Sarcoma Viral Oncogene Homolog B1 Research Articles

EGFR,KRAS,BRAFandALKgene alterations in lung adenocarcinomas: patient outcome, interplay with morphology and immunophenotype

Numerous studies have been published on single aspects of pulmonary adenocarcinoma (ADC). To comprehensively link clinically relevant ADC characteristics, we evaluated established morphological, diagnostic and predictive biomarkers in 425 resected ADCs. Morphology was reclassified. Cytokeratin-7, thyroid transcription factor (TTF)1, napsin A, thymidylate synthase and excision repair cross-complementing rodent repair deficiency complementation group-1 expression, anaplastic lymphoma kinase rearrangements as well as epidermal growth factor receptor (EGFR), V-Ki-ras2 Kirsten rat sarcoma viral oncogene homologue (KRAS) and v-Raf murine sarcoma viral oncogene homologue B1 (BRAF) mutations were analysed. All characteristics were correlated with clinical and survival parameters. Morphological ADC subtypes were significantly associated with smoking history and distinct patterns of diagnostic biomarkers. KRAS mutations were prevalent in male smokers, while EGFR mutations were associated with female sex, nonsmoking and lepidic as well as micropapillary growth patterns. TTF1 expression (hazard ratio (HR) for overall survival 0.61, p=0.021) and BRAF mutations (HR for disease-free survival 2.0, p=0.046) were found to be morphology- and stage-independent predictors of survival in multivariate analysis. Adjuvant radio-/chemotherapy, in some instances, strongly impacted on the prognostic effect of both diagnostic and predictive biomarkers. Our data draw a comprehensive picture of the prevalence and interplay of established histological and molecular ADC characteristics. These data will help to develop time- and cost-effective diagnostic and treatment algorithms for ADC.

A transient myelodysplastic/myeloproliferative neoplasm in a patient with cardio‐facio‐cutaneous syndrome and a germline BRAF mutation

A male infant, born at 32 weeks gestation by cesarean because of hydrops fetalis, presented with multiple anomalies, such as sparse and curly scalp hair, absent eyebrows, frontal bossing, an atrial septal defect, pulmonary artery stenosis, and whole myocardial thickening. He was clinically diagnosed with cardio-facio-cutaneous (CFC) syndrome, and was confirmed to have a germline V-raf murine sarcoma viral oncogene homologue B1 (BRAF) c.721 A>C mutation. At 1 month of age, he presented with a transient myelodysplastic/myeloproliferative neoplasm (MDS/MPN), which improved within a month without the administration of antineoplastic agents. This is the first report of CFC syndrome with MDS/MPN. The coexistence of MDS/MPN may be related to this BRAF c.721 A>C mutation.

A prospective study of duration of smoking cessation and colorectal cancer risk by epigenetics-related tumor classification.

The effect of duration of cigarette smoking cessation on colorectal cancer risk by molecular subtypes remains unclear. Using duplication-method Cox proportional-hazards regression analyses, we examined associations between duration of smoking cessation and colorectal cancer risk according to status of CpG island methylator phenotype (CIMP), microsatellite instability, v-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutation, or DNA methyltransferase-3B (DNMT3B) expression. Follow-up of 134,204 individuals in 2 US nationwide prospective cohorts (Nurses' Health Study (1980-2008) and Health Professionals Follow-up Study (1986-2008)) resulted in 1,260 incident rectal and colon cancers with available molecular data. Compared with current smoking, 10-19, 20-39, and ≥40 years of smoking cessation were associated with a lower risk of CIMP-high colorectal cancer, with multivariate hazard ratios (95% confidence intervals) of 0.53 (0.29, 0.95), 0.52 (0.32, 0.85), and 0.50 (0.27, 0.94), respectively (Ptrend = 0.001), but not with the risk of CIMP-low/CIMP-negative cancer (Ptrend = 0.25) (Pheterogeneity = 0.02, between CIMP-high and CIMP-low/CIMP-negative cancer risks). Differential associations between smoking cessation and cancer risks by microsatellite instability (Pheterogeneity = 0.02), DNMT3B expression (Pheterogeneity = 0.03), and BRAF (Pheterogeneity = 0.10) status appeared to be driven by the associations of CIMP-high cancer with microsatellite instability-high, DNMT3B-positive, and BRAF-mutated cancers. These molecular pathological epidemiology data suggest a protective effect of smoking cessation on a DNA methylation-related carcinogenesis pathway leading to CIMP-high colorectal cancer.

Influence of BRAF interference on SW579 cell lines in thyroid cancer

To determine the influence of v-raf murine sarcoma viral oncogene homolog B1 (BRAF) interference on SW579 cell lines in thyroid cancer. We designed 2 pairs of siRNA interference sequences, transfected them into SW579 cell line with liposome, and detected the interference with RT-PCR method. For successfully interfered cell lines, the changes in cell proliferation, cell cycle, and the expression of related proteins in mitogen-activated protein signal-regulated kinase /extracellular signal-regulated kinase (MEK/ ERK) signal pathway were detected. After 2 pairs of siRNA transfection, the expressions of BRAF mRNA and protein of SW579 cell lines were significantly inhibited (P<0.01). The proliferation was inhibited, the cell cycle was changed, G1/S phase increased, and MEK/ERK signal pathway was inhibited. Inhibition of growth and proliferation of SW579 cell lines by BRAF may be functioned by de-activating MEK/ERK signal pathway.

Cotargeting histone deacetylases and oncogenic BRAF synergistically kills human melanoma cells by necrosis independently of RIPK1 and RIPK3

Past studies have shown that histone deacetylase (HDAC) and mutant BRAF (v-Raf murine sarcoma viral oncogene homolog B1) inhibitors synergistically kill melanoma cells with activating mutations in BRAF. However, the mechanism(s) involved remains less understood. Here, we report that combinations of HDAC and BRAF inhibitors kill BRAFV600E melanoma cells by induction of necrosis. Cotreatment with the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) or panobinostat (LBH589) and the BRAF inhibitor PLX4720 activated the caspase cascade, but caspases appeared dispensable for killing, in that inhibition of caspases did not invariably block induction of cell death. The majority of dying cells acquired propidium iodide positivity instantly when they became positive for Annexin V, suggesting induction of necrosis. This was supported by caspase-independent release of high-mobility group protein B1, and further consolidated by rupture of the plasma membrane and loss of nuclear and cytoplasmic contents, as manifested by transmission electron microscopic analysis. Of note, neither the necrosis inhibitor necrostatin-1 nor the small interference RNA (siRNA) knockdown of receptor-interacting protein kinase 3 (RIPK3) inhibited cell death, suggesting that RIPK1 and RIPK3 do not contribute to induction of necrosis by combinations of HDAC and BRAF inhibitors in BRAFV600E melanoma cells. Significantly, SAHA and the clinically available BRAF inhibitor vemurafenib cooperatively inhibited BRAFV600E melanoma xenograft growth in a mouse model even when caspase-3 was inhibited. Taken together, these results indicate that cotreatment with HDAC and BRAF inhibitors can bypass canonical cell death pathways to kill melanoma cells, which may be of therapeutic advantage in the treatment of melanoma.

Epidermal growth factor receptor overexpression is a marker for adverse pathologic features in papillary thyroid carcinoma

BackgroundEpidermal growth factor receptor (EGFR) overexpression (EGFR-H) is implicated in thyroid carcinoma disease progression; however, the clinicopathologic significance of EGFR-H in tumors that harbor EGFR and/or v-Raf murine sarcoma viral oncogene homolog B1 (BRAF)(V600E) mutations is unknown. MethodsTissue microarrays from 81 patients who had undergone thyroidectomy for carcinoma from 2002–2011 were scored for EGFR expression using immunohistochemistry. Somatic mutations in EGFR exons 19 and 21 and BRAF were analyzed. Correlations between the EGFR immunohistochemistry, EGFR, and BRAF(V600E) mutations and the clinicopathologic features were assessed. ResultsEGFR-H was detected in 39.5% of carcinomas (n = 32) from patients with papillary (PTC, 46.2%, n = 18), follicular (29.6%, n = 8), and anaplastic (100.0%, n = 6) but not medullary (0.0%, n = 9) thyroid carcinoma. BRAF(V600E) mutations were identified in 22.2% of the carcinoma cases (n = 18, 15 PTCs and 3 anaplastic thyroid carcinomas). No somatic EGFR mutations were detected in any subtype. On PTC univariate analysis, EGFR-H correlated with increasing stage, extrathyroid extension, tumor capsule invasion, adverse pathologic features (any demonstration of extrathyroid extension, tumor capsule invasion, lymphovascular invasion, lymph node metastasis, and/or distant metastasis), and BRAF(V600E) mutations. On multivariate analysis, EGFR-H correlated with BRAF(V600E) mutations. In BRAF wild-type PTCs, the correlation between EGFR-H and adverse pathologic features approached statistical significance (P = 0.065). ConclusionsEGFR-H could be an important biomarker for aggressive PTCs, particularly in BRAF wild-type PTCs. Despite EGFR-H in PTC, follicular thyroid carcinoma, and anaplastic thyroid carcinoma by immunohistochemistry, somatic EGFR mutations were absent. Therefore, future investigations of EGFR should consider histologic and immunohistochemical methods, in addition to molecular profiling of thyroid carcinomas. This multimodal approach is particularly important for future clinical trials testing anti-EGFR therapy.

Combination Molecularly Targeted Drug Therapy in Metastatic Melanoma: Progress to Date

Previously characterized by a median overall survival of between 6 and 12months, metastatic melanoma now has a number of novel and effective treatment options. The ability to target the mitogen-activated protein kinase (MAPK) pathway with BRAF (v-raf murine sarcoma viral oncogene homolog B1) or MEK (mitogen-activated protein kinase kinase) inhibitors can result in rapid clinical benefit, but is too often associated with limited durability of response. Resistance inevitably develops either via reactivation of the MAPK pathway or via bypass signalling pathways, such as the PI3K (phosphoinositide 3-kinase) pathway. Combination strategies are thus appealing with an aim to overcome potential resistance mechanisms. Already, the combination of the BRAF inhibitor, dabrafenib, along with the MEK inhibitor, trametinib, has shown promising results clinically and with an improved toxicity profile. Other combination strategies with agents that target the PI3K pathway, angiogenesis, and the immune system are in development or already underway, although potential overlapping toxicities require close monitoring. The currently available molecularly targeted agents that target the MAPK pathway and development of combination therapies for treatment of metastatic melanoma are discussed in further detail.

An Update on BRAF Inhibitors and Other New Molecular Targets for the Treatment of Malignant Melanoma of the Skin

Malignant melanoma of the skin originates from mutations in melanocytes and can be lethal if unrecognized or untreated in its earlier stages. Deaths from melanoma are increasing in the United States and around the world every year. The available treatments produce low rates of response with modest survival impact. Among potential molecular targets under investigation, which are mostly in the tyrosine kinase pathway, the BRAF (V-raf murine sarcoma viral oncogene homolog B1) gene is the best studied and most frequently reported mutation in melanoma. The molecular targets for melanoma treatment, promising drugs for future melanoma treatment as well as the new molecular entities that are approved are reviewed here. Approved by FDA in 2011, vemurafenib (Zelboraf) is the first per - sonalized targeted therapy for treatment of metastatic melanoma that acts by selectively inhibiting BRAF V600E. This has opened a new avenue for the discovery of targeted drug therapies for melanoma based on the principles of pharmacogenomics.

The Ludwig Institute for Cancer Research Melbourne Melanoma Cell Line Panel

We established a range of melanoma cell lines from patient material (Table 1) termed Ludwig-Melbourne-Melanoma (LM-MEL-) followed by a unique number (method: Anaka et al., 2012). These lines have been human leucocyte antigen (HLA)-typed, their mutational status for common mutated genes in melanoma determined using the Sequenom MelCarta panel, and their transcriptomes profiled. All lines are tested for mycoplasma, and ethical approval for research purposes has been granted by the Austin Health Human Research Ethics Committee (HREC). Therapeutic options for advanced stage melanoma are limited, and despite the recent success with inhibitors of mutant v-raf murine sarcoma viral oncogene homolog B1 (BRAF) activity, long-lasting responses remain rare (Chapman et al., 2011). Immunotherapy may help overcome treatment failure, and there has been some success in the clinic with immunotherapy agents (Hodi et al., 2010). Knowledge of the antigen presenting major histocompatibility complex (MHC) class I HLA-types and antigenic proteins expressed by model systems is crucial for the preclinical testing of immunological interventions such as therapeutic cancer vaccines. Cancer-testis antigens (CTAg) and differentiation-antigens [mainly regulated by the microphtalmia-associated transcription factor (MITF)], represent two of the most studied families of potential cancer vaccine targets in melanoma (Caballero and Chen, 2009); and show various degrees of tissue-restriction and immunogenicity. The expression levels of some of these ‘immune-targetable’ genes premelanosome protein (PMEL), melan-A(MLANA), tyrosinase (TYR) and members of the melanoma antigen family (MAGE) in the LM-MEL cell lines are shown as an example in Figure 1. The complete gene-expression data and available additional clinical data of the here presented cell line panel are accessible online at ArrayExpress (http://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-1496/) and in Table S1. All cell lines are available upon request for a small handling fee or on a collaborative basis and slides for IHC from matched patient tumors and matched PBMCs/sera are accessible for some lines in limited quantities. AB is supported by a fellowship from the Cure Cancer Australia Foundation. JC and AB are supported by a grant from the Melanoma Research Alliance (MRA) and JC is supported by a practitioner fellowship from the Nation Health and Medical Research Council (NHMRC). This research was supported in part by the Cancer Council Victoria (CCV), the Austin Medical Research Foundation (AHMRF) and Operational Infrastructure Support Program Funding of the Victorian State Government. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Gossypin as a Novel Selective Dual Inhibitor of v-raf Murine Sarcoma Viral Oncogene Homolog B1 and Cyclin-Dependent Kinase 4 for Melanoma

Mutation in the BRAF gene (BRAFV600E) exists in nearly 70% of human melanomas. Targeted therapy against BRAFV600E kinase using a recently identified RAF-selective inhibitor, PLX4032, has been successful in early clinical trials. However, in patients with the normal BRAF allele (wild-type), PLX4032 is protumorigenic. This conundrum identifies the unmet need for novel therapeutic agents to target BRAFV600E kinase that are not counterproductive. We have identified gossypin, a pentahydroxy flavone, as a potent antimelanoma agent. Gossypin inhibited human melanoma cell proliferation, in vitro, in melanoma cell lines that harbor both BRAFV600E kinase and cyclin-dependent kinase 4 (CDK4) as well as in cells with BRAF wild-type allele. Gossypin inhibited kinase activities of BRAFV600E and CDK4, in vitro, possibly through direct binding of gossypin with these kinases, as confirmed by molecular docking studies. For cells harboring the BRAFV600E, gossypin inhibited cell proliferation through abrogation of the MEK-ERK-cyclin D1 pathway and in cells with BRAF wild-type allele, through attenuation of the retinoblastoma-cyclin D1 pathway. Furthermore, gossypin significantly inhibited melanoma growth in an organotypic three-dimensional skin culture mimicking human skin. Gossypin (10 and 100 mg/kg) treatment for 10 days in human melanoma (A375) cell xenograft tumors harboring BRAFV600E significantly reduced tumor volume through induction of apoptosis and increased survival rate in mice, and the effect was significantly superior to that of PLX4032 (10 mg/kg) or roscovitine 10 mg/kg. In summary, this study identified gossypin as a novel agent with dual inhibitory effects for BRAFV600E kinase and CDK4 for treatment of melanoma.

Predictive molecular pathology and its role in targeted cancer therapy: a review focussing on clinical relevance

The increasing importance of targeting drugs in the treatment of several tumor entities (breast, colon, lung, malignant melanoma (MM), lymphoma, and so on) and the necessity of a companion diagnostic (human epidermal growth factor receptor 2, Kirsten rat sarcoma viral oncogene, epidermal growth factor receptor (EGFR), v-raf murine sarcoma viral oncogene homolog B1 (BRAF), and so on) is leading to new challenges for surgical pathology. As all the biomarkers to be specifically detected are tissue based, a precise and reliable diagnostic is absolutely crucial. To meet this challenge, surgical pathology has adapted a number of molecular methods (semi-quantitative immunohistochemistry, fluorescence in situ hybridization), PCR and its multiple variants, (pyro/Sanger) sequencing, next-generation sequencing, DNA-arrays, methylation analyses, and so on) to be applicable for formalin-fixed paraffin-embedded (FFPE) tissue. To read a patients' tissue as 'deeply' as possible and to obtain information on morphological, genetic, proteomic as well as epigenetic background is the actual task of pathologists and molecular biologists in order to provide the clinicians with information relevant for individualized medicine. The intensified cooperation of clinicians and pathologists will provide the basis of improved clinical drug selection as well as guide development of new cancer gene therapies and molecularly targeted drugs by research units and the pharmaceutical industry. This review will give some information on (1) biomarker detection methods adapted to FFPE tissue, (2) the potency of predictive pathology in tumor detection and treatment and (3) the implications of pathology on the development of new drugs in molecularly targeted and gene therapies.

Frequencies and prognostic impact of RAS mutations in MLL-rearranged acute lymphoblastic leukemia in infants

Acute lymphoblastic leukemia in infants represents an aggressive malignancy associated with a high incidence (approx. 80%) of translocations involving the Mixed Lineage Leukemia (MLL) gene. Attempts to mimic Mixed Lineage Leukemia fusion driven leukemogenesis in mice raised the question whether these fusion proteins require secondary hits. RAS mutations are suggested as candidates. Earlier results on the incidence of RAS mutations in Mixed Lineage Leukemia-rearranged acute lymphoblastic leukemia are inconclusive. Therefore, we studied frequencies and relation with clinical parameters of RAS mutations in a large cohort of infant acute lymphoblastic leukemia patients. Using conventional sequencing analysis, we screened neuroblastoma RAS viral (v-ras) oncogene homolog gene (NRAS), v-Ki-ras Kirsten rat sarcoma viral oncogene homolog gene (KRAS), and v-raf murine sarcoma viral oncogene homolog B1 gene (BRAF) for mutations in a large cohort (n=109) of infant acute lymphoblastic leukemia patients and studied the mutations in relation to several clinical parameters, and in relation to Homeobox gene A9 expression and the presence of ALL1 fused gene 4-Mixed Lineage Leukemia (AF4-MLL). Mutations were detected in approximately 14% of all cases, with a higher frequency of approximately 24% in t(4;11)-positive patients (P=0.04). Furthermore, we identified RAS mutations as an independent predictor (P=0.019) for poor outcome in Mixed Lineage Leukemia-rearranged infant acute lymphoblastic leukemia, with a hazard ratio of 3.194 (95% confidence interval (CI):1.211-8.429). Also, RAS-mutated infants have higher white blood cell counts at diagnosis (P=0.013), and are more resistant to glucocorticoids in vitro (P<0.05). Finally, we demonstrate that RAS mutations, and not the lack of Homeobox gene A9 expression nor the expression of AF4-MLL are associated with poor outcome in t(4;11)-rearranged infants. We conclude that the presence of RAS mutations in Mixed Lineage Leukemia-rearranged infant acute lymphoblastic leukemia is an independent predictor for a poor outcome. Therefore, future risk-stratification based on abnormal RAS-pathway activation and RAS-pathway inhibition could be beneficial in RAS-mutated infant acute lymphoblastic leukemia patients.

Clinical implications of BRAF mutation test in colorectal cancer.

Knowledge about the clinical significance of V-Raf Murine Sarcoma Viral Oncogene Homolog B1 (BRAF) mutations in colorectal cancer (CRC) is growing. BRAF encodes a protein kinase involved with intracellular signaling and cell division. The gene product is a downstream effector of Kirsten Ras 1(KRAS) within the RAS/RAF/MAPK cellular signaling pathway. Evidence suggests that BRAF mutations, like KRAS mutations, result in uncontrolled, non-growth factor-dependent cellular proliferation. Similar to the rationale that KRAS mutation precludes effective treatment with anti-EGFR drugs. Recently, BRAF mutation testing has been introduced into routine clinical laboratories because its significance has become clearer in terms of effect on pathogenesis of CRC, utility in differentiating sporadic CRC from Lynch syndrome (LS), prognosis, and potential for predicting patient outcome in response to targeted drug therapy. In this review we describe the impact of BRAF mutations for these aspects.

Efficient detection of the V600E mutation of the BRAF gene in papillary thyroid carcinoma using multiplex allele-specific polymerase chain reaction combined with denaturing high-performance liquid chromatography

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.

Applications of nanotechnology for melanoma treatment, diagnosis, and theranostics

Melanoma is the most aggressive type of skin cancer and has very high rates of mortality. An early stage melanoma can be surgically removed, with a survival rate of 99%. However, metastasized melanoma is difficult to cure. The 5-year survival rates for patients with metastasized melanoma are still below 20%. Metastasized melanoma is currently treated by chemotherapy, targeted therapy, immunotherapy and radiotherapy. The outcome of most of the current therapies is far from optimistic. Although melanoma patients with a mutation in the oncogene v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) have an initially higher positive response rate to targeted therapy, the majority develop acquired drug resistance after 6 months of the therapy. To increase treatment efficacy, early diagnosis, more potent pharmacological agents, and more effective delivery systems are urgently needed. Nanotechnology has been extensively studied for melanoma treatment and diagnosis, to decrease drug resistance, increase therapeutic efficacy, and reduce side effects. In this review, we summarize the recent progress on the development of various nanoparticles for melanoma treatment and diagnosis. Several common nanoparticles, including liposome, polymersomes, dendrimers, carbon-based nanoparticles, and human albumin, have been used to deliver chemotherapeutic agents, and small interfering ribonucleic acids (siRNAs) against signaling molecules have also been tested for the treatment of melanoma. Indeed, several nanoparticle-delivered drugs have been approved by the US Food and Drug Administration and are currently in clinical trials. The application of nanoparticles could produce side effects, which will need to be reduced so that nanoparticle-delivered drugs can be safely applied in the clinical setting.

Heterogeneity of Epidermal Growth Factor Receptor Status and Mutations of KRAS/PIK3CA in Circulating Tumor Cells of Patients with Colorectal Cancer

Molecular characterization of circulating tumor cells (CTCs) is pivotal to increasing the diagnostic specificity of CTC assays and investigating therapeutic targets and their downstream pathways on CTCs. We focused on epidermal growth factor receptor (EGFR) and genes relevant for its inhibition in patients with colorectal cancer (CRC). We used the CellSearch® system for CTC detection in peripheral blood samples from 49 patients with metastatic CRC (mCRC) and 32 patients with nonmetastatic CRC (nmCRC). We assessed EGFR expression in 741 CTCs from 27 patients with mCRC and 6 patients with nmCRC using a fluorescein-conjugated antibody with the CellSearch Epithelial Cell Kit. DNA of a single CTC isolated by micromanipulation was propagated by whole-genome amplification and analyzed by quantitative PCR for EGFR gene amplification and sequencing for KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog), BRAF (v-raf murine sarcoma viral oncogene homolog B1), and PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit α) mutations. At least 2 CTCs were detected in 24 of 49 patients with mCRC and 7 of 32 patients with nmCRC. In 7 of 33 patients, CTCs with increased EGFR expression were identified. Heterogeneity in EGFR expression was observed between CTCs from the same patient. EGFR gene amplification was found in 7 of 26 CTCs from 3 patients. The investigated BRAF gene locus was not mutated in 44 analyzed CTCs, whereas KRAS mutations were detected in 5 of 15 CTCs from 1 patient and PIK3CA mutations in 14 of 36 CTCs from 4 patients. Molecular characterization of single CTCs demonstrated considerable intra- and interpatient heterogeneity of EGFR expression and genetic alterations in EGFR, KRAS, and PIK3CA, possibly explaining the variable response rates to EGFR inhibition in patients with CRC.

Clinical development of phosphatidylinositol 3-kinase inhibitors for cancer treatment.

The phosphatidylinositol 3-kinase (PI3K) pathway is commonly deregulated in cancer. In recent years, the results of the first phase I clinical trials with PI3K inhibitors have become available. In comparison to other targeted agents such v-raf murine sarcoma viral oncogene homolog B1 (BRAF) inhibitors in melanoma or crizotinib in anaplastic lymphoma receptor tyrosine kinase (ALK) translocated tumors, the number of objective responses to PI3K inhibitors is less dramatic. In this review we propose possible strategies to optimize the clinical development of PI3K inhibitors: by exploring the potential role of PI3K isoform-specific inhibitors in improving the therapeutic index, molecular characterization as a basis for patient selection, and the relevance of performing serial tumor biopsies to understand the associated mechanisms of drug resistance. The main focus of this review will be on PI3K isoform-specific inhibitors by describing the functions of different PI3K isoforms, the preclinical activity of selective PI3K isoform-specific inhibitors and the early clinical data of these compounds.

Successful rechallenge in two patients with BRAF-V600-mutant melanoma who experienced previous progression during treatment with a selective BRAF inhibitor

The v-raf murine sarcoma viral oncogene homolog B1 (BRAF) gene is mutated at position 600 in about 50% of melanoma. Mutant BRAF activates the downstream effectors of the RAS-RAF-MEK-MAPK pathways and is a driver oncogene in these melanoma cells. Selective BRAF-V600 inhibitors (vemurafenib, dabrafenib) have high antitumor activity against BRAF-V600-mutant melanoma with objective tumor response rates. Resistance, however, develops within less than a year in the majority of patients. Several different mechanisms have been found to mediate acquired resistance, but these do not involve the occurrence of secondary mutations in the BRAF gene. Two patients with BRAF-V600E mutant melanoma who had documented progression during treatment with dabrafenib/GSK1120212 and dabrafenib, respectively, were rechallenged with dabrafenib and vemurafenib after a treatment-free interval of 8 and 4 months during which further progression was documented. Both patients showed a marked clinical response and, in both, objective tumor regression (qualifying as a mixed and a partial response according to RECIST) was documented. These two case observations indicate that resistance to BRAF-selective inhibitors can be reversible following treatment interruption.

Molecular predictors of outcome in low-grade glioma

Recent and ongoing translational studies in neurooncology have investigated the role of molecular markers as potential predictors of outcome in patients with WHO grade I and II gliomas, commonly summarized as low-grade gliomas (LGGs). Here, we seek to highlight the most relevant molecular aberrations associated with these tumour types and update on recent findings on their potential prognostic and predictive value. So far, no biomarker discussed has any relevance for the postoperative course of disease without genotoxic treatment. Isocitrate dehydrogenase (IDH) mutations, 1p deletion or 1p/19q codeletion have the strongest prognostic impact on survival of patients with LGG, given a genotoxic treatment is provided. Recent findings from phase III clinical trials on anaplastic oligodendroglial tumours conducted in North America and Europe suggest that the addition of procarbazine, lomustine and vincristine to radiotherapy is beneficial in the treatment of anaplastic gliomas with 1p/19q codeletion. To decipher the role of 1p/19q codeletion in LGG will be challenging. Recent developments in v-raf murine sarcoma viral oncogene homolog B1 (BRAF)(V600E)-specific small molecule inhibitors and their clinical approval for other cancer types could turn BRAF(V600E) into a promising molecular predictor of outcome in pilocytic astrocytomas, given a treatment with a mutation-specific BRAF inhibitor is applied. Clinical prognostic factors such as age, tumour size and the presence or absence of clinical symptoms have long been recognized in the management of patients with LGGs. Molecular biomarkers are increasingly evolving as additional factors that facilitate diagnostics and therapeutic decision-making. However, further prospective randomized studies including multivariate analyses are needed to clearly distinguish between prognostic and predictive effects.

Progress in the detection of Kras, EGFR, BRAF mutations and its clinical application

Since a number of genes have been discovered, which are associated with the effect of drug therapy, the development of personalized medicine is enhanced.Meanwhile, personalized test played an important role in choosing proper medicine.Herein, the status of detection of V-ki-ras2 Kirsten rat sarcoma viral oncogene homolog（Kras）, epidermal growth factor receptor（EGFR）, B-Raf and v-Raf murine sarcoma viral oncogene homolog B1（BRAF） mutation is concluded, moreover, the association between the mutation test and the effect of cancer therapy is summarized.(Chin J Lab Med, 2012,35:971-977) Key words: Proto-oncogene proteins; Ras proteins; Receptor; epidermal growth factor; Personalized medicine; Mutation