Lentigo maligna (LM), a type of in situ melanoma, has a high rate of local recurrence after surgical resection. Although recurrences might represent inadequately excised incident LMs, apparent recurrences could be new primary LMs developing from distinct melanocytic clones in the same UVR-damaged anatomical region. Because distinguishing these possibilities has implications for patient management, we tested genetic relationships between incident and recurrent LMs. Incident LMs with histologically clear margins and their recurrent LMs and invasive LM melanomas were laser microdissected for DNA extraction. Tumor and matched germline DNA underwent whole-exome sequencing. Clonal relationships between incident and recurrent LMs were tested by elucidating and tracking clones within and across samples. Seven incident LMs with recurrent LM and 4 with recurrent LM melanoma were sequenced. LM/LM melanomas had a high mutational burden, a predominant UVR signature, copy number alterations, and defects in DNA repair and cell-cycle checkpoint regulator genes. All incident LMs were clonally related to their postoperative local recurrences. We therefore found that local recurrences of LM commonly arise from subclinical residual disease after surgery. The mutational landscape of recurrent LM/LM melanoma was typical of melanomas arising from chronic sun exposure but also displayed features described in late-stage melanomas.

BACKGROUND The advent of effective adjuvant therapies for patients with resected melanoma has highlighted the need to stratify patients based on risk of relapse given the cost and toxicities associated with treatment. Here we assessed circulating tumor DNA (ctDNA) to predict and monitor relapse in resected stage III melanoma. PATIENTS AND METHODS Somatic mutations were identified in 99/133 (74%) patients through tumor tissue sequencing. Personalized droplet digital PCR (ddPCR) assays were used to detect known mutations in 315 prospectively collected plasma samples from mutation-positive patients. External validation was performed in a prospective independent cohort (n = 29). RESULTS ctDNA was detected in 37 of 99 (37%) individuals. In 81 patients who did not receive adjuvant therapy, 90% of patients with ctDNA detected at baseline and 100% of patients with ctDNA detected at the postoperative timepoint relapsed at a median follow up of 20 months. ctDNA detection predicted patients at high risk of relapse at baseline [relapse-free survival (RFS) hazard ratio (HR) 2.9; 95% confidence interval (CI) 1.5-5.6; P = 0.002] and postoperatively (HR 10; 95% CI 4.3-24; P < 0.001). ctDNA detection at baseline [HR 2.9; 95% CI 1.3-5.7; P = 0.003 and postoperatively (HR 11; 95% CI 4.3-27; P < 0.001] was also associated with inferior distant metastasis-free survival (DMFS). These findings were validated in the independent cohort. ctDNA detection remained an independent predictor of RFS and DMFS in multivariate analyses after adjustment for disease stage and BRAF mutation status. CONCLUSION Baseline and postoperative ctDNA detection in two independent prospective cohorts identified stage III melanoma patients at highest risk of relapse and has potential to inform adjuvant therapy decisions.

Background: Adjuvant immunotherapy and BRAF-targeted therapies have shown improved relapse-free survival (RFS) in patients (pts) with resected melanoma. There is a critical need to develop biomarkers to stratify pts based on risk of relapse given the cost and toxicities with these therapies. Methods: Droplet digital PCR was used to detect known mutations in circulating tumour DNA (ctDNA) from 112 pts with resected stage II-III melanoma who consented to prospective serial plasma and risk-adapted F-18-fluoro-deoxyglucose positron emission tomography (FDG-PET) monitoring. External validation was performed in a prospective independent cohort. Results: In 92/112 pts who did not receive adjuvant therapy, 55/92 (60%) pts relapsed at a median follow-up of 21 months. Plasma samples were available in 67/92 pts at baseline and 59/92 pts postoperatively (median 2 weeks post surgery). ctDNA was detected at baseline and postoperatively in 21/67 (31%) pts and 15/59 (25%) pts respectively. 19/21 and 14/15 pts with ctDNA detected at baseline and the postoperative visit relapsed. Detection of ctDNA predicted patients at high risk of relapse at baseline (RFS hazard ratio [HR] 4.7; 95% confidence interval [CI] 2.3-9.6; p < 0.0001) and postoperatively (HR 9.3; 95% CI 4-22; p < 0.0001). Inferior distant metastasis-free survival (DMFS) was associated with ctDNA detection at baseline (HR 5.3; 95% CI 2.5-11; P < 0.0001) and postoperatively (HR 14; 95% CI 5.4-37; P < 0.0001). These findings were validated in an independent cohort. Postoperative ctDNA detection remained an independent predictor of both RFS (HR 8; 95% CI 3-20; P < 0.0001) and DMFS (HR 11; 95% CI 4-30; P < 0.0001) after adjustment for disease stage and BRAF mutation status. In 20/112 pts who received adjuvant therapy, 3/18 pts with a postoperative plasma sample had detectable ctDNA. Serial plasma samples were available in 2 of 3 cases and showed clearance of ctDNA post immunotherapy. At a median follow-up of 7 months, none of the pts with detectable ctDNA who received adjuvant therapy have relapsed. Conclusions: Detection of ctDNA at baseline and after surgical resection in two independent prospective cohorts identifies pts with stage II/III melanoma at highest risk of relapse with potential to guide adjuvant therapy decisions. Legal entity responsible for the study: Peter MacCallum Cancer Centre. Funding: National Health and Medical Research Council, Cancer Research UK, Wellcome Trust. Disclosure: S.K. Sandhu: Honorarium: Merck, Bristol-Myers Squibb. M. Shackleton: Travel support: BMS, Merck; Consultancy: BMS, Merck, MSD; Honoraria: BMS, Merck, MSD Research support: BMS. A. Haydon: Advisory board: Novartis, MSD. D. Gyorki: Advisory board: Amgen; Honorarium: Amgen. P. Lorigan: Consultancy/ Advisory work: Amgen, GSK, Roche, BMS, Merck, Novartis; Speaker bureau: Merck, BMS, Novartis, Roche; Support for travel: Merck, BMS, Roche. R. Marais: Research funding: Basilea Pharmaceuticals. All other authors have declared no conflicts of interest.

A prospective study explored the heterogeneous nature of metastatic melanoma using Multiplex immunohistochemistry (IHC) and flow cytometry (FACS). Multiplex IHC data quantitated immune subset number present intra-tumoral (IT) vs the tumor stroma, plus distance of immune subsets from the tumor margin (TM). In addition, mIHC showed a close association between the presence of IT CD8(+) T cells and PDL1 expression in melanoma, which was more prevalent on macrophages than on melanoma cells. In contrast, FACS provided more detailed information regarding the T cell subset differentiation, their activation status and expression of immune checkpoint molecules. Interestingly, mIHC detected significantly higher Treg numbers than FACS and showed preferential CD4(+) T cell distribution in the tumor stroma. Based on the mIHC and FACS data, we provide a model which defines metastatic melanoma immune context into four categories using the presence or absence of PDL1(+) melanoma cells and/or macrophages, and their location within the tumor or on the periphery, combined with the presence or absence of IT CD8(+) T cells. This model interprets melanoma immune context as a spectrum of tumor escape from immune control, and provides a snapshot upon which interpretation of checkpoint blockade inhibitor (CBI) therapy responses can be built.

The chemistry of benzoquinone (BQ) on TiO2(110) was examined using temperature programmed desorption (TPD), electron energy loss spectroscopy (EELS) and Auger electron spectroscopy (AES). BQ adsorbs mostly molecularly on the clean surface, although EELS demonstrates that electrons from surface Ti3+ at oxygen vacancy sites (VO) are readily oxidized by the molecule. In contrast, when the surface is covered with water, subsequently adsorbed BQ molecules that scavenge surface electrons also abstract H from surface bridging OH (OHb) groups to form hydroquinone (HQ), which desorbs at ~450 K. The ability of BQ to scavenge electrons from TiO2 also accounts for the formation of coupling products that accumulate on the surface as very stable carbon deposits, likely as polymerized aromatics.

BACKGROUND:Sentinel-lymph-node biopsy is associated with increased melanoma-specific survival (i.e., survival until death from melanoma) among patients with node-positive intermediate-thickness melanomas (1.2 to 3.5 mm). The value of completion lymph-node dissection for patients with sentinel-node metastases is not clear. METHODS:In an international trial, we randomly assigned patients with sentinel-node metastases detected by means of standard pathological assessment or a multimarker molecular assay to immediate completion lymph-node dissection (dissection group) or nodal observation with ultrasonography (observation group). The primary end point was melanoma-specific survival. Secondary end points included disease-free survival and the cumulative rate of nonsentinel-node metastasis. RESULTS:Immediate completion lymph-node dissection was not associated with increased melanoma-specific survival among 1934 patients with data that could be evaluated in an intention-to-treat analysis or among 1755 patients in the per-protocol analysis. In the per-protocol analysis, the mean (±SE) 3-year rate of melanoma-specific survival was similar in the dissection group and the observation group (86±1.3% and 86±1.2%, respectively; P=0.42 by the log-rank test) at a median follow-up of 43 months. The rate of disease-free survival was slightly higher in the dissection group than in the observation group (68±1.7% and 63±1.7%, respectively; P=0.05 by the log-rank test) at 3 years, based on an increased rate of disease control in the regional nodes at 3 years (92±1.0% vs. 77±1.5%; P<0.001 by the log-rank test); these results must be interpreted with caution. Nonsentinel-node metastases, identified in 11.5% of the patients in the dissection group, were a strong, independent prognostic factor for recurrence (hazard ratio, 1.78; P=0.005). Lymphedema was observed in 24.1% of the patients in the dissection group and in 6.3% of those in the observation group. CONCLUSIONS:Immediate completion lymph-node dissection increased the rate of regional disease control and provided prognostic information but did not increase melanoma-specific survival among patients with melanoma and sentinel-node metastases. (Funded by the National Cancer Institute and others; MSLT-II ClinicalTrials.gov number, NCT00297895 .).

Using temperature programmed desorption (TPD) and photon-stimulated desorption (PSD), we show that coadsorbates of varying binding energies on the rutile TiO2(110) surface exert a commensurate inhibiting influence on the hole-mediated photodesorption of adsorbed O2. A variety of coadsorbates (Ar, Kr, Xe, N2, CO, CO2, CH4, N2O, acetone, methanol or water) were shown to quench O2 photoactivity, with the extent correlating with the coadsorbate's gas phase basicity, which in turn determines the strength of the coadsorbate–Ti4+ bond. Coadsorbed rare gases inhibited the photodesorption of O2 by ~10–25%, whereas strongly bound species (water, methanol, and acetone) nearly completely inhibited O2 PSD. We suggest that coadsorption of these molecules inhibit the arrival probability of holes to the surface. Band-bending effects, which vary with the extent of charge transfer between the coadsorbate and the TiO2(110) surface, are not expected to be significant in the cases of the rare gases and physisorbed species. These results indicate that neutral coadsorbates can exert a significant influence on charge transfer events by altering the interfacial dipole in the vicinity of the target molecule.