Suzanne Egyhazi

Gene expression profiling spares early breast cancer patients from adjuvant therapy: derived and validated in two population-based cohorts

IntroductionAdjuvant breast cancer therapy significantly improves survival, but overtreatment and undertreatment are major problems. Breast cancer expression profiling has so far mainly been used to identify women with a poor prognosis as candidates for adjuvant therapy but without demonstrated value for therapy prediction.MethodsWe obtained the gene expression profiles of 159 population-derived breast cancer patients, and used hierarchical clustering to identify the signature associated with prognosis and impact of adjuvant therapies, defined as distant metastasis or death within 5 years. Independent datasets of 76 treated population-derived Swedish patients, 135 untreated population-derived Swedish patients and 78 Dutch patients were used for validation. The inclusion and exclusion criteria for the studies of population-derived Swedish patients were defined.ResultsAmong the 159 patients, a subset of 64 genes was found to give an optimal separation of patients with good and poor outcomes. Hierarchical clustering revealed three subgroups: patients who did well with therapy, patients who did well without therapy, and patients that failed to benefit from given therapy. The expression profile gave significantly better prognostication (odds ratio, 4.19; P = 0.007) (breast cancer end-points odds ratio, 10.64) compared with the Elston–Ellis histological grading (odds ratio of grade 2 vs 1 and grade 3 vs 1, 2.81 and 3.32 respectively; P = 0.24 and 0.16), tumor stage (odds ratio of stage 2 vs 1 and stage 3 vs 1, 1.11 and 1.28; P = 0.83 and 0.68) and age (odds ratio, 0.11; P = 0.55). The risk groups were consistent and validated in the independent Swedish and Dutch data sets used with 211 and 78 patients, respectively.ConclusionWe have identified discriminatory gene expression signatures working both on untreated and systematically treated primary breast cancer patients with the potential to spare them from adjuvant therapy.

Features associated with germline CDKN2A mutations: a GenoMEL study of melanoma-prone families from three continents

Background: The major factors individually reported to be associated with an increased frequency of CDKN2A mutations are increased number of patients with melanoma in a family, early age at melanoma diagnosis, and family members with multiple primary melanomas (MPM) or pancreatic cancer. Methods: These four features were examined in 385 families with ⩾3 patients with melanoma pooled by 17 GenoMEL groups, and these attributes were compared across continents. Results: Overall, 39% of families had CDKN2A mutations ranging from 20% (32/162) in Australia to 45% (29/65) in North America to 57% (89/157) in Europe. All four features in each group, except pancreatic cancer in Australia (p = 0.38), individually showed significant associations with CDKN2A mutations, but the effects varied widely across continents. Multivariate examination also showed different predictors of mutation risk across continents. In Australian families, ⩾2 patients with MPM, median age at melanoma diagnosis ⩽40 years and ⩾6 patients with melanoma in a family jointly predicted the mutation risk. In European families, all four factors concurrently predicted the risk, but with less stringent criteria than in Australia. In North American families, only ⩾1 patient with MPM and age at diagnosis ⩽40 years simultaneously predicted the mutation risk. Conclusions: The variation in CDKN2A mutations for the four features across continents is consistent with the lower melanoma incidence rates in Europe and higher rates of sporadic melanoma in Australia. The lack of a pancreatic cancer–CDKN2A mutation relationship in Australia probably reflects the divergent spectrum of mutations in families from Australia versus those from North America and Europe. GenoMEL is exploring candidate host, genetic and/or environmental risk factors to better understand the variation observed.

Increased cyclin D1 expression can mediate BRAF inhibitor resistance in BRAF V600E–mutated melanomas

Recent studies have shown that there is a considerable heterogeneity in the response of melanoma cell lines to MEK and BRAF inhibitors. In the current study, we address whether dysregulation of cyclin-dependent kinase 4 (CDK4) and/or cyclin D1 contribute to the BRAF inhibitor resistance of melanoma cells. Mutational screening identified a panel of melanoma cell lines that harbored both a BRAF V600E mutation and a CDK4 mutation: K22Q (1205Lu), R24C (WM39, WM46, and SK-Mel-28), and R24L (WM902B). Pharmacologic studies showed that the presence of a CDK4 mutation did not alter the sensitivity of these cell lines to the BRAF inhibitor. The only cell line with significant BRAF inhibitor resistance was found to harbor both a CDK4 mutation and a CCND1 amplification. Array comparative genomic hybridization analysis showed that CCND1 was amplified in 17% of BRAF V600E–mutated human metastatic melanoma samples, indicating the clinical relevance of this finding. As the levels of CCND1 amplification in cell lines are lower than those seen in clinical specimens, we overexpressed cyclin D1 alone and in the presence of CDK4 in a drug-sensitive melanoma line. Cyclin D1 overexpression alone increased resistance and this was enhanced when cyclin D1 and CDK4 were concurrently overexpressed. In conclusion, increased levels of cyclin D1, resulting from genomic amplification, may contribute to the BRAF inhibitor resistance of BRAF V600E–mutated melanomas, particularly when found in the context of a CDK4 mutation/overexpression. [Mol Cancer Ther 2008;7(9):2876–83]

MicroRNA Expression Profiles Associated with Mutational Status and Survival in Malignant Melanoma

Malignant cutaneous melanoma is a highly aggressive form of skin cancer. Despite improvements in early melanoma diagnosis, the 5-year survival rate remains low in advanced disease. Therefore, novel biomarkers are urgently needed to devise new means of detection and treatment. In this study, we aimed to improve our understanding of microRNA (miRNA) deregulation in melanoma development and their impact on patient survival. Global miRNA expression profiles of a set of melanoma lymph node metastases, melanoma cell lines, and melanocyte cultures were determined using Agilent array. Deregulated miRNAs were evaluated in relation with clinical characteristics, patient survival, and mutational status for BRAF and NRAS. Several miRNAs were differentially expressed between melanocytes and melanomas as well as melanoma cell lines. In melanomas, miR-193a, miR-338, and miR-565 were underexpressed in cases with a BRAF mutation. Furthermore, low expression of miR-191 and high expression of miR-193b were associated with poor melanoma-specific survival. In conclusion, our findings show miRNA dysregulation in malignant melanoma and its relation to established molecular backgrounds of BRAF and NRAS oncogenic mutations. The identification of an miRNA classifier for poor survival may lead to the development of miRNA detection as a complementary prognostic tool in clinical practice.

O6-methylguanine-DNA-methyltransferase expression and gene polymorphisms in relation to chemotherapeutic response in metastatic melanoma

In a retrospective study, O6-methylguanine-DNA-methyltransferase (MGMT) expression was analysed by immunohistochemistry using monoclonal human anti-MGMT antibody in melanoma metastases in patients receiving dacarbazine (DTIC) as single-drug therapy or as part of combination chemotherapy with DTIC–vindesine or DTIC–vindesine–cisplatin. The correlation of MGMT expression levels with clinical response to chemotherapy was investigated in 79 patients with metastatic melanoma. There was an inverse relationship between MGMT expression and clinical response to DTIC-based chemotherapy (P=0.05). Polymorphisms in the coding region of the MGMT gene were also investigated in tumours from 52 melanoma patients by PCR/SSCP and nucleotide sequence analyses. Single-nucleotide polymorphisms (SNPs) in exon 3 (L53L and L84F) and in exon 5 (I143V/K178R) were identified. There were no differences in the frequencies of these polymorphisms between these melanoma patients and patients with familial melanoma or healthy Swedish individuals. Functional analysis of variants MGMT-I143V and -I143V/K178R was performed by in vitro mutagenesis in Escherichia coli. There was no evidence that these variants decreased the MGMT DNA repair activity compared to the wild-type protein. All melanoma patients with the MGMT 53/84 polymorphism except one had tumours with high MGMT expression. There was no significant correlation between any of the MGMT polymorphisms and clinical response to chemotherapy, although an indication of a lower response rate in patients with SNPs in exon 5 was obtained. Thus, MGMT expression appears to be more related to response to chemotherapy than MGMT polymorphisms in patients with metastatic melanoma.

Biallelic deletions in INK4 in cutaneous melanoma are common and associated with decreased survival.

Purpose: Both the retinoblastoma and p53 pathways are often genetically altered in human cancers and their complex regulation is in part mediated by the three gene products p16, p14ARF, and p15 of the INK4 locus on chromosome 9p21. Partial or complete biallelic deletions of the INK4 locus have been recognized in a variety of malignant tumors, including malignant melanoma. We have in the present study measured the frequency of INK4 deletions in a large number of melanoma metastases and determined their association with clinicopathologic variables and survival data. Experimental Design: Quantitative real-time PCR, as well as fluorescence-based fragment analysis, has been used to perform measurements of the relative allelic concentrations of the INK4 genes in 112 human melanoma tumor samples from 86 patients. Results: Thirty-eight of 86 melanoma patients (44%) had metastases with biallelic losses in INK4. Ten of 20 patients with multiple metastases showed similar deletion patterns in all analyzed tumors. There was no significant association between any of the clinicopathologic variables and loss of INK4. However, loss of INK4 had an adverse effect on median survival from time of diagnosis. Patients with tumors with diploid INK4 had a median survival of 142 months, whereas those with monoallelic or biallelic loss in INK4 had a median survival of only 47 months (P = 0.006). Conclusions: Our results point to homozygous deletions in the INK4 region as being one of the most common genetic alterations in malignant cutaneous melanoma. INK4 deletions are associated with an adverse prognosis.

Coexisting NRAS and BRAF Mutations in Primary Familial Melanomas with Specific CDKN2A Germline Alterations

To the Editor Germline aberrations in the CDKN2A gene are observed in some melanoma-prone families and represent high penetrance mutations (Hussussian et al., 1994; Kamb et al., 1994). INK4A (p16) and ARF (p14) are two distinct proteins encoded by the CDKN2A locus. Loss of INK4A has been associated with unrestricted cell cycle progression through retinoblastoma (RB) protein inactivation, while loss of ARF has been linked to p53 inactivation with subsequent malfunction in cell cycle regulation, apoptosis and DNA repair (Chin et al., 2006). The NRAS and BRAF genes are commonly mutated in sporadic primary cutaneous melanomas, with mutation frequencies between 4–50% (Platz et al., 2008) and 25–80% (Platz et al., 2008), respectively. Codon 61 is the most common position of NRAS alterations in melanoma, with frequent glutamine changes to either lysine, Q61K (c.181C>A), or arginine, Q61R (c.182A>G) (Omholt et al., 2002). Mutations in this residue lock the Ras protein in the GTP-bound state with subsequent continuous activation of its downstream effectors (Platz et al., 2008) through the Ras-Raf-MEK-ERK and Ras-PI3K-Akt pathways. Approximately 90% of reported BRAF mutations occur at residue 600, which is located in the activation domain of this kinase (Thomas, 2006). Current results from melanoma cohorts show that mutations in these genes are almost always mutually exclusive (Edlundh-Rose et al., 2006; Omholt et al., 2003; Platz et al., 2008). Moreover, a high rate of BRAF mutation is found also in nevi, suggesting a role in early stages of the neoplastic process (Pollock et al., 2003). Previously, a high frequency of NRAS mutations (95%) has been reported in Swedish familial melanoma cases with germline CDKN2A alterations (Eskandarpour et al., 2003). The association of BRAF somatic mutations with MC1R germline variants indicates an influence of constitutive genotype on the preferential acquisition of specific mutations during melanoma development (Landi et al., 2006). In line with this, cooperation between RAS and CDKN2A has been shown in animal models of melanoma (Chin et al., 1997). Since there is limited information on NRAS and BRAF mutations in familial melanoma we sought to assess their mutation frequency in melanomas from patients with different CDKN2A germline alterations. The study was performed on formalin-fixed, paraffin-embedded (FFPE) primary familial cutaneous melanomas originating from Brisbane, Australia (16 samples from 15 patients) and Genoa, Italy (3 patients/samples). Clinical and pathological characteristics are shown in Table 1. One patient with a CDKN2A L32P mutation had two melanomas originating from the trunk and upper extremity, respectively, while a single melanoma was analyzed from each of the other patients. Eight different germline CDKN2A mutations were present in the patients from whom the melanomas were analyzed (Table 2). Laser capture microdissection, DNA extraction, polymerase chain reaction (PCR) amplification, single strand conformation polymorphism (SSCP) and nucleotide sequence analyses of NRAS exon 2 and BRAF exon 15 were carried out as previously described (Jovanovic et al., 2008; Omholt et al., 2002; Omholt et al., 2003). Each mutation was confirmed by two independent PCR/SSCP analyses followed by sequence analysis performed in both directions. The study was approved by the Ethics Committees of the Queensland Institute of Medical Research, University of Genoa and Karolinska Institutet. Table 1 Patient and tumor characteristics Table 2 NRAS and BRAF genotypes in familial melanomas harboring CDKN2A germline mutations Three (16%) samples had NRAS residue 61 alterations (one Q61R and two Q61K substitutions) while 7 (37%) samples had valine to glutamic acid changes in amino acid 600 of BRAF (V600E; Table 2). The NRAS mutation frequency in this study was lower than we reported previously in melanomas from Swedish families with germline CDKN2A mutations (Eskandarpour et al., 2003). The reason for this is unclear. It could possibly be attributed to different origins of studied cohorts (Sweden versus Australia/Italy) and to different CDKN2A germline alterations in these two studies (the 112Argdup founder mutation is predominant in Swedish families, which is in contrast to genotypes reported here). However, we cannot exclude that technical factors, possibly related to the fragmented nature of DNA extracted from FFPE samples, may play a role in different rates of mutation detection. Intriguingly, all 3 tumors with NRAS mutations also had BRAF V600E mutations. The presence of both NRAS and BRAF V600E mutations in the same lesions is contrary to the current consensus that such mutations are almost always mutually exclusive in melanomas and other tumor types (Davies et al., 2002; Omholt et al., 2003; Thomas et al., 2007). However, Pollock et al (2003) observed concomitant NRAS and BRAF V600E mutations in 9% of nevi and suggested this might be due to different clonal nests of cells within these tumors carrying distinct mutations, a possibility that could also explain our findings. Although intriguingly, the joint presence of both NRAS and BRAF mutations was found only in tumors from patients with CDKN2A L32P mutations. In two of these tumors, NRAS and BRAF mutations (NRAS/BRAF: Q61K/V600E and Q61R/V600E) were found in two different DNA extracts while in one case, alterations of both genes (NRAS/BRAF: Q61K/V600E) were identified in the same DNA extract. Thus far, it is recognized that a mutation in either NRAS or BRAF is sufficient for activation of the Ras-Raf-MEK-ERK pathway, with mutant RAS having a 50-fold higher activation effect than mutant BRAF (Davies et al., 2002). Although we do not have any evidence that the NRAS and BRAF mutations found in the same DNA extract were coexisting in the same cells, it is possible that the L32P mutation in CDKN2A somehow permits cellular tolerance of these dual mutations. In conclusion, the NRAS and BRAF mutation rates we observed in familial melanomas were generally lower than most previous reports in sporadic melanoma but equal to those reported for primary melanomas of similar thickness (Goel et al., 2006; Shinozaki et al., 2004). Samples that harbored INK4A L32P substitutions also had high frequency of coexisting mutations in both NRAS and BRAF. This suggests that in some instances constitutional CDKN2A mutations affect the occurrence of somatic mutations in NRAS and BRAF, although further work is needed to substantiate this hypothesis.

Apoptosis and c-jun induction by cisplatin in a human melanoma cell line and a drug-resistant daughter cell line.

Cisplatin resistance was developed in the human melanoma cell line RPMI8322 by repeated short-term exposures to cisplatin. The most resistant daughter cell line, RPMI8322/CDDP-300, was 4-fold resistant to cisplatin, and partially cross-resistant to carboplatin, melphalan and UV, but not to BCNU. RPMI8322/CDDP-300 cells showed less apoptosis after cisplatin than the parental cells. The cisplatin resistance was not paralleled by a simliar reduction in cellular cisplatin accumulation or DNA cross-links in RPMI8322/CDDP-300 cells, and these cells exhibited no increase in cellular glutathione or in mRNA encoding the DNA excision repair proteins ERCC1 and XPB. Induction of c-jun mRNA by cisplatin was considerably lower in RPMI8322/CDDP-300 cells than in RPMI8322 cells, consistent with the possibility that c-jun induction may be involved in a pathway that triggers apoptosis after exposure to DNA damaging agents. However, c-jun induction is not necessary for apoptosis, since cisplatin also induced apoptosis in A14 rat embryo fibroblasts, cells in which the c-jun gene is deleted.

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.

Analysis of O(6)-methylguanine-DNA methyltransferase in melanoma tumours in patients treated with dacarbazine-based chemotherapy.

In a retrospective study we analysed the levels of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) in melanoma metastases in patients receiving dacarbazine (DTIC) either as a single drug or as part of combination chemotherapy regimens, and related the expression levels to the clinical response to treatment. Biopsies of subcutaneous and lymph node metastases obtained before chemotherapy in 65 patients with disseminated malignant melanoma were examined for MGMT protein levels by immunohistochemistry using a monoclonal anti-human MGMT antibody. All patients received chemotherapy with DTIC, given either as a single drug or in combination with vindesine and in some cases cisplatin. DTIC as single agent was given to 44 patients, while 21 received combination chemotherapy. Objective responses to chemotherapy were seen in 12 patients, while 53 patients failed to respond to treatment. The expression of MGMT was determined according to the proportion of antibody-stained tumour cells, using a cut-off level of 50%. In 12 of the patients more than one metastasis was analysed, and in seven of these cases the MGMT expression differed between tumours in the same individual. Among the responders a larger proportion (six out of 12, 50%) had tumours containing less than 50% MGMT-positive tumour cells than among the non-responders (12 out of 53, 23%). These data are consistent with the hypothesis that MGMT contributes to resistance to DTIC-based treatment, although the difference between responders and non-responders with respect to the proportion of MGMT-positive tumour cells was not statistically significant (P = 0.077).