In most colorectal cancer (CRC) patients, outcome cannot be predicted because tumors with similar clinicopathological features can have differences in disease progression and treatment response. Therefore, a better understanding of the CRC biology is required to identify those patients who will benefit from chemotherapy and to find a more tailored therapy plan for other patients. Based on unsupervised classification of whole genome data from 188 stages I-IV CRC patients, a molecular classification was developed that consist of at least three major intrinsic subtypes (A-, B- and C-type). The subtypes were validated in 543 stages II and III patients and were associated with prognosis and benefit from chemotherapy. The heterogeneity of the intrinsic subtypes is largely based on three biological hallmarks of the tumor: epithelial-to-mesenchymal transition, deficiency in mismatch repair genes that result in high mutation frequency associated with microsatellite instability and cellular proliferation. A-type tumors, observed in 22% of the patients, have the best prognosis, have frequent BRAF mutations and a deficient DNA mismatch repair system. C-type patients (16%) have the worst outcome, a mesenchymal gene expression phenotype and show no benefit from adjuvant chemotherapy treatment. Both A-type and B-type tumors have a more proliferative and epithelial phenotype and B-types benefit from adjuvant chemotherapy. B-type tumors (62%) show a low overall mutation frequency consistent with the absence of DNA mismatch repair deficiency. Classification based on molecular subtypes made it possible to expand and improve CRC classification beyond standard molecular and immunohistochemical assessment and might help in the future to guide treatment in CRC patients.

OBJECTIVE

In this study, we assessed the specific role of BRAF(V600E) signaling in modulating the expression of immune regulatory genes in melanoma, in addition to analyzing downstream induction of immune suppression by primary human melanoma tumor-associated fibroblasts (TAF).

METHODS

Primary human melanocytes and melanoma cell lines were transduced to express WT or V600E forms of BRAF, followed by gene expression analysis. The BRAF(V600E) inhibitor vemurafenib was used to confirm targets in BRAF(V600E)-positive melanoma cell lines and in tumors from melanoma patients undergoing inhibitor treatment. TAF lines generated from melanoma patient biopsies were tested for their ability to inhibit the function of tumor antigen-specific T cells, before and following treatment with BRAF(V600E)-upregulated immune modulators. Transcriptional analysis of treated TAFs was conducted to identify potential mediators of T-cell suppression.

RESULTS

Expression of BRAF(V600E) induced transcription of interleukin 1 alpha (IL-1α) and IL-1β in melanocytes and melanoma cell lines. Further, vemurafenib reduced the expression of IL-1 protein in melanoma cell lines and most notably in human tumor biopsies from 11 of 12 melanoma patients undergoing inhibitor treatment. Treatment of melanoma-patient-derived TAFs with IL-1α/β significantly enhanced their ability to suppress the proliferation and function of melanoma-specific cytotoxic T cells, and this inhibition was partially attributable to upregulation by IL-1 of COX-2 and the PD-1 ligands PD-L1 and PD-L2 in TAFs.

CONCLUSIONS

This study reveals a novel mechanism of immune suppression sensitive to BRAF(V600E) inhibition, and indicates that clinical blockade of IL-1 may benefit patients with BRAF wild-type tumors and potentially synergize with immunotherapeutic interventions.

OBJECTIVE

Activating mutations in the tyrosine kinase domain of HER2 (ERBB2) have been described in a subset of lung adenocarcinomas (ADCs) and are mutually exclusive with EGFR and KRAS mutations. The prevalence, clinicopathologic characteristics, prognostic implications, and molecular heterogeneity of HER2-mutated lung ADCs are not well established in U.S. patients.

METHODS

Lung ADC samples (N = 1,478) were first screened for mutations in EGFR (exons 19 and 21) and KRAS (exon 2), and negative cases were then assessed for HER2 mutations (exons 19-20) using a sizing assay and mass spectrometry. Testing for additional recurrent point mutations in EGFR, KRAS, BRAF, NRAS, PIK3CA, MEK1, and AKT was conducted by mass spectrometry. ALK rearrangements and HER2 amplification were assessed by FISH.

RESULTS

We identified 25 cases with HER2 mutations, representing 6% of EGFR/KRAS/ALK-negative specimens. Small insertions in exon 20 accounted for 96% (24/25) of the cases. Compared with insertions in EGFR exon 20, there was less variability, with 83% (20/24) being a 12 bp insertion causing duplication of amino acids YVMA at codon 775. Morphologically, 92% (23/25) were moderately or poorly differentiated ADC. HER2 mutation was not associated with concurrent HER2 amplification in 11 cases tested for both. HER2 mutations were more frequent among never-smokers (P < 0.0001) but there were no associations with sex, race, or stage.

CONCLUSIONS

HER2 mutations identify a distinct subset of lung ADCs. Given the high prevalence of lung cancer worldwide and the availability of standard and investigational therapies targeting HER2, routine clinical genotyping of lung ADC should include HER2.

Immune checkpoint inhibitors have been successful across several tumor types; however, their efficacy has been uncommon and unpredictable in glioblastomas (GBM), where <10% of patients show long-term responses. To understand the molecular determinants of immunotherapeutic response in GBM, we longitudinally profiled 66 patients, including 17 long-term responders, during standard therapy and after treatment with PD-1 inhibitors (nivolumab or pembrolizumab). Genomic and transcriptomic analysis revealed a significant enrichment of PTEN mutations associated with immunosuppressive expression signatures in non-responders, and an enrichment of MAPK pathway alterations (PTPN11, BRAF) in responders. Responsive tumors were also associated with branched patterns of evolution from the elimination of neoepitopes as well as with differences in T cell clonal diversity and tumor microenvironment profiles. Our study shows that clinical response to anti-PD-1 immunotherapy in GBM is associated with specific molecular alterations, immune expression signatures, and immune infiltration that reflect the tumor's clonal evolution during treatment.

BACKGROUND

BRAF mutations act as an oncogenic driver via the mitogen-activated protein kinase (MAPK) pathway in non-small cell lung cancer (NSCLC). BRAF inhibition has shown antitumour activity in patients with BRAF(V600E)-mutant NSCLC. Dual MAPK pathway inhibition with BRAF and MEK inhibitors in BRAF(V600E)-mutant NSCLC might improve efficacy over BRAF inhibitor monotherapy based on observations in BRAF(V600)-mutant melanoma. We aimed to assess the antitumour activity and safety of dabrafenib plus trametinib in patients with BRAF(V600E)-mutant NSCLC.

METHODS

In this phase 2, multicentre, non-randomised, open-label study, we enrolled adult patients (aged ≥18 years) with pretreated metastatic stage IV BRAF(V600E)-mutant NSCLC who had documented tumour progression after at least one previous platinum-based chemotherapy and had had no more than three previous systemic anticancer therapies. Patients with previous BRAF or MEK inhibitor treatment were ineligible. Patients with brain metastases were allowed to enrol only if the lesions were asymptomatic, untreated (or stable more than 3 weeks after local therapy if treated), and measured less than 1 cm. Enrolled patients received oral dabrafenib (150 mg twice daily) plus oral trametinib (2 mg once daily) in continuous 21-day cycles until disease progression, unacceptable adverse events, withdrawal of consent, or death. The primary endpoint was investigator-assessed overall response, which was assessed by intention to treat in the protocol-defined population (patients who received second-line or later treatment); safety was also assessed in this population and was assessed at least once every 3 weeks, with adverse events, laboratory values, and vital signs graded according to the Common Terminology Criteria for Adverse Events version 4.0. The study is ongoing but no longer recruiting patients. This trial is registered with ClinicalTrials.gov, number NCT01336634.

RESULTS

Between Dec 20, 2013, and Jan 14, 2015, 59 patients from 30 centres in nine countries across North America, Europe, and Asia met eligibility criteria. Two patients who had previously been untreated due to protocol deviation were excluded; thus, 57 eligible patients were enrolled. 36 patients (63·2% [95% CI 49·3-75·6]) achieved an investigator-assessed overall response. Serious adverse events were reported in 32 (56%) of 57 patients and included pyrexia in nine (16%), anaemia in three (5%), confusional state in two (4%), decreased appetite in two (4%), haemoptysis in two (4%), hypercalcaemia in two (4%), nausea in two (4%), and cutaneous squamous cell carcinoma in two (4%). The most common grade 3-4 adverse events were neutropenia in five patients (9%), hyponatraemia in four (7%), and anaemia in three (5%). Four patients died during the study from fatal adverse events judged to be unrelated to treatment (one retroperitoneal haemorrhage, one subarachnoid haemorrhage, one respiratory distress, and one from disease progression that was more severe than typical progression, as assessed by the investigator).

CONCLUSIONS

Dabrafenib plus trametinib could represent a new targeted therapy with robust antitumour activity and a manageable safety profile in patients with BRAF(V600E)-mutant NSCLC.

BACKGROUND

GlaxoSmithKline.

Recent studies have shown that there is a considerable heterogeneity in the response of melanoma cell lines to MEK and BRAF inhibitors. In the current study, we address whether dysregulation of cyclin-dependent kinase 4 (CDK4) and/or cyclin D1 contribute to the BRAF inhibitor resistance of melanoma cells. Mutational screening identified a panel of melanoma cell lines that harbored both a BRAF V600E mutation and a CDK4 mutation: K22Q (1205Lu), R24C (WM39, WM46, and SK-Mel-28), and R24L (WM902B). Pharmacologic studies showed that the presence of a CDK4 mutation did not alter the sensitivity of these cell lines to the BRAF inhibitor. The only cell line with significant BRAF inhibitor resistance was found to harbor both a CDK4 mutation and a CCND1 amplification. Array comparative genomic hybridization analysis showed that CCND1 was amplified in 17% of BRAF V600E-mutated human metastatic melanoma samples, indicating the clinical relevance of this finding. As the levels of CCND1 amplification in cell lines are lower than those seen in clinical specimens, we overexpressed cyclin D1 alone and in the presence of CDK4 in a drug-sensitive melanoma line. Cyclin D1 overexpression alone increased resistance and this was enhanced when cyclin D1 and CDK4 were concurrently overexpressed. In conclusion, increased levels of cyclin D1, resulting from genomic amplification, may contribute to the BRAF inhibitor resistance of BRAF V600E-mutated melanomas, particularly when found in the context of a CDK4 mutation/overexpression.

The oncogene BRAF(V600E) is the most frequent genetic event in papillary thyroid carcinoma (PTC) but its prognostic impact still remains to be elucidated. We evaluated a representative series of 67 individuals with PTC who underwent total thyroidectomy. BRAF-positive tumours correlated with early recurrences (32% vs 7.6%; P=0.02) during a median postoperative follow-up period of 3 years. Interestingly, within the recurrences, a significant majority had negative radioiodine ((131)I) total body scans, predicting a poorer outcome as treatment with (131)I is not effective. This last observation led us to investigate the role of BRAF(V600E) and the MEK-ERK pathway in thyroid dedifferentiation, particularly in Na(+)/I(-) symporter (NIS) impairment, as this thyroid-specific plasma membrane glycoprotein mediates active transport of I(-) into the thyroid follicular cells. A subset of 60 PTC samples was evaluated for NIS immunoreactivity and, accordingly, we confirmed a significant low NIS expression and impaired targeting to membranes in BRAF-positive samples (3.5% vs 30%; P=0.005). Furthermore, experiments with differentiated PCCl3 thyroid cells demonstrated that transient expression of BRAF(V600E) sharply impaired both NIS expression and targeting to membrane and, surprisingly, this impairment was not totally dependent on the MEK-ERK pathway. We have concluded that BRAF(V600E) is a new prognostic factor in PTC that correlates with a high risk of recurrences and less differentiated tumours due to the loss of NIS-mediated (131)I uptake.

In the present study, DNA from 27 grade I and grade II pediatric gliomas, including ganglioglioma, desmoplastic infantile ganglioglioma, dysembryoplastic neuroepithelial tumor, and pleomorphic xanthoastrocytoma was analyzed using the Illumina 610K Beadchip SNP-based oligonucleotide array. Several consistent abnormalities, including gain of chromosome 7 and loss of 9p21 were observed. Based on our previous studies, in which we demonstrated BRAF mutations in 3 gangliogliomas, 31 tumors were screened for activating mutations in exons 11 and 15 of the BRAF oncogene or a KIAA1549-BRAF fusion product. There were no cases with a KIAA1549-BRAF fusion. A BRAF V600E mutation was detected in 14 of 31 tumors, which was not correlated with any consistent pattern of aberrations detected by the SNP array analysis. Tumors were also screened for mutations in codon 132 in exon 4 of IDH1, exons 2 and 3 of KRAS, and exons 2-9 of TP53. No mutations in KRAS or TP53 were identified in any of the samples, and there was only 1 IDH1 R132H mutation detected among the sample set. BRAF mutations constitute a major genetic alteration in this histologic group of pediatric brain tumors and may serve as a molecular target for biologically based inhibitors.

KRAS mutation is a hallmark of pancreatic ductal adenocarcinoma (PDA) but remains an intractable pharmacologic target. Consequently, defining RAS effector pathway(s) required for PDA initiation and maintenance is critical to improve treatment of this disease. Here, we show that expression of BRAF(V600E), but not PIK3CA(H1047R), in the mouse pancreas leads to pancreatic intraepithelial neoplasia (PanIN) lesions. Moreover, concomitant expression of BRAF(V600E) and TP53(R270H) result in lethal PDA. We tested pharmacologic inhibitors of RAS effectors against multiple human PDA cell lines. Mitogen-activated protein (MAP)/extracellular signal-regulated (ERK) kinase (MEK) inhibition was highly effective both in vivo and in vitro and was synergistic with AKT inhibition in most cell lines tested. We show that RAF→MEK→ERK signaling is central to the initiation and maintenance of PDA and to rational combination strategies in this disease. These results emphasize the value of leveraging multiple complementary experimental systems to prioritize pathways for effective intervention strategies in PDA.

CONCLUSIONS

PDA is diffi cult to treat, in large part, due to recurrent mutations in the KRAS gene. Here, we defi ne rational treatment approaches for the disease achievable today with existing drug combinations by thorough genetic and pharmacologic dissection of the major KRAS effector pathways, RAF→MEK→ERK and phosphoinositide 3′-kinase (PI3'K)→AKT.

BACKGROUND

TERT encodes the reverse transcriptase component of telomerase, which adds telomere repeats to chromosome ends, thus enabling cell replication. Telomerase activity is required for cell immortalization. Somatic TERT promoter mutations modifying key transcriptional response elements were recently reported in several cancers, such as melanomas and gliomas.

OBJECTIVE

The objectives of the study were: 1) to determine the prevalence of TERT promoter mutations C228T and C250T in different thyroid cancer histological types and cell lines; and 2) to establish the possible association of TERT mutations with mutations of BRAF, RAS, or RET/PTC.

METHODS

TERT promoter was PCR-amplified and sequenced in 42 thyroid cancer cell lines and 183 tumors: 80 papillary thyroid cancers (PTCs), 58 poorly differentiated thyroid cancers (PDTCs), 20 anaplastic thyroid cancers (ATCs), and 25 Hurthle cell cancers (HCCs).

RESULTS

TERT promoter mutations were found in 98 of 225 (44%) specimens. TERT promoters C228T and C250T were mutually exclusive. Mutations were present in 18 of 80 PTCs (22.5%), in 40 of 78 (51%) advanced thyroid cancers (ATC + PDTC) (P = 3 × 10(-4) vs PTC), and in widely invasive HCCs (4 of 17), but not in minimally invasive HCCs (0 of 8). TERT promoter mutations were seen more frequently in advanced cancers with BRAF/RAS mutations compared to those that were BRAF/RAS wild-type (ATC + PDTC, 67.3 vs 24.1%; P < 10(-4)), whereas BRAF-mutant PTCs were less likely to have TERT promoter mutations than BRAF wild-type tumors (11.8 vs 50.0%; P = .04).

CONCLUSIONS

TERT promoter mutations are highly prevalent in advanced thyroid cancers, particularly those harboring BRAF or RAS mutations, whereas PTCs with BRAF or RAS mutations are most often TERT promoter wild type. Acquisition of a TERT promoter mutation could extend survival of BRAF- or RAS-driven clones and enable accumulation of additional genetic defects leading to disease progression.

There has been much recent progress in our understanding of the role played by the RAS-RAF-MEK-ERK cascade in human cancer. RAS is an oncogene and this pathway is known to promote proliferation and malignant transformation. More recently, however, RAF has become the focus of attention, particularly in melanoma, where approximately 70% of cases carry mutations in the BRAF gene. The majority of the mutations in BRAF in cancer are activating, but rare mutants that cannot activate MEK have provided new insight into RAF signalling networks that exist in cancer and normal cells. Surprisingly, germline mutations in BRAF that occur in rare genetic syndromes have also recently been described. The induction of BRAF mutations in melanoma depends on the type of UV exposure that the skin receives, and some studies have suggested the existence of susceptibility loci that make it more likely that some individuals will acquire these mutations. Importantly, genetic profiling and microarray studies have provided insight into the spectrum of melanomas in which BRAF plays a role and also revealed intriguing new data that could be important for the diagnosis and treatment of human cancers.

BACKGROUND

Thyroid cancer incidence rates in the United States and globally have increased steadily over the last 40 years, primarily due to a tripling of the incidence of papillary thyroid carcinoma (PTC).

OBJECTIVE

The purpose of this study was to analyze trends in demographic, clinical, pathologic, and molecular characteristics of PTC from 1974 to 2009.

METHODS

We identified and histologically reviewed 469 consecutive cases of PTC from one US institution from 4 preselected periods (1974 to 1985, 1990 to 1992, 2000, and 2009) and assessed BRAF and RAS point mutations and RET/PTC rearrangements among 341 tumors ≥0.3 cm in size. Changes over time were analyzed using polytomous and binary logistic regression; all analyses were adjusted for age and sex.

RESULTS

During this period, the median age of patients at diagnosis increased from 37 to 53 years (P < .001) and the percentage of microcarcinomas (≤1.0 cm) increased from 33% to 51% (P < .001), whereas extrathyroidal extension and advanced tumor stage decreased from 40% to 21% (P = .005) and from 43% to 28% (P = .036), respectively. Changes in tumor histopathology showed a decrease in classic PTC and an increase in the follicular variant (P < .001). The proportion of tumors with a BRAF mutation was stable (∼46%) but increased from 50% to 77% (P = .008) within classic papillary PTCs. The proportion of tumors with RAS mutations increased from 3% to 25% and within follicular pattern tumors from 18% to 44% (P < .001). The proportion of RET/PTC rearrangements decreased from 11% to 2% (P = .038).

CONCLUSIONS

Similar to US national trends, we found an increasing age at diagnosis and greater detection of smaller-sized intrathyroidal PTCs. However, the overall proportion of BRAF mutations remained stable. Sharply rising percentages of the follicular variant histology and RAS mutations after 2000 suggest new and more recent etiologic factors. The increased incidence is not likely to be due to environmental or therapeutic radiation because the percentage of RET/PTC rearrangements decreased.

Malignant cutaneous melanoma is a highly aggressive form of skin cancer. Despite improvements in early melanoma diagnosis, the 5-year survival rate remains low in advanced disease. Therefore, novel biomarkers are urgently needed to devise new means of detection and treatment. In this study, we aimed to improve our understanding of microRNA (miRNA) deregulation in melanoma development and their impact on patient survival. Global miRNA expression profiles of a set of melanoma lymph node metastases, melanoma cell lines, and melanocyte cultures were determined using Agilent array. Deregulated miRNAs were evaluated in relation with clinical characteristics, patient survival, and mutational status for BRAF and NRAS. Several miRNAs were differentially expressed between melanocytes and melanomas as well as melanoma cell lines. In melanomas, miR-193a, miR-338, and miR-565 were underexpressed in cases with a BRAF mutation. Furthermore, low expression of miR-191 and high expression of miR-193b were associated with poor melanoma-specific survival. In conclusion, our findings show miRNA dysregulation in malignant melanoma and its relation to established molecular backgrounds of BRAF and NRAS oncogenic mutations. The identification of an miRNA classifier for poor survival may lead to the development of miRNA detection as a complementary prognostic tool in clinical practice.

The identification of somatically acquired tumor mutations is increasingly important in the clinical management of cancer because the sensitivity of targeted drugs is related to the genetic makeup of individual tumors. Thus, mutational profiles of tumors can help prioritize anticancer therapy. We report herein the development and validation of two multiplexed assays designed to detect in DNA from FFPE tissue more than 40 recurrent mutations in nine genes relevant to existing and emerging targeted therapies in lung cancer. The platform involves two methods: a screen (SNaPshot) based on multiplex PCR, primer extension, and capillary electrophoresis that was designed to assess for 38 somatic mutations in eight genes (AKT1, BRAF, EGFR, KRAS, MEK1, NRAS, PIK3CA, and PTEN) and a PCR-based sizing assay that assesses for EGFR exon 19 deletions, EGFR exon 20 insertions, and HER2 exon 20 insertions. Both the SNaPshot and sizing assays can be performed rapidly, with minimal amounts of genetic material. Compared with direct sequencing, in which mutant DNA needs to compose 25% or more of the total DNA to easily detect a mutation, the SNaPshot and sizing assays can detect mutations in samples in which mutant DNA composes 1.56% to 12.5% and 1.56% to 6.25% of the total DNA, respectively. These robust, reliable, and relatively inexpensive assays should help accelerate adoption of a genotype-driven approach in the treatment of lung cancer.

The BRAF kinase is mutated, typically Val 600→Glu (V600E), to induce an active oncogenic state in a large fraction of melanomas, thyroid cancers, hairy cell leukaemias and, to a smaller extent, a wide spectrum of other cancers. BRAF(V600E) phosphorylates and activates the MEK1 and MEK2 kinases, which in turn phosphorylate and activate the ERK1 and ERK2 kinases, stimulating the mitogen-activated protein kinase (MAPK) pathway to promote cancer. Targeting MEK1/2 is proving to be an important therapeutic strategy, given that a MEK1/2 inhibitor provides a survival advantage in metastatic melanoma, an effect that is increased when administered together with a BRAF(V600E) inhibitor. We previously found that copper (Cu) influx enhances MEK1 phosphorylation of ERK1/2 through a Cu-MEK1 interaction. Here we show decreasing the levels of CTR1 (Cu transporter 1), or mutations in MEK1 that disrupt Cu binding, decreased BRAF(V600E)-driven signalling and tumorigenesis in mice and human cell settings. Conversely, a MEK1-MEK5 chimaera that phosphorylated ERK1/2 independently of Cu or an active ERK2 restored the tumour growth of murine cells lacking Ctr1. Cu chelators used in the treatment of Wilson disease decreased tumour growth of human or murine cells transformed by BRAF(V600E) or engineered to be resistant to BRAF inhibition. Taken together, these results suggest that Cu-chelation therapy could be repurposed to treat cancers containing the BRAF(V600E) mutation.

OBJECTIVE

To assess prognostic roles of various KRAS oncogene mutations in colorectal cancer, BRAF mutation status must be controlled for because BRAF mutation is associated with poor prognosis, and almost all BRAF mutants are present among KRAS wild-type tumors. Taking into account experimental data supporting a greater oncogenic effect of codon 12 mutations compared with codon 13 mutations, we hypothesized that KRAS codon 12-mutated colorectal cancers might behave more aggressively than KRAS wild-type tumors and codon 13 mutants.

METHODS

Using molecular pathological epidemiology database of 1,261 rectal and colon cancers, we examined clinical outcome and tumor biomarkers of KRAS codon 12 and 13 mutations in 1,075 BRAF wild-type cancers (i.e., controlling for BRAF status). Cox proportional hazards model was used to compute mortality HR, adjusting for potential confounders, including stage, PIK3CA mutations, microsatellite instability, CpG island methylator phenotype, and LINE-1 methylation.

RESULTS

Compared with patients with KRAS wild-type/BRAF wild-type cancers (N = 635), those with KRAS codon 12 mutations (N = 332) experienced significantly higher colorectal cancer-specific mortality [log-rank P = 0.0001; multivariate HR, 1.30; 95% confidence interval (CI), 1.02-1.67; P = 0.037], whereas KRAS codon 13-mutated cases (N = 108) were not significantly associated with prognosis. Among the seven most common KRAS mutations, c.35G>T (p.G12V; N = 93) was associated with significantly higher colorectal cancer-specific mortality (log-rank P = 0.0007; multivariate HR, 2.00; 95% CI, 1.38-2.90, P = 0.0003) compared with KRAS wild-type/BRAF wild-type cases.

CONCLUSIONS

KRAS codon 12 mutations (in particular, c.35G>T), but not codon 13 mutations, are associated with inferior survival in BRAF wild-type colorectal cancer. Our data highlight the importance of accurate molecular characterization in colorectal cancer.

Protein-fragment complementation assays (PCAs) are widely used for investigating protein interactions. However, the fragments used are structurally compromised and have not been optimized nor thoroughly characterized for accurately assessing these interactions. We took advantage of the small size and bright luminescence of NanoLuc to engineer a new complementation reporter (NanoBiT). By design, the NanoBiT subunits (i.e., 1.3 kDa peptide, 18 kDa polypeptide) weakly associate so that their assembly into a luminescent complex is dictated by the interaction characteristics of the target proteins onto which they are appended. To ascertain their general suitability for measuring interaction affinities and kinetics, we determined that their intrinsic affinity (KD = 190 μM) and association constants (kon = 500 M(-1) s(-1), koff = 0.2 s(-1)) are outside of the ranges typical for protein interactions. The accuracy of NanoBiT was verified under defined biochemical conditions using the previously characterized interaction between SME-1 β-lactamase and a set of inhibitor binding proteins. In cells, NanoBiT fusions to FRB/FKBP produced luminescence consistent with the linear characteristics of NanoLuc. Response dynamics, evaluated using both protein kinase A and β-arrestin-2, were rapid, reversible, and robust to temperature (21-37 °C). Finally, NanoBiT provided a means to measure pharmacology of kinase inhibitors known to induce the interaction between BRAF and CRAF. Our results demonstrate that the intrinsic properties of NanoBiT allow accurate representation of protein interactions and that the reporter responds reliably and dynamically in cells.

Cancer gender disparity in incidence, disease aggressiveness and prognosis has been observed in a variety of cancers. Thyroid cancer is one of the fastest growing cancer diagnoses worldwide. It is 2.9-times more common in women than men. The less aggressive histologic subtypes of thyroid cancer are more common in women, whereas the more aggressive histologic subtypes have similar gender distribution. The gender disparity in incidence, aggressiveness and prognosis is well established for thyroid cancer but the cause of the disparity is poorly understood. The aim of this article is to evaluate the current evidence on the cause of thyroid cancer gender disparity. Dietary and environmental factors do not appear to have a significant role in thyroid cancer gender disparity. Common somatic mutations in BRAF, rearranged in transformation/papillary thyroid carcinomas (RET/PTC) and neurotrophin receptor-tyrosine kinase (NTRK) also do not account for the gender disparity in thyroid cancer. While reproductive factors would seem a logical hypothesis to account for the gender disparity, there appears to be no conclusive effect on the risk of developing thyroid cancer. Recent studies on estrogen receptor status in thyroid cancer show a difference in the receptor subtypes expressed based on the histology of thyroid cancer. Moreover, the response to estrogen is dependent on the specific estrogen receptor expressed in thyroid cancer cells. However, what determines the tumor-specific sex hormone receptor expression is unclear. No established molecular factors appear to explain gender differences in thyroid cancer. Therefore, the application of high-throughput genomic and proteomic approaches to the study of thyroid cancer gender disparity could be helpful for better understanding the molecular basis for gender differences in thyroid and other cancers.

Hsp90 is a ubiquitously expressed molecular chaperone that folds, stabilizes, and functionally regulates many cellular proteins. The benzoquinone ansamysin 17-allylamino-17-demethoxygeldanamycin (17-AAG) is an anticancer drug that disrupts Hsp90 binding to its clients, causing their degradation through the ubiquitin-dependent proteasomal pathway. The protein kinase B-RAF is mutated in approximately 7% of human cancers. The most common mutation (approximately 90%) is (V600E)B-RAF, which has constitutively elevated kinase activity, stimulates cancer cell proliferation, and promotes survival. Here, we show that (V600E)B-RAF is an Hsp90 client protein that requires Hsp90 for its folding and stability. (V600E)BRAF is more sensitive to degradation by 17-AAG treatment than (WT)B-RAF and we show that the majority of the other mutant forms of B-RAF are also sensitive to 17-AAG-mediated proteasomal degradation. Our data show that B-RAF is an important target for 17-AAG in human cancer.

BACKGROUND

Oncogenic mutations of PIK3CA, RAS (KRAS, NRAS), and BRAF have been identified in various malignancies, and activate the PI3K/AKT/mTOR and RAS/RAF/MEK pathways, respectively. Both pathways are critical drivers of tumorigenesis.

METHODS

Tumor tissues from 504 patients with diverse cancers referred to the Clinical Center for Targeted Therapy at MD Anderson Cancer Center starting in October 2008 were analyzed for PIK3CA, RAS (KRAS, NRAS), and BRAF mutations using polymerase chain reaction-based DNA sequencing.

RESULTS

PIK3CA mutations were found in 54 (11%) of 504 patients tested; KRAS in 69 (19%) of 367; NRAS in 19 (8%) of 225; and BRAF in 31 (9%) of 361 patients. PIK3CA mutations were most frequent in squamous cervical (5/14, 36%), uterine (7/28, 25%), breast (6/29, 21%), and colorectal cancers (18/105, 17%); KRAS in pancreatic (5/9, 56%), colorectal (49/97, 51%), and uterine cancers (3/20, 15%); NRAS in melanoma (12/40, 30%), and uterine cancer (2/11, 18%); BRAF in melanoma (23/52, 44%), and colorectal cancer (5/88, 6%). Regardless of histology, KRAS mutations were found in 38% of patients with PIK3CA mutations compared to 16% of patients with wild-type (wt)PIK3CA (p = 0.001). In total, RAS (KRAS, NRAS) or BRAF mutations were found in 47% of patients with PIK3CA mutations vs. 24% of patients wtPIK3CA (p = 0.001). PIK3CA mutations were found in 28% of patients with KRAS mutations compared to 10% with wtKRAS (p = 0.001) and in 20% of patients with RAS (KRAS, NRAS) or BRAF mutations compared to 8% with wtRAS (KRAS, NRAS) or wtBRAF (p = 0.001).

CONCLUSIONS

PIK3CA, RAS (KRAS, NRAS), and BRAF mutations are frequent in diverse tumors. In a wide variety of tumors, PIK3CA mutations coexist with RAS (KRAS, NRAS) and BRAF mutations.

Relatively little is known about the molecular changes that promote the formation or growth of pilocytic astrocytomas. We investigated genomic alterations in 25 pilocytic astrocytomas, including 5 supratentorial and 20 posterior fossa tumors, using oligonucleotide array comparative genomic hybridization. Large changes were identified in 7 tumors and included gains of chromosomes 5, 6, and 7 and losses of chromosomes 16, 17, 19, and 22. The most common alteration was a 1.9-MB region of low-level gain at chromosome 7q34 identified in 17 of 20 posterior fossa tumors. In most tumors, the region of gain ended within the BRAF locus and encompassed only exons that encode the BRAF kinase domain. We confirmed copy number increase at the 7q34 locus using quantitative polymerase chain reaction with primers adjacent to the HIPK2, RAB19B, and BRAF genes. Western blot analysis revealed that 3 of 6 pilocytic astrocytomas with 7q34 gain contained high levels of phosphorylated extracellular signal-related kinase (ERK) and nitrogen-activated protein kinase/ERK kinase (MEK), while 1 tumor lacking 7q34 gain and 2 normal brain specimens did not. Immunohistochemical stains of a tissue microarray containing 43 pilocytic astrocytoma identified ERK phosphorylation in 35 (81%). These data indicate that focal gains at chromosome 7q34 and increased BRAF-MEK-ERK signaling are common findings in sporadic pilocytic astrocytomas.

Although a number of genes related to melanoma development have been identified through candidate gene screening approaches, few studies have attempted to conduct such analyses on a genome-wide scale. Here we use Illumina 317K whole-genome single-nucleotide polymorphism arrays to define a comprehensive allelotype of melanoma based on loss of heterozygosity (LOH) and copy number changes in a panel of 76 melanoma cell lines. In keeping with previous reports, we found frequent LOH on chromosome arms 9p (72%), 10p (55%), 10q (55%), 9q (49%), 6q (43%), 11q (43%), and 17p (41%). Tumor suppressor genes (TSGs) can be identified through homozygous deletion (HD). We detected 174 HDs, the most common of which targeted CDKN2A (n = 33). The second highest frequency of HD occurred in PTEN (n = 8), another well known melanoma TSG. HDs were also common for PTPRD (n = 7) and HDAC4 (n = 3), TSGs recently found to be mutated or deleted in other cancer types. Analysis of other HDs and regions of LOH that we have identified might lead to the characterization of further melanoma TSGs. We noted 197 regional amplifications, including some centered on the melanoma oncogenes MITF (n = 9), NRAS (n = 3), BRAF (n = 3), and CCND1 (n = 3). Other amplifications potentially target novel oncogenes important in the development of a subset of melanomas. The numerous focal amplifications and HDs we have documented here are the first step toward identifying a comprehensive catalog of genes involved in melanoma development, some of which may be useful prognostic markers or targets for therapies to treat this disease.

BACKGROUND

The sustained clinical activity of the BRAF inhibitor vemurafenib (PLX4032/RG7204) in patients with BRAF(V600) mutant melanoma is limited primarily by the development of acquired resistance leading to tumor progression. Clinical trials are in progress using MEK inhibitors following disease progression in patients receiving BRAF inhibitors. However, the PI3K/AKT pathway can also induce resistance to the inhibitors of MAPK pathway.

RESULTS

The sensitivity to vemurafenib or the MEK inhibitor AZD6244 was tested in sensitive and resistant human melanoma cell lines exploring differences in activation-associated phosphorylation levels of major signaling molecules, leading to the testing of co-inhibition of the AKT/mTOR pathway genetically and pharmacologically. There was a high degree of cross-resistance to vemurafenib and AZD6244, except in two vemurafenib-resistant cell lines that acquired a secondary mutation in NRAS. In other cell lines, acquired resistance to both drugs was associated with persistence or increase in activity of AKT pathway. siRNA-mediated gene silencing and combination therapy with an AKT inhibitor or rapamycin partially or completely reversed the resistance.

CONCLUSIONS

Primary and acquired resistance to vemurafenib in these in vitro models results in frequent cross resistance to MEK inhibitors, except when the resistance is the result of a secondary NRAS mutation. Resistance to BRAF or MEK inhibitors is associated with the induction or persistence of activity within the AKT pathway in the presence of these drugs. This resistance can be potentially reversed by the combination of a RAF or MEK inhibitor with an AKT or mTOR inhibitor. These combinations should be available for clinical testing in patients progressing on BRAF inhibitors.