, for a false negative rate of 17·1%. Adjuvant regional radiotherapy in the setting of a negative SLNB did not affect regional recurrence (P = 0·31), providing credence to emerging evidence that regional therapy can be safely omitted in the setting of a negative SLNB. Distant relapse was noted far more frequently following a positive rather than negative SLNB (17·6% vs. 7·3%, P < 0·001).

Background The outcomes of patients with stage III cutaneous melanoma who undergo complete surgical resection can be highly variable, and estimation of individual risk of disease recurrence and mortality remains imprecise. With recent demonstrations of effective adjuvant targeted and immune checkpoint inhibitor therapy, more precise stratification of patients for costly and potentially toxic adjuvant therapy is needed. We report the utility of pre-operative circulating tumour DNA (ctDNA) in patients with high-risk stage III melanoma. Patients and methods ctDNA was analysed in blood specimens that were collected pre-operatively from 174 patients with stage III melanoma undergoing complete lymph node (LN) dissection. Cox regression analyses were used to evaluate the prognostic significance of ctDNA for distant metastasis recurrence-free survival and melanoma-specific survival (MSS). Results The detection of ctDNA in the discovery and validation cohort was 34% and 33%, respectively, and was associated with larger nodal melanoma deposit, higher number of melanoma involved LNs, more advanced stage and high lactate dehydrogenase (LDH) levels. Detectable ctDNA was significantly associated with worse MSS in the discovery [hazard ratio (HR) 2.11 P < 0.01] and validation cohort (HR 2.29, P = 0.04) and remained significant in a multivariable analysis (HR 1.85, P = 0.04). ctDNA further sub-stratified patients with AJCC stage III substage, with increasing significance observed in more advanced stage melanoma. Conclusion Pre-operative ctDNA predicts MSS in high-risk stage III melanoma patients undergoing complete LN dissection, independent of stage III substage. This biomarker may have an important role in determining prognosis and stratifying patients for adjuvant treatment.

Targeted therapies are increasingly being used to treat a variety of cancers. Their efficacy depends upon the accurate detection and targeting of a specific mutation or aberration in the tumor. All cancers, such as melanoma, are molecularly heterogeneous, with drug-resistant subclones present before the treatment or emerging as a result of targeted therapies. Here, we show intralesional molecular heterogeneity in a progressing V600E BRAF-mutant melanoma metastasis from a patient treated for 7 months with the BRAF inhibitor vemurafenib. In the single metastasis, two distinct subclones were observed, both V600E BRAFmutant and only one with an additional G13R NRAS mutation. Molecular heterogeneity even at the intralesional level shows that personalizing or adjusting therapies based on genotyping of a portion of a single lesion may not accurately depict the molecular profile or drivers of oncogenesis across the entire patient's melanoma.