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.     

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.     

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     

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.     

BRAF mutation correlates with recurrent papillary thyroid carcinoma in Chinese patients

Purpose

We investigated correlations of somatic BRAF V600E mutation and RET/PTC1 rearrangement with recurrent disease in Chinese patients with papillary thyroid carcinoma (ptc).

Methods

This prospective study included 214 patients with ptc histologically confirmed between November 2009 and May 2011 at a single institute.

Results

We found somatic BRAF V600E mutation in 68.7% and RET/PTC1 rearrangement in 25.7% of the patients. Although BRAF mutation was not significantly associated with clinicopathologic features such as patient sex or age, multicentric disease, thyroid capsule invasion, tumour stage, or nodal metastasis, it was significantly associated with recurrent disease. Multivariate analysis revealed that BRAF mutation and tumour size were independent risk factors associated with recurrent disease, with odds ratios of 9.072 and 2.387 respectively. The area under the receiver operating characteristic curve increased 8.3% when BRAF mutation was added to the traditional prognostic factors, but that effect was statistically nonsignificant (0.663 vs. 0.746, p = 0.124). RET/PTC1 rearrangement and nodal metastasis were significantly associated in all patients (p = 0.042), marginally associated in ptc patients (p = 0.051), but not associated in microptc patients (p = 0.700). RET/PTC1 rearrangement was not significantly associated with recurrent disease.

Conclusions

BRAF positivity is an independent predictor of recurrent disease in ptc.     

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.     

Analysis of Dermatologic Events in Vemurafenib‐Treated Patients With Melanoma

Background.

Vemurafenib has been approved for the treatment of patients with advanced BRAF^V600E-mutant melanoma. This report by the Vemurafenib Dermatology Working Group presents the characteristics of dermatologic adverse events (AEs) that occur in vemurafenib-treated patients, including cutaneous squamous cell carcinoma (cuSCC).

Methods.

Dermatologic AEs were assessed from three ongoing trials of BRAF^V600E mutation-positive advanced melanoma. Histologic central review and genetic characterization were completed for a subset of cuSCC lesions.

Results.

A total of 520 patients received vemurafenib. The most commonly reported AEs were dermatologic AEs, occurring in 92%–95% of patients. Rash was the most common AE (64%–75% of patients), and the most common types were rash not otherwise specified, erythema, maculopapular rash, and folliculitis. Rash development did not appear to correlate with tumor response. Photosensitivity occurred in 35%–63% of patients, and palmar-plantar erythrodysesthesia (PPE) occurred in 8%–10% of patients. The severity of rash, photosensitivity, and PPE were mainly grade 1 or 2. In all, 19%–26% of patients developed cuSCC, mostly keratoacanthomas (KAs). The majority of patients with cuSCC continued therapy without dose reduction after resection. Genetic analysis of 29 cuSCC/KA samples demonstrated HRAS mutations in 41%.

Conclusions.

Dermatologic AEs associated with vemurafenib treatment in patients with melanoma were generally manageable with supportive care measures. Dose interruptions and/or reductions were required in <10% of patients.     

Metastasis-associated MCL1 and P16 copy number alterations dictate resistance to vemurafenib in a BRAFV600E patient-derived papillary thyroid carcinoma preclinical model

BRAF^V600E mutation exerts an essential oncogenic function in many tumors, including papillary thyroid carcinoma (PTC). Although BRAF^V600E inhibitors are available, lack of response has been frequently observed. To study the mechanism underlying intrinsic resistance to the mutant BRAF^V600E selective inhibitor vemurafenib, we established short-term primary cell cultures of human metastatic/recurrent BRAF^V600E-PTC, intrathyroidal BRAF^V600E-PTC, and normal thyroid (NT). We also generated an early intervention model of human BRAF^V600E-PTC orthotopic mouse. We find that metastatic BRAF^V600E-PTC cells elicit paracrine-signaling which trigger migration of pericytes, blood endothelial cells and lymphatic endothelial cells as compared to BRAF^WT-PTC cells, and show a higher rate of invasion. We further show that vemurafenib therapy significantly suppresses these aberrant functions in non-metastatic BRAF^V600E-PTC cells but lesser in metastatic BRAF^V600E-PTC cells as compared to vehicle treatment. These results concur with similar folds of down-regulation of tumor microenvironment–associated pro-metastatic molecules, with no effects in BRAF^WT-PTC and NT cells. Our early intervention preclinical trial shows that vemurafenib delays tumor growth in the orthotopic BRAF^WT/V600E-PTC mice. Importantly, we identify high copy number gain of MCL1 (chromosome 1q) and loss of CDKN2A (P16, chromosome 9p) in metastatic BRAF^V600E-PTC cells which are associated with resistance to vemurafenib treatment. Critically, we demonstrate that combined vemurafenib therapy with BCL2/MCL1 inhibitor increases metastatic BRAF^V600E-PTC cell death and ameliorates response to vemurafenib treatment as compared to single agent treatment. In conclusion, short-term PTC and NT cultures offer a predictive model for evaluating therapeutic response in patients with PTC. Our PTC pre-clinical model suggests that combined targeted therapy might be an important therapeutic strategy for metastatic and refractory BRAF^V600E-positive PTC.     

KRAS codon 61, 146 and BRAF mutations predict resistance to cetuximab plus irinotecan in KRAS codon 12 and 13 wild-type metastatic colorectal cancer

Background:

KRAS codons 12 and 13 mutations predict resistance to anti-EGFR monoclonal antibodies (moAbs) in metastatic colorectal cancer. Also, BRAF V600E mutation has been associated with resistance. Additional KRAS mutations are described in CRC.

Methods:

We investigated the role of KRAS codons 61 and 146 and BRAF V600E mutations in predicting resistance to cetuximab plus irinotecan in a cohort of KRAS codons 12 and 13 wild-type patients.

Results:

Among 87 KRAS codons 12 and 13 wild-type patients, KRAS codons 61 and 146 were mutated in 7 and 1 case, respectively. None of mutated patients responded vs 22 of 68 wild type (P=0.096). Eleven patients were not evaluable. KRAS mutations were associated with shorter progression-free survival (PFS, HR: 0.46, P=0.028). None of 13 BRAF-mutated patients responded vs 24 of 74 BRAF wild type (P=0.016). BRAF mutation was associated with a trend towards shorter PFS (HR: 0.59, P=0.073). In the subgroup of BRAF wild-type patients, KRAS codons 61/146 mutations determined a lower response rate (0 vs 37%, P=0.047) and worse PFS (HR: 0.45, P=0.023). Patients bearing KRAS or BRAF mutations had poorer response rate (0 vs 37%, P=0.0005) and PFS (HR: 0.51, P=0.006) compared with KRAS and BRAF wild-type patients.

Conclusion:

Assessing KRAS codons 61/146 and BRAF V600E mutations might help optimising the selection of the candidate patients to receive anti-EGFR moAbs.     

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.     

BRAF mutations,microsatellite instability status and cyclin D1 expression predict metastatic colorectal patients' outcome

Background:

The significance of BRAF mutations, microsatelite instability (MSI) status and cyclin D1 expression in patients with metastatic colorectal cancer (mCRC) was evaluated.

Methods:

Primary tumours from 144 patients treated for mCRC were assessed for BRAF (V600E) mutation, MSI status and cyclin D1. The data were correlated with progression-free survival (PFS) and overall survival (OS).

Results:

BRAF mutations were detected in 10 (out of 22, 45%) patients with MSI-H tumours compared with 2 (out of 122, 1.6%) in those with microsatellite stable tumours (P<0.001). The presence of BRAF mutations was correlated with cyclin D1 overexpression (7 out of 26 patients, 58% vs 5 out of 118 patients, 14% P=0.001). Patients with BRAF-mutated primary tumours had a significantly decreased PFS (2.7 vs 9.8 months; P<0.001) and median OS (14 vs 30 months; P<0.001) than patients with wild-type (wt) tumours. Patients with MSI-H and BRAF-mutated tumours experienced significantly lower PFS (3.1 vs 11.4 months; P=0.008) and OS (14.5 vs 35.5 months; P=0.004) than patients with MSI-H and BRAF wt tumours. Similarly, BRAF mutations and cyclin D1 overexpression were correlated with decreased PFS (3.1 vs 8.6 months; P=0.03) and OS (17.8 vs 39.2 months; P=0.01).

Conclusion:

BRAF V600E mutations are associated with MSI-H status and cyclin D1 overexpression and characterize a subgroup of patients with poor prognosis.     

Circulating Tumor DNA Measurement by Picoliter Droplet-Based Digital PCR and Vemurafenib Plasma Concentrations in Patients with Advanced <Emphasis Type="Italic">BRAF-</Emphasis>Mutated Melanoma

Background

Circulating tumor DNA (ctDNA) has been reported as a prognostic marker in melanoma. In

Objective

We aimed to investigate the relationship between vemurafenib plasma concentrations and the ctDNA plasma concentration during follow-up of

Patients and Methods

Eleven patients treated with single-agent vemurafenib for advanced BRAF V600-mutant melanoma were analyzed in an exploratory monocentric study. The vemurafenib plasma concentration was measured by liquid chromatography. ctDNA was extracted from plasma samples and the ctDNA concentration was evaluated using picoliter droplet-based digital PCR with Taqman^® detection probes targeting the BRAF p.V600E/K mutation and wild-type BRAF sequences.

Results

At baseline, plasma ctDNA was detectable in 72% (n = 8/11) of patients and the ctDNA concentration decreased in 88% of these patients (p = 0.026). The ctDNA concentration at baseline was associated with overall survival (hazard ratio = 2.61, 95% CI 1.04–6.56; p = 0.04).

Conclusions

This study demonstrates the relevance of vemurafenib plasma monitoring during the follow-up of metastatic melanoma patients. Plasma drug monitoring and ctDNA concentrations could be combined to monitor tumor evolution in melanoma patients treated with anti-

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Comparison of high resolution melting analysis, pyrosequencing, next generation sequencing and immunohistochemistry to conventional Sanger sequencing for the detection of p.V600E and non-p.V600E BRAF mutations

Background

The approval of vemurafenib in the US 2011 and in Europe 2012 improved the therapy of not resectable or metastatic melanoma. Patients carrying a substitution of valine to glutamic acid at codon 600 (p.V600E) or a substitution of valine to leucine (p.V600K) in BRAF show complete or partial response. Therefore, the precise identification of the underlying somatic mutations is essential. Herein, we evaluate the sensitivity, specificity and feasibility of six different methods for the detection of BRAF mutations.

Methods

Samples harboring p.V600E mutations as well as rare mutations in BRAF exon 15 were compared to wildtype samples. DNA was extracted from formalin-fixed paraffin-embedded tissues by manual micro-dissection and automated extraction. BRAF mutational analysis was carried out by high resolution melting (HRM) analysis, pyrosequencing, allele specific PCR, next generation sequencing (NGS) and immunohistochemistry (IHC). All mutations were independently reassessed by Sanger sequencing. Due to the limited tumor tissue available different numbers of samples were analyzed with each method (82, 72, 60, 72, 49 and 82 respectively).

Results

There was no difference in sensitivity between the HRM analysis and Sanger sequencing (98%). All mutations down to 6.6% allele frequency could be detected with 100% specificity. In contrast, pyrosequencing detected 100% of the mutations down to 5% allele frequency but exhibited only 90% specificity. The allele specific PCR failed to detect 16.3% of the mutations eligible for therapy with vemurafenib. NGS could analyze 100% of the cases with 100% specificity but exhibited 97.5% sensitivity. IHC showed once cross-reactivity with p.V600R but was a good amendment to HRM.

Conclusion

Therefore, at present, a combination of HRM and IHC is recommended to increase sensitivity and specificity for routine diagnostic to fulfill the European requirements concerning vemurafenib therapy of melanoma patients.     

Dabrafenib plus Trametinib: a Review in Advanced Melanoma with a <Emphasis Type="Italic">BRAF</Emphasis><Superscript><Emphasis Type="Italic">V600</Emphasis></Superscript> Mutation

The BRAF inhibitor dabrafenib (Tafinlar^®) and the MEK inhibitor trametinib (Mekinist^®) are indicated, as monotherapy or in combination with each other, for the treatment of patients with unresectable or metastatic melanoma with a BRAF ^V600 mutation. This article reviews the therapeutic efficacy and tolerability of combination treatment with dabrafenib and trametinib in this indication and summarizes relevant pharmacological data. Dabrafenib plus trametinib significantly prolonged progression-free survival (PFS) and overall survival (OS), improved objective response rates (ORRs) and preserved health-related quality of life (HR-QOL) to a greater extent than dabrafenib (in the double-blind COMBI-d study) and vemurafenib (in the open-label COMBI-v study) in two large, randomized, phase III studies in treatment-naïve patients with unresectable or metastatic melanoma with BRAF ^V600E/K mutation. Limited treatment benefit with the combination was also seen in patients who had progressed on prior BRAF inhibitor therapy, as indicated by ORRs of ≤ 15 % and stable disease in ≤ 50 % of patients in small phase I and II studies. Combination therapy did not increase overall toxicity relative to dabrafenib or vemurafenib monotherapy, with most adverse events (AEs) mild or moderate in severity and generally manageable. Fewer skin-related AEs (e.g. cutaneous malignancies, hyperkeratinosis and hand-foot syndrome) were reported with combination therapy than with dabrafenib or vemurafenib, probably because of reduced paradoxical activation of the MAPK pathway. Thus, dabrafenib plus trametinib provides an important treatment option for patients with BRAF ^V600 mutation-positive unresectable or metastatic melanoma.

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KRAS mutations in primary tumours and post-FOLFOX metastatic lesions in cases of colorectal cancer

Background:

KRAS mutations are predictive markers for the efficacy of anti-EGFR antibody therapies in patients with metastatic colorectal cancer. Although the mutational status of KRAS is reportedly highly concordant between primary and metastatic lesions, it is not yet clear whether genotoxic chemotherapies might induce additional mutations.

Methods:

A total of 63 lesions (23 baseline primary, 18 metastatic and 24 post-treatment metastatic) from 21 patients who were treated with FOLFOX as adjuvant therapy for stage III/IV colorectal cancer following curative resection were examined. The DNA samples were obtained from formalin-fixed paraffin-embedded specimens, and KRAS, NRAS, BRAF and PIK3CA mutations were evaluated.

Results:

The numbers of primary lesions with wild-type and mutant KRAS codons 12 and 13 were 8 and 13, respectively. The mutational status of KRAS remained concordant between the primary tumours and the post-FOLFOX metastatic lesions, irrespective of patient background, treatment duration and disease-free survival. Furthermore, the mutational statuses of the other genes evaluated were also concordant between the primary and metastatic lesions.

Conclusion:

Because the mutational statuses of predictive biomarker genes were not altered by FOLFOX therapy, specimens from both primary tumours and post-FOLFOX tumour metastases might serve as valid sources of DNA for known genomic biomarker testing.     

Optimising the combination dosing strategy of abemaciclib and vemurafenib in BRAF-mutated melanoma xenograft tumours

Background:

Resistance to BRAF inhibition is a major cause of treatment failure for BRAF-mutated metastatic melanoma patients. Abemaciclib, a cyclin-dependent kinase 4 and 6 inhibitor, overcomes this resistance in xenograft tumours and offers a promising drug combination. The present work aims to characterise the quantitative pharmacology of the abemaciclib/vemurafenib combination using a semimechanistic pharmacokinetic/pharmacodynamic modelling approach and to identify an optimum dosing regimen for potential clinical evaluation.

Methods:

A PK/biomarker model was developed to connect abemaciclib/vemurafenib concentrations to changes in MAPK and cell cycle pathway biomarkers in A375 BRAF-mutated melanoma xenografts. Resultant tumour growth inhibition was described by relating (i) MAPK pathway inhibition to apoptosis, (ii) mitotic cell density to tumour growth and, under resistant conditions, (iii) retinoblastoma protein inhibition to cell survival.

Results:

The model successfully described vemurafenib/abemaciclib-mediated changes in MAPK pathway and cell cycle biomarkers. Initial tumour shrinkage by vemurafenib, acquisition of resistance and subsequent abemaciclib-mediated efficacy were successfully captured and externally validated. Model simulations illustrate the benefit of intermittent vemurafenib therapy over continuous treatment, and indicate that continuous abemaciclib in combination with intermittent vemurafenib offers the potential for considerable tumour regression.

Conclusions:

The quantitative pharmacology of the abemaciclib/vemurafenib combination was successfully characterised and an optimised, clinically-relevant dosing strategy was identified.     

The expression profile of integrin receptors and osteopontin in thyroid malignancies varies depending on the tumor progression rate and presence of BRAF V600E mutation

Thyroid cancer (TC) is one of the most common malignancy of the human endocrine system. BRAF V600E mutation is the most frequent genetic alteration of papillary carcinoma, the most frequent TC, which effects RAS-RAF-MEK intracellular signaling pathway. These alterations in RAS-RAF-MEK pathway lead to changes in expression levels of cell membrane integrin receptors and their ligand - extracellular matrix protein osteopontin, which in turn increases the metastatic potential of tumor cells. Thus, integrins and their ligand osteopontin can be considered as potential biomarkers of tumor progression and aggressive tumor phenotypes.The aim of the study was to evaluate the expression levels of integrin receptors ITGA2, ITGA3, ITGAV, ITGA6, ITGA9, ITGB1, ITGB3 and their ligands OPNa, OPNb in the thyroid cancer with different BRAF V600E mutation status.

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.