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.

List of scientific papers

I. Johan Falkenius, Joakim Lundeberg, Hemming Johansson, Rainer Tuominen, Marianne Frostvik-Stolt, Johan Hansson, Suzanne Egyhazi Brage. High expression of glycolytic and pigment proteins is associated with worse clinical outcome in stage III melanoma. Melanoma Research. 2013 Dec; 23(6):452-60.
https://doi.org/10.1097/CMR.0000000000000027

II. Johan Falkenius, Hemming Johansson, Rainer Tuominen, Marianne Frostvik Stolt, Johan Hansson, Suzanne Egyhazi Brage. 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. BMC Cancer. 2017 Aug 29;17(1):584.
https://doi.org/10.1186/s12885-017-3577-x

III. Johan Falkenius, Johanna Keskitalo, Lena Kanter, Marianne Frostvik Stolt, Hemming Johansson, Veronica Höiom, Johan Hansson, Suzanne Egyhazi Brage. Prognostic impact of the proportion of BRAF mutated alleles, Ki67 expression, presence of TILs and extent of ulceration on recurrence-free survival in ulcerated primary cutaneous melanoma. [Submitted]

IV. Johan Falkenius, Marianne Frostvik Stolt, Lena Kanter, Hemming Johansson, Veronica Höiom, Johan Hansson, Suzanne Egyhazi Brage. Presence of TILs with expression of CD8 and FOXP3 as well as high expression of GAPDHS in tumour cells is associated with favourable clinical outcome in ulcerated primary cutaneous melanoma. [Manuscript]