BACKGROUND

Mutually exclusive mutations of RET, RAS, or BRAF are present in about 70% of papillary thyroid carcinomas, whereas only the latter two are seen in poorly differentiated and anaplastic cancers. Although the signal output common to these oncoproteins is ERK, a recent report showed that only BRAF mutations consistently predicted responsiveness to MAPK kinase (MEK) inhibitors.

OBJECTIVE

Here we investigated whether sensitivity to MEK inhibition was determined by oncogene status in 13 human thyroid cancer cell lines: four with BRAF mutations, four RAS, one RET/PTC1, and four wild type.

RESULTS

Growth of BRAF (+) cells was inhibited by the MEK antagonist PD0325901 with an IC(50) of less than 5 nm. By contrast, RAS, RET/PTC1, or wild-type cells had IC(50) of 4 nm to greater than 1000 nm. Sensitivity was not predicted by coexisting mutations in PIK3CA or by PTEN status. Similar effects were obtained with the MEK inhibitor AZD6244. PD0325901 induced a sustained G1/S arrest in BRAF (+) but not BRAF (-) lines. PD0325901 was equipotent at inhibiting pERK1/2 after 2 h, regardless of genetic background, but pERK rebounded at 24 h in most lines. MEK inhibitor resistance was associated with partial refractoriness of pERK to further inhibition by the compounds. AZD6244 was more potent at inhibiting growth of NPA (BRAF +) than Cal62 (KRAS +) xenografts.

CONCLUSIONS

Thyroid cancers with BRAF mutation are preferentially sensitive to MEK inhibitors, whereas tumors with other MEK-ERK effector pathway gene mutations have variable responses, either because they are only partially dependent on ERK and/or because feedback responses elicit partial refractoriness to MEK inhibition.

The role of aberrant tumor suppressor gene methylation in the aggressiveness of papillary thyroid cancer (PTC) has not been documented. By showing promoter methylation-induced gene silencing in PTC-derived cell lines, we first demonstrated the functional consequence of methylation of several recently identified tumor suppressor genes, including those for tissue inhibitor of metalloproteinase-3 (TIMP3), SLC5A8, death-associated protein kinase (DAPK) and retinoic acid receptor beta2 (RARbeta2). We then investigated the role of methylation of these genes in the aggressiveness of PTC by examining the relationship of their aberrant methylation to clinicopathological characteristics and BRAF mutation in 231 primary PTC tumors. Methylation of TIMP3, SLC5A8 and DAPK was significantly associated with several aggressive features of PTC, including extrathyroidal invasion, lymph node metastasis, multifocality and advanced tumor stages. Methylation of these genes was also significantly associated with BRAF mutation in PTC, either individually or collectively in various combinations. Methylation of these genes, either individually or collectively, occurred more frequently in more aggressive classical and tall-cell PTC subtypes than in less aggressive follicular-variant PTC, with the latter known to infrequently harbor BRAF mutation. Several other tumor suppressor genes investigated were not methylated. These results suggest that aberrant methylation and hence silencing of TIMP3, SLC5A8, DAPK and RARbeta2, in association with BRAF mutation, may be an important step in PTC tumorigenesis and progression.

BACKGROUND

Reolysin is reovirus serotype 3-Dearing strain, a double-stranded replication-competent RNA non-enveloped icosahedral virus. It induces cytopathic and anti-cancer effects in cells with an activated ras pathway due to inhibition of the dsRNA-activated protein kinase.

METHODS

This was a single center dose escalation trial of Reolysin administered intravenously every 4 weeks in doses ranging from 1 x 10(8) to 3 x 10(10) tissue culture infective dose (TCID)(50). Serum for neutralizing antibody, and serum, stool, saliva, and urine for viral shedding were collected. Tumor samples were analyzed for activating mutations in the ras and braf oncogenes.

RESULTS

Eighteen patients received 27 doses of Reolysin in 6 dose cohorts accomplishing a 300 fold dose escalation without a protocol-defined dose limiting toxicity. Drug related grade 2 toxicities included fatigue and fever (1 patient each). All patients developed neutralizing antibody during the course of the study. Viral shedding was observed in 6 patients. One patient with anthracycline and taxane refractory breast cancer experienced a partial response (PR) and her tumor had a ras G12A mutation. Biopsy from her chest wall mass showed evidence of necrosis and viral replication by electron microscopy. Overall clinical benefit (1 PR + 7 stable disease) rate was 45%, and appeared higher in patients with viral shedding (67%) than those without (33%).

CONCLUSIONS

Reolysin administered monthly as a one-hour infusion is safe and well-tolerated even in multiple doses. Reolysin has anti-tumor activity as a single agent warranting further evaluation, including in combination with chemotherapy. Viral shedding may suggest intrapatient replication yielding a benefit and should be studied carefully in future studies.

The B-type Raf kinase (BRAF) protein is a serine/threonine kinase that has an important role in cellular proliferation, differentiation, and programmed cell death. The BRAF gene has been recently found to be mutated in human carcinomas, predominantly in malignant melanoma. The aim of this study was to investigate the frequency of the BRAF mutation in papillary thyroid carcinoma (PTC) of Koreans through direct DNA sequencing of the polymerase chain reaction (PCR)- amplified exon 15 with clinicopathological features. Seventy paraffin-embedded conventional papillary carcinomas in the thyroid gland were evaluated. The BRAF missense mutation at V599E was found in 58 of 70 PTCs (83%). The frequency of our series was much higher than the frequencies of other PTC series (36 - 69%). The frequency of nodal metastasis was also significantly higher in the BRAF mutation group (p= 0.048). These results suggest that the BRAF mutation is involved in the carcinogenesis in most conventional PTCs, especially those occurring in Koreans, and this is a potentially valuable marker for the evaluation of prognosis of patients with PTC. These findings support the specific inhibitors of BRAF being promising targets for the disease outcome.

BACKGROUND

Addition of a MEK inhibitor to a BRAF inhibitor enhances tumour growth inhibition, delays acquired resistance, and abrogates paradoxical activation of the MAPK pathway in preclinical models of BRAF-mutated melanoma. We assessed the safety and efficacy of combined BRAF inhibition with vemurafenib and MEK inhibition with cobimetinib in patients with advanced BRAF-mutated melanoma.

METHODS

We undertook a phase 1b study in patients with advanced BRAF(V600)-mutated melanoma. We included individuals who had either recently progressed on vemurafenib or never received a BRAF inhibitor. In the dose-escalation phase of our study, patients received vemurafenib 720 mg or 960 mg twice a day continuously and cobimetinib 60 mg, 80 mg, or 100 mg once a day for either 14 days on and 14 days off (14/14), 21 days on and 7 days off (21/7), or continuously (28/0). The primary endpoint was safety of the drug combination and to identify dose-limiting toxic effects and the maximum tolerated dose. Efficacy was a key secondary endpoint. All patients treated with vemurafenib and cobimetinib were included in safety and efficacy analyses (intention-to-treat). The study completed accrual and all analyses are final. This study is registered with ClinicalTrials.gov, number NCT01271803.

RESULTS

129 patients were treated at ten dosing regimens combining vemurafenib and cobimetinib: 66 had recently progressed on vemurafenib and 63 had never received a BRAF inhibitor. Dose-limiting toxic effects arose in four patients. One patient on a schedule of vemurafenib 960 mg twice a day and cobimetinib 80 mg once a day 14/14 had grade 3 fatigue for more than 7 days; one patient on a schedule of vemurafenib 960 mg twice a day and cobimetinib 60 mg once a day 21/7 had a grade 3 prolongation of QTc; and two patients on a schedule of vemurafenib 960 mg twice a day and cobimetinib 60 mg 28/0 had dose-limiting toxic effects-one developed grade 3 stomatitis and fatigue and one developed arthralgia and myalgia. The maximum tolerated dose was established as vemurafenib 960 mg twice a day in combination with cobimetinib 60 mg 21/7. Across all dosing regimens, the most common adverse events were diarrhoea (83 patients, 64%), non-acneiform rash (77 patients, 60%), liver enzyme abnormalities (64 patients, 50%), fatigue (62 patients, 48%), nausea (58 patients, 45%), and photosensitivity (52 patients, 40%). Most adverse events were mild-to-moderate in severity. The most common grade 3 or 4 adverse events were cutaneous squamous-cell carcinoma (12 patients, 9%; all grade 3), raised amounts of alkaline phosphatase (11 patients, 9%]), and anaemia (nine patients, 7%). Confirmed objective responses were recorded in ten (15%) of 66 patients who had recently progressed on vemurafenib, with a median progression-free survival of 2·8 months (95% CI 2·6-3·4). Confirmed objective responses were noted in 55 (87%) of 63 patients who had never received a BRAF inhibitor, including six (10%) who had a complete response; median progression-free survival was 13·7 months (95% CI 10·1-17·5).

CONCLUSIONS

The combination of vemurafenib and cobimetinib was safe and tolerable when administered at the respective maximum tolerated doses. The combination has promising antitumour activity and further clinical development is warranted in patients with advanced BRAF(V600)-mutated melanoma, particularly in those who have never received a BRAF inhibitor; confirmatory clinical testing is ongoing.

BACKGROUND

F Hoffmann-La Roche/Genentech.

The development of targeted therapies has revolutionized the treatment of cancer patients. The identification of "druggable" oncogenic kinases and the creation of small-molecule inhibitors designed to specifically target these mutant kinases have become an important therapeutic paradigm across several different malignancies. Often these inhibitors induce dramatic clinical responses in molecularly defined cohorts. However, resistance to such targeted therapies is an inevitable consequence of this therapeutic approach. Resistance can be either primary (de novo) or acquired. Mechanisms leading to primary resistance may be categorized as tumor intrinsic factors or as patient/drug-specific factors. Acquired resistance may be mediated by target gene modification, activation of "bypass tracks" that serve as compensatory signaling loops, or histologic transformation. This brief review is a snapshot of the complex problem of therapeutic resistance, with a focus on resistance to kinase inhibitors in EGF receptor mutant and ALK rearranged non-small cell lung cancer, BRAF-mutant melanoma, and BCR-ABL-positive chronic myeloid leukemia. We describe specific mechanisms of primary and acquired resistance and then review emerging strategies to delay or overcome drug resistance.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumor of the gastrointestinal tract. Soon after GIST was recognized as a tumor driven by a KIT or platelet-derived growth factor receptor mutation, it became the first solid tumor target for tyrosine kinase inhibitor therapies. More recently, alternative molecular mechanisms for GIST pathogenesis have been discovered. These are related to deficiencies in the succinate dehydrogenase complex, NF1-gene alterations in connection with neurofibromatosis type 1 tumor syndrome, and mutational activation of the BRAF oncogene in very rare cases.

BACKGROUND

An increasing number of studies show that genetic markers can aid in refining prognostic information and predicting the benefit from systemic therapy. Our goal was to develop a high throughput, cost-effective and simple methodology for the detection of clinically relevant hot spot mutations in colon cancer.

METHODS

The Maldi-Tof mass spectrometry platform and OncoCarta panel from Sequenom were used to profile 239 colon cancers and 39 metastatic lymph nodes from NSABP clinical trial C-07 utilizing routinely processed FFPET (formalin-fixed paraffin-embedded tissue).

RESULTS

Among the 238 common hot-spot cancer mutations in 19 genes interrogated by the OncoCarta panel, mutations were detected in 7 different genes at 26 different nucleotide positions in our colon cancer samples. Twenty-four assays that detected mutations in more than 1% of the samples were reconfigured into a new multiplexed panel, termed here as ColoCarta. Mutation profiling was repeated on 32 mutant samples using ColoCarta and the results were identical to results with OncoCarta, demonstrating that this methodology was reproducible. Further evidence demonstrating the validity of the data was the fact that the mutation frequencies of the most common colon cancer mutations were similar to the COSMIC (Catalog of Somatic Mutations in Cancer) database. The frequencies were 43.5% for KRAS, 20.1% for PIK3CA, and 12.1% for BRAF. In addition, infrequent mutations in NRAS, AKT1, ABL1, and MET were detected. Mutation profiling of metastatic lymph nodes and their corresponding primary tumors showed that they were 89.7% concordant. All mutations found in the lymph nodes were also found in the corresponding primary tumors, but in 4 cases a mutation was present in the primary tumor only.

CONCLUSIONS

This study describes a high throughput technology that can be used to interrogate DNAs isolated from routinely processed FFPET and identifies the specific mutations that are common to colon cancer. The development of this technology and the ColoCarta panel may provide a mechanism for rapid screening of mutations in clinically relevant genes like KRAS, PIK3CA, and BRAF.

BACKGROUND

ClinicalTrials.gov: NSABP C-07: NCT00004931.

We show that BRAF(V600E) initiates an alternative pathway to colorectal cancer (CRC), which progresses through a hyperplasia/adenoma/carcinoma sequence. This pathway underlies significant subsets of CRCs with distinctive pathomorphologic/genetic/epidemiologic/clinical characteristics. Genetic and functional analyses in mice revealed a series of stage-specific molecular alterations driving different phases of tumor evolution and uncovered mechanisms underlying this stage specificity. We further demonstrate dose-dependent effects of oncogenic signaling, with physiologic Braf(V600E) expression being sufficient for hyperplasia induction, but later stage intensified Mapk-signaling driving both tumor progression and activation of intrinsic tumor suppression. Such phenomena explain, for example, the inability of p53 to restrain tumor initiation as well as its importance in invasiveness control, and the late stage specificity of its somatic mutation. Finally, systematic drug screening revealed sensitivity of this CRC subtype to targeted therapeutics, including Mek or combinatorial PI3K/Braf inhibition.

BACKGROUND

Osteosarcoma (OS) is the most common primary bone tumour in children and young adults. Despite improved prognosis, metastatic or relapsed OS remains largely incurable and no significant improvement has been observed in the last 20 years. Therefore, the search for alternative agents in OS is mandatory.

RESULTS

We investigated phospho-ERK 1/2, MCL-1, and phospho-Ezrin/Radixin/Moesin (P-ERM) as potential therapeutic targets in OS. Activation of these pathways was shown by immunohistochemistry in about 70% of cases and in all OS cell lines analyzed. Mutational analysis revealed no activating mutations in KRAS whereas BRAF gene was found to be mutated in 4/30 OS samples from patients. Based on these results we tested the multi-kinase inhibitor sorafenib (BAY 43-9006) in preclinical models of OS. Sorafenib inhibited OS cell line proliferation, induced apoptosis and downregulated P-ERK1/2, MCL-1, and P-ERM in a dose-dependent manner. The dephosphorylation of ERM was not due to ERK inhibition. The downregulation of MCL-1 led to an increase in apoptosis in OS cell lines. In chick embryo chorioallantoic membranes, OS supernatants induced angiogenesis, which was blocked by sorafenib and it was also shown that sorafenib reduced VEGF and MMP2 production. In addition, sorafenib treatment dramatically reduced tumour volume of OS xenografts and lung metastasis in SCID mice.

CONCLUSIONS

In conclusion, ERK1/2, MCL-1 and ERM pathways are shown to be active in OS. Sorafenib is able to inhibit their signal transduction, both in vitro and in vivo, displaying anti-tumoural activity, anti-angiogenic effects, and reducing metastatic colony formation in lungs. These data support the testing of sorafenib as a potential therapeutic option in metastatic or relapsed OS patients unresponsive to standard treatments.

The RAS/BRAF/MEK/ERK mitogen-activated protein kinase (MAPK) pathway is emerging as a key modulator of melanoma initiation and progression. However, a variety of clinical studies indicate that inhibiting the MAPK pathway is insufficient per se to effectively kill melanoma cells. Here, we report on a genetic and pharmacologic approach to identify survival factors responsible for the resistance of melanoma cells to MEK/ERK antagonists. In addition, we describe a new tumor cell-selective means to bypass this resistance in vitro and in vivo. By generating a panel of isogenic cell lines with specific defects in the apoptotic machinery, we found that the ability of melanoma cells to survive in the absence of functional MEK relies on an ERK-independent expression of the antiapoptotic factor Mcl-1 (and to a lesser extent, Bcl-x(L) and Bcl-2). Using computer-based modeling, we developed a novel Bcl-2 homology domain 3 (BH3) mimetic. This compound, named TW-37, is the first rationally designed small molecule with high affinity for Mcl-1, Bcl-x(L), and Bcl-2. Mechanistic analyses of the mode of action of TW-37 showed a synergistic tumor cell killing in the presence of MEK inhibitors. Importantly, TW-37 unveiled an unexpected role of the MAPK pathway in the control of reactive oxygen species (ROS). This function was critical to prevent the activation of proapoptotic functions of p53 in melanoma cells, but surprisingly, it was dispensable for normal melanocytes. Our results suggest that this MAPK-dependent ROS/p53 feedback loop is a point of vulnerability of melanoma cells that can be exploited for rational drug design.

BRAF, a cellular oncogene and effector of RAS-mediated signaling, is activated by mutation in approximately 60% of melanomas. Most of these mutations consist of a V600E substitution resulting in constitutive kinase activation. Mutant BRAF thus represents an important therapeutic target in melanoma. In an effort to produce a pre-clinical model of mutant BRAF function in melanoma, we have generated a mouse expressing BRAF V600E targeted to melanocytes. We show that in these transgenic mice, widespread benign melanocytic hyperplasia with histological features of nevi occurs, with biochemical evidence of senescence. Melanocytic hyperplasia progresses to overt melanoma with an incidence dependent on BRAF expression levels. Melanomas show CDKN2A loss, and genetic disruption of the CDKN2A locus greatly enhances melanoma formation, consistent with collaboration between BRAF activation and CDKN2A loss suggested from studies of human melanoma. The development of melanoma also involves activation of the Mapk and Akt signaling pathways and loss of senescence, findings that faithfully recapitulate those seen in human melanomas. This murine model of mutant BRAF-induced melanoma formation thus provides an important tool for identifying further genetic alterations that cooperates with BRAF and that may be useful in enhancing susceptibility to BRAF-targeted therapeutics in melanoma.

OBJECTIVE

To determine the prevalence of colorectal polyps of different types in an unselected population, and to correlate the morphological diagnoses with BRAF mutation analysis.

METHODS

Cases of colorectal polyps diagnosed at endoscopy were retrieved from the files of Southern.IML Pathology. All slides were reviewed and the lesions classified histologically. A diagnosis of sessile serrated adenoma was made even if the characteristic features were present only focally. If there was more than one polyp of a particular type in any patient, one lesion was chosen at random so that the results represent the number of patients with each type of polyp rather than the total number of polyps. A proportion of the polyps was subjected to BRAF mutation analysis.

RESULTS

A total of 1479 patients were identified. Non-serrated ("conventional") adenomas were found in 964 patients (65%), hyperplastic polyps in 437 (30%), sessile serrated adenomas in 57 (3.9%), traditional serrated adenomas in 11 (0.7%) and mixed hyperplastic adenomatous polyps in 10 (0.7%). BRAF V600E mutation analysis was performed in 148 selected cases; mutations were found in 44/49 (90%) of lesions diagnosed as sessile serrated adenoma, in 10/34 (29%) of hyperplastic polyps of microvesicular type, in 4/11 (36%) of traditional serrated adenomas, in 10/10 (100%) of mixed hyperplastic adenomatous polyps, and in 2/42 (5%) of "conventional" adenomas.

CONCLUSIONS

Sessile serrated adenomas are encountered commonly in routine endoscopy practice. The histological diagnosis correlates strongly with the presence of BRAF mutation.

Gastrointestinal stromal tumors (GISTs) are mesenchymal tumors characterized by mutations of KIT or PDGFRA. The objectives of this study were to evaluate BRAF mutations in GISTs and then to correlate BRAF mutational status in the tumor with clinical parameters, with B-raf expression, and with activation of some cellular pathways. BRAF mutation was screened in 321 GISTs with 70 wild-type GISTs. BRAF V600E was detected in 9 (13%) of 70 wild-type GISTs. No mutations were detected in GISTs bearing KIT or PDGFRA mutations. BRAF V600E detection in the tumor does not induce a higher expression of the B-raf protein or the preferential activation of the p42/44 mitogen-activated protein kinase (MAPK) signaling pathway compared with GISTs without the BRAF mutation. In comparison with the GIST group with KIT or PDGFRA mutation or the wild-type GIST group without BRAF mutation, the wild-type GIST group with a BRAF mutation is not different in terms of B-raf expression or the p44/42 MAPK- or AKT-activated signaling pathway.

OBJECTIVE

Imaging with [(18)F]fluorodeoxyglucose (FDG) -positron emission tomography (PET) allows early recognition of a response to agents that target key driver mutations in human cancer. We aimed to determine the metabolic response rate to vemurafenib in patients with advanced BRAF-mutant melanoma.

METHODS

Baseline and day 15 FDG-PET was evaluated in 31 patients with advanced melanoma treated in a phase I study of dose escalation of vemurafenib (PLX06-02), which included four patients treated at subtherapeutic doses and 24 patients treated at 960 mg twice a day, which is the maximum-tolerated dose of vemurafenib.

RESULTS

All 27 patients treated at potentially therapeutic levels had at least a partial metabolic response, and three patients achieved a complete metabolic response. In the 27 patients, there was an 80% ± 3% reduction in the maximum standardized uptake value (SUVmax) of target lesions and an 87% ± 3% decrease in the percentage of injected dose (%ID) in all identified disease sites. There was a positive correlation between %ID in all identified disease and target-lesion SUVmax (r(2) = 0.66; P < .001) that indicated a significant homogeneity of the response between lesions in individual patients. Although no relationship was found between the reduction in target lesion SUVmax and best response according to RECIST (Response Evaluation Criteria in Solid Tumors), there was a trend for patients with greater reductions in uptake of FDG to have longer progression-free survival.

CONCLUSIONS

FDG-PET is a useful marker of an early biologic response to vemurafenib. Little variability in PET response was found between lesions in individual patients, which suggested minimal intrapatient molecular heterogeneity. FDG-PET is a useful tool for the evaluation of the biologic impact of inhibiting mutant BRAF and may allow for the more effective development of novel agents.

Aberrant activation of Ras signaling is a common finding in human glioblastomas. To determine the contribution of Ras gene mutations to this aberration, we screened 94 glioblastomas for mutations in the three Ras family genes NRAS, KRAS and HRAS. All tumors were additionally analyzed for mutations in BRAF, which encodes a Ras-regulated serine/threonine kinase with oncogenic properties. Mutation analysis of the entire coding regions of NRAS and KRAS, as well as the known mutation hot-spot sites in HRAS, identified somatic point mutations in two glioblastomas, both affecting codon 12 of NRAS (c.35G>A, p.G12D). Three additional tumors carried BRAF mutations altering the known hot-spot codon 599 (c.1796T>A, p.V599E). None of these five glioblastomas showed amplification of the EGFR or PDGFRA genes, while three of the tumors, including two with NRAS and one with BRAF mutation, demonstrated PTEN missense mutations or loss of PTEN mRNA expression. Taken together, our data suggest activating mutations in NRAS or BRAF as a molecular alteration that contributes to aberrant Ras signaling in a small fraction of glioblastomas.

BACKGROUND

Around 50% of cutaneous melanomas harbor the BRAF(V600E) mutation and can be treated with BRAF inhibitors. DNA carrying this mutation can be released into circulation as cell-free BRAF(V600E) (cfBRAF(V600E)). Droplet digital PCR (ddPCR) is an analytically sensitive technique for quantifying small concentrations of DNA. We studied the plasma concentrations of cfBRAF(V600E) by ddPCR in patients with melanoma during therapy with BRAF inhibitors.

METHODS

Plasma concentrations of cfBRAF(V600E) were measured in 8 controls and 20 patients with advanced melanoma having the BRAF(V600E) mutation during treatment with BRAF inhibitors at baseline, first month, best response, and progression.

RESULTS

The BRAF(V600E) mutation was detected by ddPCR even at a fractional abundance of 0.005% in the wild-type gene. Agreement between tumor tissue BRAF(V600E) and plasma cfBRAF(V600E) was 84.3%. Baseline cfBRAF(V600E) correlated with tumor burden (r = 0.742, P < 0.001). cfBRAF(V600E) concentrations decreased significantly at the first month of therapy (basal median, 216 copies/mL; Q1-Q3, 27-647 copies/mL; first response median, 0 copies/mL; Q1-Q3, 0-49 copies/mL; P < 0.01) and at the moment of best response (median, 0 copies/mL; Q1-Q3, 0-33 copies/mL; P < 0.01). At progression, there was a significant increase in the concentration of cfBRAF(V600E) compared with best response (median, 115 copies/mL; Q1-Q3, 3-707 copies/mL; P = 0.013). Lower concentrations of basal cfBRAF(V600E) were significantly associated with longer overall survival and progression-free survival (27.7 months and 9 months, respectively) than higher basal concentrations (8.6 months and 3 months, P < 0.001 and P = 0.024, respectively).

CONCLUSIONS

cfBRAF(V600E) quantification in plasma by ddPCR is useful as a follow-up to treatment response in patients with advanced melanoma.

OBJECTIVE

KRAS mutations are predictive of nonresponse to anti-EGFR therapies in metastatic colorectal cancer (mCRC). However, only 50% of nonmutated patients benefit from them. KRAS-mutated subclonal populations nondetectable by conventional methods have been suggested as the cause of early progression. Molecular analysis technology with high sensitivity and precision is required to test this hypothesis.

METHODS

From two cohorts of patients with mCRC, 136 KRAS, NRAS, and BRAF wild-type tumors with sufficient tumor material to perform highly sensitive picodroplet digital PCR (dPCR) and 41 KRAS-mutated tumors were selected. All these patients were treated by anti-EGFR therapy. dPCR was used for KRAS or BRAF mutation screening and compared with qPCR. Progression-free survival (PFS) and overall survival (OS) were analyzed according to the KRAS-mutated allele fraction.

RESULTS

In addition to the confirmation of the 41 patients with KRAS-mutated tumors, dPCR also identified KRAS mutations in 22 samples considered as KRAS wild-type by qPCR. The fraction of KRAS-mutated allele quantified by dPCR was inversely correlated with anti-EGFR therapy response rate (P < 0.001). In a Cox model, the fraction of KRAS-mutated allele was associated with worse PFS and OS. Patients with less than 1% of mutant KRAS allele have similar PFS and OS than those with wild-type KRAS tumors.

CONCLUSIONS

This study suggests that patients with mCRC with KRAS-mutated subclones (at least those with a KRAS-mutated subclones fraction lower or equal to 1%) had a benefit from anti-EGFR therapies.

OBJECTIVE

Preclinical and early clinical studies have demonstrated that initial therapy with combined BRAF and MEK inhibition is more effective in BRAF(V600)-mutant melanoma than single-agent BRAF inhibitors. This study assessed the safety and efficacy of dabrafenib and trametinib in patients who had received prior BRAF inhibitor treatment.

METHODS

In this open-label phase I/II study, we evaluated the pharmacology, safety, and efficacy of dabrafenib and trametinib. Here, we report patients treated with combination therapy after disease progression with BRAF inhibitor treatment administered before study enrollment (part B; n = 26) or after cross-over at progression with dabrafenib monotherapy (part C; n = 45).

RESULTS

In parts B and C, confirmed objective response rates (ORR) were 15% (95% CI, 4% to 35%) and 13% (95% CI, 5% to 27%), respectively; an additional 50% and 44% experienced stable disease ≥ 8 weeks, respectively. In part C, median progression-free survival (PFS) was 3.6 months (95% CI, 2 to 4), and median overall survival was 11.8 months (95% CI, 8 to 25) from cross-over. Patients who previously received dabrafenib ≥ 6 months had superior outcomes with the combination compared with those treated < 6 months; median PFS was 3.9 (95% CI, 3 to 7) versus 1.8 months (95% CI, 2 to 4; hazard ratio, 0.49; P = .02), and ORR was 26% (95% CI, 10% to 48%) versus 0% (95% CI, 0% to 15%).

CONCLUSIONS

Dabrafenib plus trametinib has modest clinical efficacy in patients with BRAF inhibitor-resistant melanoma. This regimen may be a therapeutic strategy for patients who previously benefited from BRAF inhibitor monotherapy ≥ 6 months but demonstrates minimal efficacy after rapid progression with BRAF inhibitor therapy.

BACKGROUND

Acquired resistance to BRAF inhibitors (BRAFi) is a near-universal phenomenon caused by numerous genetic and non-genetic alterations. In this study, we evaluated the spectrum, onset, pattern of progression, and subsequent clinical outcomes associated with specific mechanisms of resistance.

METHODS

We compiled clinical and genetic data from 100 patients with 132 tissue samples obtained at progression on BRAFi therapy from 3 large, previously published studies of BRAFi resistance. These samples were subjected to whole-exome sequencing and/or polymerase chain reaction-based genetic testing.

RESULTS

Among 132 samples, putative resistance mechanisms were identified in 58%, including NRAS or KRAS mutations (20%), BRAF splice variants (16%), BRAF(V600E/K) amplifications (13%), MEK1/2 mutations (7%), and non-mitogen-activated protein kinase pathway alterations (11%). Marked heterogeneity was observed within tumors and patients; 18 of 19 patients (95%) with more than one progression biopsy had distinct/unknown drivers of resistance between samples. NRAS mutations were associated with vemurafenib use (p = 0.045) and intracranial metastases (p = 0.036), and MEK1/2 mutations correlated with hepatic progression (p = 0.011). Progression-free survival and overall survival were similar across resistance mechanisms. The median survival after disease progression was 6.9 months, and responses to subsequent BRAF and MEK inhibition were uncommon (2 of 15; 13%). Post-progression outcomes did not correlate with specific acquired BRAFi-resistance mechanisms.

CONCLUSIONS

This is the first study to systematically characterise the clinical implications of particular acquired BRAFi-resistance mechanisms in patients with BRAF-mutant melanoma largest study to compile the landscape of resistance. Despite marked heterogeneity of resistance mechanisms within patients, NRAS mutations correlated with vemurafenib use and intracranial disease involvement.

BRAF V600E mutation has been identified in up to 2/3 of pleomorphic xanthoastrocytomas (PXAs), World Health Organization grade II, as well as in varying percentages of PXAs with anaplastic features (PXA-A), gangliogliomas, extracerebellar pilocytic astrocytomas, and, rarely, giant cell glioblastoma multiforme (GC-GBMs). GC-GBMs and epithelioid GBMs (E-GBMs) can be histologically challenging to distinguish from PXA-A. We undertook this study specifically to address whether these 2 tumor types also showed the mutation. We tested our originally reported cohort of 8 E-GBMs and 2 rhabdoid GBMs (R-GBM) as well as 5 new E-GBMs (1 pediatric, 4 adult) and 9 GC-GBMs (2 pediatric, 7 adult) (n=24) for BRAF V600E mutational status. Twenty-one of 24 had sufficient material for IDH-1 immunostaining, which is usually absent in PXAs, PXA-As, and primary GBMs but present in secondary GBMs. Patients ranged in age from 4 to 67 years. BRAF V600E mutation was identified in 7/13 of E-GBMs, including 3 of our original cases; patients with mutation were aged 10 to 50 years. None of the 9 GC-GBMs or 2 R-GBMs manifested this mutation, including pediatric patients. The sole secondary E-GBM was the single case manifesting positive IDH-1 immunoreactivity. A high percentage of E-GBMs manifest BRAF V600E mutation, paralleling PXAs. All R-GBMs and GC-GBMs were negative, although larger multi-institutional cohorts will have to be tested to extend this result. BRAF V600E mutational analyses should be performed on E-GBMs, particularly in all pediatric and young-aged adults, given the potential for BRAF inhibitor therapy in this subset of GBM patients.

Sequencing complete tumor genomes and exomes has sparked the cancer field's interest in mutation signatures for identifying the tumor's carcinogen. This review and meta-analysis discusses signatures and their proper use. We first distinguish between a mutagen's canonical mutations—deviations from a random distribution of base changes to create a pattern typical of that mutagen—and the subset of signature mutations, which are unique to that mutagen and permit inference backward from mutations to mutagen. To verify UV signature mutations, we assembled literature datasets on cells exposed to UVC, UVB, UVA, or solar simulator light (SSL) and tested canonical UV mutation features as criteria for clustering datasets. A confirmed UV signature was: ≥60% of mutations are C→T at a dipyrimidine site, with ≥5% CC→TT. Other canonical features such as a bias for mutations on the nontranscribed strand or at the 3' pyrimidine had limited application. The most robust classifier combined these features with criteria for the rarity of non-UV canonical mutations. In addition, several signatures proposed for specific UV wavelengths were limited to specific genes or species; UV's nonsignature mutations may cause melanoma BRAF mutations; and the mutagen for sunlight-related skin neoplasms may vary between continents.

We previously reported that colon carcinomas, adenomas, and hyperplastic polyps exhibiting a serrated histology were very likely to possess BRAF mutations, whereas when these same advanced colonic lesions exhibited non-serrated histology, they were wild type for BRAF; among hyperplastic polyps, KRAS mutations were found mainly in a non-serrated variant. On this basis, we predicted that hyperplastic aberrant crypt foci (ACF), a putative precancerous lesion found in the colon, exhibiting a serrated phenotype would also harbor BRAF mutations and that non-serrated ACF would not. In contrast, KRAS mutations would be found more often in the non-serrated ACF. We examined 55 ACF collected during screening colonoscopy from a total of 28 patients. Following laser capture microdissection, DNA was isolated, and mutations in BRAF and KRAS were determined by direct PCR sequencing. When hyperplastic lesions were further classified into serrated and non-serrated histologies, there was a strong inverse relationship between BRAF and KRAS mutations: a BRAF(V600E) mutation was identified in 10 of 16 serrated compared with 1 of 33 non-serrated lesions (P = 0.001); conversely, KRAS mutations were present in 3 of 16 serrated compared with 14 of 33 non-serrated lesions. Our finding of a strong association between BRAF mutations and serrated histology in hyperplastic ACF supports the idea that these lesions are an early, sentinel, or a potentially initiating step on the serrated pathway to colorectal carcinoma.

BACKGROUND

Colorectal cancer is a heterogeneous disease with multiple underlying genetic mutations causing different clinical phenotypes. Mutation in the BRAF oncogene is a key step in malignant transformation within the methylator pathway to colorectal cancer. However, there is a paucity of information about BRAF mutant colorectal tumors.

OBJECTIVE

This study defines the clinical characteristics and oncologic outcome associated with colorectal cancer BRAF mutations.

METHODS

Colorectal adenocarcinomas from a single-institution frozen-tumor biobank were studied. Genomic DNA was isolated and analyzed for mutations in the BRAF oncogene by polymerase chain reaction amplification followed by direct sequencing. A sample was classified as mutant if any of the tested loci were mutated. Patient and tumor characteristics were recorded including patient age, sex, tumor location, tumor differentiation, and microsatellite instability.

METHODS

Statistical associations with BRAF mutant tumors were determined by the Fisher exact probability test, χ test, or Wilcoxon analysis. Kaplan-Meier estimates and multivariate Cox regression analysis were performed for overall survival.

RESULTS

Four hundred seventy-five colorectal adenocarcinomas were included in the study population; 56 samples harbored a BRAF mutation (12%). There were significant differences between BRAF wild-type and mutant tumors in age (66 vs 75 years, p = 0.004), female sex (44% vs 71%, p < 0.001), proximal tumor location (44% vs 95%, p < 0.001), and frequency of microsatellite instability (16% vs 76%, p < 0.001). There was no difference in cancer stage between BRAF mutant and wild-type populations. Survival data were analyzed for 322 patients with stage I to III disease, and patients with a BRAF mutation had decreased overall survival than those without a mutation (p = 0.018). With the use of Cox regression analysis, BRAF mutation conferred a worse overall survival (HR 1.79, CI 1.05-3.05, p = 0.03) independent of microsatellite instability status.

CONCLUSIONS

BRAF mutations in colorectal cancers are associated with distinct clinical characteristics and worse prognosis.