BACKGROUND

Although several studies undoubtedly demonstrated that BRAF mutation is an important genetic event in the pathogenesis of papillary thyroid carcinoma (PTC), its prognostic significance and correlation with less differentiated states remains unclear. It has been suggested that the discrepancy may be at least partially due to the insufficient number of cases analyzed, epidemiologic factors, and a combination of different variants of PTC included in these studies.

METHODS

In this context, the prevalence of the BRAF mutation in a Brazilian cohort of PTCs (n = 120) was first assessed and correlated with clinicopathologic features. The BRAF exon 15 mutation was evaluated by direct sequencing. Furthermore, using quantitative polymerase chain reaction, the issue of whether the expression level of the iodide-metabolizing genes (NIS and TSHR) was correlated with BRAF mutational status was investigated.

RESULTS

A high prevalence of the BRAF mutation was found in PTC cases (48%). The BRAF mutation was found to be significantly associated with the classic variant of PTC (66%; P < .0001), although it was found in the follicular variant as well (21%). Subtype stratification demonstrated that the BRAF V600E mutation was associated with tumor size, extrathyroid invasion, the presence of lymph node metastasis and risk of disease recurrence, and mortality in patients with the classic variant of PTC. Moreover, the expression levels of NIS and TSHR were remarkably lower in PTCs harboring the BRAF V600E mutation.

CONCLUSIONS

These findings provide further evidence that BRAF might be associated with a more aggressive phenotype and less differentiated state due to decreased expression of iodide-metabolizing genes. The search for a BRAF mutation in the current study population appears to be valuable for predicting prognosis and guiding management in patients with PTC.

During a clinical trial of the tyrosine kinase inhibitor dasatinib for advanced non-small cell lung cancer (NSCLC), one patient responded dramatically and remains cancer-free 4 years later. A comprehensive analysis of his tumor revealed a previously undescribed, kinase-inactivating BRAF mutation ((Y472C)BRAF); no inactivating BRAF mutations were found in the nonresponding tumors taken from other patients. Cells transfected with (Y472C)BRAF exhibited CRAF, MEK (mitogen-activated or extracellular signal-regulated protein kinase kinase), and ERK (extracellular signal-regulated kinase) activation-characteristics identical to signaling changes that occur with previously known kinase-inactivating BRAF mutants. Dasatinib selectively induced senescence in NSCLC cells with inactivating BRAF mutations. Transfection of other NSCLC cells with these BRAF mutations also increased these cells' dasatinib sensitivity, whereas transfection with an activating BRAF mutation led to their increased dasatinib resistance. The sensitivity induced by (Y472C)BRAF was reversed by the introduction of a BRAF mutation that impairs RAF dimerization. Dasatinib inhibited CRAF modestly, but concurrently induced RAF dimerization, resulting in ERK activation in NSCLC cells with kinase-inactivating BRAF mutations. The sensitivity of NSCLC with kinase-impaired BRAF to dasatinib suggested synthetic lethality of BRAF and an unknown dasatinib target. Inhibiting BRAF in NSCLC cells expressing wild-type BRAF likewise enhanced these cells' dasatinib sensitivity. Thus, the patient's BRAF mutation was likely responsible for his tumor's marked response to dasatinib, suggesting that tumors bearing kinase-impaired BRAF mutations may be exquisitely sensitive to dasatinib. Moreover, the potential synthetic lethality of combination therapy including dasatinib and BRAF inhibitors may lead to additional therapeutic options against cancers with wild-type BRAF.

Approximately one-half of advanced (unresectable or metastatic) melanomas harbor a mutation in the BRAF gene, with V600E being the most common mutation. Targeted therapy with BRAF and MEK inhibitors is associated with significant long-term treatment benefit in patients with BRAF V600-mutated melanoma. Therefore, molecular testing for BRAF mutations is a priority in determining the course of therapy. A literature search was performed using MEDLINE/PubMed and scientific congress databases using the terms 'BRAF,' 'mutation,' and 'cancer/tumor.' These results were filtered to include manuscripts that focused on diagnostic tests for determining BRAF mutation status. Numerous BRAF testing methods were identified, including DNA-based companion diagnostic tests and DNA- and protein-based laboratory-developed tests. Herein we review the characteristics of each method and highlight the strengths and weaknesses that should be considered before use and when interpreting results for each patient. Molecular profiling has shown that mutation load increases with melanoma tumor progression and that unique patterns of genetic changes and evolutionary trajectories for different melanoma subtypes can occur. Discordance in the BRAF mutational status between primary and metastatic lesions, as well as intratumoral heterogeneity, is known to occur. Additionally, the development of acquired resistance to combination BRAF and MEK inhibitor therapy is still a formidable obstacle. Therefore, tumor heterogeneity and the development of acquired resistance have important implications for molecular testing and ultimately the treatment of patients with advanced-stage melanoma. Overall, this information may help community oncologists more accurately and effectively interpret results of diagnostic tests within the context of recent data characterizing melanoma tumor progression.

BACKGROUND

BRAF V600E mutation plays a negative prognostic role in metastatic colorectal cancer (mCRC), leading to a median Progression Free Survival (PFS) of 4-6months with first-line conventional treatments. Our group recently reported in a retrospective exploratory analysis of a phase II trial that FOLFOXIRI (5-FU/LV+Oxaliplatin+Irinotecan) plus bevacizumab might allow to achieve remarkable results in terms of PFS and Overall Survival (OS) also in this poor-prognosis subgroup. The aim of this work was to prospectively validate our retrospective finding.

METHODS

This phase II trial was designed to detect an increase in 6month-Progression Free Rate (6m-PFR) from 45% to 80% in a population of BRAF mutant mCRC patients treated with first-line FOLFOXIRI plus bevacizumab. Secondary end-points were PFS, OS, response rate (RR) and the analysis of outcome parameters in the pooled population consisting of both retrospectively and prospectively included patients. This trial is registered with ClinicalTrials.gov, number NCT01437618.

RESULTS

Two-hundred-fourteen potentially eligible mCRC patients were screened for BRAF mutational status. Fifteen BRAF mutant patients (7%) were included in the validation cohort. At a median follow up of 25.7months, 6m-PFR was 73%. Median PFS and OS were 9.2 and 24.1months, respectively. In the pooled population, at a median follow up of 40.4months, 6m-PFR was 84%. Median PFS and OS were 11.8 and 24.1months, respectively. Overall RR and disease control rate were 72% and 88%, respectively.

CONCLUSIONS

Lacking randomised trials in this specific molecular subgroup, FOLFOXIRI plus bevacizumab might be a reasonable option for the first-line treatment of BRAF mutant mCRC patients.

OBJECTIVE

In 2009, the Evaluation of Genomic Applications in Practice and Prevention recommended that all colorectal cancers (CRCs) be screened for Lynch syndrome (LS) through microsatellite instability (MSI) or immunohistochemistry (IHC). No studies report how this process is implemented on a health system-wide basis.

METHODS

Since 2004, Cleveland Clinic has screened CRC specimens with MSI/IHC. Between January 2004 and July 2007, MSI/IHC results went only to the colorectal surgeon (approach 1). Between August 2007 and June 2008, colorectal surgeons and a genetic counselor received the MSI/IHC results, and the counselor e-mailed the colorectal surgeon regarding appropriate patients for genetic counseling (GC) referral (approach 2). After July 2008, the colorectal surgeon and counselor received MSI/IHC results, but the counselor contacted the patient to facilitate referral (approach 3). In approaches 2 and 3, patients were presumed to have sporadic CRC if the tumor lacked MLH1 expression and was also BRAF mutated or if the patient was diagnosed at age greater than 72 years and had no cancer family history.

RESULTS

Abnormal MSI/IHC results occurred in 178 (16%) of 1,108 patients. In approach 1, 21 (55%) of 38 patients with abnormal MSI/IHC were referred for GC, 12 (32%) of 38 underwent GC, and 10 (26%) of 38 underwent genetic testing (GT). In approach 2, nine (82%) of 11 patients were referred for GC, seven (64%) of 11 underwent GC, and five (45%) of 11 underwent GT. In approach 3, 56 (100%) of 56 patients were referred for GC, 40 (71%) of 56 underwent GC, and 37 (66%) of 56 underwent GT. Time from referral to GC was 10-fold quicker in approach 3 than approach 1.

CONCLUSIONS

Implementation of universal MSI/IHC with GC/GT, along with effective multidisciplinary communication and plans of responsibility for patient contact, resulted in increased identification of patients with LS.

OBJECTIVE

To develop gene expression profiles that characterise KRAS-, BRAF- or PIK3CA-activated- tumours, and to explore whether these profiles might be helpful in predicting the response to the epidermal growth factor receptor (EGFR) pathway inhibitors better than mutation status alone.

METHODS

Fresh frozen tumour samples from 381 colorectal cancer (CRC) patients were collected and mutations in KRAS, BRAF and PIK3CA were assessed. Using microarray data, three individual oncogenic and a combined model were developed and validated in an independent set of 80 CRC patients, and in a dataset from metastatic CRC patients treated with cetuximab.

RESULTS

175 tumours (45.9%) harboured oncogenic mutations in KRAS (30.2%), BRAF (11.0%) and PIK3CA (11.5%). Activating mutation signatures for KRAS (75 genes), for BRAF (58 genes,) and for PIK3CA (49 genes) were developed. The development of a combined oncogenic pathway signature-classified tumours as 'activated oncogenic', or as 'wildtype-like' with a sensitivity of 90.3% and a specificity of 61.7%. The identified signature revealed other mechanisms that can activate ERK/MAPK pathway in KRAS, BRAF and PIK3CA wildtype patients. The combined signature is associated with response to cetuximab treatment in patients with metastatic CRC (HR 2.51, p<0.0009).

CONCLUSIONS

A combined oncogenic pathway signature allows the identification of patients with an active EGFR-signalling pathway that could benefit from downstream pathway inhibition.

A low sodium iodide symporter (NIS) expression has been shown in papillary thyroid carcinomas (PTCs) harboring the BRAFV600E mutation. In the present study, we analyzed the mRNA expression of thyroid differentiation genes, glucose transporter (GLUT)-1 and GLUT-3, in 78 PTCs according to the presence of BRAFV600E or RET/PTC rearrangements. We found BRAFV600E and RET/PTC rearrangements in 35.8 and 19.4% of PTCs respectively. The mRNA expression of NIS and thyroperoxidase (TPO) genes were significantly lower (P<0.0001 and P=0.004 respectively) in BRAFV600E-positive PTC with respect to non-mutated samples. In support of this result, immunohistochemistry showed that the percentage of NIS-positive cells was significantly lower (P=0.005) in BRAFV600E-mutated PTC (mean 53.5%) than in negative cases (mean 72.6%). In contrast, no difference either in NIS or in any other thyroid differentiation genes' mRNA expression was found in PTC with or without RET/PTC rearrangements. When GLUT-1 and GLUT-3 mRNA expression was considered, no correlation was found either in BRAFV600E- nor in RET/PTC-mutated cases. In conclusion, this study confirmed the presence of a genetic alteration of BRAF and/or RET oncogenes in 64% of PTC cases and revealed a significant correlation of BRAFV600E mutation with a lower expression of both NIS and TPO. This latter finding could indicate that an early dedifferentiation process is present at the molecular level in BRAFV600E-mutated PTC, thus suggesting that the previously demonstrated poor prognostic significance of BRAFV600E mutation could be related to the dedifferentiation process more than to a more advanced stage at diagnosis.

A major early event in papillary thyroid carcinogenesis is constitutive activation of the mitogen-activated protein kinase signaling pathway caused by alterations of a single gene, typically rearrangements of the RET and NTRK1 genes or point mutations in the BRAF and RAS genes. In childhood papillary thyroid cancer, regardless of history of radiation exposure, RET/PTC rearrangements are a major event. Conversely, in adult-onset papillary thyroid cancer among the general population, the most common molecular event is BRAF(V600E) point mutation, not RET/PTC rearrangements. To clarify which gene alteration, chromosome aberration, or point mutation preferentially occurs in radiation-associated adult-onset papillary thyroid cancer, we have performed molecular analyses on RET/PTC rearrangements and BRAF(V600E) mutation in 71 papillary thyroid cancer cases among atomic bomb survivors (including 21 cases not exposed to atomic bomb radiation), in relation to radiation dose as well as time elapsed since atomic bomb radiation exposure. RET/PTC rearrangements showed significantly increased frequency with increased radiation dose (P(trend) = 0.002). In contrast, BRAF(V600E) mutation was less frequent in cases exposed to higher radiation dose (P(trend) < 0.001). Papillary thyroid cancer subjects harboring RET/PTC rearrangements developed this cancer earlier than did cases with BRAF(V600E) mutation (P = 0.03). These findings were confirmed by multivariate logistic regression analysis. These results suggest that RET/PTC rearrangements play an important role in radiation-associated thyroid carcinogenesis.

OBJECTIVE

The homeodomain transcription factor CDX2 is a relatively specific immunohistochemical marker for gastrointestinal carcinoma. However, no study has comprehensively examined the relationship between CDX2 expression in colon cancer and clinical, pathologic, prognostic, and molecular features, including microsatellite instability and CpG island methylator phenotype (CIMP).

METHODS

Utilizing 621 colorectal cancers with clinical outcome and molecular data, CDX2 loss was detected in 183 (29%) tumors by immunohistochemistry.

RESULTS

In multivariate logistic regression analysis, CDX2 loss was associated with female gender [odds ratio (OR), 3.32; P < 0.0001], CIMP-high (OR, 4.42; P = 0.0003), high tumor grade (OR, 2.69; P = 0.0085), stage IV disease (OR, 2.03; P = 0.019), and inversely with LINE-1 hypomethylation (for a 30% decline; OR, 0.33; P = 0.0031), p53 expression (OR, 0.55; P = 0.011), and beta-catenin activation (OR, 0.60; P = 0.037), but not with body mass index, tumor location, microsatellite instability, BRAF, KRAS, PIK3CA, p21, or cyclooxygenase-2. CDX2 loss was not independently associated with patient survival. However, the prognostic effect of CDX2 loss seemed to differ according to family history of colorectal cancer (P(interaction) = 0.0094). CDX2 loss was associated with high overall mortality (multivariate hazard ratio, 2.40; 95% CI, 1.28-4.51) among patients with a family history of colorectal cancer; no such association was present (multivariate hazard ratio, 0.97; 95% CI, 0.66-1.41) among patients without a family history of colorectal cancer.

CONCLUSIONS

CDX2 loss in colorectal cancer is independently associated with female gender, CIMP-high, high-level LINE-1 methylation, high tumor grade, and advanced stage. CDX2 loss may be associated with poor prognosis among patients with a family history of colorectal cancer.

BACKGROUND

The BRAF(V600E) mutation is the most common genetic alteration found in papillary thyroid cancer (PTC). Recent studies show that this mutation occurs more frequently in patients with PTC showing aggressive clinicopathologic features. The aim of the present study was to evaluate the prevalence of the BRAF(V600E) mutation in tumor samples and its association with high-risk clinicopathologic features prospectively.

METHODS

From February 2009 to January 2010, 547 PTC patients who underwent surgery in Seoul National University Hospital were enrolled in the study. Polymerase chain reaction was used to amplify exon 15 of the BRAF gene from paraffin-embedded thyroid tumor specimens, followed by direct sequencing to detect the BRAF(V600E) mutation. Both univariate and multivariate analyses were performed to analyze associations between the BRAF(V600E) mutation and clinicopathologic features.

RESULTS

The BRAF(V600E) mutation was found in 381/547 (69.7%) patients with primary PTC. The BRAF(V600E) mutation was significantly associated with age (≥ 45 years), tumor size (>1 cm), extrathyroidal extension, and cervical lymph node metastases (P < 0.05). Multiple logistic regression showed that it was significantly associated with gender (OR = 1.834; 95% CI 1.021-3.463), tumor size (OR = 1.972; 95% CI 1.250-3.103), and extra-thyroidal extension (OR = 2.428; 95% CI 1.484-3.992), but not with age, multifocality, lymph node metastases, and advanced disease stage. The proportion of BRAF(V600E) mutation was significantly associated with the number of high-risk factors of tumor recurrence (P < 0.001).

CONCLUSIONS

The BRAF(V600E) mutation was associated with high-risk clinicopathologic characteristics in patients with PTC. The BRAF(V600E) mutation may be a potential prognostic factor in PTC patients.

This study investigated the cytotoxicity and mechanism of action of AS703026, a novel, selective, orally bioavailable MEK1/2 inhibitor, in human multiple myeloma (MM). AS703026 inhibited growth and survival of MM cells and cytokine-induced osteoclast differentiation more potently (9- to 10-fold) than AZD6244. Inhibition of proliferation induced by AS703026 was mediated by G0-G1 cell cycle arrest and was accompanied by reduction of MAF oncogene expression. AS703026 further induced apoptosis via caspase 3 and Poly ADP ribose polymerase (PARP) cleavage in MM cells, both in the presence or absence of bone marrow stromal cells (BMSCs). Importantly, AS703026 sensitized MM cells to a broad spectrum of conventional (dexamethasone, melphalan), novel or emerging (lenalidomide, perifosine, bortezomib, rapamycin) anti-MM therapies. Significant tumour growth reduction in AS703026- vs. vehicle-treated mice bearing H929 MM xenograft tumours correlated with downregulated pERK1/2, induced PARP cleavage, and decreased microvessels in vivo. Moreover, AS703026 (<200 nmol/l) was cytotoxic against the majority of tumour cells tested from patients with relapsed and refractory MM (84%), regardless of mutational status of RAS and BRAF genes. Importantly, BMSC-induced viability of MM patient cells was similarly blocked within the same dose range. Our results therefore support clinical evaluation of AS703026, alone or in combination with other anti-MM agents, to improve patient outcome.

Tumor screening for Lynch syndrome is recommended in all or most patients with colorectal cancer (CRC). In metastatic CRC, sequencing of RAS/BRAF is necessary to guide clinical management. We hypothesized that a next-generation sequencing (NGS) panel that identifies RAS/BRAF and other actionable mutations could also reliably identify tumors with DNA mismatch repair protein deficiency (MMR-D) on the basis of increased mutational load.

We identified all CRCs that underwent genomic mutation profiling with a custom NGS assay (MSK-IMPACT) between March 2014 and July 2015. Tumor mutational load, with exclusion of copy number changes, was determined for each case and compared with MMR status as determined by routine immunohistochemistry.

Tumors from 224 patients with unique CRC analyzed for MMR status also underwent MSK-IMPACT. Thirteen percent (n = 28) exhibited MMR-D by immunohistochemistry. Using the 341-gene assay, 100% of the 193 tumors with < 20 mutations were MMR-proficient. Of 31 tumors with ≥ 20 mutations, 28 (90%) were MMR-D. The three remaining tumors were easily identified as being distinct from the MMR-D tumors with>> 150 mutations each. Each of these tumors harbored the P286R hotspot POLE mutation consistent with the ultramutator phenotype. Among MMR-D tumors, the median number of mutations was 50 (range, 20 to 90) compared with six (range, 0 to 17) in MMR-proficient/POLE wild-type tumors (P < .001). With a mutational load cutoff of ≥ 20 and < 150 for MMR-D detection, sensitivity and specificity were both 1.0 (95% CI, 0.93 to 1.0).

A cutoff for mutational load can be identified via multigene NGS tumor profiling, which provides a highly accurate means of screening for MMR-D in the same assay that is used for tumor genotyping.

To gain a better understanding of the genetic variants associated with carboplatin-induced cytotoxicity in individuals of African descent, we present a step-wise approach integrating genotypes, gene expression, and sensitivity of HapMap cell lines to carboplatin. Cell lines derived from 30 trios of African descent (YRI) were used to develop a preclinical model to identify genetic variants and gene expression that contribute to carboplatin-induced cytotoxicity. Cytotoxicity was determined as cell growth inhibition at increasing concentrations of carboplatin for 72 h. Gene expression of 89 HapMap YRI cell lines was determined using the Affymetrix GeneChip Human Exon 1.0 ST Array. Single nucleotide polymorphism genotype and the percent survival at different treatment concentrations along with carboplatin IC50 were linked through whole genome association. A second association test was done between single nucleotide polymorphism genotype and gene expression, and linear regression was then used to capture those genes whose expression correlated to drug sensitivity phenotypes. This approach allows us to identify genetic variants that significantly associate with sensitivity to the cytotoxic effects of carboplatin through their effect on gene expression. We found a gene (GPC5) whose expression is important in all carboplatin treatment concentrations as well as many genes unique to either low (e.g., MAPK1) or high (e.g., BRAF, MYC, and BCL2L1) concentrations of drug. Our whole genome approach enables us to evaluate the contribution of genetic and gene expression variation to a wide range of cellular phenotypes. The identification of concentration specific genetic signatures allows for potential integration of pharmacokinetics, pharmacodynamics, and pharmacogenetics in tailoring chemotherapy.

Deregulation of the RAS-RAF-mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)-ERK signaling cascade is often caused by somatic mutations in genes encoding proteins which influence the activity of this pathway and include NRAS, KRAS2, FLT3, PTPN11, and BRAF. We report the first comprehensive mutational screen of key exons of these genes in a large cohort of unselected acute lymphoblastic leukemia (ALL) cases at diagnosis (n = 86) and in a more selected cohort at disease recurrence (n = 47) using the sensitive method of denaturing high-performance liquid chromatography. We show that somatic mutations that deregulate the pathway constitute one of the most common genetic aberrations in childhood ALL (cALL), being found in 35% of diagnostic and 25% of relapse samples. In matched presentation/relapse pairs, mutations predominating at relapse could be shown to be present at very low levels at diagnosis using allele-specific PCR, thus implicating the mutated clone in disease progression. Importantly, in primary samples, we show that mutations are associated with activated ERK and differential cytotoxicity to MEK-ERK inhibitors was shown for some patients. Inhibitors of the pathway, which are currently undergoing clinical trial, may be a novel therapeutic option for cALL, particularly at relapse.

Although anti-EGFR therapy has established efficacy in metastatic colorectal cancer, only 10-20% of unselected patients respond. This is partly due to KRAS and BRAF mutations, which are currently assessed in the primary tumor. To improve patient selection, assessing mutation status in circulating tumor cells (CTCs), which possibly better represent metastases than the primary tumor, could be advantageous. We investigated the feasibility of KRAS and BRAF mutation detection in colorectal CTCs by comparing three sensitive methods and compared mutation status in matching primary tumor, liver metastasis and CTCs. CTCs were isolated from blood drawn from 49 patients before liver resection using CellSearch™. DNA and RNA was isolated from primary tumors, metastases and CTCs. Mutations were assessed by co-amplification at lower denaturation temperature-PCR (Transgenomic™), real-time PCR (EntroGen™) and nested Allele-Specific Blocker (ASB-)PCR and confirmed by Sanger sequencing. In 43 of the 49 patients, tissue RNA and DNA was of sufficient quantity and quality. In these 43 patients, discordance between primary and metastatic tumor was 23% for KRAS and 7% for BRAF mutations. RNA and DNA from CTCs was available from 42 of the 43 patients, in which ASB-PCR was able to detect the most mutations. Inconclusive results in patients with low CTC counts limited the interpretation of discrepancies between tissue and CTCs. Determination of KRAS and BRAF mutations in CTCs is challenging but feasible. Of the tested methods, nested ASB-PCR, enabling detection of KRAS and BRAF mutations in patients with as little as two CTCs, seems to be superior.

OBJECTIVE

Cetuximab improves survival in patients with K-ras wild-type advanced colorectal cancer. We examined the predictive and prognostic significance of additional biomarkers in this setting, in particular BRAF, PIK3CA, and PTEN.

METHODS

Available colorectal tumor samples were analyzed from the CO.17 study. BRAF mutations were identified in tumor-derived DNA by direct sequencing and PIK3CA mutations were identified using a high-resolution melting screen with confirmation by sequencing. PTEN expression by immunohistochemistry (IHC) was performed on tissue microarrays. For each biomarker, prognostic and predictive effects were examined using a Cox model with tests for treatment-biomarker interaction.

RESULTS

A total of 572 patients with pretreated colorectal cancer were randomly assigned to receive cetuximab or best supportive care (BSC). Of 401 patients assessed for BRAF status, 13 (3.2%) had mutations. Of 407 patients assessed for PIK3CA status, 61 (15%) had mutations. Of 205 patients assessed for PTEN, 148 (72%) were negative for IHC expression. None of BRAF, PIK3CA, or PTEN was prognostic for overall or progression-free survival in the BSC arm. None was predictive of benefit from cetuximab, either in the whole study population or the K-ras wild-type subset. In the K-ras wild-type subgroup, the overall survival adjusted HR according to BRAF mutation status was 1.39 (interaction P = 0.69), PIK3CA mutation status HR = 0.79 (interaction P = 0.63), and PTEN expression HR = 0.75 (interaction P = 0.61).

CONCLUSIONS

In chemotherapy-refractory colorectal cancer, neither PIK3CA mutation status nor PTEN expression were prognostic, nor were they predictive of benefit from cetuximab. Evaluation of predictive significance of BRAF mutations requires a larger sample size.

Programmed cell-death 1 ligand 1 (PD-L1) is a member of the B7/CD28 family of proteins that control T-cell activation. Many tumors can upregulate expression of PD-L1, inhibiting antitumor T-cell responses and avoiding immune surveillance and elimination. We have identified and characterized MEDI4736, a human IgG1 monoclonal antibody that binds with high affinity and specificity to PD-L1 and is uniquely engineered to prevent antibody-dependent cell-mediated cytotoxicity. In vitro assays demonstrate that MEDI4736 is a potent antagonist of PD-L1 function, blocking interaction with PD-1 and CD80 to overcome inhibition of primary human T-cell activation. In vivo MEDI4736 significantly inhibits the growth of human tumors in a novel xenograft model containing coimplanted human T cells. This activity is entirely dependent on the presence of transplanted T cells, supporting the immunological mechanism of action for MEDI4736. To further determine the utility of PD-L1 blockade, an anti-mouse PD-L1 antibody was investigated in immunocompetent mice. Here, anti-mouse PD-L1 significantly improved survival of mice implanted with CT26 colorectal cancer cells. The antitumor activity of anti-PD-L1 was enhanced by combination with oxaliplatin, which resulted in increased release of HMGB1 within CT26 tumors. Taken together, our results demonstrate that inhibition of PD-L1 function can have potent antitumor activity when used as monotherapy or in combination in preclinical models, and suggest it may be a promising therapeutic approach for the treatment of cancer. MEDI4736 is currently in several clinical trials both alone and in combination with other agents, including anti-CTLA-4, anti-PD-1, and inhibitors of IDO, MEK, BRAF, and EGFR.

The incidence of colorectal cancer (CRC) increases with age and early onset indicates an increased likelihood for genetic predisposition for this disease. The somatic genetics of tumor development in relation to patient age remains mostly unknown. We have examined the mutation status of five known cancer critical genes in relation to age at diagnosis, and compared the genomic complexity of tumors from young patients without known CRC syndromes with those from elderly patients. Among 181 CRC patients, stratified by microsatellite instability status, DNA sequence changes were identified in KRAS (32%), BRAF (16%), PIK3CA (4%), PTEN (14%) and TP53 (51%). In patients younger than 50 years (n = 45), PIK3CA mutations were not observed and TP53 mutations were more frequent than in the older age groups. The total gene mutation index was lowest in tumors from the youngest patients. In contrast, the genome complexity, assessed as copy number aberrations, was highest in tumors from the youngest patients. A comparable number of tumors from young (<50 years) and old patients (>70 years) was quadruple negative for the four predictive gene markers (KRAS-BRAF-PIK3CA-PTEN); however, 16% of young versus only 1% of the old patients had tumor mutations in PTEN/PIK3CA exclusively. This implies that mutation testing for prediction of EGFR treatment response may be restricted to KRAS and BRAF in elderly (>70 years) patients. Distinct genetic differences found in tumors from young and elderly patients, whom are comparable for known clinical and pathological variables, indicate that young patients have a different genetic risk profile for CRC development than older patients.

KRAS and BRAF mutations lead to the constitutive activation of EGFR signaling through the oncogenic Ras/Raf/Mek/Erk pathway. Currently, KRAS is the only potential biomarker for predicting the efficacy of anti-EGFR monoclonal antibodies (mAb) in colorectal cancer (CRC). However, a recent report suggested that the use of cetuximab was associated with survival benefit among patients with p.G13D-mutated tumors. Furthermore, although the presence of mutated BRAF is one of the most powerful prognostic factors for advanced and recurrent CRC, it remains unknown whether patients with BRAF-mutated tumors experience a survival benefit from treatment with anti-EGFR mAb. Thus, the prognostic or predictive relevance of the KRAS and BRAF genotype in CRC remains controversial despite several investigations. Routine KRAS/BRAF screening of pathological specimens is required to promote the appropriate clinical use of anti-EGFR mAb and to determine malignant phenotypes in CRC. The significance of KRAS/BRAF mutations as predictive or prognostic biomarkers should be taken into consideration when selecting a KRAS/BRAF screening assay. This article will review the spectrum of KRAS/BRAF genotype and the impact of KRAS/BRAF mutations on the clinicopathological features and prognosis of patients with CRC, particularly when differentiating between the mutations at KRAS codons 12 and 13. Furthermore, the predictive role of KRAS/BRAF mutations in treatments with anti-EGFR mAb will be verified, focusing on KRAS p.G13D and BRAF mutations.

Fusions of the RET and ROS1 protein tyrosine kinase oncogenes with several partner genes were recently identified as new targetable genetic aberrations in cases of non-small cell lung cancer (NSCLC) lacking activating EGFR, KRAS, ALK, BRAF, or HER2 oncogene aberrations. RET and ROS1 fusion-positive tumors are mainly observed in young, female, and/or never smoking patients. Studies based on in vitro and in vivo (i.e., mouse) models and studies of several fusion-positive patients indicate that inhibiting the kinase activity of the RET and ROS1 fusion proteins is a promising therapeutic strategy. Accordingly, there are several ongoing clinical trials aimed at examining the efficacy of tyrosine kinase inhibitors (TKIs) against RET and ROS1 proteins in patients with fusion-positive lung cancer. Other gene fusions (NTRK1, NRG1, and FGFR1/2/3) that are targetable by existing TKIs have also been identified in NSCLCs. Options for personalized lung cancer therapy will be increased with the help of multiplex diagnosis systems able to detect multiple druggable gene fusions.

Tumor genomic instability and selective treatment pressures result in clonal disease evolution; molecular stratification for molecularly targeted drug administration requires repeated access to tumor DNA. We hypothesized that circulating plasma DNA (cpDNA) in advanced cancer patients is largely derived from tumor, has prognostic utility, and can be utilized for multiplex tumor mutation sequencing when repeat biopsy is not feasible. We utilized the Sequenom MassArray System and OncoCarta panel for somatic mutation profiling. Matched samples, acquired from the same patient but at different time points were evaluated; these comprised formalin-fixed paraffin-embedded (FFPE) archival tumor tissue (primary and/or metastatic) and cpDNA. The feasibility, sensitivity, and specificity of this high-throughput, multiplex mutation detection approach was tested utilizing specimens acquired from 105 patients with solid tumors referred for participation in Phase I trials of molecularly targeted drugs. The median cpDNA concentration was 17 ng/ml (range: 0.5-1600); this was 3-fold higher than in healthy volunteers. Moreover, higher cpDNA concentrations associated with worse overall survival; there was an overall survival (OS) hazard ratio of 2.4 (95% CI 1.4, 4.2) for each 10-fold increase in cpDNA concentration and in multivariate analyses, cpDNA concentration, albumin, and performance status remained independent predictors of OS. These data suggest that plasma DNA in these cancer patients is largely derived from tumor. We also observed high detection concordance for critical 'hot-spot' mutations (KRAS, BRAF, PIK3CA) in matched cpDNA and archival tumor tissue, and important differences between archival tumor and cpDNA. This multiplex sequencing assay can be utilized to detect somatic mutations from plasma in advanced cancer patients, when safe repeat tumor biopsy is not feasible and genomic analysis of archival tumor is deemed insufficient. Overall, circulating nucleic acid biomarker studies have clinically important multi-purpose utility in advanced cancer patients and further studies to pursue their incorporation into the standard of care are warranted.

The protein kinase BRAF, a component of the RAS/RAF/mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK signaling pathway, regulates cell fate in response to extracellular signals. Activating mutations in BRAF occur in approximately 70% of human melanomas. The active proteins stimulate constitutive pathway signaling, proliferation, and survival. Thus, inhibition of BRAF signaling in melanoma cells causes cell cycle arrest and induces cell death through apoptosis, validating BRAF as an important therapeutic target. Here, we show that the apoptosis induced by inhibition of BRAF signaling in melanoma cells can be prevented if the cells are treated with tumor necrosis factor (TNF)-alpha. This allows the cells to recover from the inhibition of BRAF signaling and reenter the cell cycle. This effect occurs due to a specific TNF-alpha and BRAF interaction because TNF-alpha does not prevent cell death in the presence of cisplatin, nitrogen mustard or thapsigargin. Furthermore, the cytokines Fas ligand, TNF-related apoptosis-inducing ligand, interleukin (IL)-1, and IL-6 do not prevent cell death when BRAF signaling is inhibited. The survival mechanism requires nuclear factor-kappaB (NF-kappaB) transcription factor activity, which is strongly induced by TNF-alpha in these cells. These findings suggest that drugs that target the BRAF/MEK pathway could be combined with agents that target TNF-alpha and/or NF-kappaB signaling to provide exciting new therapeutic opportunities for the treatment of melanoma.

Malignant melanoma originates in melanocytes, the pigment-producing cells of the skin and eye, and is one of the most deadly human cancers with no effective cure for metastatic disease. Like many other cancers, melanoma has both environmental and genetic components. For more than 20 years, the melanoma genome has been subject to extensive scrutiny, which has led to the identification of several genes that contribute to melanoma genesis and progression. Three molecular pathways have been found to be nearly invariably dysregulated in melanocytic tumors, including the RAS-RAF-MEK-ERK pathway (through mutation of BRAF, NRAS or KIT), the p16 INK4A-CDK4-RB pathway (through mutation of INK4A or CDK4) and the ARF-p53 pathway (through mutation of ARF or TP53). Less frequently targeted pathways include the PI3K-AKT pathway (through mutation of NRAS, PTEN or PIK3CA) and the canonical Wnt signaling pathway (through mutation of CTNNB1 or APC). Beyond the specific and well-characterized genetic events leading to activation of proto-oncogenes or inactivation of tumor suppressor genes in these pathways, systematic high-resolution genomic analysis of melanoma specimens has revealed recurrent DNA copy number aberrations as well as perturbations of DNA methylation patterns. Melanoma provides one of the best examples of how genomic analysis can lead to a better understanding of tumor biology. We review current knowledge of the genes involved in the development of melanoma and the molecular pathways in which these genes operate.