Discovery and validation of prognostic markers for cutaneous melanoma

During the last years there has been an alarming increase in incidence of cutaneous melanoma (CMM) which is the deadliest form of skin cancer. There has been a paradigm shift in treatment of disseminated stage IV disease since the introduction of novel systemic therapies with significant survival benefits. Recently adjuvant therapy has been introduced for stage III CMM. Hence new options to prevent relapses for high-risk categories in stage I-III CMM have appeared. However, the currently used prognostic factors are not enough to identify all high-risk patients. Improved prognostic tools are required to define patient groups with an increased risk of developing metastatic disease who should be offered adjuvant therapies. The overall aim of these studies was to identify and validate prognostic markers for CMM using microarray, qPCR, DNA sequencing and immunohistochemistry. In papers I-II a tumor set of regional lymph node metastases (n=42) from two stage III CMM patient groups with extremely different disease specific survival after lymph node dissection was used, ≤13months respectively ≥60 months. In papers III-IV a consecutive cohort of ulcerated primary stage I-II CMM tumors (n=71) was used. In paper I gene expression profiling of tumors from stage III CMM patients identified glycolysis and pigment related gene ontology (GO) categories among the top five GO categories in which overexpression was associated with short survival. GAPDHS was identified as a novel candidate prognostic factor in CMM. Further validation was done on selected genes from these GO categories, three glycolytic genes (GAPDHS, GAPDH and PKM2) and one pigment-related gene (TYRP1), at the protein level. High expression of at least two out of four proteins was found to be of independent adverse prognostic significance. In paper II a prognostic biomarker panel was found to identify patients with a favorable prognosis in stage III CMM. By combining high expression of CD8+ and FOXP3+ immune cells, low expression of Ki67 and BRAF wildtype status a significant independent association with favorable clinical outcome was found, with the best result observed when three out of four factors were present. When adjusting for the previously identified panel in paper I the result remained significant. In paper III there was a significant association with longer recurrence-free survival (RFS) in ulcerated stage I-II CMM when at least two out of four factors were present in a panel of BRAF wildtype/low proportion of BRAF mutated alleles, minor ulceration, low proliferation and high presence of TILs, in the multivariate analysis adjusted for Breslow thickness. In paper IV the presence of TILs was found to be a stronger predictor for RFS in combination with the expression of CD8, FOXP3 and GAPDHS. When at least three of these four factors were present/expressed a significant association with longer RFS was found in multivariate analyses. GAPDHS has been discovered to be a potential prognostic factor in both paper I and IV but may have different functions in stage I-III CMM, a so called moonlighting protein. This thesis highlights the strength of using a panel of biomarkers instead of using a single biomarker to identify patients with high risk for relapse in stage I-III CMM

Presence of immune cells, low tumor proliferation and wild type BRAF mutation status is associated with a favourable clinical outcome in stage III cutaneous melanoma

Abstract Background The variable prognosis in stage III cutaneous melanoma is partially due to unknown prognostic factors. Improved prognostic tools are required to define patients with an increased risk of developing metastatic disease who might benefit from adjuvant therapies. The aim was to examine if cellular immune markers in association with tumor proliferation rate and BRAF mutation status have an impact on prognosis in stage III melanoma. Methods We have used two sets of case series with stage III disease: 23 patients with short survival (≤ 13 months) and 19 patients with long survival (≥ 60 months). Lymph node metastases were analyzed for Ki67, CD8 and FOXP3 protein expression using immunohistochemistry. BRAF mutation status was analyzed in a previous study on the same samples. Results Low tumor proliferation rate was significantly associated with a better prognosis (p = 0.013). Presence of FOXP3+ T cells was not correlated to adverse clinical outcome. A highly significant trend for a longer survival was found in the presence of an increasing number of markers; CD8+ and FOXP3+ T cells, low tumor proliferation and BRAF wildtype status (p = 0.003). Presence of at least three of these four markers was found to be an independent favorable prognostic factor (OR 19.4, 95% CI 1.9-197, p = 0.012), when adjusting for ulceration and number of lymph node metastases. Proliferation alone remained significant in multivariate analyses (OR 26.1, 95% CI 2.0-344, p = 0.013) but with a wider confidence interval. This panel still remained independent when also adjusting for a previously identified prognostic glycolytic-pigment panel. Conclusions We have demonstrated that presence of immune cells in association with tumor proliferation and BRAF mutation status may further contribute to identify stage III melanoma patients with high risk of relapse

Survival after introduction of adjuvant treatment in stage III melanoma: a nationwide registry-based study

Background Adjuvant treatments with PD-1 and BRAF+MEK inhibitors statistically significantly prolong recurrence-free survival in stage III cutaneous melanoma. Yet, the effect on overall survival is still unclear. Based on recurrence-free survival outcomes, these treatments have been approved and widely implemented. The treatments have considerable side effects and costs, and overall survival effect remains a highly anticipated outcome. Methods Clinical and histopathological parameters were obtained from the Swedish Melanoma Registry for patients diagnosed with stage III melanoma between 2016 and 2020. The patients were divided depending on if they were diagnosed before or from July 2018, based on the timepoint when adjuvant treatment was introduced in Sweden. Patients were followed up until the end of 2021. In this cohort study, melanoma-specific and overall survival were calculated using the Kaplan-Meier method and Cox-regression analyses. Results There were 1371 patients diagnosed with stage III primary melanoma in Sweden in 2016-2020. The 2-year overall survival rates, comparing the 634 patients in the precohort and the 737 in the postcohort, were 84.3% (95% confidence interval [CI] = 81.4% to 87.3%) and 86.1% (95% CI = 83.4% to 89.0%), respectively, with an adjusted hazard ratio of 0.91 (95% CI = 0.70 to 1.19, P = .51). Further, no statistically significant overall or melanoma-specific survival differences were seen when comparing the precohort and the postcohort in different subgroups for age, sex, or tumor characteristics. Conclusions In this nationwide population-based and registry-based study, no survival benefit was detected in patients diagnosed before or after the implementation of adjuvant treatment in stage III melanoma. These findings encourage a careful assessment of the current recommendations on adjuvant treatment.Funding Agencies|Regional Cancer Centers (RCC) in Sweden; Swedish Cancer society [20 0156 F, 21 1486 Pj]; Region Stockholm [20200638]; Cancer Research Funds of Radiumhemmet [194092, 224023]; [2021-YF0069]</p

A phase Ia/Ib trial of the DNA-dependent protein kinase inhibitor (DNA-PKi) M3814 in combination with radiotherapy in patients with advanced solid tumors

e14048 Background: DNA-PK, with its protein subunits, Ku70 and Ku80, regulates one of the major pathways responsible for repair of double-strand breaks in DNA that are induced by radiotherapy (RT) and some chemotherapeutic agents (CT). Therefore, the combined strategy of RT with a DNA-PKi is promising. The purpose of this phase I trial is to explore the safety, tolerability, pharmacokinetic (PK) profile, and clinical activity of M3814 administered together with RT and chemo-RT (CRT). Methods: Patients (pts) with tumors or metastasis in the head and neck region or thorax in need of palliative RT (10 x 3 Gy) are eligible for the dose escalation part Ia, with a starting dose of 100 mg once daily. Dose escalation decisions are aided by the Bayesian logistic regression model with overdose control. Dose-limiting toxicity (DLT) is evaluated up to 3 weeks after RT. Rich PK sampling is taken during treatment. Tumor evaluation is performed every 6 weeks up to 6 months and every third month thereafter. Results: As of January 2017, 7 pts had been enrolled into the part Ia dose escalation arm and treated with 100 mg daily. The most frequent adverse events (AEs) were fatigue, constipation, decreased appetite, dry mouth, dysphagia, headache, oral pain, radiation skin injury, and mucositis in more than 1 pt (20%). No pts discontinued due to AE. Two episodes of grade 3 mucositis lasting > 7 days were reported; one of these was classified as a DLT, and both pts recovered without sequelae. Two of 7 pts enrolled had local tumor control at +300 days. PK analysis demonstrated high exposure variability. Conclusions: M3814 at the 100 mg daily dose was tolerable as palliative treatment and dose escalation is currently at 200 mg. In part Ib of the study, two separate dose escalation arms will enroll pts with either head and neck squamous cell carcinoma (SCCHN; US only) or non-small cell lung cancer (EU and US) to receive CRT 2 Gy x 30-35 over 6-7 weeks with curative intent; the SCCHN cohort is now open for enrollment. For these two cohorts, the DLT window will be 12 weeks. Clinical trial information: NCT02516813