Correlation of BRAF and NRAS mutation status with outcome,site of distant metastasis and response to chemotherapy in metastatic melanoma

Background:

The prognostic significance of BRAF and NRAS mutations in metastatic melanoma patients remains uncertain, with several studies reporting conflicting results, often biased by the inclusion of patients treated with BRAF and MEK (MAPK) inhibitors. We therefore interrogated a historical cohort of patients free of the confounding influence of MAPK inhibitor therapy.

Methods:

Patients with available archival tissue first diagnosed with metastatic melanoma between 2002 and 2006 were analysed. Mutational analysis was performed using the OncoCarta Panel. Patient characteristics, treatment outcome and survival were correlated with BRAF/NRAS mutation status.

Results:

In 193 patients, 92 (48%) melanomas were BRAF-mutant, 39 (20%) were NRAS-mutant and 62 (32%) were wild-type for BRAF/NRAS mutations (wt). There was no difference in response to chemotherapy based on mutation status (35–37%). The distant disease-free interval (DDFI) was significantly shorter in patients with wt melanoma (27.9 months vs 35.1 for BRAF and 49.1 for NRAS) although this was not significant in multivariate analysis. Survival from stage IV melanoma diagnosis was not significantly different based on mutation status. The DDFI was significantly shorter in patients with BRAF^V600K/R versus BRAF^V600E melanoma in univariate and multivariate analyses.

Conclusions:

BRAF and NRAS mutation status does not influence survival in metastatic melanoma.     

Mutation landscape in melanoma patients clinical implications of heterogeneity of BRAF mutations

Background:

The detection of V600E BRAF mutations has fundamental clinical consequences as the treatment option with BRAF inhibitors such as vemurafenib or dabrafenib yields response rates of ∼48%. Heterogeneity with respect to BRAF mutation in different metastases has been described in single cases. As this has important implications for the determination of BRAF status and treatment of patients, it is essential to acquire more data.

Methods:

A total of 300 tumour samples from 187 melanoma patients were analysed for BRAF mutations by pyrosequencing. Equivocal results were confirmed by capillary sequencing. Clinical data with respect to melanoma type, tumour site and survival were summarised for 53 patients with multiple analysed tumour samples (2–13 per patient).

Results:

BRAF mutations were found in 84 patients (44.9%) and 144 tumour samples (48%) with BRAF mutations in 45.5% of primary tumours and 51.3% of metastases, respectively. In 10 out of 53 patients (18.9%) where multiple samples were analysed results were discordant with respect to mutation findings with wild-type and mutated tumours in the same patient. Mutations did not appear more frequently over the course of disease nor was its occurrence associated with a specific localisation of metastases.

Conclusion:

As heterogeneity with respect to BRAF mutation status is detected in melanoma patients, subsequent testing of initially wild-type patients can yield different results and thus make BRAF inhibitor therapy accessible. The role of heterogeneity in testing and for clinical response to therapy with a BRAF inhibitor needs to be further investigated.     

Polyclonality of BRAF mutations in primary melanoma and the selection of mutant alleles during progression

Background:

Oncogenic BRAF mutation had been considered to be a founder event in the formation of melanocytic tumours; however, we recently argued against this notion by showing marked polyclonality of BRAF mutations in acquired melanocytic nevi (Lin et al, J Natl Cancer Inst., 2009; 101:1423–7). Here, we tested whether similar heterogeneity of BRAF mutations exists in primary melanomas.

Methods:

We isolated and sequenced single melanoma cells from five primary melanoma tissues using antibodies against human high-molecular-weight melanoma-associated antigen. We also examined 10 primary melanomas by the sensitive Mutector assay detecting the BRAF^V600E mutation, as well as by cloning and sequencing of separated alleles. Furthermore, we estimated the frequency of BRAF mutant alleles in paired samples of primary tumour and recurrence or metastasis in three patients.

Results:

Single-cell mutation analyses revealed that four of five primary melanomas contained both BRAF-wild-type and BRAF-mutant tumour cells. Tumour heterogeneity in terms of BRAF mutations was also shown in 8 of 10 primary melanomas. Selection of BRAF mutant alleles during progression was demonstrated in all the three patients.

Conclusion:

Acquisition of a BRAF mutation is not a founder event, but may be one of the multiple clonal events in melanoma development, which is selected for during the progression.     

BRAFV600E protein expression and outcome from BRAF inhibitor treatment in BRAFV600E metastatic melanoma

Background:

To examine the association between level and patterns of baseline intra-tumoural BRAF^V600E protein expression and clinical outcome of BRAF^V600E melanoma patients treated with selective BRAF inhibitors.

Methods:

Fifty-eight BRAF^V600E metastatic melanoma patients treated with dabrafenib or vemurafenib on clinical trials had pre-treatment tumour BRAF^V600E protein expression immunohistochemically (IHC) assessed using the BRAF V600E mutant-specific antibody VE1. Sections were examined for staining intensity (score 1–3) and percentage of immunoreactive tumour cells, and from this an immunoreactive score (IRS) was derived (intensity × per cent positive/10). The presence of intra-tumoural heterogeneity for BRAF^V600E protein expression was also assessed. BRAF^V600E expression was correlated with RECIST response, time to best response (TTBR), progression-free survival (PFS) and overall survival (OS).

Results:

Expression was generally high (median IRS 28 (range 5–30)) and homogeneous (78%). Expression of mutated protein BRAF^V600E as measured by intensity, per cent immunoreactive cells, or IRS did not correlate with RECIST response, TTBR, PFS or OS, including on multivariate analysis. Heterogeneity of staining was seen in 22% of cases and did not correlate with outcome.

Conclusion:

In the current study population, IHC-measured pre-treatment BRAF^V600E protein expression does not predict response or outcome to BRAF inhibitor therapy in BRAF^V600E metastatic melanoma patients.     

Allele frequencies of BRAFV600 mutations in primary melanomas and matched metastases and their relevance for BRAF inhibitor therapy in metastatic melanoma

Background

The detection of BRAFV600 mutations in patients with metastatic melanoma is important because of the availability of BRAF inhibitor therapy. However, the clinical relevance of the frequency of BRAFV600 mutant alleles is unclear.

Patients and Methods

Allele frequencies of BRAFV600 mutations were analyzed by ultra-deep next-generation sequencing in formalin-fixed, paraffin-embedded melanoma tissue (75 primary melanomas and 88 matched metastases). In a second study, pretreatment specimens from 76 patients who received BRAF inhibitors were retrospectively analyzed, and BRAFV600 allele frequencies were correlated with therapeutic results.

Results

Thirty-five patients had concordantly BRAF-positive and 36 (48%) patients had concordantly BRAF-negative primary melanomas and matched metastases, and four patients had discordant samples with low allele frequencies (3.4–5.2%). Twenty-six of 35 patients with concordant samples had BRAFV600E mutations, three of whom had additional mutations (V600K in two patients and V600R in one) and nine patients had exclusively non-V600E mutations (V600K in eight patients and V600E -c.1799_1800TG > AA- in one patient). The frequency of mutated BRAFV600 alleles was similar in the primary melanoma and matched metastasis in 27/35 patients, but differed by >3-fold in 8/35 of samples. BRAFV600E allele frequencies in pretreatment tumor specimens were not significantly correlated with treatment outcomes in 76 patients with metastatic melanoma who were treated with BRAF inhibitors.

Conclusions

BRAFV600 mutation status and allele frequency is consistent in the majority of primary melanomas and matched metastases. A small subgroup of patients has double mutations. BRAFV600 allele frequencies are not correlated with the response to BRAF inhibitors.     

Detecting BRAF Mutations in Formalin-Fixed Melanoma: Experiences with Two State-of-the-Art Techniques

Background

Melanoma is characterized by a high frequency of BRAF mutations. It is unknown if the BRAF mutation status has any predictive value for therapeutic approaches such as angiogenesis inhibition.

Patients and Methods

We used 2 methods to analyze the BRAF mutation status in 52 of 62 melanoma patients. Method 1 (mutation-specific real-time PCR) specifically detects the most frequent BRAF mutations, V600E and V600K. Method 2 (denaturing gel gradient electrophoresis and direct sequencing) identifies any mutations affecting exons 11 and 15.

Results

Eighteen BRAF mutations and 15 wild-type mutations were identified with both methods. One tumor had a double mutation (GAA) in codon 600. Results of 3 samples were discrepant. Additional mutations (V600M, K601E) were detected using method 2. Sixteen DNA samples were analyzable with either method 1 or method 2. There was a significant association between BRAF V600E mutation and survival.

Conclusion

Standardized tissue fixation protocols are needed to optimize BRAF mutation analysis in melanoma. For melanoma treatment decisions, the availability of a fast and reliable BRAF V600E screening method may be sufficient. If other BRAF mutations in exons 11 and 15 are found to be of predictive value, a combination of the 2 methods would be useful.Key Words: BRAF mutations, V600E, Mutation detection methods, Melanoma     

Consolidation electrochemotherapy with bleomycin in metastatic melanoma during treatment with dabrafenib

Background

Small molecules that inhibit V600 mutated BRAF protein, such as vemurafenib and dabrafenib, are effective in treatment of metastatic melanoma.

Case report

We here describe the clinical course of a V600E BRAF mutated metastatic melanoma patient with systemic disease, who developed tumor progression on superficial soft-tissue metastases during treatment with dabrafenib. Bleomycin electrochemotherapy during dabrafenib treatment was administered to control the soft-tissue progressing metastases and ensured sustained local control without significant toxicity.

Conclusions

The new combined approach maintained the patient quality of life and allowed for the prosecution of the target therapy, which proved to be still effective on systemic disease, up to 17 months.     

BRAF mutations in thyroid tumors from an ethnically diverse group

Background

The molecular etiology of thyroid carcinoma (TC) and other thyroid diseases which may present malignant precursor lesions is not fully explored yet. The purpose of this study was to estimate frequency, type and clinicopathological value of BRAF exon 15 mutations in different types of cancerous and non-cancerous thyroid lesions originating in an ethnically diverse population.

Methods

BRAF exon 15 was sequenced in 381 cases of thyroid lesions including Hashimoto´s thyroiditis, nodular goiters, hyperplastic nodules, follicular adenomas (FA), papillary TC (PTC), follicular variant PTC (FVPTC), microcarcinomas of PTC (micro PTC; tumor size ≤ 1 cm), follicular TC (FTC), and non-well differentiated TC (non-WDTC).

Results

We identified BRAF mutations in one of 69 FA, 72 of 115 (63%) PTC, seven of 42 (17%) FVPTC, 10 of 56 (18%) micro PTC, one of 17 (6%) FTC, and one of eight (13%) non-WDTC. Most of the cases showed the common V600E mutation. One case each of PTC, FVPTC, and FTC harbored a K601E mutation. A novel BRAF mutation was identified in a FA leading to deletion of threonine at codon 599 (p.T599del). A rare 3-base pair insertion was detected in a stage III PTC resulting in duplication of threonine at codon 599 (p.T599dup). Patients with PTC harboring no BRAF mutation (BRAF^wt) were on average younger than those with a BRAF mutation (BRAF^mut) in the PTC (36.6 years vs. 43.8 years). Older age (≥ 45 years) in patients with PTC was significantly associated with tumor size ≥ 4 cm (P = 0.018), vessel invasion (P = 0.004), and distant metastasis (P = 0.001). Lymph node (LN) involvement in PTC significantly correlated with tumor size (P = 0.044), and vessel invasion (P = 0.013). Of notice, taken the whole TC group, family history of thyroid disease positively correlated with capsular invasion (P = 0.025).

Conclusions

Older age is manifold associated with unfavorable tumor markers in our series. The K601E identified in a PTC, FVPTC, and FTC seems to be more distributed among different histological types of TC than previously thought. The T599del is a yet undescribed mutation and the rare T599dup has not been reported as a mutation in PTC so far.     

Recovery of phospho-ERK activity allows melanoma cells to escape from BRAF inhibitor therapy

Background:

Resistance to BRAF inhibitors is an emerging problem in the melanoma field. Strategies to prevent and overcome resistance are urgently required.

Methods:

The dynamics of cell signalling, BrdU incorporation and cell-cycle entry after BRAF inhibition was measured using flow cytometry and western blot. The ability of combined BRAF/MEK inhibition to prevent the emergence of resistance was demonstrated by apoptosis and colony formation assays and in 3D organotypic cell culture.

Results:

BRAF inhibition led to a rapid recovery of phospho-ERK (pERK) signalling. Although most of the cells remained growth arrested in the presence of drug, a minor population of cells retained their proliferative potential and escaped from BRAF inhibitor therapy. A function for the rebound pERK signalling in therapy escape was demonstrated by the ability of combined BRAF/MEK inhibition to enhance the levels of apoptosis and abrogate the onset of resistance.

Conclusion:

Combined BRAF/MEK inhibition may be one strategy to prevent the emergence of drug resistance in BRAF-V600E-mutated melanomas.     

The combination of axitinib followed by paclitaxel/carboplatin yields extended survival in advanced BRAF wild-type melanoma: results of a clinical/correlative prospective phase II clinical trial

Background:

Simultaneous chemotherapy with vascular endothelial growth factor (VEGF) inhibition has not shown additional benefit over chemotherapy alone in advanced melanoma. We tested administration of the potent VEGF inhibitor axitinib followed by paclitaxel/carboplatin to determine whether enhanced tumour proliferation during axitinib withdrawal leads to sustained chemosensitivity.

Methods:

We conducted a prospective phase II trial in metastatic melanoma patients with ECOG performance status 0–1 and normal organ function. Axitinib 5 mg PO b.i.d. was taken on days 1–14 of each 21-day treatment cycle, and carboplatin (AUC=5) with paclitaxel (175 mg m⁻²) was administered on day 1 starting with cycle 2. 3′-Deoxy-3′-¹⁸F-fluorothymidine (¹⁸F-FLT)-PET scans were performed in five patients to assess tumour proliferation on days 1, 14, 17, and 20 of cycle 1. Molecular profiling for BRAF was performed for all patients with cutaneous, acral, or mucosal melanoma.

Results:

The treatment was well tolerated. The most common grade 3 AEs were hypertension, neutropenia, and anaemia. Grade 4 non-haematologic AEs were not observed. Four of five patients completing ¹⁸F-FLT-PET scans showed increases (23–92%) in SUV values during the axitinib holiday. Of 36 evaluable patients, there were 8 confirmed PRs by Response Evaluation Criteria in Solid Tumors. Overall, 20 patients had SD and 8 had PD as the best response. The median PFS was 8.7 months and the median overall survival was 14.0 months. Five BRAF^V600E/K patients had significantly worse PFS than patients without these mutations.

Conclusions:

Axitinib followed by carboplatin and paclitaxel was well tolerated and effective in BRAF wild-type metastatic melanoma. 3′-Deoxy-3′-¹⁸F-fluorothymidine-PET scans showed increased proliferation during axitinib withdrawal.     

A comparison of ARMS and DNA sequencing for mutation analysis in clinical biopsy samples

Background

We have compared mutation analysis by DNA sequencing and Amplification Refractory Mutation System™ (ARMS™) for their ability to detect mutations in clinical biopsy specimens.

Methods

We have evaluated five real-time ARMS assays: BRAF 1799T>A, [this includes V600E and V600K] and NRAS 182A>G [Q61R] and 181C>A [Q61K] in melanoma, EGFR 2573T>G [L858R], 2235-2249del15 [E746-A750del] in non-small-cell lung cancer, and compared the results to DNA sequencing of the mutation ''hot-spots'' in these genes in formalin-fixed paraffin-embedded tumour (FF-PET) DNA.

Results

The ARMS assays maximised the number of samples that could be analysed when both the quality and quantity of DNA was low, and improved both the sensitivity and speed of analysis compared with sequencing. ARMS was more robust with fewer reaction failures compared with sequencing and was more sensitive as it was able to detect functional mutations that were not detected by DNA sequencing. DNA sequencing was able to detect a small number of lower frequency recurrent mutations across the exons screened that were not interrogated using the specific ARMS assays in these studies.

Conclusions

ARMS was more sensitive and robust at detecting defined somatic mutations than DNA sequencing on clinical samples where the predominant sample type was FF-PET.     

Impact of mutational status on survival in low-grade serous carcinoma of the ovary or peritoneum

Background:

Low-grade serous carcinoma of the ovary or peritoneum is a distinct, well- recognized histologic subtype characterized by young age at diagnosis, relative chemoresistance, and prolonged overall survival. Common mutations reported to be found within this subtype include KRAS and BRAF.

Methods:

Using clinical information of patients from our IRB-approved registry and tissue from a subset of these patients, we performed mutational analysis for KRAS and BRAF using the direct Sanger sequencing technique and correlated findings with the clinical outcome, overall survival (OS).

Results:

In 79 cases, patients with KRAS or BRAF mutations (n=21) had a significantly better OS than those with wild-type KRAS or BRAF (n=58) (106.7 months (95% CI, 50.6, 162.9) vs 66.8 months (95% CI, 43.6, 90.0)), respectively (P=0.018).

Conclusions:

Mutational status appears to be a potential prognostic factor in low-grade serous carcinoma of the ovary or peritoneum.     

BRAFV600E melanoma cells secrete factors that activate stromal fibroblasts and enhance tumourigenicity

Background:

Melanoma, the most lethal form of skin cancer, is responsible for over 80% of all skin cancer deaths and is highly metastatic, readily spreading to the lymph nodes or metastasising to other organs. The frequent genetic mutation found in metastatic melanoma, BRAF^V600E, results in constitutive activation of the mitogen-activated protein kinase pathway.

Methods:

In this study, we utilised genetically engineered melanoma cell lines and xenograft mouse models to investigate how BRAF^V600E affected cytokine (IL-1β, IL-6, and IL-8) and matrix metalloproteinase-1 (MMP-1) expression in tumour cells and in human dermal fibroblasts.

Results:

We found that BRAF^V600E melanoma cells expressed higher levels of these cytokines and of MMP-1 than wild-type counterparts. Further, conditioned medium from the BRAF^V600E melanoma cells promoted the activation of stromal fibroblasts, inducing expression of SDF-1 and its receptor CXCR4. This increase was mitigated when the conditioned medium was taken from melanoma cells treated with the BRAF^V600E specific inhibitor, vemurafenib.

Conclusions:

Our findings highlight the role of BRAF^V600E in activating the stroma and suggest a mechanistic link between BRAF^V600E and MMP-1 in mediating melanoma progression and in activating adjacent fibroblasts in the tumour microenvironment.     

Efficacy of first-line systemic treatment in correlation with BRAF V600E and different KRAS mutations in metastatic colorectal cancer - a single institution retrospective analysis

Background

KRAS mutation status in codons 12 and 13 is recognized as a predictive factor for resistance to anti-EGFR monoclonal antibodies. Despite having a wild type KRAS (wt-KRAS), not all patients with wt-KRAS respond to anti-EGFR antibody treatment. Additional mechanisms of resistance may activate mutations of the other main EGFR effectors pathway. Consequently, other molecular markers in colorectal cancer are needed to be evaluated to predict the response to therapy.

Patients and methods

In this retrospective study, objective responses (OR), time to progression (TTP), overall survival (OS) were analyzed in 176 metastatic colorectal cancer (mCRC) patients treated with first-line chemotherapy in combination with monoclonal antibodies in respect of KRAS status in codons 12 and 13 and BRAF mutational status.

Results

The KRAS mutations were found in 63 patients (35.8 %), the KRAS mutation in codon 12 in 53 patients (30.1%) and the KRAS mutation in codon 13 in 10 patients (5.7%). The BRAF V600E mutation was detected in 13 of 176 patients (7.4%). In the subgroup of mCRC patients having wt-KRAS and wild type BRAF (wt-BRAF), the objective response rates were higher (OR 54.0% ,CR 14.7%, PR 39.3%) than in the patients with wt-KRAS and mt-BRAF (OR 38.5%,CR 15.4%, PR 23.1%), the difference was not statistically significant (p= 0.378). Median OS in patients with wt-KRAS wt-BRAF, and in patients with wt-KRAS mt-BRAF, was 107.4 months and 45 months, respectively. The difference was statistically significant (p= 0.042). TTP in patients with wt-KRAS wt-BRAF, and in patients with wt-KRAS mt-BRAF, was 16 months and 12 months, respectively. The difference was not statistically significant (p= 0.558).

Conclusions

Patients with BRAF V600E mutation have statistically significantly worse prognosis than the patients with wt-BRAF and progress earlier during treatment. The definitive role of the BRAF V600E mutation as a prognostic and predictive factor for the response to anti-EGFR monoclonal antibodies needs to be analyzed in large prospective clinical studies.     

Adult pilocytic astrocytomas: clinical features and molecular analysis

Background

Adult pilocytic astrocytomas (PAs) are rare and have an aggressive clinical course compared with pediatric patients. Constitutive Ras/RAF/MAPK signaling appears to be an important oncogenic event in sporadic PA. We evaluated clinical data and molecular profiles of adult PAs at our institution.

Methods

We identified 127 adult PAs in our institutional database. Cases with available tissue were tested for BRAF-KIAA1549 fusion/duplication (B-K fusion) by fluorescence in situ hybridization and submitted for mutation profiling using the Sequenom mutation profiling panel. Subgroup analyses were performed based on clinical and molecular data.

Results

The majority of adult PAs are supratentorial. Twenty-two percent of cases had an initial pathologic diagnosis discordant with the diagnosis made at our institution. Recurrence was seen in 42% of cases, and 13% of patients died during follow-up. Adjuvant radiotherapy following surgical resection was associated with a statistically significant decrease in progression-free survival (P = .004). B-K fusion was identified in 20% (9 of 45) of patients but was not associated with outcome. No BRAF V600E mutations (0 of 40 tested) were found.

Conclusion

This was the largest single institution series of adult PA. A significant proportion of adult PAs follow an aggressive clinical course. Our results support a period of observation following biopsy or surgical resection. B-K fusion in adult PA does not influence outcome, and BRAF V600E mutation appears to be a very rare event. Further study of tumor biology and optimal treatment is needed, given a more aggressive clinical behavior.     

Promoter methylation of Wnt5a is associated with microsatellite instability and BRAF V600E mutation in two large populations of colorectal cancer patients

Background:

In colorectal cancer (CRC), tumour microsatellite instability (MSI) status and CpG island methylator phenotype (CIMP) status are indicators of patient outcome, but the molecular events that give rise to these outcomes remain largely unknown. Wnt5a is a critical regulator of non-canonical Wnt activity and promoter hypermethylation of this gene has emerging prognostic roles in CRC; however the frequency and prognostic significance of this epigenetic event have not been explored in the context of colorectal tumour subtype. Consequently, we investigated the frequency and prognostic significance of Wnt5a methylation in a large cohort of MSI-stratified CRCs.

Methods:

Methylation was quantified in a large cohort of 1232 colorectal carcinomas from two clinically distinct populations from Canada. Associations were examined between methylation status and clinicopathlogical features, including tumour MSI status, BRAF V600E mutation, and patient survival.

Results:

In Ontario, Wnt5a methylation was strongly associated with MSI tumours after adjustment for age, sex, and tumour location (odds ratio (OR)=4.2, 95% confidence interval (CI)=2.4–7.4, P<10⁻⁶) and with BRAF V600E mutation, a marker of CIMP (OR=12.3, 95% CI=6.9–21.7, P<10⁻¹⁷), but was not associated with patient survival. Concordant results were obtained in Newfoundland.

Conclusion:

Methylation of Wnt5a is associated with distinct tumour subtypes, strengthening the evidence of an epigenetic-mediated Wnt bias in CRC.     

Phase I clinical trial of the Src inhibitor dasatinib with dacarbazine in metastatic melanoma

Background:

Src inhibitors sensitise melanoma cells to chemotherapy in preclinical models. The combination of dasatinib and dacarbazine was tested in a phase I trial in melanoma.

Methods:

Patients had ECOG performance status 0–2 and normal organ function. Dacarbazine was administered on day 1 and dasatinib on day 2 through 19 of each 21-day cycle. Both were escalated from 50 mg b.i.d. of dasatinib and 800 mg m⁻² of dacarbazine. Available pre-treatment biopsies were sequenced for BRAF, NRAS, and C-Kit mutations.

Results:

Dose-limiting toxicity was reached at dasatinib 70 mg b.i.d./dacarbazine 1000 mg m⁻², and was predominantly haematological. In 29 patients receiving dasatinib 70 mg b.i.d., the objective response rate (ORR) was 13.8%, the clinical benefit rate (ORR+SD) was 72.4%, the 6-month progression-free survival (PFS) was 20.7%, and the 12-month overall survival (OS) was 34.5%. Two out of three patients who were wild type for BRAF, NRAS, and c-KIT mutations had confirmed partial responses, and one had a minor response.

Conclusion:

The recommended phase II dose is dasatinib70 mg b.i.d with dacarbazine 800 mg m⁻². PFS and OS data for dasatinib at 70 mg b.i.d. with dacarbazine compared favourably with historical controls. Preliminary data support evaluating tumour mutation status further as a biomarker of response.     

TERT promoter mutations in ocular melanoma distinguish between conjunctival and uveal tumours

Background:

Recently, activating mutations in the TERT promoter were identified in cutaneous melanoma. We tested a cohort of ocular melanoma samples for similar mutations.

Methods:

The TERT promoter region was analysed by Sanger sequencing in 47 uveal (ciliary body or choroidal) melanomas and 38 conjunctival melanomas.

Results:

Mutations of the TERT promoter were not identified in uveal melanomas, but were detected in 12 (32%) conjunctival melanomas. Mutations had a UV signature and were identical to those found in cutaneous melanoma.

Conclusion:

Mutations of TERT promoter with UV signatures are frequent in conjunctival melanomas and favour a pathogenetic kinship with cutaneous melanomas. Absence of these mutations in uveal melanomas emphasises their genetic distinction from cutaneous and conjunctival melanomas.     

Episodic Src activation in uveal melanoma revealed by kinase activity profiling

Background:

The RAS/RAF/MEK/ERK pathway is involved in the balance between melanocyte proliferation and differentiation. The same pathway is constitutively activated in cutaneous and uveal melanoma (UM) and related to tumour growth and survival. Whereas mutant BRAF and NRAS are responsible for the activation of the RAS/RAF/MEK/ERK pathway in most cutaneous melanoma, mutations in these genes are usually absent in UM.

Methods:

We set out to explore the RAS/RAF/MEK/ERK pathway and used mitogen-activated protein kinase profiling and tyrosine kinase arrays.

Results:

We identified Src as a kinase that is associated with ERK1/2 activation in UM. However, low Src levels and reduced ERK1/2 activation in metastatic cell lines suggest that proliferation in metastases can become independent of Src and RAS/RAF/MEK/ERK signalling. Inhibition of Src led to the growth reduction of primary UM cultures and cell lines, whereas metastatic cell line growth was only slightly reduced.

Conclusion:

We identified Src as an important kinase and a potential target for treatment in primary UM. Metastasis cell lines seemed largely resistant to Src inhibition and indicate that in metastases treatment, a different approach may be required.