We reviewed the anti-cancer effects of DCA, an orphan drug long used as an investigational treatment for various acquired and congenital disorders of mitochondrial intermediary metabolism. Inhibition by DCA of mitochondrial pyruvate dehydrogenase kinases and subsequent reactivation of the pyruvate dehydrogenase complex and oxidative phosphorylation is the common mechanism accounting for the drug's anti-neoplastic effects. At least two fundamental changes in tumor metabolism are induced by DCA that antagonize tumor growth, metastases and survival: the first is the redirection of glucose metabolism from glycolysis to oxidation (reversal of the Warburg effect), leading to inhibition of proliferation and induction of caspase-mediated apoptosis. These effects have been replicated in both human cancer cell lines and in tumor implants of diverse germ line origin. The second fundamental change is the oxidative removal of lactate, via pyruvate, and the co-incident buffering of hydrogen ions by dehydrogenases located in the mitochondrial matrix. Preclinical studies demonstrate that DCA has additive or synergistic effects when used in combination with standard agents designed to modify tumor oxidative stress, vascular remodeling, DNA integrity or immunity. These findings and limited clinical results suggest that potentially fruitful areas for additional clinical trials include 1) adult and pediatric high grade astrocytomas; 2) BRAF-mutant cancers, such as melanoma, perhaps combined with other pro-oxidants; 3) tumors in which resistance to standard platinum-class drugs alone may be overcome with combination therapy; and 4) tumors of endodermal origin, in which extensive experimental research has demonstrated significant anti-proliferative, pro-apoptotic effects of DCA, leading to improved host survival.

PLX4032/vemurafenib is a first-in-class small-molecule BRAF(V600E) inhibitor with clinical activity in patients with BRAF mutant melanoma. Nevertheless, drug resistance develops in treated patients, and strategies to overcome primary and acquired resistance are required. To explore the molecular mechanisms involved in primary resistance to PLX4032, we investigated its effects on cell proliferation and signaling in a panel of 27 genetically characterized patient-derived melanoma cell lines. Cell sensitivity to PLX4032 was dependent on BRAF(V600E) and independent from other gene alterations that commonly occur in melanoma such as PTEN loss, BRAF, and MITF gene amplification. Two cell lines lacking sensitivity to PLX4032 and harboring a different set of genetic alterations were studied as models of primary resistance. Treatment with the MEK inhibitor UO126 but not with PLX4032 inhibited cell growth and ERK activation. Resistance to PLX4032 was maintained after CRAF down-regulation by siRNA indicating alternative activation of MEK-ERK signaling. Genetic characterization by multiplex ligation-dependent probe amplification and analysis of phosphotyrosine signaling by MALDI-TOF mass spectrometry analysis revealed the activation of MET and SRC signaling, associated with the amplification of MET and of CTNNB1 and CCND1 genes, respectively. The combination of PLX4032 with drugs or siRNA targeting MET was effective in inhibiting cell growth and reducing cell invasion and migration in melanoma cells with MET amplification; similar effects were observed after targeting SRC in the other cell line, indicating a role for MET and SRC signaling in primary resistance to PLX4032. Our results support the development of classification of melanoma in molecular subtypes for more effective therapies.

Most colorectal cancers (CRCs) containing activated BRAF (BRAF[V600E]) have a CpG island methylator phenotype (CIMP) characterized by aberrant hypermethylation of many genes, including the mismatch repair gene MLH1. MLH1 silencing results in microsatellite instability and a hypermutable phenotype. Through an RNAi screen, here we identify the transcriptional repressor MAFG as the pivotal factor required for MLH1 silencing and CIMP in CRCs containing BRAF(V600E). In BRAF-positive human CRC cell lines and tumors, MAFG is bound at the promoters of MLH1 and other CIMP genes, and recruits a corepressor complex that includes its heterodimeric partner BACH1, the chromatin remodeling factor CHD8, and the DNA methyltransferase DNMT3B, resulting in hypermethylation and transcriptional silencing. BRAF(V600E) increases BRAF/MEK/ERK signaling resulting in phosphorylation and elevated levels of MAFG, which drives DNA binding. Analysis of transcriptionally silenced CIMP genes in KRAS-positive CRCs indicates that different oncoproteins direct the assembly of distinct repressor complexes on common promoters.

Next-generation sequencing (NGS) has enabled genome-wide personalized oncology efforts at centers and companies with the specialty expertise and infrastructure required to identify and prioritize actionable variants. Such approaches are not scalable, preventing widespread adoption. Likewise, most targeted NGS approaches fail to assess key relevant genomic alteration classes. To address these challenges, we predefined the catalog of relevant solid tumor somatic genome variants (gain-of-function or loss-of-function mutations, high-level copy number alterations, and gene fusions) through comprehensive bioinformatics analysis of >700,000 samples. To detect these variants, we developed the Oncomine Comprehensive Panel (OCP), an integrative NGS-based assay [compatible with <20 ng of DNA/RNA from formalin-fixed paraffin-embedded (FFPE) tissues], coupled with an informatics pipeline to specifically identify relevant predefined variants and created a knowledge base of related potential treatments, current practice guidelines, and open clinical trials. We validated OCP using molecular standards and more than 300 FFPE tumor samples, achieving >95% accuracy for KRAS, epidermal growth factor receptor, and BRAF mutation detection as well as for ALK and TMPRSS2:ERG gene fusions. Associating positive variants with potential targeted treatments demonstrated that 6% to 42% of profiled samples (depending on cancer type) harbored alterations beyond routine molecular testing that were associated with approved or guideline-referenced therapies. As a translational research tool, OCP identified adaptive CTNNB1 amplifications/mutations in treated prostate cancers. Through predefining somatic variants in solid tumors and compiling associated potential treatment strategies, OCP represents a simplified, broadly applicable targeted NGS system with the potential to advance precision oncology efforts.

Understanding the profile of oncogene and tumor suppressor gene mutations with their interactions and impact on the prognosis of multiple myeloma (MM) can improve the definition of disease subsets and identify pathways important in disease pathobiology. Using integrated genomics of 1273 newly diagnosed patients with MM, we identified 63 driver genes, some of which are novel, including IDH1, IDH2, HUWE1, KLHL6, and PTPN11 Oncogene mutations are significantly more clonal than tumor suppressor mutations, indicating they may exert a bigger selective pressure. Patients with more driver gene abnormalities are associated with worse outcomes, as are identified mechanisms of genomic instability. Oncogenic dependencies were identified between mutations in driver genes, common regions of copy number change, and primary translocation and hyperdiploidy events. These dependencies included associations with t(4;14) and mutations in FGFR3, DIS3, and PRKD2; t(11;14) with mutations in CCND1 and IRF4; t(14;16) with mutations in MAF, BRAF, DIS3, and ATM; and hyperdiploidy with gain 11q, mutations in FAM46C, and MYC rearrangements. These associations indicate that the genomic landscape of myeloma is predetermined by the primary events upon which further dependencies are built, giving rise to a nonrandom accumulation of genetic hits. Understanding these dependencies may elucidate potential evolutionary patterns and lead to better treatment regimens.

Approximately 200 BRAF mutant alleles have been identified in human tumours. Activating BRAF mutants cause feedback inhibition of GTP-bound RAS, are RAS-independent and signal either as active monomers (class 1) or constitutively active dimers (class 2). Here we characterize a third class of BRAF mutants-those that have impaired kinase activity or are kinase-dead. These mutants are sensitive to ERK-mediated feedback and their activation of signalling is RAS-dependent. The mutants bind more tightly than wild-type BRAF to RAS-GTP, and their binding to and activation of wild-type CRAF is enhanced, leading to increased ERK signalling. The model suggests that dysregulation of signalling by these mutants in tumours requires coexistent mechanisms for maintaining RAS activation despite ERK-dependent feedback. Consistent with this hypothesis, melanomas with these class 3 BRAF mutations also harbour RAS mutations or NF1 deletions. By contrast, in lung and colorectal cancers with class 3 BRAF mutants, RAS is typically activated by receptor tyrosine kinase signalling. These tumours are sensitive to the inhibition of RAS activation by inhibitors of receptor tyrosine kinases. We have thus defined three distinct functional classes of BRAF mutants in human tumours. The mutants activate ERK signalling by different mechanisms that dictate their sensitivity to therapeutic inhibitors of the pathway.

BACKGROUND

In their previous analysis of papillary thyroid carcinomas (PTCs) from an Ukrainian-American cohort that was exposed to iodine-131 ((131) I) from the Chernobyl accident, the authors identified RET/PTC rearrangements and other driver mutations in 60% of tumors.

METHODS

In this study, the remaining mutation-negative tumors from that cohort were analyzed using RNA sequencing (RNA-Seq) and reverse transcriptase-polymerase chain reaction to identify novel chromosomal rearrangements and to characterize their relation with radiation dose.

RESULTS

The ETS variant gene 6 (ETV6)-neurotrophin receptor 3 (NTRK3) rearrangement (ETV6-NTRK3) was identified by RNA-Seq in a tumor from a patient who received a high (131) I dose. Overall, the rearrangement was detected in 9 of 62 (14.5%) post-Chernobyl PTCs and in 3 of 151 (2%) sporadic PTCs (P = .019). The most common fusion type was between exon 4 of ETV6 and exon 14 of NTRK3. The prevalence of ETV6-NTRK3 rearrangement in post-Chernobyl PTCs was associated with increasing (131) I dose, albeit at borderline significance (P = .126). The group of rearrangement-positive PTCs (ETV6-NTRK3, RET/PTC, PAX8-PPARγ) was associated with significantly higher dose response compared with the group of PTCs with point mutations (BRAF, RAS; P < .001). In vitro exposure of human thyroid cells to 1 gray of (131) I and γ-radiation resulted in the formation of ETV6-NTRK3 rearrangement at a rate of 7.9 × 10(-6) cells and 3.0 × 10(-6) cells, respectively.

CONCLUSIONS

The authors report the occurrence of ETV6-NTRK3 rearrangements in thyroid cancer and demonstrate that this rearrangement is significantly more common in tumors associated with exposure to (131) I and has a borderline significant dose response. Moreover, ETV6-NTRK3 rearrangement can be directly induced in thyroid cells by ionizing radiation in vitro and, thus, may represent a novel mechanism of radiation-induced carcinogenesis.

The clinical benefits of BRAF inhibition in patients with advanced-stage BRAF-mutant melanoma are now well established. Although the emergence of cutaneous squamous-cell carcinomas (SCCs) and secondary melanomas in patients on BRAF-inhibitor therapy have been well described, reports are emerging of additional secondary premalignant and malignant events, including RAS-mutant leukaemia, the metastatic recurrence of RAS-mutant colorectal cancer and the development of gastric and colonic polyps. In most cases, paradoxical MAPK activation--resulting from the BRAF-inhibitor-mediated homodimerization and heterodimerization of nonmutant RAF isoforms--seems to underlie the development of these secondary tumours. Although evidence supports that therapy with the simultaneous administration of BRAF and MEK inhibitors abrogates the onset of treatment-induced SCCs, whether combination treatment will limit the emergence of all BRAF-inhibitor-driven pathologies is unclear. In this Review, we describe the clinical and mechanistic manifestations of secondary cancers that have thus far been observed to arise as a consequence of BRAF inhibition. We discuss the concept of pre-existing populations of partly transformed cells with malignant potential that might be present in various organ systems, and the rationale for novel therapeutic strategies for the management of BRAF-inhibitor-induced neoplasia.

The development of personalized medicine with a focus on novel targeted therapies has supplanted the one-size-fits-all approach to the treatment of many cancers, including non-small cell lung cancer. Targeted therapies, if given to a patient subpopulation enriched by the presence of relevant molecular targets, can often abrogate cell signaling that perpetuates cancer progression. Critical targets activating procancer pathways include, but are not limited to, epidermal growth factor receptor (EGFR), hepatocyte growth factor receptor (MET), vascular endothelial growth factor (VEGF), VEGF receptor, GTPase KRAS (KRAS), receptor tyrosine protein kinase erbB-2 (HER2), echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (PIK3CA), serine/threonine-protein kinase B-raf (BRAF), and insulin-like growth factor 1 receptor (IGF-1R). Some target-directed therapies, such as epidermal growth factor receptor tyrosine kinase inhibitors and anti-VEGF monoclonal antibody, have already been approved for clinical use. Others, such as those targeted to MET, VEGFR, HER2, PIK3CA, and IGF-1R, are in clinical testing. This review describes molecular targets in non-small cell lung cancer that are in development or being clinically applied and their implications for developing novel anticancer therapies for this previously refractory malignancy.

Pediatric glioblastoma (pedGBM) is amongst the most common malignant brain tumors of childhood and carries a dismal prognosis. In contrast to adult GBM, few molecular prognostic markers for the pediatric counterpart have been established. We, therefore, investigated the prognostic significance of genomic and epigenetic alterations through molecular analysis of 202 pedGBM (1-18 years) with comprehensive clinical annotation. Routinely prepared formalin-fixed paraffin-embedded tumor samples were assessed for genome-wide DNA methylation profiles, with known candidate genes screened for alterations via direct sequencing or FISH. Unexpectedly, a subset of histologically diagnosed GBM (n = 40, 20 %) displayed methylation profiles similar to those of either low-grade gliomas or pleomorphic xanthoastrocytomas (PXA). These tumors showed a markedly better prognosis, with molecularly PXA-like tumors frequently harboring BRAF V600E mutations and 9p21 (CDKN2A) homozygous deletion. The remaining 162 tumors with pedGBM molecular signatures comprised four subgroups: H3.3 G34-mutant (15 %), H3.3/H3.1 K27-mutant (43 %), IDH1-mutant (6 %), and H3/IDH wild-type (wt) GBM (36 %). These subgroups were associated with specific cytogenetic aberrations, MGMT methylation patterns and clinical outcomes. Analysis of follow-up data identified a set of biomarkers feasible for use in risk stratification: pedGBM with any oncogene amplification and/or K27M mutation (n = 124) represents a particularly unfavorable group, with 3-year overall survival (OS) of 5 %, whereas tumors without these markers (n = 38) define a more favorable group (3-year OS ~70 %).Combined with the lower grade-like lesions, almost 40 % of pedGBM cases had distinct molecular features associated with a more favorable outcome. This refined prognostication method for pedGBM using a molecular risk algorithm may allow for improved therapeutic choices and better planning of clinical trial stratification for this otherwise devastating disease.

The mitogen-activated protein kinases (MAPK) pathway, often known as the RAS-RAF-MEK-ERK signal cascade, functions to transmit upstream signals to its downstream effectors to regulate physiological process such as cell proliferation, differentiation, survival and death. As the most frequently mutated signaling pathway in human cancer, targeting the MAPK pathway has long been considered a promising strategy for cancer therapy. Substantial efforts in the past decades have led to the clinical success of BRAF and MEK inhibitors. However, the clinical benefits of these inhibitors are compromised by the frequently occurring acquired resistance due to cancer heterogeneity and genomic instability. This review briefly introduces the key protein kinases involved in this pathway as well as their activation mechanisms. We also generalize the correlations between mutations of MAPK members and human cancers, followed by a summarization of progress made on the development of small molecule MAPK kinases inhibitors. In particular, this review highlights the potential advantages of ERK inhibitors in overcoming resistance to upstream targets and proposes that targeting ERK kinase may hold a promising prospect for cancer therapy.

Monoclonal antibodies against immune checkpoint blockade have proven to be a major success in the treatment of melanoma. The programmed death receptor-1 ligand-1 (PD-L1) expression on melanoma cells is believed to have an inhibitory effect on T cell responses and to be an important escape mechanism from immune attack. Previous studies have shown that PD-L1 can be expressed constitutively or can be induced by IFN-γ secreted by infiltrating lymphocytes. In the present study we have investigated the mechanism underlying these two modes of PD-L1 expression in melanoma cells including cells that had acquired resistance to the BRAF inhibitor vemurafenib. PD-L1 expression was examined by flow cytometry and immunoblotting. Specific inhibitors and siRNA knockdown approaches were used to examine the roles of the RAF/ MEK, PI3K, NF-κB, STAT3 and AP1/ c-Jun pathways. IFN-γ inducible expression of PD-L1 was dependent on NF-κB as shown by inhibition with BMS-345541, an inhibitor of IκB and the BET protein inhibitor I-BET151, as well as by siRNA knockdown of NF-κB subunits. We were unable to implicate the BRAF/MEK pathway as major regulators in PD-L1 expression on vemurafenib resistant cells. Similarly the PI3K/AKT pathway and the transcription factors STAT3 and c-Jun had only minor roles in IFN-γ induced expression of PD-L1. The mechanism underlying constitutive expression remains unresolved. We suggest these results have significance in selection of treatments that can be used in combination with monoclonal antibodies against PD1, to enhance their effectiveness and to reduce inhibitory effects melanoma cells have against cytotoxic T cell activity.

Heterogeneous expression of melanocytic antigens occurs frequently in melanomas and represents a potent barrier to immunotherapy. We previously showed that coordinated losses of several melanocytic antigens are generally attributable to down-regulation of antigen gene expression rather than irreversible mutation. Treatment of melanoma cells with mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitors blocks ERK activation and increases steady-state levels of mRNAs and corresponding protein expression for the melanocytic antigens Melan-A/MART-1, gp100, and tyrosinase. Although the degree of MEK inhibitor enhancement of antigen expression varied among different cell lines irrespective of their antigen expression status, all showed detectable responses. Notably, the antigen-enhancing effects of the MEK inhibitors could not be attributed to the master melanocytic regulator MITF-M. Because MAPK pathway activation via constitutively active mutant forms of BRAF is common in melanomas, correlation between BRAF function and antigen expression was investigated. No simple correlation of endogenous BRAF mutational status and antigen levels was observed, but transient overexpression of V600E BRAF increased ERK activation and reduced Melan-A/MART-1 levels in antigen-positive cell lines. These data indicate that whereas multiple factors may regulate antigen expression in melanomas, enhancement of MAPK signaling can act as a negative influence. Blocking such signaling with MEK inhibitors accordingly augments antigen levels, thereby enhancing Melan-A/MART-1-specific cytotoxic T-cell responses to antigen-negative cells following MEK inhibition treatment. Consequently, MAPK inhibition may assist targeting of melanomas for immunotherapy.

LY3009120 is a pan-RAF and RAF dimer inhibitor that inhibits all RAF isoforms and occupies both protomers in RAF dimers. Biochemical and cellular analyses revealed that LY3009120 inhibits ARAF, BRAF, and CRAF isoforms with similar affinity, while vemurafenib or dabrafenib have little or modest CRAF activity compared to their BRAF activities. LY3009120 induces BRAF-CRAF dimerization but inhibits the phosphorylation of downstream MEK and ERK, suggesting that it effectively inhibits the kinase activity of BRAF-CRAF heterodimers. Further analyses demonstrated that LY3009120 also inhibits various forms of RAF dimers including BRAF or CRAF homodimers. Due to these unique properties, LY3009120 demonstrates minimal paradoxical activation, inhibits MEK1/2 phosphorylation, and exhibits anti-tumor activities across multiple models carrying KRAS, NRAS, or BRAF mutation.

The majority of human colorectal cancers (CRCs) are initiated by mutations arising in the adenomatous polyposis coli (APC) tumour suppressor gene. However, a new class of non-APC mutated CRCs has been defined that have a serrated histopathology and carry the (V600E)BRAF oncogene. Here we have investigated the pathogenesis of serrated CRCs by expressing (V600E)Braf in the proliferative cells of the mouse gastrointestinal tract. We show that the oncogene drives an initial burst of Mek-dependent proliferation, leading to the formation of hyperplastic crypts. This is associated with β-catenin nuclear localization by a mechanism involving Mapk/Erk kinase (Mek)-dependent, Akt-independent phosphorylation of Gsk3β. However, hyperplastic crypts remain dormant for prolonged periods due to the induction of crypt senescence accompanied by upregulation of senescence-associated β-galactosidase and p16(Ink4a). We show that tumour progression is associated with down-regulation of p16(Ink4a) through enhanced CpG methylation of exon 1 and knockout of Cdkn2a confirms this gene is a barrier to tumour progression. Our studies identify (V600E)BRAF as an early genetic driver mutation in serrated CRCs and indicate that, unlike APC-mutated cancers, this subtype arises by the bypassing of a (V600E)Braf driven oncogene-induced senescence programme.

The ERK1/2 MAPK pathway is a critical signaling system that mediates ligand-stimulated signals for the induction of cell proliferation, differentiation, and cell survival. Studies have shown that this pathway is constitutively active in several human malignancies and may be involved in the pathogenesis of these tumors. In the present study we examined the ERK1/2 pathway in cell lines derived from epithelial and granulosa cell tumors, two distinct forms of ovarian cancer. We show that ERK1 and ERK2 are constitutively active and that this activation results from both MAPK kinase-dependent and independent mechanisms and is correlated with elevated BRAF expression. MAPK phosphatase 1 (MKP-1) expression, which is involved in ERK1/2 deactivation, is down-regulated in the cancer cells, thus further contributing to ERK hyperactivity in these cells. Treatment of these cancer cell lines with the proteasome inhibitor ZLLF-CHO increased MKP-1 but not MKP-2 expression and decreased ERK1/2 phosphorylation. More importantly, silencing of ERK1/2 protein expression using RNA interference led to the complete suppression of tumor cell proliferation. These results provide evidence that the ERK pathway plays a major role in ovarian cancer pathogenesis and that down-regulation of this master signaling pathway is highly effective for the inhibition of ovarian tumor growth.

We show that imatinib, nilotinib, and dasatinib possess weak off-target activity against RAF and, therefore, drive paradoxical activation of BRAF and CRAF in a RAS-dependent manner. Critically, because RAS is activated by BCR-ABL, in drug-resistant chronic myeloid leukemia (CML) cells, RAS activity persists in the presence of these drugs, driving paradoxical activation of BRAF, CRAF, MEK, and ERK, and leading to an unexpected dependency on the pathway. Consequently, nilotinib synergizes with MEK inhibitors to kill drug-resistant CML cells and block tumor growth in mice. Thus, we show that imatinib, nilotinib, and dasatinib drive paradoxical RAF/MEK/ERK pathway activation and have uncovered a synthetic lethal interaction that can be used to kill drug-resistant CML cells in vitro and in vivo.

OBJECTIVE

The recent concept that oncogenes responsible for thyroid neoplastic transformation are able to elicit an inflammatory protumourigenic microenvironment raises interest in further studies on papillary thyroid cancer (PTC) associated with thyroid autoimmunity.

METHODS

The clinical and molecular features, and the expression of inflammation-related genes, were investigated in a large series of PTCs with and without associated thyroiditis (groups A, n = 128 and B, n = 215).

RESULTS

The two groups did not show significant differences in clinical and prognostic features, whereas they harboured a significantly different genetic background (P = 0.001), with RET/PTC1 being more represented in PTCs associated with autoimmunity, and BRAF(V600E) in patients with PTC alone. A RET/PTC rearrangement was also found in 41% of non-neoplastic thyroiditis tissues, contralateral to tumours harbouring either RET/PTC or BRAF mutations. The expression of genes encoding CCL20, CXCL8 and l-selectin was significantly higher in PTC specimens (either with RET/PTC, BRAF(V600E) or unknown genetic lesion) compared with normal thyroid samples. On the contrary, thyroiditis showed l-selectin expression levels even higher than PTCs, but CCL20 and CXCL8 levels comparable with normal tissues.

CONCLUSIONS

The present data extend the knowledge about the tight relationships among oncogenes, thyroiditis and thyroid cancer. A different genetic background among PTCs with and without associated autoimmunity has been firstly demonstrated. The strong association between RET/PTC1 and thyroiditis points to a critical role of this oncoprotein in the modulation of the autoimmune response. Moreover, preliminary expression studies, indicating enhanced expression of inflammatory molecules in PTCs, suggest a proinflammatory, nonautoimmune relationship between thyroiditis and thyroid cancer.

We used a novel method based on allele-specific quantitative polymerase chain reaction (Intplex) for the analysis of circulating cell.free DNA (ccfDNA) to compare total ccfDNA and KRAS- or BRAF-mutated ccfDNA concentrations in blood samples from mice xenografted with the human SW620 colorectal cancer (CRC) cell line and from patients with CRC. Intplex enables single-copy detection of variant alleles down to a sensitivity of ≥0.005 mutant to wild-type ratio. The proportion of mutant allele corresponding to the percentage of tumor-derived ccfDNA was elevated in xenografted mice with KRAS homozygous mutation and varied highly from 0.13% to 68.7% in samples from mutation-positive CRC patients (n = 38). Mutant ccfDNA alleles were quantified in the plasma of every patient at stages II/III and IV with a mean of 8.4% (median, 8.4%) and 21.8% (median, 12.4%), respectively. Twelve of 38 (31.6%) and 5 of 38 (13.2%) samples showed a mutation load higher than 25%and 50%, respectively. This suggests that an important part of ccfDNA may originate from tumor cells. In addition, we observed that tumor-derived (mutant) ccfDNA was more fragmented than ccfDNA from normal tissues. This observation suggests that the form of tumor-derived and normal ccfDNA could differ. Our approach revealed that allelic dilution is much less pronounced than previously stated, considerably facilitating the noninvasive molecular analysis of tumors.

Almost 50% of metastatic melanoma patients harbor a BRAF(V600) mutation and the introduction of BRAF inhibitors has improved their treatment options. BRAF inhibitors vemurafenib and dabrafenib achieved improved overall survival over chemotherapy and have been approved for the treatment of BRAF-mutated metastatic melanoma. However, most patients develop mechanisms of acquired resistance and about 15% of them do not achieve tumor regression at all, due to intrinsic resistance to therapy. Moreover, early adaptive responses limit the initial efficacy of BRAF inhibition, leading mostly to incomplete responses that may favor the selection of a sub-population of resistant clones and the acquisition of alterations that cause tumor regrowth and progressive disease. The purpose of this paper is to review the mechanisms of resistance to therapy with BRAF inhibitors and to discuss the strategies to overcome them based on pre-clinical and clinical evidences.

OBJECTIVE

During colorectal cancer pathogenesis, mutations and epigenetic events cause neoplastic behavior in epithelial cells by deregulating the Wnt, Ras-Raf-extracellular signal-regulated kinase (ERK), and transforming growth factor (TGF)-beta-signaling pathways, among others. The TGF-beta-signaling pathway is often inactivated in colon cancer cells by mutations in the gene encoding the TGF-beta receptor TGFBR2. The RAS-RAF-ERK pathway is frequently up-regulated in colon cancer via mutational activation of KRAS or BRAF. We assessed how these pathways interact in vivo and affect formation of colorectal tumors.

METHODS

We analyzed intestinal tumors that arose in mice that express an oncogenic (active) form of Kras and that have Tgfbr2 inactivations-2 common molecular events observed in human colorectal tumors. LSL-KrasG12D mice were crossed with Villin-Cre;Tgfbr2E2flx/E2flx mice, which do not express Tgfbr2 in the intestinal epithelium.

RESULTS

Neither inactivation of Tgfbr2 nor expression of oncogenic Kras alone was sufficient to induce formation of intestinal neoplasms. Histologic abnormalities arose in mice that expressed Kras, but only the combination of Tgfbr2 inactivation and Kras activation led to intestinal neoplasms and metastases. The cancers arose via a beta-catenin-independent mechanism; the epidermal growth factor-signaling pathway was also activated. Cells in the resulting tumors proliferated at higher rates, expressed decreased levels of p15, and expressed increased levels of cyclin D1 and cdk4, compared with control cells.

CONCLUSIONS

A combination of inactivation of the TGF-beta-signaling pathway and expression of oncogenic Kras leads to formation of invasive intestinal neoplasms through a beta-catenin-independent pathway; these adenocarcinomas have the capacity to metastasize.

Cetuximab (Erbitux®) targets the epidermal growth factor receptor (EGFR) and is approved for treatment of colorectal and head and neck cancer. Despite wide expression of EGFR, only a subgroup of cancer patients responds to cetuximab therapy. In the present study we assessed the cetuximab response in vivo of 79 human patient-derived xenografts originating from five tumour histotypes. We analysed basic tumour characteristics including EGFR expression and activation, mutational status of KRAS, BRAF and NRAS, the expression of EGFR ligands and the activation of HER3 (ErbB3) and the hepatocyte growth factor receptor MET. Based on these results, a cetuximab response score including positive and negative factors affecting therapeutic response is proposed. Positive factors are high expression and activation of EGFR and its ligands epiregulin or amphiregulin, negative factors are markers for downstream pathway activation independent of EGFR. In cetuximab resistant NSCL adenocarcinoma LXFA 526 and LXFA 1647, overexpression due to gene amplification and strong activation of MET was identified. Knock-down of MET by siRNA in the corresponding cell lines showed that anchorage-independent growth and migration are dependent on MET. MET knock down sensitized LXFA 526L and LXFA 1647L to EGF. Combined treatments of a MET inhibitor and cetuximab were additive. Therefore, combination therapy of cetuximab and a MET inhibitor in selected lung cancer patients could be of high clinical significance.

BRAF rearrangements and BRAF V600E point mutations are recurring events in pediatric low-grade gliomas. However, their clinical significance, including possible interactions between these markers and other glioma biomarkers, is unclear. In this study a retrospective cohort of 198 pediatric low-grade gliomas (including 40 treated with adjuvant therapy) was analyzed for BRAF rearrangements, BRAF V600E, p16/CDKN2A deletion, p53 expression, and MIB1 proliferation index. In tumors with BRAF rearrangement, homozygous p16 deletion correlated with shorter progression-free survival (P = .04). A high MIB1 proliferation index trended toward worse response to adjuvant radiotherapy compared to BRAF-rearranged, p16-intact tumors (P = .08). On multivariate analysis, the 2 most consistently powerful independent adverse prognostic markers were midline location (P = .0001) and p16 deletion (P = .03). Tumors with BRAF V600E had a strong trend toward an increased risk for progression (hazard ratio = 2.48, P = .07), whereas those with BRAF rearrangement had a milder trend toward reduced risk (hazard ratio = .54, P = .15). These data suggest that p16 deletion adversely impacts the outcomes of BRAF-driven gliomas, that high proliferation index may be a better marker of progression risk than BRAF, that BRAF rearrangement and BRAF V600E might not necessarily produce comparable outcomes, and that none of these markers is stronger than tumor location in determining prognosis in pediatric low-grade gliomas.

The anti-epidermal growth factor receptor (anti-EGFR) monoclonal antibodies cetuximab and panitumumab have been demonstrated to be new therapeutic options for metastatic colorectal cancer (mCRC). Oncogenic activation of intracellular signalling pathways downstream of EGFR has a major role in colorectal carcinogenesis but has also been reported to be an important mechanism of resistance to anti-EGFR antibodies. Among the activating mutations found in colorectal cancers, tumour KRAS mutations, which are found in approximately 40% of the cases, have been widely demonstrated as a major predictive marker of resistance to cetuximab or panitumumab, therefore, opening the way to individualized treatment for patients with mCRC. Other oncogenic mutations, such as BRAF or PIK3CA mutations or loss of PTEN expression, may also be additional interesting predictive markers of response to anti-EGFR monoclonal antibodies but required further evaluation before being incorporated in clinical practice. The identification of these molecular markers involved in the resistance of anti-EGFR antibodies will allow the development of new therapies that should target 'escape mechanisms' used by tumours to circumvent a pathway that has been pharmacologically blocked by anti-EGFR.