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.

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.

MGMT promoter methylation is associated with temozolomide response and prolonged progression-free survival in disseminated cutaneous melanoma.

To investigate the predictive and prognostic value of O(6) -methylguanine DNA methyltransferase (MGMT) inactivation by analyses of promoter methylation in pretreatment tumor biopsies from patients with cutaneous melanoma treated with dacarbazine (DTIC) or temozolomide (TMZ) were performed. The patient cohorts consisted of Belgian and Swedish disseminated melanoma patients. Patients were subdivided into those receiving single-agent treatment with DTIC/TMZ (cohort S, n = 74) and those treated with combination chemotherapy including DTIC/TMZ (cohort C, n = 79). Median follow-up was 248 and 336 days for cohort S and cohort C, respectively. MGMT promoter methylation was assessed by three methods. The methylation-related transcriptional silencing of MGMT mRNA expression was assessed by real-time RT-PCR. Response to chemotherapy and progression-free survival (PFS) and overall survival were correlated to MGMT promoter methylation status. MGMT promoter methylation was detected in tumor biopsies from 21.5 % of the patients. MGMT mRNA was found to be significantly lower in tumors positive for MGMT promoter methylation compared to tumors without methylation in both treatment cohorts (p < 0.005). DTIC/TMZ therapy response rate was found to be significantly associated with MGMT promoter methylation in cohort S (p = 0.0005), but did not reach significance in cohort C (p = 0.16). Significantly longer PFS was observed among patients with MGMT promoter-methylated tumors (p = 0.002). Multivariate Cox regression analysis identified presence of MGMT promoter methylation as an independent variable associated with longer PFS. Together, this implies that MGMT promoter methylation is associated with response to single-agent DTIC/TMZ and longer PFS in disseminated cutaneous melanoma.

Metabolic markers and HSP60 in chemonaive serous solid ovarian cancer versus ascites.

OBJECTIVE: Metabolic pathway alterations in cancer are thought to be dependent upon tumor type-specific oncogenic activation and local nutrient and oxygen supply during disease progression. In serous ovarian cancer, the typical peritoneal spread of disease is caused by shedding of tumor cells into the abdominal cavity, often along with ascites formation. Not much is known about the metabolic features of these detached serous tumor cells. In this study, we investigate the messenger RNA (mRNA) expression of GAPDH (glycolytic glyceraldehyde 3-phosphate dehydrogenase) and PKM2 (pyruvate kinase isoform M2), ATP5B (mitochondrial ß-F1-ATPase), and heat shock protein 60 in matched serous solid tumor and corresponding ascites. MATERIALS/METHODS: Fresh samples from solid tumor and corresponding ascites were prospectively collected from 40 patients undergoing primary surgery for suspected advanced ovarian cancer. Of these, 25 met the study eligibility criteria, that is, stage IIC to IV disease of the serous (24) or endometrioid (1) subtype with solid and ascites specimens containing 50% or more tumor cells and with good quality and quantity mRNA yield. All but 2 patients (92%) had type II disease. GAPDH, PKM2, ATP5B, and HSP60 mRNA expressions were assessed by real-time polymerase chain reaction. For each marker, the mRNA expression in solid tumor was pairwise compared with the corresponding expression in ascites using the Wilcoxon matched pairs signed rank sum test. RESULTS: In contrast to our hypothesis, the mRNA expression of analyzed metabolic markers and HSP60 did not significantly differ between matched solid tumor and malignant ascites. CONCLUSIONS: Our results indicate that further expression changes in genes related to glycolysis or oxidative phosphorylation are not a prerequisite for serous cancer cell survival after detachment.

Metabolic markers GAPDH, PKM2, ATP5B and BEC-index in advanced serous ovarian cancer.

BACKGROUND: A deregulated energy metabolism is a hallmark of malignant disease that offers possible future targets for treatment. We investigated the prognostic value of the glycolytic enzymes glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and pyruvate kinase type M2 (PKM2), mitochondrial ß-F1-ATPase (ATP5B) and the bioenergetic cellular (BEC) index in advanced ovarian cancer. METHODS: Fresh tumor samples were prospectively collected from 123 patients undergoing primary surgery for suspected advanced ovarian cancer. Of these, 57 met the eligibility criteria; stage IIC-IV, serous or endometrioid subtype, specimens containing ≥ 50% tumor cells and patients receiving platinum-based chemotherapy. An adequate amount of mRNA could be extracted in all but one case, with a resultant study population of 56 patients. Eighty-six percent of cases had serous tumors, and 93% were grade 2-3. GAPDH, PKM2 and ATP5B mRNA- and protein expression was assessed by real-time PCR and immunohistochemistry. We estimated the association with platinum-free interval (PFI) and overall survival (OS) by Cox proportional hazards models. Median follow-up was 60 months. RESULTS: High GAPDH mRNA levels (HR 2.1, 95% CI 1.0-4.5) and low BEC-index (HR 0.47, 95% CI 0.23-0.95) were both independently associated with shorter PFI. Median PFI for patients with high GAPDH mRNA was 5.0 months compared to 10.1 months for low expression cases (p = 0.031). Similarly, median PFI for patients with low BEC-index based on mRNA was 5.3 months compared to 9.8 months for high BEC-index cases (p = 0.028). CONCLUSIONS: High GAPDH or low BEC-index mRNA expression indicate early disease progression in advanced serous ovarian cancer.

PTENP1-AS contributes to BRAF inhibitor resistance and is associated with adverse clinical outcome in stage III melanoma.

BRAF inhibitors (BRAFi) selectively target oncogenic BRAFV600E/K and are effective in 80% of advanced cutaneous malignant melanoma cases carrying the V600 mutation. However, the development of drug resistance limits their clinical efficacy. Better characterization of the underlying molecular processes is needed to further improve treatments. We previously demonstrated that transcription of PTEN is negatively regulated by the PTEN pseudogene antisense RNA, PTENP1-AS, and here we investigated the impact of this transcript on clinical outcome and BRAFi resistance in melanoma. We observed that increased expression levels of PTENP1-AS in BRAFi resistant cells associated with enrichment of EZH2 and H3K27me3 at the PTEN promoter, consequently reducing the expression levels of PTEN. Further, we showed that targeting of the PTENP1-AS transcript sensitized resistant cells to BRAFi treatment and that high expression of PTENP1-AS in stage III melanoma correlated with poor survival. Collectively, the data presented here show that PTENP1-AS is a promising target for re-sensitizing cells to BRAFi and also a possible prognostic marker for clinical outcome in stage III melanoma.

Silencing FLI or targeting CD13/ANPEP lead to dephosphorylation of EPHA2, a mediator of BRAF inhibitor resistance, and induce growth arrest or apoptosis in melanoma cells.

A majority of patients with BRAF-mutated metastatic melanoma respond to therapy with BRAF inhibitors (BRAFi), but relapses are common owing to acquired resistance. To unravel BRAFi resistance mechanisms we have performed gene expression and mass spectrometry based proteome profiling of the sensitive parental A375 BRAF V600E-mutated human melanoma cell line and of daughter cell lines with induced BRAFi resistance. Increased expression of two novel resistance candidates, aminopeptidase-N (CD13/ANPEP) and ETS transcription factor FLI1 was observed in the BRAFi-resistant daughter cell lines. In addition, increased levels of the previously reported resistance mediators, receptor tyrosine kinase ephrine receptor A2 (EPHA2) and the hepatocyte growth factor receptor MET were also identified. The expression of these proteins was assessed in matched tumor samples from melanoma patients obtained before BRAFi and after disease progression. MET was overexpressed in all progression samples while the expression of the other candidates varied between the individual patients. Targeting CD13/ANPEP by a blocking antibody induced apoptosis in both parental A375- and BRAFi-resistant daughter cells as well as in melanoma cells with intrinsic BRAFi resistance and led to dephosphorylation of EPHA2 on S897, previously demonstrated to cause inhibition of the migratory capacity. AKT and RSK, both reported to induce EPHA2 S897 phosphorylation, were also dephosphorylated after inhibition of CD13/ANPEP. FLI1 silencing also caused decreases in EPHA2 S897 phosphorylation and in total MET protein expression. In addition, silencing of FLI1 sensitized the resistant cells to BRAFi. Furthermore, we show that BRAFi in combination with the multi kinase inhibitor dasatinib can abrogate BRAFi resistance and decrease both EPHA2 S897 phosphorylation and total FLI1 protein expression. This is the first report presenting CD13/ANPEP and FLI1 as important mediators of resistance to BRAF inhibition with potential as drug targets in BRAFi refractory melanoma.

Transcriptional Profiling of Breast Cancer Metastases Identifies Liver Metastasis-Selective Genes Associated with Adverse Outcome in Luminal A Primary Breast Cancer.

PURPOSE: The complete molecular basis of the organ-specificity of metastasis is elusive. This study aimed to provide an independent characterization of the transcriptional landscape of breast cancer metastases with the specific objective to identify liver metastasis-selective genes of prognostic importance following primary tumor diagnosis. EXPERIMENTAL DESIGN: A cohort of 304 women with advanced breast cancer was studied. Associations between the site of recurrence and clinicopathologic features were investigated. Fine-needle aspirates of metastases (n = 91) were subjected to whole-genome transcriptional profiling. Liver metastasis-selective genes were identified by significance analysis of microarray (SAM) analyses and independently validated in external datasets. Finally, the prognostic relevance of the liver metastasis-selective genes in primary breast cancer was tested. RESULTS: Liver relapse was associated with estrogen receptor (ER) expression (P = 0.002), luminal B subtype (P = 0.01), and was prognostic for an inferior postrelapse survival (P = 0.01). The major variation in the transcriptional landscape of metastases was also associated with ER expression and molecular subtype. However, liver metastases displayed unique transcriptional fingerprints, characterized by downregulation of extracellular matrix (i.e., stromal) genes. Importantly, we identified a 17-gene liver metastasis-selective signature, which was significantly and independently prognostic for shorter relapse-free (P < 0.001) and overall (P = 0.001) survival in ER-positive tumors. Remarkably, this signature remained independently prognostic for shorter relapse-free survival (P = 0.001) among luminal A tumors. CONCLUSIONS: Extracellular matrix (stromal) genes can be used to partition breast cancer by site of relapse and may be used to further refine prognostication in ER positive primary breast cancer.

Contrasting breast cancer molecular subtypes across serial tumor progression stages: biological and prognostic implications.

The relevance of the intrinsic subtypes for clinical management of metastatic breast cancer is not comprehensively established. We aimed to evaluate the prevalence and prognostic significance of drifts in tumor molecular subtypes during breast cancer progression. A well-annotated cohort of 304 women with advanced breast cancer was studied. Tissue microarrays of primary tumors and synchronous lymph node metastases were constructed. Conventional biomarkers were centrally assessed and molecular subtypes were assigned following the 2013 St Gallen guidelines. Fine-needle aspirates of asynchronous metastases were transcriptionally profiled and subtyped using PAM50. Discordant expression of individual biomarkers and molecular subtypes was observed during tumor progression. Primary luminal-like tumors were relatively unstable, frequently adopting a more aggressive subtype in the metastases. Notably, loss of ER expression and a luminal to non-luminal subtype conversion was associated with an inferior post-recurrence survival. In addition, ER and molecular subtype assessed at all tumor progression stages were independent prognostic factors for post-recurrence breast cancer mortality in multivariable analyses. Our results demonstrate that drifts in tumor molecular subtypes may occur during tumor progression, conferring adverse consequences on outcome following breast cancer relapse.

Real-time RT-PCR for the determination of topoisomerase II mRNA level in leukaemic cells.

We developed a real-time RT-PCR assay for the quantification of topoisomerase II (topo II) mRNA level. It was applied on peripheral leukaemic cells from 23 patients with acute myelogenous leukaemia (AML) and 23 with chronic lymphocytic leukaemia (CLL). RNA template dilutions from 0.25 to 25ng per reaction were used as standard curves for topo IIalpha, beta and the internal control 18S rRNA. About 57% (26/46) and 26% (12/46) of the specimens had detectable topo IIbeta and alpha mRNA, respectively. The correlation between these two factors was rho=0.7 and P=0.0001. No relationship between topo IIalpha or beta mRNA level and response to chemotherapy was found in AML patients (n=19 assessable for response). Our method is rapid and convenient for quantification of topo IIalpha and beta mRNA levels, and could be suitable for investigation in a larger population.

High expression of glycolytic and pigment proteins is associated with worse clinical outcome in stage III melanoma.

There are insufficient numbers of prognostic factors available for prediction of clinical outcome in patients with stage III malignant cutaneous melanoma, even when known adverse pathological risk factors, such as macrometastasis, number of lymph node metastases, and ulceration are taken into consideration. The aim of this study was therefore to identify additional prognostic factors to better predict patients with a high risk of relapse, thus enabling us to better determine the need for adjuvant treatment in stage III disease. An RNA oligonucleotide microarray study was performed on first regional lymph node metastases in 42 patients with stage III melanoma: 23 patients with short-term survival (≤ 13 months) and 19 with long-term survival (≥ 60 months), to identify genes associated with clinical outcome. Candidate genes were validated by real-time PCR and immunohistochemical analysis. Several gene ontology (GO) categories were highly significantly differentially expressed including glycolysis (GO: 0006096; P<0.001) and the pigment biosynthetic process (GO: 0046148; P<0.001), in which overexpression was associated with short-disease-specific survival. Three overexpressed glycolytic genes, GAPDHS, GAPDH, and PKM2, and two pigment-related genes, TYRP1 and OCA2, were selected for validation. A significant difference in GAPDHS protein expression between short- and long-term survivors (P=0.021) and a trend for PKM2 (P=0.093) was observed in univariate analysis. Positive expression of at least two of four proteins (GAPDHS, GAPDH, PKM2, TYRP1) in immunohistochemical analysis was found to be an independent adverse prognostic factor for disease-specific survival (P=0.011). Our results indicate that this prognostic panel in combination with established risk factors may contribute to an improved prediction of patients with a high risk of relapse.

Association of MITF and other melanosome-related proteins with chemoresistance in melanoma tumors and cell lines.

Previous studies in cell lines have suggested a role for melanosomes and related protein trafficking pathways in melanoma drug response. We have investigated the expression of six proteins related to melanosomes and melanogenesis (MITF, GPR143, gp100/PMEL, MLANA, TYRP1, and RAB27A) in pretreatment metastases from melanoma patients (n = 52) with different response to dacarbazine/temozolomide. Microphthalmia-associated transcription factor (MITF) and G-protein coupled receptor 143 (GPR143) showed significantly higher expression in nonresponders compared with responders. The premelanosome protein (gp100/PMEL) has been indicated previously in resistance to cisplatin in melanoma cells, but the expression levels of gp100/PMEL showed no association with response to dacarbazine/temozolomide in our clinical material. We also investigated the effects on chemosensitivity of siRNA inhibition of gp100/PMEL in the MNT-1 melanoma cell line. As expected from the study of the tumor material, no effect was detected with respect to response to temozolomide. However, knockdown of gp100/PMEL sensitized the cells to both paclitaxel and cisplatin. Overall, our results suggest that MITF, and several MITF-regulated factors, are associated with resistance to chemotherapy in melanoma and that different MITF targets can be of importance for different drugs.