Marianne Frostvik Stolt

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 O6‐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.

HSP60 predicts survival in advanced serous ovarian cancer.

Objective Heat shock protein 60 (HSP60) plays an essential role in malignant cell survival. We evaluated the prognostic and treatment predictive value of HSP60 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 fulfilled the eligibility criteria, that is, International Federation of Gynecology and Obstetrics stage IIC-IV, serous/endometrioid tumors, platinum-based chemotherapy, and specimens with 50% tumor cells or greater. Heat shock protein 60 mRNA and protein expression was determined by real-time polymerase chain reaction and immunohistochemistry. We estimated the association between HSP60 and overall survival (OS) and platinum-free interval (PFI) by Cox proportional hazards models and its relationship with treatment response by Fisher’s exact test. Median follow-up was 60 months. Results High HSP60 mRNA expression was associated with shorter OS (hazard ratio [HR], 3.4; 95% confidence interval [CI], 1.3–8.5) and PFI (HR, 3.3; 95% CI, 1.5–7.2). Likewise, high HSP60 protein expression was associated with shorter OS (HR, 3.2; 95% CI, 1.5–7.1) and PFI (HR, 2.6; 95% CI, 1.3–5.3). Median survival for patients with high HSP60 protein expression was 31 months compared with 55 months for low expression cases (P = 0.016). The impact on OS and PFI was even stronger in the subgroup of grade 3 serous tumors. All patients with low HSP60 levels responded to first-line chemotherapy. Conclusion Heat shock protein 60 may identify groups of advanced serous ovarian cancer with different prognosis and treatment response.

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.

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. Clin Cancer Res; 22(1); 146–57. ©2015 AACR.

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 β-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.

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.

Abstract B28: PTENpg1 antisense RNA mediates PTEN suppression in vemurafenib resistance and predicts clinical outcome in melanoma patients

Approximately 50% of cutaneous melanomas carry activating mutations in the serine/threonine protein kinase BRAF. Vemurafenib is a highly potent inhibitor of BRAFV600E and prolongs survival in ~80% of melanoma patients carrying the mutation. Unfortunately, almost all patients develop resistance within 6-8 months of starting treatment. The molecular mechanism explaining this scenario is largely unclear, but loss of PTEN expression, has been suggested. In this study we reveal a role for the PTEN pseudogene encoded antisense RNA (PTENpg1 asRNA) in epigenetic suppression of PTEN in melanoma cell lines resistant to vemurafenib. Resistant cell lines showed increased PTENpg1 asRNA expression while PTEN expression was suppressed. ChIP analysis showed enrichment of the histone modification enzyme EZH2 and subsequent enrichment of H3K27me3 at the PTEN promoter in the resistant cell line. Total cellular levels of EZH2, DNMT3a and H3k27me3 were, however, unaffected thus indicating specific recruitment of these factors to the PTEN promoter in the resistant cells by a mechanism that is dictated by the increased expression of PTENpg1 asRNA. In addition, we show that PTEN is reactivated by the depletion of EZH2 and DNMT3a and that this reactivation of PTEN re-sensitizes the cells to vemurafenib treatment. Finally, we show that PTENpg1 asRNA could be a promising prognostic marker of clinical outcome of melanoma patients. PTENpg1 asRNA expression levels were measured in samples from patients that had not received any oncological treatment and were classified with stage III melanomas. We found that high PTENpg1 asRNA expression correlates with short-term survival in melanoma patients. In conclusions, the PTENpg1 asRNA appears to be an important player in vemurafenib resistance and could be an attractive therapeutic target. In addition, PTENpg1 asRNA is potentially a promising prognostic marker for clinical outcome for melanoma patients. Citation Format: Linda Vidarsdottir, Alireza Azimi, Jason Serviss, Christian Ingvar, Goran Jonsson, Hakan Olsson, Marianne Frostvik Stolt, Johan Hansson, Suzanne Egyhazi Brage, Dan Grander, Per Johnsson. PTENpg1 antisense RNA mediates PTEN suppression in vemurafenib resistance and predicts clinical outcome in melanoma patients. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr B28.