673

BRAF

B-Raf proto-oncogene, serine/threonine kinase;BRAF;B-Raf proto-oncogene, serine/threonine kinase

94 kDa B-raf protein|B-Raf proto-oncogene serine/threonine-protein kinase (p94)|murine sarcoma viral (v-raf) oncogene homolog B1|proto-oncogene B-Raf|serine/threonine-protein kinase B-raf|v-raf murine sarcoma viral oncogene homolog B|v-raf murine sarcoma

7q34

protein-coding

PURPOSE Cetuximab or panitumumab are effective in 10% to 20% unselected metastatic colorectal cancer (CRC) patients. KRAS mutations account for approximately 30% to 40% patients who are not responsive. The serine-threonine kinase BRAF is the principal effector of KRAS. We hypothesized that, in KRAS wild-type patients, BRAF mutations could have a predictive/prognostic value. PATIENTS AND METHODS We retrospectively analyzed objective tumor responses, time to progression, overall survival (OS), and the mutational status of KRAS and BRAF in 113 tumors from cetuximab- or panitumumab-treated metastatic CRC patients. The effect of the BRAF V600E mutation on cetuximab or panitumumab response was also assessed using cellular models of CRC. Results KRAS mutations were present in 30% of the patients and were associated with resistance to cetuximab or panitumumab (P = .011). The BRAF V600E mutation was detected in 11 of 79 patients who had wild-type KRAS. None of the BRAF-mutated patients responded to treatment, whereas none of the responders carried BRAF mutations (P = .029). BRAF-mutated patients had significantly shorter progression-free survival (P = .011) and OS (P < .0001) than wild-type patients. In CRC cells, the introduction of BRAF V600E allele impaired the therapeutic effect of cetuximab or panitumumab. Treatment with the BRAF inhibitor sorafenib restored sensitivity to panitumumab or cetuximab of CRC cells carrying the V600E allele. CONCLUSION BRAF wild-type is required for response to panitumumab or cetuximab and could be used to select patients who are eligible for the treatment. Double-hit therapies aimed at simultaneous inhibition of epidermal growth factor receptor and BRAF warrant exploration in CRC patients carrying the V600E oncogenic mutation.

Part of the inherited susceptibility to colorectal cancer (CRC) is caused by the coinheritance of common low risk variants. E-cadherin (CDH1) has an established role in CRC; somatic inactivation of CDH1 is a common early event, and germline mutations can cause early-onset CRC. The -160C>A promoter variant (rs16260) of CDH1 has been reported to influence CDH1 transcription and thereby represents a strong candidate for a predisposition locus. To examine this proposition, we conducted a two-staged association study based on genotyping a total of 5,679 CRC cases and 5,412 controls for rs16260. CDH1-160C>A genotype was associated with CRC risk (p(trend) = 0.001). Compared to common homozygotes, the odds ratios (ORs) of CRC associated with heterozygous and homozygote variant genotype were 0.90 (95% confidence interval [CI]: 0.84-0.97) and 0.81 (95% CI: 0.71-0.93), respectively. In combination with the previously identified 8q21, 8q24, 10p14, 11q, 15q13.3 and 18q21 risk variants, the risk of CRC increases with an increasing numbers of variant alleles for the 7 loci (OR(per allele) = 1.16; 95% CI: 1.13-1.19; p(trend) = 1.68 x 10(-34)). These data indicate CDH1-160C>A is a risk factor for CRC, and because a high proportion of the European population are carriers of at-risk genotypes, the variant is likely to contribute substantially to the development of CRC. Furthermore, our study underscores the importance of conducting association studies using large sample series to demonstrate polymorphic variants conferring modest relative risks.

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.

BACKGROUND: We address the prognostic and predictive value of KRAS, PIK3CA and BRAF mutations for clinical outcomes in response to active agents in the treatment of metastatic colorectal cancer (mCRC). METHODS: We determined KRAS, BRAF and PIK3CA mutations in tumours from 168 patients treated for mCRC at two institutions. All patients received 5-FU-based first-line chemotherapy and treatment outcome was analysed retrospectively. RESULTS: KRAS, BRAF and PIK3CA mutations were present in 62 (37%), 13 (8%) and 26 (15%) cases, respectively. Multivariate analysis uncovered BRAF mutation as an independent prognostic factor for decreased survival (hazard ratio (HR) 4.0, 95% confidence interval (CI) 2.1-7.6). In addition, patients with BRAF-mutant tumours had significantly lower progression-free survival (PFS: HR 4.0, 95% CI 2.2-7.4) than those whose tumors that carried wild-type BRAF. Among 92 patients treated using chemotherapy and cetuximab as salvage therapy, KRAS mutation was associated with lack of response (P=0.002) and shorter PFS (P=0.09). BRAF (P=0.0005) and PIK3CA (P=0.01) mutations also predicted reduced PFS in response to cetuximab salvage therapy. CONCLUSIONS: These results underscore the potential of mutational profiling to identify CRCs with different natural histories or treatment responses. The adverse significance of BRAF mutation should inform patient selection and stratification in clinical trials.

BACKGROUND: Following the discovery that mutant KRAS is associated with resistance to anti-epidermal growth factor receptor (EGFR) antibodies, the tumours of patients with metastatic colorectal cancer are now profiled for seven KRAS mutations before receiving cetuximab or panitumumab. However, most patients with KRAS wild-type tumours still do not respond. We studied the effect of other downstream mutations on the efficacy of cetuximab in, to our knowledge, the largest cohort to date of patients with chemotherapy-refractory metastatic colorectal cancer treated with cetuximab plus chemotherapy in the pre-KRAS selection era. METHODS: 1022 tumour DNA samples (73 from fresh-frozen and 949 from formalin-fixed, paraffin-embedded tissue) from patients treated with cetuximab between 2001 and 2008 were gathered from 11 centres in seven European countries. 773 primary tumour samples had sufficient quality DNA and were included in mutation frequency analyses; mass spectrometry genotyping of tumour samples for KRAS, BRAF, NRAS, and PIK3CA was done centrally. We analysed objective response, progression-free survival (PFS), and overall survival in molecularly defined subgroups of the 649 chemotherapy-refractory patients treated with cetuximab plus chemotherapy. FINDINGS: 40.0% (299/747) of the tumours harboured a KRAS mutation, 14.5% (108/743) harboured a PIK3CA mutation (of which 68.5% [74/108] were located in exon 9 and 20.4% [22/108] in exon 20), 4.7% (36/761) harboured a BRAF mutation, and 2.6% (17/644) harboured an NRAS mutation. KRAS mutants did not derive benefit compared with wild types, with a response rate of 6.7% (17/253) versus 35.8% (126/352; odds ratio [OR] 0.13, 95% CI 0.07-0.22; p<0.0001), a median PFS of 12 weeks versus 24 weeks (hazard ratio [HR] 1.98, 1.66-2.36; p<0.0001), and a median overall survival of 32 weeks versus 50 weeks (1.75, 1.47-2.09; p<0.0001). In KRAS wild types, carriers of BRAF and NRAS mutations had a significantly lower response rate than did BRAF and NRAS wild types, with a response rate of 8.3% (2/24) in carriers of BRAF mutations versus 38.0% in BRAF wild types (124/326; OR 0.15, 95% CI 0.02-0.51; p=0.0012); and 7.7% (1/13) in carriers of NRAS mutations versus 38.1% in NRAS wild types (110/289; OR 0.14, 0.007-0.70; p=0.013). PIK3CA exon 9 mutations had no effect, whereas exon 20 mutations were associated with a worse outcome compared with wild types, with a response rate of 0.0% (0/9) versus 36.8% (121/329; OR 0.00, 0.00-0.89; p=0.029), a median PFS of 11.5 weeks versus 24 weeks (HR 2.52, 1.33-4.78; p=0.013), and a median overall survival of 34 weeks versus 51 weeks (3.29, 1.60-6.74; p=0.0057). Multivariate analysis and conditional inference trees confirmed that, if KRAS is not mutated, assessing BRAF, NRAS, and PIK3CA exon 20 mutations (in that order) gives additional information about outcome. Objective response rates in our series were 24.4% in the unselected population, 36.3% in the KRAS wild-type selected population, and 41.2% in the KRAS, BRAF, NRAS, and PIK3CA exon 20 wild-type population. INTERPRETATION: While confirming the negative effect of KRAS mutations on outcome after cetuximab, we show that BRAF, NRAS, and PIK3CA exon 20 mutations are significantly associated with a low response rate. Objective response rates could be improved by additional genotyping of BRAF, NRAS, and PIK3CA exon 20 mutations in a KRAS wild-type population. FUNDING: Belgian Federation against cancer (Stichting tegen Kanker).

Numerous clinical studies have shown that anti-EGFR therapies are effective only in a subset of patients with colorectal cancer. Even though mutations in the KRAS gene have been confirmed as negative predictors of the response to EGFR-targeted therapies, not all KRAS wild-type (wt-KRAS) patients will respond to treatment. Recent studies have demonstrated that additionally wild-type BRAF (wt-BRAF) genotype is required for response to panitumumab or cetuximab, suggesting that BRAF genotype criteria should be used together with KRAS genotype for selecting the patients who are about to benefit from the anti-EGFR therapy. In this study, 239 samples obtained from 215 patients with metastatic colorectal cancer were tested for the presence of the seven most common mutations in the KRAS gene and the V600E mutation in the BRAF gene. Among the tested patients, 53.8% of patients had wt-KRAS genotype and 46.2% were KRAS mutants. Around five percent (5.1%) of the tested patients bore the V600E mutation in BRAF gene. All the patients showing to have the V600E mutation in BRAF were wt-KRAS. The concordance of KRAS and BRAF mutational status between primary and metastatic tumor tissue samples was 100%. We have shown that the proportions of mutated and non-mutated KRAS in Slovene patients, as well as the proportion of V600E mutations in BRAF is similar to genotyping results reported by other authors. The tested seven KRAS mutations on codons 12 and 13 were mutually exclusive with the V600E mutation in the BRAF gene. Summing up the results about the KRAS and the BRAF mutation carriers from our study, the portion of potentially non-responsive patients for the anti-EGFR treatment is 51.3%.

Epidemiologic studies have evaluated the association between BRAF mutations and resistance to the treatment of anti-EGFR monoclonal antibodies (MoAb) in patients with metastatic colorectal cancer (mCRC). However, the results are still inconclusive. To derive a more precise estimation of the relationship, we performed this meta-analysis. A total of 11 studies were included in the final meta-analysis. There were seven studies for unselected mCRC patients and four studies for patients with wild type KRAS mCRC. Among unselected mCRC patients, BRAF V600E mutation was detected in 48 of 546 primary tumors (8.8%). The objective response rate (ORR) of patients with mutant BRAF was 29.2% (14/48), whereas the ORR of patients with wild-type BRAF was 33.5% (158/472).The overall RR for ORR of mutant BRAF patients over wild-type BRAF patients was 0.86 (95% CI=0.57-1.30; P=0.48). For patients with KRAS wild-type mCRC, BRAF V600E mutation was detected in 40 of 376 primary tumors (10.6%). The ORR of patients with mutant BRAF was 0.0% (0/40), whereas the ORR of patients with wild-type BRAF was 36.3% (122/336). The pooled RR of mutant BRAF patients over wild-type BRAF patients was 0.14 (95% CI=0.04-0.53; P=0.004). In conclusion, this meta-analysis provides evidence that BRAF V600E mutation is associated with lack of response in wild-type KRAS mCRC treated with anti-EGFR MoAbs. BRAF mutation may be used as an additional biomarker for the selection of mCRC patients who might benefit from anti-EGFR MoAbs therapy.

BACKGROUND: Oncogenic activation of EGF-signalling pathway is central to the progression of colorectal cancer. The use of mutations of the KRAS codons 12 and 13 as a selection biomarker for anti-endothelial growth factor receptor (EGFR) monoclonal antibody treatment is at present the first major step towards individualised treatment for patients with metastatic colorectal cancer. The impact of BRAF V600E mutation is not well documented. PATIENTS AND METHODS: A total of 803 metastatic cancer samples from colorectal cancer patients were explored for KRAS exon 2 and BRAF exon 15 mutations. BRAF mutated samples were characterized for mismatch repair function. RESULTS: Overall, 344 tumours were mutated, with 34 of them involving BRAF mutations (8 of microsatellite instability type). No specificity was found according to gender, age at diagnosis and tumour localisation. CONCLUSION: A complete analysis of KRAS, BRAF and PIK3CA status may identify approximately 10-15% additional patients who are unlikely to respond an EGFR-targeted monoclonal antibody and who may benefit from prospective and specific new biomarker-driven studies.

BACKGROUND: It is hypothesized that BRAF mutant cancers represent a discrete subset of metastatic colorectal cancer (CRC) defined by poorer survival. This study investigates whether BRAF mutant CRC is further defined by a distinct pattern of metastatic spread and explores the impact of BRAF mutation and microsatellite instability (MSI) on prognosis in metastatic CRC. METHODS: By using prospective clinical data and molecular analyses from 2 major centers (Royal Melbourne Hospital and The University of Texas MD Anderson cancer Center), patients with known BRAF mutation status were analyzed for clinical characteristics, survival, and metastatic sites. RESULTS: The authors identified 524 metastatic CRC patients where BRAF mutation status was known; 57 (11%) were BRAF mutant tumors. BRAF mutant tumors were significantly associated with right-sided primary tumor, MSI, and poorer survival (median, 10.4 months vs 34.7 months, P < .001). A distinct pattern of metastatic spread was observed in BRAF mutant tumors, namely higher rates of peritoneal metastases (46% vs 24%, P = .001), distant lymph node metastases (53% vs 38%, P = .008), and lower rates of lung metastases (35% vs 49%, P = .049). In additional survival analyses, MSI tumors had significantly poorer survival compared with microsatellite stable tumors (22.1 months vs 11.1 months, P = .017), but this difference was not evident in the BRAF mutant population. CONCLUSIONS: The pattern of metastatic spread observed in this study further defines BRAF mutant CRC as a discrete disease subset. The authors demonstrated that, unlikely early stage disease, MSI is associated with poorer survival in metastatic CRC, although this is driven by its association with BRAF mutation.

Anti-EGFR monoclonal antibodies (anti-EGFRmAb) serve in the treatment of metastatic colorectal cancer (mCRC), but patients with a mutation in KRAS/BRAF and nearly one-half of those without the mutation fail to respond. We performed microRNA (miRNA) analysis to find miRNAs predicting anti-EGFRmAb efficacy. Of the 99 mCRC patients, we studied differential miRNA expression by microarrays from primary tumors of 33 patients who had wild-type KRAS/BRAF and third- to sixth-line anti-EGFRmAb treatment, with/without irinotecan. We tested the association of each miRNA with overall survival (OS) by the Cox proportional hazards regression model. Significant miR-31* up-regulation and miR-592 down-regulation appeared in progressive disease versus disease control. miR-31* expression and down-regulation of its target genes SLC26A3 and ATN1 were verified by quantitative reverse transcriptase polymerase chain reaction. Clustering of patients based on miRNA expression revealed a significant difference in OS between patient clusters. Members of the let-7 family showed significant up-regulation in the patient cluster with poor OS. Additionally, miR-140-5p up-regulation and miR-1224-5p down-regulation were significantly associated with poor OS in both cluster analysis and the Cox proportional hazards regression model. In mCRC patients with wild-type KRAS/BRAF, miRNA profiling can efficiently predict the benefits of anti-EGFRmAb treatment. Larger series of patients are necessary for application of these miRNAs as predictive/prognostic markers.

PURPOSE: Unlike cetuximab, there is a paucity of biomarkers for bevacizumab as predictors of outcome in metastatic colorectal cancer (mCRC) patients. Obviously exploring the worth of some potential markers in this setting is warranted. The purpose of this study was to investigate the predictive value of the presence of K-RAS and B-RAF mutations on the outcome of patients with mCRC treated with FOLFIRI and bevacizumab combination therapy. METHODS: A total of 172 patients with mCRC were evaluated. K-RAS and B-RAF mutations were analyzed by quantitative PCR. Median progression-free survival (PFS) and overall survival (OS) were compared utilizing chi-square and Mann-Whitney U tests, respectively. RESULTS: Forty-four percent (N=77) of the patients were found to harbor K-RAS mutations and 6 (7.5%) were positive for B-RAF mutations. In baseline no difference in PFS and OS was observed between the groups with or without K-RAS mutation. No relationship was established between K-RAS and B-RAF mutation status and baseline CEA and CA19-9 tumor markers levels. CONCLUSION: K-RAS and B-RAF mutations do not seem to be predictive of treatment outcome as potential biomarkers for bevacizumab therapy in mCRC. However, not only the presence of K-RAS and B-RAF mutations but also the different biological behavior of the various subtypes of mutations should be considered as potential determinants in the final outcome of this disease.

OBJECTIVE: This study aimed to evaluate the predictive and prognostic roles of BRAF mutation in patients with metastatic colorectal cancer (mCRC) treated with anti-epidermal growth factor receptor (EGFR) monoclonal antibodies (MoAbs). METHODS: Computer searches of the literature on BRAF mutation in mCRC patients were performed. Studies with objective response rate (ORR) to anti-EGFR MoAbs and/or overall survival (OS) and progression-free survival (PFS) with different BRAF gene expression in mCRC patients were eligible. RESULTS: A total of 19 studies including 2875 patients was enrolled in the meta-analysis. BRAF mutation was detected in 246 patients. The ORR was 18.4% (40/217) in mutant BRAF group and 41.7% (831/1993) in the wild-type BRAF group. The overall risk ratio (RR) for the ORR of BRAF mutation patients compared with wild-type BRAF patients was 0.58 (95% confidence intervals [CI] 0.35-0.94, P = 0.027). The median PFS of patients with BRAF mutation was significantly shorter than that of patients with wild-type BRAF (hazard ratio [HR] 2.98, 95% CI 2.07-4.27, P < 0.001) and the median OS of patients with BRAF mutation was also significantly shorter than that of those with wild-type BRAF (HR 2.85, 95% CI 2.31-3.52, P < 0.001). CONCLUSION: BRAF mutation is associated with poor response to anti-EGFR MoAbs and it is an adverse prognostic biomarker of the survival of patients with mCRC.

OBJECTIVE: Preoperative fine needle aspiration biopsy (FNAB) has become the initial diagnostic method for papillary thyroid carcinoma (PTC). Identification of cytomorphologic factors predicting lymph node metastasis (LNM) is clinically important for determining the appropriate treatment regimen due to the high rate of lymph node involvement in PTC at the time of diagnosis. Hobnail features (HF) have previously been described as potential histomorphologic features in the histological examination of PTC. This study evaluated the value of HF as a predictor of LNM. STUDY DESIGN: Histologically confirmed FNABs (n = 111) of papillary thyroid microcarcinoma prepared by the liquid-based method were enrolled. Along with other cytomorphologic parameters, HF were evaluated for their value in predicting LNM. RESULTS: Although HF were closely correlated with cytoplasmic vacuoles of tumor cells and background macrophages (p < 0.05), which were considered diagnostic indicators of PTC with cystic changes, HF were only found to be significantly correlated with LNM (p = 0.008). The BRAF(V660E) mutation was not associated with LNM. All combinations including HF were revealed as stronger predictors of LNM (odds ratios: 2.254-2.524, p < 0.05). CONCLUSIONS: HF, a distinctive cytomorphologic feature, may be used as a factor predicting LNM in preoperative FNAB of PTC.

BACKGROUND: The liver is the most common target organ in the metastasis of colorectal cancer (CRC). Synchronous liver metastases may confer a poorer prognosis than metachronous metastases, and genetic alterations and an inflammatory response have also been associated with a poor prognosis in cases of a liver metastasis arising from CRC. However, few studies have examined the relationship between KRAS mutations and inflammatory status in CRC, especially with respect to liver metastases. METHODS: The effect of the activated mitogen-activated protein kinase pathway and another protein involved in inflammation, C-reactive protein, in liver metastases were examined. We aimed to determine the impact of the CRP-specific single nucleotide polymorphism (SNP) rs7553007 in liver metastasis on the CRC-specific survival (CSS) of patients after colorectal liver metastasectomy. RESULTS: We found no significant differences in genotype distributions and allele frequencies at the CRP SNP rs7553007 between CRC patients with liver metastasis and the control group. CSS rates were low in the subgroup of patients with synchronous metastasis with the A-allele (A/A and A/G) at rs7553007 or mutated KRAS/BRAF in liver metastatic specimens. Furthermore, the CRP SNP rs7553007 (hazard ratio [HR] = 1.101; 95% confidence interval [CI] = 1.011-1.200; P = 0.027) and KRAS/BRAF mutations (HR = 2.377; 95%

There is conflicting evidence on the predictive role of KRAS status when cetuximab is added to oxaliplatin-based regimens. This study investigated the impact of KRAS, NRAS, BRAF, PI3KCA and TP53 status on outcome of elderly metastatic colorectal cancer patients enrolled in TEGAFOX-E (cetuximab, oxaliplatin and oral uracil/ftorafur--UFT) phase II study. Twenty-eight patients were enrolled and all were evaluable for safety and activity. Twenty-three specimens were analysed for KRAS, BRAF, NRAS, PI3KCA and TP53 mutational status by means of polymerase chain reaction and correlated with objective response, progression-free survival and overall survival. An evident increase of response rate was noted in KRAS/NRAS wild-type cases (70 versus 33%, P = 0.198). KRAS/NRAS wild-type status showed an independent association with a longer progression-free survival (44 versus 9 weeks, P = 0.009). Considering the combined assessment of BRAF, KRAS/NRAS and TP53, a trend towards an increase of response rate was noted in patients without mutations (83 versus 33%, P = 0.063). Moreover, patients with all wild-type genes had significantly longer progression-free survival than patients with any mutation (48 versus 10 weeks, P = 0.007). As a single biomarker, only KRAS/NRAS proteins maintained an independent value for outcome prediction. Patients with KRAS/NRAS, BRAF and TP53 wild-type tumours could derive the maximal benefits from treatment with cetuximab, oxaliplatin and UFT.

BACKGROUND: To prospectively evaluate the usefulness of the BRAFV600E mutation detection in daily clinical practice in patients with metastatic Colorectal cancer (mCRC). PATIENTS AND METHODS: 504 mCRC patients treated with systemic chemotherapy +/- biologics were analyzed. RESULTS: A statistically significant higher incidence of the BRAF mutation was observed in patients with ECOG-PS 2 (p=0.001), multiple metastatic sites (p=0.002),> 65 years old (p=0.004), primary tumors located in the colon (p<0.001), high-grade tumors (p=0.001) and in those with mucinous features (p=0.037). Patients with BRAFV600E mutated tumors had a statistically significantly reduced progression-free survival (PFS) compared to wild-type (wt) ones (4.1 and 11.6 months, respectively; p<0.001) and overall survival (OS) (14.0 vs. 34.6 months, respectively; p<0.001). In the multivariate analysis the BRAFV600E mutation emerged as an independent factor associated with reduced PFS (HR: 4.1, 95% CI 2.7-6.2; p<0.001) and OS (HR: 5.9, 95% CI 3.7-9.5; p<0.001). Among the 273 patients treated with salvage cetuximab or panitumumab, the BRAFV600E mutation was correlated with reduced PFS (2.2 vs. 6.0 months; p<0.0001) and OS (4.3 vs. 17.4 months; p<0.0001). CONCLUSIONS: The presence of BRAFV600E-mutation in mCRC characterizes a subgroup of patients with distinct biologic, clinical and pathological features and is associated with very poor patients prognosis.

BACKGROUND: Selective BRAF inhibitors have shown dramatic results with regard to improving outcome in patients with melanoma. Testing the BRAF status in matched primary and metastatic melanomas to optimize individual targeted therapy is not well investigated. METHODS: Extended BRAF testing using PCR for 9 mutations and VE1 immunohistochemistry for BRAF V600E detection on 95 lesions including 40 primary melanomas with their matched metastases (n = 42), recurrences (n = 9) and second primaries (n = 4) was performed. Nine patients had multiple metastases. RESULTS: V600E was the only identified mutation type; 35.4% of primary vs. 18.9% of metastatic melanomas. The overall primary-metastatic BRAF status discordance rate was 32.3% using PCR and 27.5% with immunohistochemistry, and was significantly more frequent in primary lesions with mutant BRAF (67%). Males and patients with metastasis to lymph nodes were less likely to be discordant compared to females and those with metastasis to other sites (p = 0.023). Discordant BRAF mutation status was predicted by multivariate binary logistic regression: the presence of a mutant BRAF in the primary melanoma [OR (95% C.I.) = 23.4 (2.4-229.7)] and female gender [OR = 10.6 (1.08-95)]. Inter-metastases BRAF concordance was 100% (6 comparisons). CONCLUSION: A high discordant rate implies the need for clinical trials addressing the response to targeted therapy in patients with discordant BRAF statuses between their primary and metastatic lesions.

BACKGROUND: Given the high incidence of metastatic esophageal squamous cell carcinoma, especially in Asia, we screened for the presence of somatic mutations using OncoMap platform with the aim of defining subsets of patients who may be potential candidate for targeted therapy. METHODS AND MATERIALS: We analyzed 87 tissue specimens obtained from 80 patients who were pathologically confirmed with esophageal squamous cell carcinoma and received 5-fluoropyrimidine/platinum-based chemotherapy. OncoMap 4.0, a mass-spectrometry based assay, was used to interrogate 471 oncogenic mutations in 41 commonly mutated genes. tumor specimens were prepared from primary cancer sites in 70 patients and from metastatic sites in 17 patients. In order to test the concordance between primary and metastatic sites from the patient for mutations, we analyzed 7 paired (primary-metastatic) specimens. All specimens were formalin-fixed paraffin embedded tissues and tumor content was >70%. RESULTS: In total, we have detected 20 hotspot mutations out of 80 patients screened. The most frequent mutation was PIK3CA mutation (four E545K, five H1047R and one H1047L) (N = 10, 11.5%) followed by MLH1 V384D (N = 7, 8.0%), TP53 (R306, R175H and R273C) (N = 3, 3.5%), BRAF V600E (N = 1, 1.2%), CTNNB1 D32N (N = 1, 1.2%), and EGFR P733L (N = 1, 1.2%). Distributions of somatic mutations were not different according to anatomic sites of esophageal cancer (cervical/upper, mid, lower). In addition, there was no difference in frequency of mutations between primary-metastasis paired samples. CONCLUSIONS: Our study led to the detection of potentially druggable mutations in esophageal SCC which may guide novel therapies in small subsets of esophageal cancer patients.

We have recently reported that activation of the Raf-1/mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase 1/2 (MEK1/2)/ERK1/2 signaling cascade in gastrointestinal carcinoid cell line (BON) alters cellular morphology and neuroendocrine phenotype. The mechanisms by which Raf-1 mediates these changes in carcinoid cells are unclear. Here, we report that activation of the Raf-1 signaling cascade in BON cells induced the expression of focal adhesion kinase (FAK) protein, suppressed the production of neuroendocrine markers, and resulted in significant decreases in cellular adhesion and migration. Importantly, inactivation of MEK1/2 by 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene or abolition of FAK induction in Raf-1-activated BON cells by targeted siRNA led to reversal of the Raf-1-mediated reduction in neuroendocrine markers and cellular adhesion and migration. Phosphorylation site-specific antibodies detected the phosphorylated FAK(Tyr407), but not FAK(Tyr397), in these Raf-1-activated cells, indicating that FAK(Tyr407) may be associated with changes in the neuroendocrine phenotype. Overexpression of constitutively active FAK plasmids (wild-type FAK or FAK(Tyr397) mutant) into BON cells reduced neuroendocrine markers, whereas the FAK(Tyr407) mutant plasmid did not show any decrease in the levels of neuroendocrine markers, indicating that phosphorylation of FAK at the Tyr(407) residue may be important for these effects. Our results showed for the first time that FAK is an essential downstream effector of the Raf-1/MEK1/2/ERK1/2 signaling cascade and negatively regulated the neuroendocrine and metastatic phenotype in BON cells.

PURPOSE: The purpose of the study was to clarify the incidence of B-raf oncogene (BRAF) mutations in primary cutaneous melanomas, their relation to tumor progression, and effect on disease outcome. Somatic mutations of BRAF kinase, a component of the Ras-mitogen-activated protein/extracellular signal-regulated kinase kinase-mitogen-activated protein kinase pathway, are frequently reported (>65%) in nevi and malignant melanomas. EXPERIMENTAL DESIGN: We assessed BRAF mutation frequency in exons 11 and 15 in primary (n = 59) and metastatic (n = 68) melanomas. Direct sequencing of PCR products was performed on DNA isolated and purified from microdissected tumors. RESULTS: Eighteen mutations (31%) at exon 15 were detected in primary melanoma with a significantly (P = 0.001) higher frequency in patients < 60 years old. Incidence of BRAF mutation did not correlate with Breslow thickness. Presence of BRAF mutation of primary tumors did not effect overall disease-free survival. BRAF mutation frequency in metastatic lesions was 57% and significantly (P = 0.0024) higher than primary melanomas. CONCLUSIONS: The study suggests that BRAF mutation may be acquired during development of metastasis but is not a significant factor for primary tumor development and disease outcome.

mutation of BRAF has been proposed to contribute to melanoma development. However, it remains unclear whether or not BRAF mutation is associated with any particular stage of melanoma progression. tumor biopsy specimens from patients with melanoma were analyzed to determine whether the frequency of BRAF mutation in metastatic melanoma differed from primary melanoma. BRAF mutation was present in 15 of 23 (61%) patients with primary melanoma and in 7 of 12 (58%) patients with metastatic melanoma. These results suggest that BRAF mutation in melanoma is most likely to occur prior to the development of metastatic disease.

Downstream of Ras, the serine/threonine kinase B-raf has been reported to be mutated, amongst other carcinomas, in a substantial subset of primary melanomas, with a preponderance of mutations within the kinase domain, including the activating V599E and V599K transitions. We investigated a representative series of 54 resection specimens of melanoma lymph node metastases for the presence of mutations within the activation segment (exon 15) of the B-raf kinase domain by polymerase chain reaction (PCR) and single-strand conformational polymorphism (SSCP) gel electrophoresis. Sequencing of cloned PCR-SSCP amplicons resulted in 24 (44%) samples harbouring somatic mutations, which is not significantly different from the mutation frequency found in recently investigated primary cutaneous melanomas (Deichmann M, Thome M, Benner A, Naher H. B-raf exon 15 mutations are common in primary melanoma resection specimens but not associated with clinical outcome. Oncology 2004; 66:411-419). The activating mutation T1796A was present in 20 (83%) of these resection specimens, followed in frequency by the oncogenic g1795A mutation in five (21%) cases. With regard to the B-raf protein sequence, the acidic amino acid transitions V599E and V599K were predicted in 15 (62%) and five (21%) of the 24 positive metastases, respectively. The detection of mutations at this hot spot codon was significantly associated with subsequent visceral metastasis (P=0.03; Fisher's exact test). During the transition from primary melanomas (see reference above) to lymph node metastases, the spectrum of B-raf mutations narrows significantly (P=0.00047). The oncogenic B-raf mutations V599E and V599K, as early events in melanocyte transformation, persist throughout metastasis with important prognostic implications.

OBJECTIVE: To investigate the expression of breast cancer metastasis suppressor 1 (BRMS1) and CD44v6 protein in supraglottic cancer and to evaluate its clinical significance. METHOD: The expression of BRMS1 protein and CD44v6 protein were examined by using immunohistochemical method in 70 cases of paraffin-embedded supraglottic cancer tissues and their surrounding laryngeal normal mucosa tissues (LNT). RESULT: The expression of BRMS1 protein in LNT of supraglottic cancer was positive, and the positive rate was 85.7% (60/70); in tumor tissue was negative or lower expression, and the positive rate was 35.7% (25/70). The expression of CD44v6 protein in tumor tissue of supraglottic cancer was positive, the positive rate was 82.9% (58/70), in LNT was negative. There was a significant difference in BRMS1 and CD44v6 protein expression between the supraglottic cancer tissue and LNT (P<0.01). The expression of BRMS1 and CD44v6 protein had correlation with clinical stage and pathologic differentiation and cervical lymph node metastasis of supraglottic cancer (P<0.01). No correlation was found between the two proteins expression and sex and age (P>0.05). The expression of BRMS1 protein was related to the expression of CD44v6 protein (r = -0.9042, P<0.01). Calculated by Kaplan-Meier method, there is no survival difference at 3-year between the group with positive BRMS1 protein expression and the group with negative BRMS1 protein expression in tumor tissues (P>0.05), there is a significant survival difference at 3-year between the group with positive CD44v6 protein expression and the group with negative CD44v6 protein expression in tumor tissues (P<0.05). CONCLUSION: The expression of BRMS1 protein in supraglottic cancer is significantly decreased and the expression of CD44v6 protein in supraglottic cancer is significantly increased. The expression of BRMS1 protein and CD44v6 protein has a close relationship with pathologic differentiation and clinical stage and cervical lymph node metastasis of supraglottic cancer. Combined detection of the expression of them in supraglottic cancer may provide a significant parameter to judge the cervical lymph node metastasis of supraglottic cancer.

PURPOSE: To assess the frequency and type of oncogenic BRAF mutations in metastatic melanoma and correlate BRAF status with clinicopathologic features and outcome. PATIENTS AND METHODS: Consecutive BRAF-tested Australian patients with metastatic melanoma (n = 197) were observed prospectively. A comprehensive range of clinicopathologic variables were correlated with BRAF mutation status, and a survival analysis was conducted. RESULTS: Forty-eight percent of patients had a BRAF mutation; 70 patients (74%) had V600E, 19 (20%) had V600K, and six (6%) had other genotypes. Other than age at diagnosis of distant metastasis (median age, 56 v 63 years for BRAF-mutant v BRAF wild-type patients, respectively; P < .001), there was no significant difference in clinical features of patients with metastatic melanoma by mutation status. Features of the antecedent primary melanoma significantly associated with a BRAF mutation (P < .05) were histopathologic subtype, presence of mitoses, single or occult primary melanoma, truncal location, and age at diagnosis of primary tumor metastasis was not significantly different between BRAF-mutant and BRAF wild-type patients; however, the median survival of patients with newly diagnosed metastatic melanoma was 5.7 months for BRAF-mutant patients not treated with a BRAF inhibitor, 8.5 months for BRAF wild-type patients, and not reached for BRAF-mutant patients treated with a BRAF inhibitor. CONCLUSION: V600K mutations comprised 20% of BRAF mutations. Characteristics of the antecedent primary melanoma and age at diagnosis differed in BRAF-mutant and BRAF wild-type patients. The presence of mutant BRAF had no impact on the disease-free interval from diagnosis of first-ever melanoma to first distant metastasis; however, it may have impacted survival thereafter.

Melanoma is the most deadly tumor of the skin, and systemic therapies for the advanced stage are still limited. Recent genetic analyses have revealed the molecular diversity of melanoma and potential therapeutic targets. By screening a cohort of 142 primary nonepithelial tumors, we discovered that about 10% of melanoma cases (4/39) harbored an IDH1 or IDH2 mutation. These mutations were found to coexist with BRAF or KIT mutation, and all IDH1 mutations were detected in metastatic lesions. BRAF-mutated melanoma cells, additionally expressing the cancer-related IDH1 mutant, acquired increased colony-forming and in vivo growth activities and showed enhanced activation of the MAPK and STAT3 pathways. Genome-wide gene expression profiling demonstrated that mutant IDH1 affected the expression of a set of genes. Especially, it caused the induction of growth-related transcriptional regulators (Jun, N-myc, Atf3) and the reduction of Rassf1 and two dehydrogenase genes (Dhrs1 and Adh5), which may be involved in the carcinogenesis of IDH1-mutated tumors. Our analyses demonstrate that IDH1 mutation works with other oncogenic mutations and could contribute to the metastasis in melanoma.

PURPOSE: To determine the occurrence of BRAF V600E gene mutations and copy number changes of all autosome arms and genes known to be frequently altered in tumorigenesis in primary and metastatic conjunctival melanomas (CoMs). METHODS: DNA (200 ng) was analyzed by three multiplex ligation-dependent probe amplification assays (P027 uveal melanoma, P036 human telomere, and P206 spitzoid melanoma). RESULTS: Eight of 16 primary tumor samples and 4 of 6 metastatic samples showed BRAF V600E gene mutations. CDKN1A and RUNX2 (both 6p21.2) were amplified in 11 and 16 of 21 primary CoMs, respectively. In metastatic CoMs, MLH1 (3p22.1) and TIMP2 (17q25.3) were frequently amplified, and MGMT (20q26.3) and ECHS1 (10q26.3) were frequently deleted. The BDH (3q), FLJ20265 (4p), OPRL1 (20q), and PAO (10q) genes, representing the telomeres of their respective chromosome arms in the P036 assay, were frequently amplified in metastatic CoMs. No statistically significant associations were identified between BRAF mutation or CDKN1A or RUNX2 amplification and sex, age, histologic cell type, or patient survival. CONCLUSIONS: No copy number changes were associated exclusively with metastatic CoMs. However, further investigation of the role of CDKN1A and RUNX2 in CoMs development and that of MLH1, TIMP2, MGMT, and ECHS1 in metastatic CoMs is warranted. Validation of the observed gene and chromosome arm copy number changes in a larger cohort of primary and metastatic CoMs is necessary to identify the patients at highest risk for CoMs metastasis.

Understanding the molecular alterations that confer cancer cells with motile, metastatic properties is needed to improve patient survival. Here, we report that p38gamma motogen-activated protein kinase regulates breast cancer cell motility and metastasis, in part, by controlling expression of the metastasis-associated small GTPase RhoC. This p38gamma-RhoC regulatory connection was mediated by a novel mechanism of modulating RhoC ubiquitination. This relationship persisted across multiple cell lines and in clinical breast cancer specimens. Using a computational mechanical model based on the finite element method, we showed that p38gamma-mediated cytoskeletal changes are sufficient to control cell motility. This model predicted novel dynamics of leading edge actin protrusions, which were experimentally verified and established to be closely related to cell shape and cytoskeletal morphology. Clinical relevance was supported by evidence that elevated expression of p38gamma is associated with lower overall survival of patients with breast cancer. Taken together, our results offer a detailed characterization of how p38gamma contributes to breast cancer progression. Herein we present a new mechanics-based analysis of cell motility, and report on the discovery of a leading edge behavior in motile cells to accommodate modified cytoskeletal architecture. In summary, these findings not only identify a novel mechanism for regulating RhoC expression but also advance p38gamma as a candidate therapeutic target.

PURPOSE: Certain clinicopathologic features correlate with BRAF mutation status in melanoma including younger age and primary subtype. This study sought to determine the BRAF mutation status by age-decade and whether BRAF-mutant genotypes correlated with clinicopathologic features and outcome in patients with metastatic melanoma. METHODS: A prospectively assembled cohort of Australian patients were followed from diagnosis of metastatic melanoma (N = 308). Clinicopathologic variables were correlated with BRAF mutational status, genotype, and survival. RESULTS: Forty-six percent of patients had a BRAF mutation; 73% V600E, 19% V600K, and 8% other genotypes. An inverse relationship existed between BRAF mutation prevalence and age-decade (P < 0.001). All patients <30 years and only 25% >/= 70 years had BRAF-mutant melanoma. Amongst BRAF-mutant melanoma, the frequency of non-V600E genotypes (including V600K) increased with increasing age. Non-V600E genotypes comprised <20% in patients <50 years and >40% in those >/= 70 years. A higher degree of cumulative sun-induced damage correlated with V600K but not V600E melanoma (P = 0.002). The disease-free interval from diagnosis of primary melanoma to first distant metastasis was shorter for patients with V600K compared with V600E melanoma (17.4 vs. 39.2 months, P = 0.048), with no difference in survival thereafter. In patients BRAF tested at diagnosis of metastatic melanoma, one year survival from diagnosis of metastasis was significantly longer for patients with BRAF-mutant melanoma treated with an inhibitor (83%), than those not treated with an inhibitor (29%, P < 0.001), or patients with BRAF wild-type melanoma (37%, P < 0.001). CONCLUSION: Different genotypes exist within BRAF-mutant metastatic melanoma, representing biologically and clinically discrete subtypes, suggesting distinct etiology and behavior.

AIM: Approximately 40-60% of melanomas from Caucasian populations carry activating mutations in the BRAF oncogene, with the most common being the p.Val600Glu (V600E) hotspot mutation in exon 15. The aim of the present study was to investigate the frequency of the less common p.Val600Lys (V600K) mutation in metastatic melanoma from a high incidence region. METHOD: Dideoxy sequencing and fluorescent single strand conformation analysis were used to screen for mutations in exon 15 of BRAF in 183 cases of metastatic melanoma. RESULTS: The overall incidence of BRAF mutation (89/183, 49%) was very similar to other large studies of Caucasian populations. However, the frequency of the p.Val600Lys mutation was higher than in most other studies and comprised almost one-third of all BRAF mutations in our cohort (27/89, 30%). CONCLUSION: BRAF p.Val600Lys mutations were present at a relatively high frequency in this cohort of metastatic melanoma patients (27/183, 15%). Assays used to screen for BRAF mutations in the clinic should be robust enough to detect the p.Val600Lys mutation, as this may have therapeutic implications.

Brain metastases are a major cause of morbidity and mortality in patients with advanced melanoma. With the development of targeted agents for the treatment of metastatic melanoma, a great deal of interest has focused on whether selective BRAF inhibitors may play a role in the treatment of brain metastases in lieu of or in addition to surgery and/or radiation therapy. However, relatively little is known about the intracranial effectiveness of vemurafenib, the only US Food and Drug Administration-approved selective BRAF V600E inhibitor, because patients with brain metastases have historically been excluded from vemurafenib clinical trials. We describe 3 patients with BRAF V600E mutation metastatic melanoma in whom treatment with vemurafenib resulted in prompt extracranial disease response but progression of metastatic disease in the brain. Further, we discuss possible mechanisms responsible for the suboptimal central nervous system response observed in these patients and alternative therapies for patients with melanoma metastatic to the brain.

PURPOSE: To evaluate the effects of BRAF inhibition on the tumor microenvironment in patients with metastatic melanoma. EXPERIMENTAL DESIGN: Thirty-five biopsies were collected from 16 patients with metastatic melanoma pretreatment (day 0) and at 10 to 14 days after initiation of treatment with either BRAF inhibitor alone (vemurafenib) or BRAF + MEK inhibition (dabrafenib + trametinib) and were also taken at time of progression. Biopsies were analyzed for melanoma antigens, T-cell markers, and immunomodulatory cytokines. RESULTS: Treatment with either BRAF inhibitor alone or BRAF + MEK inhibitor was associated with an increased expression of melanoma antigens and an increase in CD8+ T-cell infiltrate. This was also associated with a decrease in immunosuppressive cytokines [interleukin (IL)-6 and IL-8] and an increase in markers of T-cell cytotoxicity. Interestingly, expression of exhaustion markers TIM-3 and PD1 and the immunosuppressive ligand PDL1 was increased on treatment. A decrease in melanoma antigen expression and CD8 T-cell infiltrate was noted at time of progression on BRAF inhibitor alone and was reversed with combined BRAF and MEK inhibition. CONCLUSIONS: Together, these data suggest that treatment with BRAF inhibition enhances melanoma antigen expression and facilitates T-cell cytotoxicity and a more favorable tumor microenvironment, providing support for potential synergy of BRAF-targeted therapy and immunotherapy. Interestingly, markers of T-cell exhaustion and the immunosuppressive ligand PDL1 are also increased with BRAF inhibition, further implying that immune checkpoint blockade may be critical in augmenting responses to BRAF-targeted therapy in patients with melanoma.

We studied the behaviour of three novel human sporadic melanoma cell lines (hmel1, hmel9, hmel11) extracted from tumors with different degrees of malignancy, concerning the cell signalling pathways controlled by MC1R, BRAF, NRAS and beta-catenins. The novel cell lines were compared to metastatic cell lines (HBL, LND1), wild type (wt) for MC1R and BRAF genes, that have been extensively characterised and were used as control. All the novel cell lines have silent or no MC1R mutations even though MC1R signalling is severely impaired. Conversely, they harbour BRAF mutations at the V600 residue. These mutations determine a constitutive ERK phosphorylation in all the three cell lines. Our new melanoma cell lines were BRAF mutated in hetero- and homozygosis, even with a wild type MC1R, and unresponsive to NDP-MSH treatment. Quantity and subcellular localization of beta-catenin were analyzed in both novel and control cell lines. In HBL and LND1 there were high levels of beta-catenin distributed in the cytoplasm/nucleus, while in the novel melanoma cell lines beta-catenins were less abundant and seemed to be located at the plasma membrane/cytoplasm and absent in the nucleus. We sequenced beta-catenin cDNA for all the melanoma cell lines, and found mutations in HBL, LND1 and hmel1, while hmel9 and hmel11 were wt. We found that beta-catenin levels were not influenced by the RAS/RAF/MAPK pathway because inhibition with PD98059 (a MEK inhibitor) did not produce any effect on beta-catenin stability and/or localization.

PURPOSE To assess pharmacodynamic effects and intrinsic and acquired resistance mechanisms of the BRAF inhibitor vemurafenib in BRAF(V600)-mutant melanoma, leading to an understanding of the mechanism of action of vemurafenib and ultimately to optimization of metastatic melanoma therapy. METHODS In the phase II clinical study NP22657 (BRIM-2), patients received oral doses of vemurafenib (960 mg twice per day). Serial biopsies were collected to study changes in mitogen-activated protein kinase (MAPK) signaling, cell-cycle progression, and factors causing intrinsic or acquired resistance by immunohistochemistry, DNA sequencing, or somatic mutation profiling. Results Vemurafenib inhibited MAPK signaling and cell-cycle progression. An association between the decrease in extracellular signal-related kinase (ERK) phosphorylation and objective response was observed in paired biopsies (n = 22; P = .013). Low expression of phosphatase and tensin homolog showed a modest association with lower response. Baseline mutations in MEK1(P124) coexisting with BRAF(V600) were noted in seven of 92 samples; their presence did not preclude objective tumor responses. Acquired resistance to vemurafenib associated with reactivation of MAPK signaling as observed by elevated ERK1/2 phosphorylation levels in progressive lesions and the appearance of secondary NRAS(Q61) mutations or MEK1(Q56P) or MEK1(E203K) mutations. These two activating MEK1 mutations had not previously been observed in vivo in biopsies of progressive melanoma tumors. CONCLUSION Vemurafenib inhibits tumor proliferation and oncogenic BRAF signaling through the MAPK pathway. Acquired resistance results primarily from MAPK reactivation driven by the appearance of secondary mutations in NRAS and MEK1 in subsets of patients. The data suggest that inhibition downstream of BRAF should help to overcome acquired resistance.

Primary cutaneous folliculotropic melanoma has been described rarely, and there are even fewer published cases of folliculotropic metastases. We report a 54-year-old man with history of primary cutaneous melanoma of the right posterior shoulder, Breslow depth of 3.4 mm, with one positive sentinel lymph node, negative full axillary dissection, and no extranodal metastases at initial staging. Three years later, he presented with an asymptomatic isolated 2-mm blue-black papule on the scalp and was found to have widespread metastatic melanoma involving lymph nodes, liver, adrenal glands, subcutaneous tissue, skeleton, and lung. Histopathologic examination of the scalp lesion demonstrated a tumor nodule composed of sheets and nests of large round to polygonal cells centered about a hair follicle and within follicular epithelium. BRAF V600E gene mutation was documented in this lesion, and the patient received vemurafenib, with dramatic improvement noted on positron emission tomography scan after 2 months of treatment, soon followed by development of extensive metastases, including to brain. Although BRAF mutations have been found in primary and metastatic melanomas of the skin, and in melanoma metastases of extracutaneous sites, to our knowledge, this is the first case of a BRAF mutation documented in folliculotropic metastatic melanoma.

The Bcl-2 family member Mcl-1 is essential for melanoma survival; however, the influence of oncogenic BRAF signalling remains elusive. In this study, Mcl-1 splice variant expression was determined in a panel of melanoma cell lines in relation to BRAF mutational status. Mcl-1L mRNA expression was increased in melanoma cells compared with primary melanocytes with significantly increased mRNA and protein expression observed in BRAF(V600E) mutant melanoma cells. Although no change in Mcl-1S mRNA was observed, Mcl-1S protein expression also increased in BRAF mutant melanoma cells. Additionally, while over-expression of mutant BRAF(V600E) increased both Mcl-1L and Mcl-1S expression, inhibition of hyperactive BRAF signalling resulted in decreased Mcl-1L expression. These studies suggest that the regulation of Mcl-1 expression by BRAF signalling is increased by oncogenic activation of BRAF, revealing a mechanism of apoptotic resistance which may be overcome by the use of more specifically targeted Mcl-1 inhibitors.

PURPOSE: This study characterized the multiple-dose pharmacokinetics of vemurafenib 240-960 mg twice daily (bid) in BRAF(V600E) mutation-positive metastatic melanoma patients, using the commercial formulation (240-mg microprecipitated bulk powder film-coated tablets). METHODS: Melanoma patients (N = 52) were randomly allocated to four vemurafenib dose cohorts (240, 480, 720, or 960 mg bid for 14 days). After the day 15 morning dose, doses were interrupted until day 22, at which point patients were restarted on vemurafenib. Serial pharmacokinetic samples were collected after the morning dose on days 1, 9, and 15; trough pharmacokinetic samples were collected on day 2. RESULTS: Vemurafenib concentration increased with multiple doses to steady state at day 15; C(max), AUC(0-8h), and AUC(0-168h) increased between 3.3- and 3.8-fold across the fourfold dose range tested. Statistical analysis indicated dose proportionality across the dose range of 240-960 mg bid. Day 15 mean accumulation ratios (ratio of AUC(0-8h) on day 15/AUC(0-8h) on day 1) ranged from ~19 to 25 across cohorts. At steady state, the peak-to-trough ratio for vemurafenib exhibited a relatively flat concentration-time profile throughout the bid dosing interval. During dose interruption (days 15-22), mean vemurafenib trough concentrations decreased to minimal levels; vemurafenib exhibited a mean terminal phase half-life of 31.5-38.4 h. CONCLUSIONS: Vemurafenib plasma concentration accumulates with multiple bid doses of 240 mg. Vemurafenib exposure (AUC and C(max)) is dose proportional over the 240- to 960-mg bid dose range and exhibits constant drug levels over the bid dosing interval.

Concern regarding the presence of intertumoral heterogeneity of BRAF mutation status in patients with metastatic melanoma has led to uncertainty surrounding which specimens should preferentially undergo BRAF testing. We sought to examine the extent of intrapatient heterogeneity of BRAF(V600E) protein expression in patients with multiple tumors. Sixty-four patients with 171 tumors at various stages of disease progression had tumor BRAF(V600E) protein expression immunohistochemically (IHC) assessed using the BRAF(V600E) mutant-specific antibody VE1. Melanoma sections were examined for staining intensity (score 0 to 3), the presence of intratumoral heterogeneity, and concordance with molecular BRAF genotype. Intrapatient, intertumoral heterogeneity of BRAF(V600E) expression was also assessed by comparing VE1 staining on different tumors within the same patient. All specimens from 64 patients displayed complete intertumoral homogeneity of BRAF(V600E) expression status, and all tumors had concordant molecular and IHC BRAF status. Only 1 patient demonstrated >1 level of staining intensity heterogeneity between specimens. Intratumoral heterogeneity of staining intensity was not observed in any specimen. IHC-measured BRAF(V600E) protein expression displays complete intertumoral homogeneity, minimal intertumoral intensity heterogeneity, and no intratumoral heterogeneity in metastatic melanoma patients in various stages of disease progression. Our results suggest that, provided there is adequate quantity of viable tumor cells and minimal admixture of nontumor cells, testing any melanoma sample from a patient with metastatic disease will accurately determine BRAF status for treatment planning.

BACKGROUND: The orally available BRAF kinase inhibitor vemurafenib, compared with dacarbazine, shows improved response rates, progression-free survival (PFS), and overall survival in patients with metastatic melanoma that has a BRAF(V600) mutation. We assessed vemurafenib in patients with advanced metastatic melanoma with BRAF(V600) mutations who had few treatment options. METHODS: In an open-label, multicentre study, patients with untreated or previously treated melanoma and a BRAF(V600) mutation received oral vemurafenib 960 mg twice a day. The primary endpoint was safety. All analyses were done on the safety population, which included all patients who received at least one dose of vemurafenib. This report is the third interim analysis of this study. This study is registered with ClinicalTrials.gov, number NCT01307397. FINDINGS: Between March 1, 2011, and Jan 31, 2013, 3226 patients were enrolled in 44 countries. 3222 patients received at least one dose of vemurafenib (safety population). At data cutoff, 868 (27%) patients were on study treatment and 2354 (73%) had withdrawn, mainly because of disease progression. Common adverse events of all grades included rash (1592 [49%]), arthralgia (1259 [39%]), fatigue (1093 [34%]), photosensitivity reaction (994 [31%]), alopecia (826 [26%]), and nausea (628 [19%]). 1480 (46%) patients reported grade 3 or 4 adverse events, including cutaneous squamous cell carcinoma (389 [12%]), rash (155 [5%]), liver function abnormalities (165 [5%]), arthralgia (106 [3%]), and fatigue (93 [3%]). Grade 3 and 4 adverse events were reported more frequently in patients aged 75 years and older (n=257; 152 [59%, 95%

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.

The relationship between BRAF mutations and the patient clinical profile is still under question. The objective of the present study was to correlate the BRAF mutation status in primary and metastatic melanomas with the clinicopathological profile, disease-free (DFS) and overall survival (OS). A total of 367 melanoma samples from 278 patients were screened for their BRAF status using a combination of allele-specific amplification and DNA sequencing. Two or three tissue samples from the same patient were available for 74 patients. The clinicopathological characteristics were tested for their association with the BRAF mutation using the Fisher's or Pearson's chi2 test. Log-rank tests and Cox models were used for survival analyses. BRAF mutation was found in 152 samples (41.4%). Ten of the 74 patients with several tissue samples (13.5%) had discordant BRAF mutation results. BRAF-mutated patients were significantly younger at the time of primary melanoma and first diagnosis of metastasis than BRAF wild-type patients but with no difference in DFS and OS. According to our results, a primary melanoma with BRAF mutation is not associated with a more aggressive illness.

We have recently reported that activation of the Raf-1/mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase 1/2 (MEK1/2)/ERK1/2 signaling cascade in gastrointestinal carcinoid cell line (BON) alters cellular morphology and neuroendocrine phenotype. The mechanisms by which Raf-1 mediates these changes in carcinoid cells are unclear. Here, we report that activation of the Raf-1 signaling cascade in BON cells induced the expression of focal adhesion kinase (FAK) protein, suppressed the production of neuroendocrine markers, and resulted in significant decreases in cellular adhesion and migration. Importantly, inactivation of MEK1/2 by 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene or abolition of FAK induction in Raf-1-activated BON cells by targeted siRNA led to reversal of the Raf-1-mediated reduction in neuroendocrine markers and cellular adhesion and migration. Phosphorylation site-specific antibodies detected the phosphorylated FAK(Tyr407), but not FAK(Tyr397), in these Raf-1-activated cells, indicating that FAK(Tyr407) may be associated with changes in the neuroendocrine phenotype. Overexpression of constitutively active FAK plasmids (wild-type FAK or FAK(Tyr397) mutant) into BON cells reduced neuroendocrine markers, whereas the FAK(Tyr407) mutant plasmid did not show any decrease in the levels of neuroendocrine markers, indicating that phosphorylation of FAK at the Tyr(407) residue may be important for these effects. Our results showed for the first time that FAK is an essential downstream effector of the Raf-1/MEK1/2/ERK1/2 signaling cascade and negatively regulated the neuroendocrine and metastatic phenotype in BON cells.

INTRODUCTION: HER2 (ErbB2/neu) is a member of the ErbB family of four structurally related receptors of tyrosine kinase activity. Overexpression of ErbB-1 (EGFR) and HER2 is found in many human cancers, but the presence of these genes mutations determines the effectiveness of EGFR and HER2 tyrosine kinase inhibitors in the therapy of non-small cell lung cancer (NSCLC). MATERIAL AND METHODS: To search for insertions of the HER2 gene in exon 20 in 150 brain metastases of non-small cell lung cancer patients, we used a PCR technique based on analysis of amplified DNA fragment lengths. We also compared the HER2 mutational status with clinicopathologic features and the presence of EGFR and BRAF mutations. RESULTS: HER2 mutation was present in one male, non-smoking patient with low differentiated adenocarcinoma (0.67% of all patients and 1.5% of patients with adenocarcinoma). The mutations of EGFR and BRAF genes were not found in HER2-mutated patient. CONCLUSIONS: The literature data suggests that patients with HER2 mutations may be sensitive to tyrosine kinase inhibitors of both EGFR and HER2 receptors (e.g. afatinib). Therefore, the identification of new driver mutations in NSCLC can improve the quality of patient care by enabling the use of correct molecularly targeted therapies.

Adenosquamous carcinoma, a rare tumor of the skin with few reported cases in the literature, has not been studied with p63, cytokeratin 5/6 and cytokeratin 7. We stained nine such tumors with these markers. Histologically, the tumors showed superficial, atypical islands of keratinocytes in close association with islands displaying glandular differentiation. Clinically, lesions favored the head and trunk, and a subset of cases showed aggressive behavior. All tumors marked with p63 and cytokeratin 5/6, substantiating that diffuse positivity with these stains is supportive of a primary cutaneous origin. Six tumors stained focally in luminal areas with cytokeratin 7. Recognition of adenosquamous carcinoma is important for appropriate therapy, and stains for p63 and cytokeratin 5/6 may be helpful in ruling out metastatic adenocarcinoma.

OBJECTIVES: metastatic leptomeningeal spread from spinal cord gangliogliomas (GGs) is exceedingly rare. METHODS: Two adult women, aged 27 and 51 years, died of massive disseminations of cervicothoracic GGs 4 and 6 years, respectively, after initial diagnoses; full autopsies were performed. BRAF status was assessed by VE1 immunohistochemistry (IHC), Sanger sequencing, and a single-nucleotide base extension assay (SNaPshot, Applied Biosystems, Princeton, NJ). RESULTS: The 27-year-old underwent two biopsies, chemotherapy, radiation, and ventriculoperitoneal shunt placement; she developed craniospinal and peritoneal dissemination. Autopsy confirmed shunt-mediated peritoneal metastases, microscopic bone marrow involvement, and profuse spinal and supratentorial leptomeningeal and parenchymal spread. The 51-year-old underwent two resections, radiation, and chemotherapy and developed pancytopenia with biopsy-proven bony metastases 15 months before death. Autopsy demonstrated leptomeningeal, subpial, and subependymal metastases. The tumors in both primary and metastatic sites were BRAF negative by VE1 IHC and two different mutational analyses. This compared with negative BRAF results for an additional four nonmetastatic adult nonsupratentorial GGs and in our study. CONCLUSIONS: We document two rare cases of massively metastatic spinal cord GGs in adult patients who were negative for BRAF V600E mutations via multiple methods.

CONTEXT: The role of the T1799A BRAF mutation in lymph node metastasis of papillary thyroid cancer (PTC) is not clear. OBJECTIVE: Our objective was to explore the relationship between BRAF mutation and lymph node metastasis of PTC by examining the mutation in both the primary tumors and their paired lymph node metastases. DESIGN: We isolated genomic DNA from primary thyroid tumors and paired lymph node metastases and performed direct sequencing of exon 15 of the BRAF gene mutation that carries the T1799A mutation. RESULTS: In a series of 33 cases, 21 harbored the T1799A mutation in the primary tumors, and 17 (81%) of them harbored the same mutation also in the paired lymph node metastases. Twelve cases did not harbor the T1799A mutation in the primary tumors, among which nine cases also did not harbor BRAF mutation in the lymph node-metastasized tumors, whereas the other three did harbor the T1799A mutation in lymph node-metastasized tumor tissues. A novel tandem TG1799-1800AA mutation within one allele was found in a lymph node-metastasized tumor but not in the primary tumor. This mutation results in the change of codon 600 (GTG) of the gene to GAA with the consequent amino acid change (V600E) in the B-type Raf (BRAF) protein, same as that caused by the T1799A mutation alone. CONCLUSION: The high prevalence of BRAF mutation in lymph node-metastasized PTC tissues from BRAF mutation-positive primary tumors and the possible de novo formation of BRAF mutation in lymph node-metastasized PTC were consistent with a role of BRAF mutation in facilitating the metastasis and progression of PTC in lymph nodes.

OBJECTIVE: To define the role of BRAF gene mutation in the progression of papillary thyroid carcinoma. SUMMARY BACKGROUND DATA: BRAF gene mutation is frequently detected in papillary thyroid carcinoma. Its role in pathogenesis or progression is under investigation. METHODS: Patients who underwent thyroidectomy and sentinel lymph node biopsy for papillary thyroid cancer were accrued. BRAF mutation was assessed in primary tumors and matched sentinel lymph nodes by a quantitative real-time PCR assay. RESULTS: Tissue specimens from 103 consecutive patients were evaluated. BRAF mutation of the primary tumor was detected in 34 (33%) patients. In 26 of 34 (76%) patients with BRAF mutation, concomitant lymph node metastasis was detected. On the contrary, in 69 patients with BRAF mutation-negative primary tumors, only 12 (17%) patients had lymph node metastasis (chi, P < 0.0001). BRAF mutation was detected in 20 of 26 (77%) lymph node metastases matched to BRAF mutation-positive primary tumors; it was not detected in lymph node metastases matched to BRAF mutation-negative primary tumors. Univariate analysis identified age, stage, tumor size, and BRAF mutation as prognostic factors for lymph node metastasis. In multivariate analysis, only BRAF mutation remained a significant prognostic factor for lymph node metastasis (odds ratio = 10.8, 95% confidence interval, 3.5-34.0, P < 0.0001). CONCLUSIONS: BRAF mutation may be a key genetic factor for the metastatic progression of papillary thyroid carcinoma. The study demonstrates that this gene mutation is a significant risk factor for locoregional lymph node metastasis and has potential utility as a surrogate marker.

Papillary thyroid carcinoma (PTC) frequently presents as a multifocal process. To study the importance of separating independent primary (IP) from intrathyroid metastatic (ITM) PTC, we examined 19 molecular markers on 42 separate tumors from 18 multifocal PTC cases. In 12 of 18 (66.7%) cases, including 6 of 12 (50%) papillary microcarcinoma cases, the same or similar profile of loss of heterozygosities (LOH) and v-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutation was demonstrated, indicating that they were from the same primary and represented ITM. Different profiles of LOHs and BRAF mutation were detected in separate tumors of 6 of 18 cases, indicating that they represented IP. Patients with ITM, including papillary microcarcinoma, had significantly increased lymph node metastasis. The frequencies of LOHs of 17q21, 17p13, 10q23, and 22q13 were higher in tumors with lymph node metastasis, suggesting that these LOHs may be important in increased lymph node metastasis. LOH of 9p21 was found at the highest frequency in PTC (53.8%), followed by 1p36 (46.2%), 10q23 (34.6%), and 22q13 (34.6%). Papillary microcarcinoma had acquired similar genomic mutations as conventional PTC, but higher frequencies of mutations of BRAF, 1p36, 18q, and 22q13 were found in the larger PTC, suggesting that they might play a role in the aggressiveness of PTC. Different profiles of mutations were observed in conventional, follicular variants, and diffuse sclerosing variant of PTC, which might influence the different morphological appearances and clinical courses. In conclusion, molecular analysis can separate multifocal IP PTC from ITM PTC, and may be more important than tumor size in predicting lymph node metastasis, aggressiveness, and prognosis of PTC.

BACKGROUND: It has been reported that patients with papillary thyroid carcinoma (PTC) have a high incidence of background Hashimoto thyroiditis (HT); however, the linkage of HT to PTC is controversial. Recent studies have shown that the prevalence of activating point mutations in BRAFV600E is much higher (73-86%) in Korea than in Western countries (29-69%), and associated with a poor prognosis in PTC. The purpose of the present study was to investigate the frequency of the BRAFV600E mutation in PTC with and without HT, and to determine clinical and pathological features that were associated with concomitant HT and PTC. METHODS: Fine-needle aspiration slides from 101 patients with surgically confirmed PTC were studied. The DNA was extracted from the atypical cells that were scraped from slides. It was then analyzed for the BRAFV600E mutation by pyrosequencing. In addition, the presence of background HT in surgical specimens and other clinical and pathological features of the patients were characterized. RESULTS: HT was present in 37 (36.6%) of the patients. The BRAFV600E mutation was present in 27 (72.9%) of patients with HT but was present in 61 (95.3%) of patients without HT ( p#0.01). The inverse correlation of concurrent HT with the BRAFV600E mutation was significant for both males and females ( p < 0.01). The presence of background HT was not associated with tumor size, extrathyroidal invasion, lymph node (LN) metastasis, or tumor stage. The patients were younger in the group without background HT (44.1 +/- 13.2 vs. 49.8 +/- 13.9, p 1/40.05). The BRAFV600E mutation was present in 88 (87.1%) of the 101 patients with PTC. The presence of the BRAFV600E mutation was significantly associated with LN metastasis ( p < 0.02; odds ratio, 6.24; 95% confidence interval, 1.51-25.79). CONCLUSION: In Korean patients with PTC, the BRAFV600E mutation is associated with a lower frequency of background HT and a high frequency of LN metastasis.

CONTEXT: The BRAF(T1799A) transversion is the most frequent morphotype-specific somatic mutation in papillary thyroid carcinoma (PTC). The ability to detect this mutation in the circulation could aid in diagnosis and follow-up of PTC patients. OBJECTIVE: Our objective was to develop and clinically validate a sensitive and specific assay for the detection of BRAF(T1799A) in blood samples from PTC patients. DESIGN: We developed an allele-specific real-time PCR method for the detection of BRAF(T1799A) in blood samples and studied prospectively blood samples from 193 patients with thyroid cancer (173 PTC, 20 non-PTC) attending for routine follow-up. The results of molecular testing were correlated with disease status and thyroglobulin measurements. BRAF(T1799A) status of the original tumor samples was also confirmed, where available. RESULTS: The assay had a detection sensitivity of fewer than one heterozygote BRAF(T1799A)-carrying cell per 100,000 diploid cells, without detectable cross-reactivity between wild-type BRAF and BRAF(T1799A). Circulating BRAF(T1799A) was detected in 20 of 173 PTC patients and in none of the 20 non-PTC patients. BRAF(T1799A)-positive samples contained between one in 326 and fewer than one in 100,000 copies of BRAF(T1799A). Tissue BRAF status correlated with blood BRAF status, whereas BRAF(T1799A) positivity in blood correlated with the presence of active disease at the time of the blood draw, with eight of the 38 PTC patients with persistent/recurrent disease being positive for circulating BRAF(T1799A) (relative risk vs. circulating BRAF(T1799A)-negative, 2.55; P < 0.04). CONCLUSIONS: BRAF(T1799A) can be detected in the blood of PTC patients with residual or metastatic disease and may provide diagnostic information.

OBJECTIVE: To investigate whether BRAF mutation of papillary thyroid carcinoma (PTC) has different predictive values for regional lymph node (LN) metastasis according to tumor size. STUDY DESIGN: Prospective cohort study. Setting. A tertiary hospital. SUBJECTS AND METHODS: From January 2009 to August 2009, the authors prospectively enrolled 102 consecutive patients with unifocal PTC based on the findings of ultrasonography-guided fine-needle aspiration biopsy (FNAB). BRAF mutation was tested on preoperative FNAB specimens. Total thyroidectomy and bilateral central neck dissection (+/- lateral neck dissection) was performed for all patients. Among 102 patients, 71 who were confirmed to have unifocal PTC by the surgical pathology were finally selected. The 71 patients were classified into 3 groups according to their tumor size: group I, 0.5 cm and 1 cm. LN metastasis was evaluated in the surgical specimen as a dependent variable. The authors investigated whether BRAF mutation is predictive of LN metastasis in each group. RESULTS: Overall, BRAF mutation was a significant predictor of LN metastasis (P = .045). When patients were classified into 3 groups, frequency of LN metastasis increased with tumor size: 4.8%, 50.0%, and 66.7% (P < .001). However, the frequency of BRAF mutation was not different among 3 groups: 61.9%, 56.3%, and 72.2% (P = .536). BRAF mutation was predictive of LN metastasis only in group II (P = .026). CONCLUSION: BRAF mutation of PTC may have differential predictive values for LN metastasis, according to tumor size.

OBJECTIVE: To look for genetic mutations that might predict central compartment lymph node metastasis (LNM) in papillary thyroid carcinoma (PTC) using strict criteria for N0 and N1 disease. DESIGN: We identified patients with PTC from our institution's pathology archives. Strict criteria were used for assessing the presence or the absence of central neck LNM. Disease was classified as N0 only if a comprehensive ipsilateral and pretracheal central neck dissection was performed and if pathological analysis revealed no evidence of LNM. Primary tumor samples were analyzed for a panel of known or suspected PTC-associated molecular markers, including BRAF, RET -PTC, KRAS, NRAS, HRAS, PIK3CA, and their variants. SETTING: Academic medical center. PATIENTS: Three hundred eighty-nine patients with PTC. MAIN OUTCOME MEASURE: Molecular mutations in tumors with and without LNM. RESULTS: Of 389 identified cases, 209 fit the inclusion criteria, with 158 classified as node positive (N1) and 51 as node negative (N0). The follicular variant histological type was present in 7 of 158 N1 tumors (4.4%) and 24 of 51 N0 tumors (47.1%) and thus was strongly associated with lack of central neck metastasis in this study (odds ratio, 0.05; 95% CI, 0.02-0.14). Predictive factors for central LNM included extracapsular extension, angiolymphatic invasion, and higher T stage (T3 and T4). The BRAF mutation was more prevalent in the classic PTC histological type than the follicular variant. None of the molecular marker mutations that were analyzed in this study, including the BRAF mutation, predicted LNM in classic PTC. CONCLUSIONS: Positive risk factors for central LNM include male sex, extracapsular extension, angiolymphatic invasion, and advanced T stage. The follicular variant histological type has a significantly lower incidence of central neck metastasis. In contrast to recent studies, the BRAF mutation was not significantly associated with central neck LNM from PTC when using a strict definition of a central neck dissection.

Papillary thyroid cancer (PTC) constitutes more than 90% of the thyroid cancers. MAP kinase/ERK pathway plays an important role in the development of several cancers. BRAF which is a member of Raf-kinase family activates this way. BRAF gene activating mutations lead to neoplastic transformation in thyroid follicle cells. In PTC, this mutation itself is a poor prognostic sign independent of other clinicopathological characteristics. We evaluated BRAF(V600E) mutation and clinical-pathological characteristics in Turkish population with PTC. We assessed 109 patients with PTC (88 female, 21 male). The average age was 38.7 +/- 9.9 (17-71). BRAF(V600E) mutation was detected using polymerase chain reaction and fluorescent melting curve analysis. The results show that BRAF(V600E) mutation rate was found in 39.45% of our patients. We observed that BRAF(V600E) mutation was significantly higher in men, in tumors larger than 1 cm in size, and in patients with classical PTC. Moreover, statistically significant correlations of BRAF(V600E) with indicators of tumor aggressiveness such as thyroid capsular invasion, multifocality, lymph node metastasis, and extrathyroidal spread were found. Patient groups below and over the age of 45 did not differ in mutation frequency. Patients with micro-PTC were evaluated separately, it was found that BRAF(V600E) mutation was more frequent in the classic type and that lymph node metastasis rate significantly increased when the mutation was present. We concluded that BRAF(V600E) was correlated with indicators of tumor aggressiveness in our study population. This fact is taken into consideration in treatment and follow-up of our patients with PTC and positive BRAF(V600E) mutation.

BACKGROUND: Lungs and bones are the most common sites for distant metastases from papillary thyroid cancer (PTC). Metastases to the pancreas are extremely rare. Here we present a man with pancreatic metastases from PTC, report our experience with sorafenib therapy, and discuss the role of endoscopic ultrasound (EUS)-guided biopsy in its diagnosis. PATIENT FINDINGS: A 56-year-old man underwent total thyroidectomy, right-modified neck dissection, and radioactive iodine (RAI) remnant ablation for PTC at age 47 years (in 2002). Between 2002 and 2007, he had three more neck surgeries, two RAI therapies, and external beam radiotherapy for persistent and subsequently metastatic PTC. In 2008, a computed tomography/positron emission tomography (CT/PET) scan showed an 18F-fluorodeoxyglucose (FDG)-avid pancreatic focus. Magnetic resonance imaging (MRI) revealed a pancreatic nodule at the same location. An EUS-guided biopsy confirmed the diagnosis of pancreatic metastasis from PTC, and molecular studies showed positive BRAF(V600E) mutation. He was treated with sorafenib for 6 months. Although a lung CT scan done 2 months after initiation of sorafenib suggested stability of the disease, MRI studies done at 3 and 6 months showed clear progression with an increase in the size of the lung and pancreatic metastases. Subsequently, he developed liver, bone, and omental metastases. He died in July 2011, 9 years and 8 months after the initial diagnosis of PTC and 20 months after discovery of the pancreatic metastasis. SUMMARY: A middle-aged man with PTC developed lung metastases despite multiple surgeries and RAI therapies. Seven years after the initial diagnosis, a pancreatic metastasis was accidentally discovered. Both the metastasis and the primary thyroid tumor are positive for BRAF(V600E) mutation. The lung and pancreatic metastases progressed while the patient was receiving sorafenib for 6 months, and the patient died 20 months after diagnosis of pancreatic metastasis. CONCLUSION: PTC rarely metastasizes to the pancreas. In this patient, an FDG PET scan and EUS-guided biopsy played important roles in the diagnosis. PTC metastases to the pancreas usually occur in otherwise advanced disease. In the patient presented here, sorafenib may have slowed disease progression but the overall utility of tyrosine kinase inhibitors in pancreatic metastases from PTC is not clear.

BACKGROUND: Papillary thyroid carcinoma (PTC) is one of the most frequent endocrine malignancies. In most cases, it often presents as multifocal tumor. It has been reported that multifocal tumors are associated with elevated risk of lymph node and distant metastases. Multifocality is also one of the factors predicting prognosis. Recent studies show that BRAFV600E mutation occurs more frequently in aggressive PTC. The purpose of this study was to evaluate BRAFV600E status and clinicopathological features in multiple and solitary PTC. METHODS: We performed a retrospective study to analyze 512 PTC cases who received surgery, including 376 solitary PTCs and 136 multiple PTCs. RESULTS: Multiple PTC is more related to lymph node metastasis and vascular invasion than solitary PTC. However, the distant metastasis rate and 10-year survival rate showed no difference between these two groups. BRAFV600E mutation status was more frequent in multiple PTC patients with lymph node metastasis and late stage at diagnosis. CONCLUSION: BRAFV600E mutation is most commonly associated with extra-thyroidal extension and lymph node metastasis in PTC. Multiple PTC patients with young age, large tumors and BRAFV600E mutation should be followed carefully. Our study provides useful information for PTC patients' followup and treatment.

Clinicians have long sought to characterize biological markers of neoplasia as objective indicators of tumor presence, pathogenicity, and prognosis. Armed with data that correlate biomarker activity with disease presence and progression, clinicians can develop treatment strategies that address risks of disease recurrence or persistence and progression. The B-type Raf kinase (BRAF V600E) mutation in exon 15 of the BRAF gene has been noted to be a putative prognostic marker of the most prevalent form of thyroid cancer, papillary thyroid cancer (PTC)--a tumor type with high proclivity for recurrence or persistence. There has been a remarkable interest in determining the association of BRAF mutation with PTC recurrence or persistence. Using many new studies that have been published recently, we performed a meta-analysis to investigate correlations of BRAF mutation status with PTC prognosis, focusing on the recurrence or persistence of the disease after initial treatment. The study was based on published studies included in the PubMed and Embase databases addressing the BRAF mutation and the frequency of recurrence of PTC. We selected studies with data that enabled measurement of the risk ratio for recurrent disease. We also analyzed the factors that are classically known to be associated with recurrence. These factors included lymph node metastasis, extrathyroidal extension, distant metastasis, and American Joint Committee on cancer (AJCC) stages III/IV. We used 14 articles that included an analysis of these factors as well as PTC recurrence data, with a total of 2470 patients from 9 different countries. The overall prevalence of the BRAF mutation was 45%. The risk ratios in BRAF mutation-positive patients were 1.93 (95% confidence interval [CI], 1.61-2.32; Z = 7.01; p < 0.00001) for PTC recurrence, 1.32 (95% CI, 1.20-1.45; Z = 5.73; p < 0.00001) for lymph node metastasis, 1.71 (95% CI, 1.50-1.94; Z = 8.09; p < 0.00001) for extrathyroidal extension, 0.95 (95% CI, 0.63-1.44; Z = 0.23; p = 0.82) for distant metastasis, and 1.70 (95% CI, 1.45-1.99; Z = 6.46; p < 0.00001) for advanced stage AJCC III/IV. Thus, in this meta-analysis, the BRAF mutation in PTC was significantly associated with PTC recurrence, lymph node metastasis, extrathyroidal extension, and advanced stage AJCC III/IV. Patients with PTC harboring mutated BRAF are likely to demonstrate factors that are associated with an increased risk for recurrence of the disease, offering new prospects for optimizing and tailoring initial treatment strategies to prevent recurrence.

PURPOSE: This study was designed to examine whether available preoperative clinical parameters, including B-type Raf kinase (BRAF) V600E mutation status, can identify patients at risk for central compartment lymph node metastasis (CLNM). METHODS: Under an institutional review board-approved protocol, we conducted a single-center, retrospective review of all patients who had initial thyroidectomy for histologic papillary thyroid carcinoma (PTC) during 2010. The presence of CLNM was examined for correlation with available preoperative clinical parameters, including tumor size, gender, age, and BRAF mutation status. RESULTS: Cervical lymph node resection and molecular testing on FNAB or histopathologic specimen was performed on a consecutive series of 156 study patients with histologic PTC. Overall, CLNM was diagnosed in 37% and 46% were BRAF-mutation-positive. BRAF positivity was the only clinical parameter associated with CLNM (BRAF, p=0.002; tumor size>/=2 cm, p=0.16; male gender, p=0.1; age>/=45 years, p=0.3) and remained an independent predictor of CLNM on multiple logistic regression analysis (odds ratio (OR) 3.2, p=0.001). The PPV and NPV of BRAF positivity for CLNM was 50 and 74%, respectively. When restricting the analysis to the subset of 38 patients who had molecular testing performed preoperatively on FNAB, the PPV and NPV of BRAF positivity for CLNM was 47 and 91%, respectively, and BRAF positivity was still a significant predictor of CLNM on both univariate (OR 8.4, p=0.01) and multivariate (OR 9.7, p=0.02) analyses. CONCLUSIONS: Of the commonly used clinical parameters available preoperatively, the BRAF V600E mutation is the only independent predictor of CLNM in PTC and can be utilized to guide the extent of initial surgery.

BACKGROUND: Some have proposed using V600E BRAF mutation status to dictate the surgical management of patients with papillary thyroid cancer (PTC). However, well-designed studies examining BRAF association with aggressive clinicopathologic features of PTC, including the presence of lymph node metastases (LNM), in patients who have undergone routine central lymph node dissection (CLND), are lacking. METHODS: Under institutional review board approval, 63 patients diagnosed with PTC on fine-needle aspiration who underwent total thyroidectomy and CLND were included. BRAF mutation status was determined in fresh frozen or intraoperative fine-needle aspiration samples with a colorimetric assay. Associations between BRAF mutation status and clinicopathologic features of PTC were examined using Chi-square and multivariate logistic regression analyses. RESULTS: BRAF mutation was found to be significantly associated with race only on Chi-square analysis. BRAF mutation was not found to be significantly associated with the presence of LNM (P = .167). On multivariate analysis, only size and venous/lymphatic invasion were significantly associated with LNM. CONCLUSION: This small series underscores the prematurity in utilizing BRAF mutation status to determine the surgical management of patients with PTC, specifically whether or not to perform a CLND.

The impact of metastasized cervical lymph nodes (CLN) identified on central neck dissection (CND) on the recurrence/persistence of papillary thyroid cancer (PTC) and the extent of CND needed to reduce recurrence/persistence have not been firmly established. To assess the impact of CLN metastasis and BRAF mutation on the recurrence/persistence of PTC and the potential of BRAF mutation in assisting CND. Analyses of 379 consecutive patients with PTC who underwent thyroidectomy with (n=243) or without CND (n=136) at a tertiary-care academic hospital during the period 2001-2010 for their clinicopathological outcomes and BRAF mutation status. Increasingly aggressive tumor characteristics were found as the extent of CND was advanced following conventional risk criteria from non-CND to limited CND to formal CND. Disease recurrence/persistence rate also sharply rose from 4.7% to 15.7% and 40.5% in these CND settings respectively (P<0.0001). CLN metastasis rate rose from 18.0 to 77.3% from limited CND to formal CND (P<0.0001). An increasing rate of BRAF mutation was also found from less to more extensive CND. A strong association of CLN metastasis and BRAF mutation with disease recurrence/persistence was revealed on Kaplan-Meier analysis and BRAF mutation strongly predicted CLN metastasis. CLN metastases found on CND are closely associated with disease recurrence/persistence of PTC, which are both strongly predicted by BRAF mutation. Current selection of PTC patients for CND is appropriate but higher extent of the procedure, once selected, is needed to reduce disease recurrence, which may be defined by combination use of preoperative BRAF mutation testing and conventional risk factors of PTC.

BACKGROUND: Papillary thyroid carcinoma (PTC) represents one of the most frequent endocrine malignancies. Several factors have been found to be involved in determining the outcome of treatment for patients with PTC. Large tumor size, diagnosis at an early age, extra-thyroidal invasion, aggressive histological variants, and distant metastases are the most important determinants of a poor outcome. BRAF(V600E) mutation has been found to be a major genetic alteration in PTC. This study aimed to evaluate progression in patients with multifocal and solitary PTC. METHODS: We performed a retrospective study to analyze 368 patients with PTC who underwent surgery, including 282 patients with solitary PTC and 86 patients with multifocal PTC. The status of BRAF(V600E) mutation in all tumor foci from multifocal PTC was detected. RESULTS: Our study suggested that multifocal PTC was more related to lymph node metastasis and vascular invasion than solitary PTC. However, the distant metastasis rate and 10-year survival rate showed no difference between these two groups. The number of tumor foci did not affect progression of disease in multifocal PTC patients. Lymph node metastasis in multifocal PTC patients was associated with larger tumors, diagnosis at early stage, and extra-thyroidal invasion. CONCLUSION: The status of BRAF(V600E) mutation was more frequent in multifocal PTC patients with lymph node metastasis and diagnosis at later age.

BACKGROUND: BRAF(V600E) mutation is correlated with local aggressive clinicopathological features in papillary thyroid carcinoma; yet the relationship between this genetic variation and distant papillary thyroid carcinoma metastasis was unclear. This study aimed to investigate whether BRAF(V600E) is predictive for distant metastasis in the Chinese population. METHODS: One hundred and seven patients with papillary thyroid carcinoma were enrolled in this study, including 43 patients with distant metastasis and 64 patients without. Quantitative real-time polymerase chain reaction was used to detect BRAF(V600E) mutation, while immunohistochemistry was performed to detect vascular endothelial growth factor (VEGF) expression. The associations between distant metastasis and BRAF(V600E) mutation, and VEGF expression as well as local clinicopathological factors were determined. RESULTS: A total of 28.6% of the patients in the distant metastasis group harbored BRAF(V600E) mutation, which was significantly lower than in the without distant metastasis group (68.8%, P < 0.001). BRAF(V600E) mutation was negatively correlated with positive VEGF expression (P = 0.001). Furthermore, 52.2% of the patients with distant metastasis exhibited VEGF expression, compared with 25.0% of those without. Higher levels of VEGF expression were also observed in the distant metastasis group. tumor size, extra-thyroid invasion, and BRAF(V600E) mutation were independent predictors for distant metastasis according to multivariate analysis (odds ratios were 2.8, 12.4, and 0.3; 95%

IMPORTANCE: Papillary thyroid carcinoma (PTC) is the most common endocrine neoplasm. B-type raf kinase (BRAF) V600E mutation has been proposed as a negative prognostic indicator in PTC, and patients harboring it should receive more aggressive initial therapy. OBJECTIVE: To assess the significance of BRAF V600E mutation in PTC in the largest US sample to date. DESIGN: We identified patients from our institution's pathology archives diagnosed as having PTC and meeting criteria for BRAF mutation testing. Medical records were analyzed for BRAF status (positive or negative) and a list of standardized clinicopathologic features. PARTICIPANTS: A total of 429 patients with PTC at an academic medical center. MAIN OUTCOMES AND MEASURES: Clinicopathologic features in patients with PTC with and without BRAF mutation. RESULTS: Of 429 cases with PTC, 314 (73.2%) were positive for the BRAF mutation and 115 (26.8%) tested negative. BRAF mutation was significantly associated with tumor margin positivity (P = .03) and lymph node metastasis (P = .002) on univariate analysis but not on multivariate study. BRAF mutation was a predictor of male sex (odds ratio [OR], 3.2; 95% CI, 1.4-7.2), total thyroidectomy (OR, 2.6; 95% CI, 1.1-6.2), and a negative predictor of follicular variant PTC (OR, 0.1; 95% CI, 0.1-0.4). There was no significant association between BRAF positivity and tumor multicentricity, lymphovascular invasion, extranodal extension, central neck involvement, advanced stage (stage III or IV), and distant metastasis. CONCLUSIONS AND RELEVANCE: BRAF V600E mutation has been extensively studied in relation to negative prognostic indicators in PTC, with no consistent relationship emerging. Two recent meta-analyses showed an overall association between BRAF status and aggressive disease features and called for tailoring treatment plans in patients accordingly. In this, the largest US study to date, BRAF status was not significantly associated with most clinicopathologic features suggestive of more aggressive disease.

BACKGROUND: The prognostic role of the T1799A BRAF mutation is controversial. We investigated the protein expression in papillary thyroid carcinoma (PTCs) samples harboring the specific mutation using proteomic tools. MATERIALS AND METHODS: We performed two-dimensional gel electrophoresis to identify differential protein expression regarding lymph node metastasis (LNM). Proteins were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Immunohistochemical staining was performed for 38 PTCs harboring the mutation. We validated the association between these proteins and clinicopathological factors in a test set of 121 PTCs. RESULTS: The expression of vimentin was increased in PTCs with LNM, but the one for HSP60, SOD2 and PEBP1 was increased in samples without LNM. HSP60 protein was up-regulated in PTCs without LNM (84.2% vs. 36.8%. p=0.003) and in PTCs without LNM harboring the mutation (58% vs. 41.8%. p=0.003) in the test set as shown by immunohistochemical staining. CONCLUSION: HSP60 protein expression may inhibit LNM in PTCs harboring the BRAF mutation and may be a useful prognostic marker.

mutations in the V-raf murine sarcoma viral oncogene homolog B1 gene (BRAF) play an important role in the pathogenesis of papillary thyroid cancer (PTC). In this study, a BRAF V600E mutation was detected in formalin-fixed and paraffin-embedded PTC samples using multiplex allele-specific polymerase chain reaction and denaturing high-performance liquid chromatography. The sensitivity of the assays was validated in two cell lines, K1 and RO82-W-1, which were respectively positive and negative for the mutation. The method enabled detection of very low concentrations (~1%) of the mutation, with positive findings obtained in 119 of the 187 samples (63.6%). The mutation was not detected in 20 cases of benign thyroid diseases. The presence of the mutation was significantly associated with ages >45 years, the tumor-nodes-metastasis stage, region of metastasis, and clinical outcome (P < 0.05). Although no correlation was found between the mutation and lymph node metastasis, the proportions of patients carrying a mutation differed significantly between central and lateral cervical lymph node metastasis (P < 0.05). Multivariate logistic regression analysis also indicated the presence of mutation, tumor size, and cervical lymph node metastasis to be independent predictors for recurrence and distant metastasis. We conclude that a high proportion of PTC cases likely harbors the BRAF V600E mutation. This mutation can be used as an independent factor for predicting the recurrence and distal metastasis of PTC tumors.

Utilizing BRAF(V600E) mutation as a marker may reduce unnecessary prophylactic central neck dissection (pCND) in clinically nodal negative (cN0) neck for small (metastasis (CNM) and its contribution to the overall prediction after adjusting for other significant preoperative clinical factors in small PTC. Primary tumor tissue (paraffin-embedded) from 845 patients with small classical cN0 PTC who underwent pCND was tested for BRAF mutation. Clinicopathologic factors were compared between those with and without BRAF. BRAF was evaluated to see if it was an independent factor for CNM. Prediction scores were generated using logistic regression models and their predictability was measured by the area under the ROC curve (AUC). The prevalence of BRAF was 628/845 (74.3%) while the rate of CNM was 285/845 (33.7%). Male sex (odds ratio (OR): 2.68, 95% CI: 1.71-4.20), large tumor size (OR: 2.68, 95% CI: 1.80-4.00), multifocality (OR: 1.49, 95% CI: 1.07-2.09), lymphovascular permeation (OR: 10.40, 95% CI: 5.18-20.88), and BRAF (OR: 1.65, 95% CI: 1.10-2.46) were significant independent predictors of CNM, while coexisting Hashimoto's thyroiditis (OR: 0.56, 95% CI: 0.40-0.80) was an independent protective factor. The AUC for prediction score based on tumor size and male sex was similar to that of prediction score based on tumor size, male sex, and BRAF status (0.68 vs 0.69, P=0.60). Although BRAF was an independent predictor of CNM, knowing its status did not substantially improve the overall prediction. A simpler prediction score based on male sex and tumor size might be sufficient.

BACKGROUND: Human cytomegalovirus (CMV) has been detected in the thyroid gland and thyroid tumors. CMV infection may activate the mitogen-activated protein kinase pathway, of which aberrant activation is frequently associated with BRAF mutation in papillary thyroid cancer. METHODS: A total of 45 paired tumorous and adjacent non-neoplastic tissue samples, including 5 follicular adenoma and 40 papillary thyroid cancer, were obtained during thyroidectomy. BRAF mutational status was determined using direct sequencing. The presence of CMV DNA was determined using conventional PCR and quantitative real-time PCR. CMV protein in the tissue samples were evaluated with Western blot analysis. RESULTS: BRAF mutation was identified in the cancerous part of 31 (78%) papillary thyroid cancers. Papillary cancer with BRAF mutation was significantly associated with a larger tumor size (P = 0.045), extrathyroidal invasion (P = 0.012), lymph node metastasis (P = 0.008), and a higher TNM stage (P = 0.044). CMV DNA and protein were not detected in any studied samples. CONCLUSIONS: Our results suggest no association between CMV infection and papillary thyroid cancer.

Intratumoral heterogeneity is widely recognized as an important determinant of a cancer's initial response and its subsequent resistance to targeted therapy. BRAF V600E mutation, common in papillary thyroid carcinoma (PTC), is helpful in fine needle aspiration diagnosis of thyroid nodules and is being evaluated for targeted therapies. This study was designed to assess the presence of BRAF mutation heterogeneity within primary PTCs and between paired primary and metastatic lesions. Genetic heterogeneity was evaluated in 47 PTCs (38 differentiated papillary thyroid carcinomas and 9 poorly differentiated PTCs with anaplastic areas). The differentiated papillary thyroid carcinomas included 16 cases with regional lymph node metastases at thyroidectomy and 9 cases with recurrent metastases to regional lymph nodes more than 5 years post thyroidectomy. Genetic heterogeneity of BRAF was studied by comparing the mutation status in different samples of tumor as follows: (a) 2 separate areas (each >1.5 cm in diameter) within the primary tumor, (b) a more than 1.5 cm area of primary carcinoma and a second 5 mm area simulating a fine needle aspiration sample from a different portion of the primary tumor, (c) primary carcinoma and its lymph node metastasis at thyroidectomy, (d) primary carcinoma and the recurrent metastasis, and (e) differentiated and anaplastic areas in the primary carcinoma. BRAF mutation status was concordant in 95.2% of the 62 paired samples. Discordant BRAF status was detected in only 4.8% of the pairs studied and most frequently involved cases with recurrent metastasis thus suggesting a need for additional testing of these lesions before instituting therapy.

The aim of our study was to find a specific imaging (contrast-enhanced ultrasound, CEUS) to detect extracapsular extension and cervical lymph node metastases (ECE-LNM) that associated with BRAF protein expression in papillary thyroid carcinoma (PTC). Preoperative utrasonography (US) or CEUS was performed in the diagnosis of extracapsular extension (ECE) in 317 patients with 369 PTC. BRAF protein status was tested on the primary tumor and lymph node involvement. The diagnostic accuracy of CEUS and US was evaluated after thyroid surgery. The association between CEUS and BRAF expression were then analyzed to investigate the diagnostic value of ECE-LNM in PTC. The sensitivity and specificity of CEUS were higher than those in US in the diagnosis of ECE in patients with PTC (91.1, 86.5 vs 49, 55 %). BRAF protein overexpression were significantly associated with ECE (P = 0.0003) and lymph node metastasis (LNM) positive cases (P = 0.0014). The results of CEUS, not US, have a significant correlation with BRAF expression status in PTC samples (P < 0.0001). Associated with BRAF protein expression status, the routine preoperative CEUS could have a good value in the diagnosis of ECE-LNM in PTC and facilitate a surgeon to improve further clinical management.