The role of germline alterations in the DNA damage response genes BRIP1 and BRCA2 in melanoma susceptibility.

We applied a targeted sequencing approach to identify germline mutations conferring a moderately to highly increased risk of cutaneous and uveal melanoma. Ninety-two high-risk melanoma patients were screened for inherited variation in 120 melanoma candidate genes. Observed gene variants were filtered based on frequency in reference populations, cosegregation with melanoma in families and predicted functional effect. Several novel or rare genetic variants in genes involved in DNA damage response, cell-cycle regulation and transcriptional control were identified in melanoma patients. Among identified genetic alterations was an extremely rare variant (minor allele frequency of 0.00008) in the BRIP1 gene that was found to cosegregate with the melanoma phenotype. We also found a rare nonsense variant in the BRCA2 gene (rs11571833), previously associated with cancer susceptibility but not with melanoma, which showed weak association with melanoma susceptibility in the Swedish population. Our results add to the growing knowledge about genetic factors associated with melanoma susceptibility and also emphasize the role of DNA damage response as an important factor in melanoma etiology. © 2016 Wiley Periodicals, Inc.

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.

Comparison of true non-contrast and virtual non-contrast images in the characterization of renal lesions using detector-based spectral CT.

OBJECTIVES: Renal lesions are sometimes incidentally detected during computed tomography (CT) examinations in which an unenhanced series is not included, preventing the lesions from being fully characterized. The aim of this study was to investigate the feasibility to use virtual non-contrast (VNC) images, acquired using a detector-based dual-energy CT, for the characterization of renal lesions. METHODS: Twenty-seven patients (12 women) underwent a renal CT scan, including a non-contrast, an arterial, and a venous phase contrast-enhanced series, using a detector-based dual-energy CT scanner. VNC images were reconstructed from the venous contrast-enhanced series. The mean attenuation values of 65 renal lesions in both the VNC and true non-contrast (TNC) images were measured and compared quantitatively. Three radiologists blindly assessed all lesions using either VNC or TNC images in combination with contrast-enhanced images. RESULTS: Sixteen patients had cystic lesions, five had angiomyolipoma (AML), and six had suspected renal cell carcinomas (RCC). Attenuation values in VNC and TNC images were strongly correlated (ρ = 0.7, mean difference -6.0 ± 13 HU). The largest differences were found for unenhanced high-attenuation lesions. Radiologists classified 86% of the lesions correctly using VNC images. CONCLUSIONS: In 70% of the patients, incidentally detected renal lesions could be accurately characterized using VNC images, resulting in less patient burden and a reduction in radiation exposure. ADVANCES IN KNOWLEDGE: This study shows that renal lesions can be accurately characterized using VNC images acquired by detector-based dual-energy CT, which is in agreement with previous studies using dual-source and rapid X-ray tube potential switching technique.

Prognostic significance of tumor iNOS and COX-2 in stage III malignant cutaneous melanoma.

PURPOSE: New prognostic markers are needed for malignant melanoma. Inducible nitric oxide synthase (iNOS) and cyclooxygenase type 2 (COX-2) have been described to correlate with progression of melanoma. Moreover, activating mutations in BRAF/NRAS oncogenes are often detected in melanoma. The BRAF/NRAS mutation status and expression of COX-2 and iNOS were examined to compare their prognostic value for overall survival (OS) in stage III malignant cutaneous melanoma. EXPERIMENTAL DESIGN: The expression of iNOS and COX-2 in metastatic lymph nodes from 21 rapidly progressing (OS from date of diagnosis of stage III disease < or =14 months) and 17 slowly progressing (OS > or =60 months) stage III cutaneous melanoma patients was examined by immunohistochemistry. The presence of BRAF/NRAS mutations was analyzed using direct DNA sequencing. Chi2 exact trend test and logistic regression analysis were used for statistical analysis. RESULTS: Both iNOS (P = 0.002) and COX-2 (P = 0.048) alone significantly predicted OS. The BRAF/NRAS mutation status did not significantly differ between patient groups, although iNOS significantly (P = 0.013) correlated with BRAF mutation frequency. Furthermore, the odds ratio (OR) with respect to OS of iNOS (OR = 10.4) was higher than that of COX-2 (OR = 5.6) and was stable in the multivariate analysis of OS together with disease stage IIIB/C, ulceration, number of metastatic lymph nodes, and Breslow tumor thickness. CONCLUSION: Our data show that iNOS is an independent and stronger prognostic factor for OS in stage III malignant cutaneous melanoma than COX-2.

KIT, NRAS, BRAF and PTEN mutations in a sample of Swedish patients with acral lentiginous melanoma.

BACKGROUND: Acral lentiginous melanoma (ALM) accounts for <10% of all melanomas in Caucasians. Although the involvement of KIT, NRAS and BRAF mutations is well known in ALM, the impact of these mutations on clinicopathological features has not been established. OBJECTIVE: To define the KIT, NRAS, BRAF and PTEN mutation frequencies in Swedish patients with ALM and to evaluate the impact of mutation status on patient and tumor characteristics. METHODS: Tumor cells were microdissected from 88 primary ALMs and 16 paired metastases and analyzed for KIT, NRAS and BRAF mutations. A subset of 25 ALMs was also evaluated for PTEN mutations. RESULTS: BRAF mutations were identified in 17% of the primary ALMs. Both NRAS and KIT mutations were found at a similar frequency of 15%. Only one of the ALMs that were screened for PTEN harbored a mutation (4%). The KIT, NRAS and BRAF mutation status in paired primary and metastatic ALMs was identical. Patients with BRAF mutated tumors were significantly younger (57 years) than those with BRAF wild-type tumors (73 years, p=0.028). BRAF mutations were significantly more common in females (p=0.011) and more often found in tumors located on the feet (p=0.039). Anatomical site was an independent prognostic factor for overall survival; patients with ALMs on the hands or under fingernails had a better prognosis than those with tumors on the feet or under toenails (p=0.025). CONCLUSION: Our results confirm the presence of KIT, NRAS and BRAF mutations in ALM and provide evidence that mutations in these genes occur at similar frequencies. Our results also show that PTEN is mutated in a small subset of ALM tumors.

MC1R variation and melanoma risk in the Swedish population in relation to clinical and pathological parameters.

The genetic background of cutaneous malignant melanoma (CMM) includes both germ line aberrations in high-penetrance genes, like CDKN2A, and allelic variation in low-penetrance genes like the melanocortin-1 receptor gene, MC1R. Red-hair colour associated MC1R alleles (RHC) have been associated with red hair, fair skin and risk of CMM. We investigated MC1R and CDKN2A variation in relation to phenotype, clinical factors and CMM risk in the Swedish population. The study cohort consisted of sporadic primary melanoma patients, familial melanoma patients and a control group. An allele-dose dependent increase in melanoma risk for carriers of variant MC1R alleles (after adjusting for phenotype), with an elevated risk among familial CMM patients, was observed. This elevated risk was found to be significantly associated with an increased frequency of dysplastic nevi (DN) among familial patients compared to sporadic patients. MC1R variation was found to be less frequent among acral lentiginous melanomas (ALM) and dependent on tumour localisation. No association was found between CDKN2A gene variants and general melanoma risk. Two new variants in the POMC gene were identified in red haired individuals without RHC alleles.

Lack of cytoplasmic ERK activation is an independent adverse prognostic factor in primary cutaneous melanoma.

The aim of this study was to estimate the impact on survival of NRAS and BRAF mutations and activation of Akt and extracellular signal-regulated kinase (ERK) in primary melanomas. A cohort of 57 primary cutaneous T1-2 melanoma tumors was analyzed. Mutation frequency for both genes was 61% (NRAS 26% and BRAF 39%). In a univariate analysis, shorter overall survival was associated with the presence of ulceration (P=0.001) and BRAF exon 15 mutations (P=0.005) as well as the absence of nuclear activation of Akt (P=0.022) and of cytoplasmic activation of ERK (P=0.003). Unexpectedly, ulceration was a significant adverse prognostic factor only in melanomas with BRAF mutations, whereas there was no effect of ulceration on overall survival in tumors with wild-type BRAF. A multivariate analysis showed that significant independent adverse survival prognostic markers were absence of cytoplasmic activation of ERK (P=0.007) and ulceration (P=0.008), whereas BRAF exon 15 mutation status showed a nonsignificant trend (P=0.066). The absence of cytoplasmic ERK activation in poor prognosis T1-2 melanomas may be associated with activation of some other uncharacterized pathway leading to tumor progression and adverse outcome. Immunohistochemical analysis of cytoplasmic phosphorylated ERK could be used as a prognostic marker in primary melanomas if confirmed in another data set.