The RAS/RAF/MEK/ERK signaling pathway has emerged as a major player in the induction and maintenance of melanoma, particularly the protein kinase BRAF, mutated in approximately 44% of melanoma cases. The availability of new drugs affecting the components of this pathway and pathways that may cooperate with MAPK signaling, means that targeted therapies are fast becoming a real option in the clinical management of melanoma. The authors discuss what they learned from clinical trials using first- and second-generation inhibitors to this pathway.

Mitogen-activated protein kinase (MAPK) pathway activation is a frequent event in human cancer and is often the result of activating mutations in the BRAF and RAS oncogenes. Targeted inhibitors of BRAF and its downstream effectors are in various stages of preclinical and clinical development. These agents offer the possibility of greater efficacy and less toxicity than current therapies for tumors driven by oncogenic mutations in the MAPK pathway. Early clinical results with the BRAF-selective inhibitor PLX4032 suggest that this strategy will prove successful in a select group of patients whose tumors are driven by V600E BRAF. Relief of physiologic feedback upon pathway inhibition may, however, attenuate drug response and contribute to the development of acquired resistance. An improved understanding of the adaptive response of cancer cells to MAPK pathway inhibition may thus aid in the identification of those patients most likely to respond to targeted pathway inhibitors and provide a rational basis for tailored combination strategies.

BACKGROUND

Juvenile pilocytic astrocytomas (JPA), a subgroup of low-grade astrocytomas (LGA), are common, heterogeneous and poorly understood subset of brain tumours in children. Chromosomal 7q34 duplication leading to fusion genes formed between KIAA1549 and BRAF and subsequent constitutive activation of BRAF was recently identified in a proportion of LGA, and may be involved in their pathogenesis. Our aim was to investigate additional chromosomal unbalances in LGA and whether incidence of 7q34 duplication is associated with tumour type or location.

RESULTS

Using Illumina-Human-Hap300-Duo and 610-Quad high-resolution-SNP-based arrays and quantitative PCR on genes of interest, we investigated 84 paediatric LGA. We demonstrate that 7q34 duplication is specific to sporadic JPA (35 of 53 - 66%) and does not occur in other LGA subtypes (0 of 27) or NF1-associated-JPA (0 of 4). We also establish that it is site specific as it occurs in the majority of cerebellar JPA (24 of 30 - 80%) followed by brainstem, hypothalamic/optic pathway JPA (10 of 16 - 62.5%) and is rare in hemispheric JPA (1 of 7 - 14%). The MAP-kinase pathway, assessed through ERK phosphorylation, was active in all tumours regardless of 7q34 duplication. Gain of function studies performed on hTERT-immortalised astrocytes show that overexpression of wild-type BRAF does not increase cell proliferation or baseline MAPK signalling even if it sensitises cells to EGFR stimulation.

CONCLUSIONS

Our results suggest that variants of JPA might arise from a unique site-restricted progenitor cell where 7q34 duplication, a hallmark of this tumour-type in association to MAPK-kinase pathway activation, potentially plays a site-specific role in their pathogenesis. Importantly, gain of function abnormalities in components of MAP-Kinase signalling are potentially present in all JPA making this tumour amenable to therapeutic targeting of this pathway.

The epidermal growth factor receptor (EGFR) plays an important role in tumorigenesis and tumor progression of colorectal cancer (CRC). As a result, the EGFR has evolved as a relevant target in the treatment of metastatic CRC. KRAS serves as a mediator between extracellular ligand binding and intracellular transduction of signals from the EGFR to the nucleus. The presence of activating KRAS mutations has been identified as a potent predictor of resistance to EGFR-directed antibodies such as cetuximab or panitumumab. These agents should therefore be applied only in tumors with a wild-type status of the KRAS gene. Further parameters of resistance are lack of EGFR amplification, PTEN loss or BRAF mutation. However, they are less well studied or associated with less consistent data and therefore require prospective analyses before integration into clinical decision making. Future studies need to identify patterns of single or multiple mutations to further increase the power of patient selection for anti-EGFR therapy. While molecular parameters help to predict treatment efficacy upfront, skin toxicity has been accepted as an independent predictor of response during exposure to anti-EGFR therapy.

The "cancerized field" concept posits that cancer-prone cells in a given tissue share an oncogenic mutation, but only discreet clones within the field initiate tumors. Most benign nevi carry oncogenic BRAF(V600E) mutations but rarely become melanoma. The zebrafish crestin gene is expressed embryonically in neural crest progenitors (NCPs) and specifically reexpressed in melanoma. Live imaging of transgenic zebrafish crestin reporters shows that within a cancerized field (BRAF(V600E)-mutant; p53-deficient), a single melanocyte reactivates the NCP state, revealing a fate change at melanoma initiation in this model. NCP transcription factors, including sox10, regulate crestin expression. Forced sox10 overexpression in melanocytes accelerated melanoma formation, which is consistent with activation of NCP genes and super-enhancers leading to melanoma. Our work highlights NCP state reemergence as a key event in melanoma initiation.

Metastatic melanoma has historically been considered as one of the most therapeutically challenging malignancies. However, for the first time after decades of basic research and clinical investigation, new drugs have produced major clinical responses. The discovery of BRAF mutations in melanoma created the first opportunity to develop oncogene-directed therapy in this disease and led to the development of compounds that inhibit aberrant BRAF activity. A decade later, vemurafenib, an orally available and well-tolerated selective BRAF inhibitor, ushered in a new era of molecular treatments for advanced disease. Additional targets have been identified, and novel agents that impact on various signaling pathways or modulate the immune system hold the promise of a whole new therapeutic landscape for patients with metastatic melanoma. One of the major thrusts in melanoma therapy is now focused on understanding and targeting the network of signal transduction pathways and on attacking elements that underlie the tumor's propensity for growth and chemoresistance. In this article, we review the novel targeted anticancer approaches that are under consideration in melanoma treatment.

AZD6244 (ARRY 142886) is a potent and selective mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor currently in early clinical trials. We examined the activity of AZD6244 in a panel of non-small cell lung cancer and a panel of cell lines representing many cancer types using in vitro growth assays. AZD6244 induced G(0)-G(1) cell cycle arrest in sensitive cell lines that primarily included cells containing the BRAF V600E mutation. In these cells, G(0)-G(1) arrest is accompanied by the up-regulation of the cell cycle inhibitors p21(WAF1) and p27(Kip1) and down-regulation of cyclin D1. In the majority of cell lines tested, including those with K-ras or non-V600E BRAF mutations, AZD6244 induced the accumulation of phospho-MEK, an effect not observed in the most sensitive BRAF V600E-containing cells. Accumulation of phospho-MEK in non-V600E-containing cell lines is due to abrogation of negative feedback pathways. BRAF V600E disrupts negative feedback signaling, which results in enhanced baseline phospho-MEK expression. Exogenous expression of BRAF V600E disrupts feedback inhibition but does not sensitize cells to AZD6244. Specific suppression of endogenous BRAF V600E does not confer resistance to AZD6244 but enhances sensitivity to AZD6244. Thus, our findings show that BRAF V600E marks cells with an in vitro requirement for MAPK signaling to support proliferation. These cells are exquisitely sensitive to AZD6244 (IC(50), <100 nmol/L), have high baseline levels of phospho-MEK, and lack feedback inhibition between ERK and Raf. These data suggest an approach to identifying cells that may be sensitive to AZD6244 and other MEK inhibitors.

Children with Down syndrome (DS) have a greatly increased risk of acute megakaryoblastic leukemia (AMKL) and acute lymphoblastic leukemia (ALL). Both DS-AMKL and the related transient myeloproliferative disorder (TMD) have GATA1 mutations as obligatory, early events. To identify mutations contributing to leukemogenesis in DS-ALL, we undertook sequencing of candidate genes, including FLT3, RAS, PTPN11, BRAF, and JAK2. Sequencing of the JAK2 pseudokinase domain identified a specific, acquired mutation, JAK2R683, in 12 (28%) of 42 DS-ALL cases. Functional studies of the common JAK2R683G mutation in murine Ba/F3 cells showed growth factor independence and constitutive activation of the JAK/STAT signaling pathway. High-resolution SNP array analysis of 9 DS-ALL cases identified additional submicroscopic deletions in key genes, including ETV6, CDKN2A, and PAX5. These results infer a complex molecular pathogenesis for DS-ALL leukemogenesis, with trisomy 21 as an initiating or first hit and with chromosome aneuploidy, gene deletions, and activating JAK2 mutations as complementary genetic events.

An emerging concept in melanoma biology is that of dynamic, adaptive phenotype switching, where cells switch from a highly proliferative, poorly invasive phenotype to a highly invasive, less proliferative one. This switch may hold significant implications not just for metastasis, but also for therapy resistance. We demonstrate that phenotype switching and subsequent resistance can be guided by changes in expression of receptors involved in the noncanonical Wnt5A signaling pathway, ROR1 and ROR2. ROR1 and ROR2 are inversely expressed in melanomas and negatively regulate each other. Furthermore, hypoxia initiates a shift of ROR1-positive melanomas to a more invasive, ROR2-positive phenotype. Notably, this receptor switch induces a 10-fold decrease in sensitivity to BRAF inhibitors. In patients with melanoma treated with the BRAF inhibitor vemurafenib, Wnt5A expression correlates with clinical response and therapy resistance. These data highlight the fact that mechanisms that guide metastatic progression may be linked to those that mediate therapy resistance.

CONCLUSIONS

These data show for the fi rst time that a single signaling pathway, the Wnt signaling pathway, can effectively guide the phenotypic plasticity of tumor cells, when primed to do so by a hypoxic microenvironment. Importantly, this increased Wnt5A signaling can give rise to a subpopulation of highly invasive cells that are intrinsically less sensitive to novel therapies for melanoma, and targeting the Wnt5A/ROR2 axis could improve the efficacy and duration of response for patients with melanoma on vemurafenib.

Activating BRAF(V600E) (Val600Glu) mutations are found in about 1-2% of lung adenocarcinomas, which might provide an opportunity for targeted treatment in these patients. Dabrafenib is an oral selective inhibitor of BRAF kinase. We did a trial to assess the clinical activity of dabrafenib in patients with advanced non-small-cell lung cancer (NSCLC) positive for the BRAF(V600E) mutation.

In this phase 2, multicentre, non-randomised, open-label study, we enrolled previously treated and untreated patients with stage IV metastatic BRAF(V600E)-positive NSCLC. Patients received oral dabrafenib 150 mg twice daily. The primary endpoint was investigator-assessed overall response, which was assessed in patients who had received at least one dose of dabrafenib; safety was also assessed in this population. The study is ongoing but not enrolling patients in this cohort. This trial is registered with ClinicalTrials.gov, number NCT01336634.

Between Aug 3, 2011, and Feb 25, 2014, 84 patients were enrolled, six of whom had not previously received systemic treatment for NSCLC. 26 of the 78 previously treated patients achieved an investigator-assessed overall response (33% [95% CI 23-45]). Four of the six previously untreated patients had an objective response. One patient died from an intracranial haemorrhage that was judged by the investigator to be due to the study drug. Serious adverse events were reported in 35 (42%) of 84 patients. The most frequent grade 3 or worse adverse events were cutaneous squamous-cell carcinoma in ten (12%), asthenia in four (5%), and basal-cell carcinoma in four (5%).

Dabrafenib showed clinical activity in BRAF(V600E)-positive NSCLC. Our findings suggest that dabrafenib could represent a treatment option for a population of patients with limited therapeutic options.

GlaxoSmithKline.

Recent studies have demonstrated that BRAF(V600E) mutation is a common event in papillary thyroid carcinoma and a majority of these lesions have shown a direct relationship between BRAF(V600E) mutation and aggressive characteristics, including a worse patient prognosis. However, there are no studies from Japan regarding this issue in a large series with adequate postoperative follow-up periods. We investigated BRAF(V600E) mutation in 631 patients with papillary carcinoma having median follow-up periods of 83 months. The prevalence of BRAF(V600E) mutation was 38.4%, and the rate was higher in carcinoma larger than 1.0 cm but did not successively increase with tumor size. Furthermore, the prevalence did not significantly increase in cases demonstrating high-risk biological features such as clinically apparent lymph node metastasis, massive extrathyroid extension, advanced age, distant metastasis at surgery, and advanced Stage. The disease-free survival of patients with BRAF(V600E) mutation did not differ from that of those without BRAF(V600E) mutation. These findings indicate that, although BRAF(V600E) mutation may play some roles in local carcinoma development, there is no evidence that BRAF(V600E) mutation significantly reflects the aggressive characteristics and poor prognosis of patients with papillary carcinoma in Japan.

The impact of KRAS mutations on cetuximab sensitivity in epidermal growth factor receptor fluorescence in situ hybridisation-positive (EGFR FISH+) metastatic colorectal cancer patients (mCRC) has not been previously investigated. In the present study, we analysed KRAS, BRAF, PI3KCA, MET, and IGF1R in 85 mCRC treated with cetuximab-based therapy in whom EGFR status was known. KRAS mutations (52.5%) negatively affected response only in EGFR FISH+ patients. EGFR FISH+/KRAS mutated had a significantly lower response rate (P=0.04) than EGFR FISH+/KRAS wild type patients. Four EGFR FISH+ patients with KRAS mutations responded to cetuximab therapy. BRAF was mutated in 5.0% of patients and none responded to the therapy. PI3KCA mutations (17.7%) were not associated to cetuximab sensitivity. Patients overexpressing IGF1R (74.3%) had significantly longer survival than patients with low IGF1R expression (P=0.006), with no difference in response rate. IGF1R gene amplification was not detected, and only two (2.6%) patients, both responders, had MET gene amplification. In conclusion, KRAS mutations are associated with cetuximab failure in EGFR FISH+ mCRC, even if it does not preclude response. The rarity of MET and IGF1R gene amplification suggests a marginal role in primary resistance. The potential prognostic implication of IGF1R expression merits further evaluation.

BACKGROUND

Mutations in the Kirsten Ras (KRAS) oncogene are common in colorectal cancer (CRC). The role of KRAS-mutation status as a prognostic factor, however, is unclear. We evaluated the relationship between KRAS-mutation status and CRC survival, considering heterogeneity in this association by tumour and patient characteristics.

METHODS

The population-based study included individuals diagnosed with CRC between 1998-2007 in Western Washington State. Tumour specimens were tested for KRAS exon 2 mutations, the BRAF p.V600E mutation, and microsatellite instability (MSI). We used Cox regression to estimate hazard ratios (HR) and 95% confidence intervals (CI) for the association between KRAS-mutation status and disease-specific and overall survival. Stratified analyses were conducted by age, sex, tumour site, stage, and MSI. We conducted additional analyses combining KRAS-mutation, BRAF-mutation, and MSI status.

RESULTS

Among 1989 cases, 31% had KRAS-mutated CRC. Kirsten Ras (KRAS)-mutated CRC was associated with poorer disease-specific survival (HR=1.37, 95% CI: 1.13-1.66). This association was not evident in cases who presented with distant-stage CRC. Cases with KRAS-wild-type/BRAF-wild-type/MSI-high CRC had the most favourable prognosis; those with CRC exhibiting a KRAS- or BRAF-mutation and no MSI had the poorest prognosis. Patterns were similar for overall survival.

CONCLUSIONS

Kirsten Ras (KRAS)-mutated CRC was associated with statistically significantly poorer survival after diagnosis than KRAS-wild-type CRC.

BACKGROUND

The microsatellite instability-high (MSI-H) phenotype, present in 15% of early colorectal cancer (CRC), confers good prognosis. MSI-H metastatic CRC is rare and its impact on outcomes is unknown. We describe survival outcomes and the impact of chemotherapy, metastatectomy, and BRAF V600E mutation status in the largest reported cohort of MSI-H metastatic colorectal cancer (CRC).

METHODS

A retrospective review of 55 MSI-H metastatic CRC patients from two institutions, Royal Melbourne Hospital (Australia) and The University of Texas MD Anderson Cancer Center (United States), was conducted. Statistical analyses utilized Kaplan-Meier method, Log-rank test, and Cox proportional hazards models.

RESULTS

Median age was 67 years (20-90), 58% had poor differentiation, and 45% had stage IV disease at presentation. Median overall survival (OS) from metastatic disease was 15.4 months. Thirteen patients underwent R0/R1 metastatectomies, with median OS from metastatectomy 33.8 months. Thirty-one patients received first-line systemic chemotherapy for metastatic disease with median OS from the start of chemotherapy 11.5 months. No statistically significant difference in progression-free survival or OS was seen between fluoropyrimidine, oxaliplatin, or irinotecan based chemotherapy. BRAF V600E mutation was present in 14 of 47 patients (30%). BRAF V600E patients demonstrated significantly worse median OS; 10.1 versus 17.3 months, P = 0.03. In multivariate analyses, BRAF V600E mutants had worse OS (HR 4.04; P = 0.005), while patients undergoing metastatectomy (HR 0.11; P = <0.001) and patients who initially presented as stage IV disease had improved OS (HR 0.27; P = 0.003).

CONCLUSIONS

Patients with MSI-H metastatic CRC do not appear to have improved outcomes. BRAF V600E mutation is a poor prognostic factor in MSI-H metastatic CRC.

OBJECTIVE

We conducted a basket clinical trial to assess the feasibility of such a design strategy and to independently evaluate the effects of multiple targeted agents against specific molecular aberrations in multiple histologic subtypes concurrently.

METHODS

We enrolled patients with advanced non-small-cell lung cancer (NSCLC), small-cell lung cancer, and thymic malignancies who underwent genomic characterization of oncogenic drivers. Patients were enrolled onto a not-otherwise-specified arm and treated with standard-of-care therapies or one of the following five biomarker-matched treatment groups: erlotinib for EGFR mutations; selumetinib for KRAS, NRAS, HRAS, or BRAF mutations; MK2206 for PIK3CA, AKT, or PTEN mutations; lapatinib for ERBB2 mutations or amplifications; and sunitinib for KIT or PDGFRA mutations or amplification.

RESULTS

Six hundred forty-seven patients were enrolled, and 88% had their tumors tested for at least one gene. EGFR mutation frequency was 22.1% in NSCLC, and erlotinib achieved a response rate of 60% (95% CI, 32.3% to 83.7%). KRAS mutation frequency was 24.9% in NSCLC, and selumetinib failed to achieve its primary end point, with a response rate of 11% (95% CI, 0% to 48%). Completion of accrual to all other arms was not feasible. In NSCLC, patients with EGFR mutations had the longest median survival (3.51 years; 95% CI, 2.89 to 5.5 years), followed by those with ALK rearrangements (2.94 years; 95% CI, 1.66 to 4.61 years), those with KRAS mutations (2.3 years; 95% CI, 2.3 to 2.17 years), those with other genetic abnormalities (2.17 years; 95% CI, 1.3 to 2.74 years), and those without an actionable mutation (1.85 years; 95% CI, 1.61 to 2.13 years).

CONCLUSIONS

This basket trial design was not feasible for many of the arms with rare mutations, but it allowed the study of the genetics of less common malignancies.

UNASSIGNED

Whole-exome sequencing (WES) has the potential to reveal tumor and germline mutations of clinical relevance, but the diagnostic yield for pediatric patients with solid tumors is unknown.

UNASSIGNED

To characterize the diagnostic yield of combined tumor and germline WES for children with solid tumors.

UNASSIGNED

Unselected children with newly diagnosed and previously untreated central nervous system (CNS) and non-CNS solid tumors were prospectively enrolled in the BASIC3 study at a large academic children's hospital during a 23-month period from August 2012 through June 2014. Blood and tumor samples underwent WES in a certified clinical laboratory with genetic results categorized on the basis of perceived clinical relevance and entered in the electronic health record.

UNASSIGNED

Clinical categorization of somatic mutations; frequencies of deleterious germline mutations related to patient phenotype and incidental medically-actionable mutations.

UNASSIGNED

Of the first 150 participants (80 boys and 70 girls, mean age, 7.4 years), tumor samples adequate for WES were available from 121 patients (81%). Somatic mutations of established clinical utility (category I) were reported in 4 (3%) of 121 patients, with mutations of potential utility (category II) detected in an additional 29 (24%) of 121 patients. CTNNB1 was the gene most frequently mutated, with recurrent mutations in KIT, TSC2, and MAPK pathway genes (BRAF, KRAS, and NRAS) also identified. Mutations in consensus cancer genes (category III) were found in an additional 24 (20%) of 121 tumors. Fewer than half of somatic mutations identified were in genes known to be recurrently mutated in the tumor type tested. Diagnostic germline findings related to patient phenotype were discovered in 15 (10%) of 150 cases: 13 pathogenic or likely pathogenic dominant mutations in adult and pediatric cancer susceptibility genes (including 2 each in TP53, VHL, and BRCA1), 1 recessive liver disorder with hepatocellular carcinoma (TJP2), and 1 renal diagnosis (CLCN5). Incidental findings were reported in 8 (5%) of 150 patients. Most patients harbored germline uncertain variants in cancer genes (98%), pharmacogenetic variants (89%), and recessive carrier mutations (85%).

UNASSIGNED

Tumor and germline WES revealed mutations in a broad spectrum of genes previously implicated in both adult and pediatric cancers. Combined reporting of tumor and germline WES identified diagnostic and/or potentially actionable findings in nearly 40% of newly diagnosed pediatric patients with solid tumors.

BACKGROUND

Appropriate patient selection is needed for targeted therapies that are efficacious only in patients with specific genetic alterations. We aimed to define subgroups of patients with candidate driver genes in patients with non-small cell lung cancer.

METHODS

Patients with primary lung cancer who underwent clinical genetic tests at Guangdong General Hospital were enrolled. Driver genes were detected by sequencing, high-resolution melt analysis, qPCR, or multiple PCR and RACE methods.

RESULTS

524 patients were enrolled in this study, and the differences in driver gene alterations among subgroups were analyzed based on histology and smoking status. In a subgroup of non-smokers with adenocarcinoma, EGFR was the most frequently altered gene, with a mutation rate of 49.8%, followed by EML4-ALK (9.3%), PTEN (9.1%), PIK3CA (5.2%), c-Met (4.8%), KRAS (4.5%), STK11 (2.7%), and BRAF (1.9%). The three most frequently altered genes in a subgroup of smokers with adenocarcinoma were EGFR (22.0%), STK11 (19.0%), and KRAS (12.0%). We only found EGFR (8.0%), c-Met (2.8%), and PIK3CA (2.6%) alterations in the non-smoker with squamous cell carcinoma (SCC) subgroup. PTEN (16.1%), STK11 (8.3%), and PIK3CA (7.2%) were the three most frequently enriched genes in smokers with SCC. DDR2 and FGFR2 only presented in smokers with SCC (4.4% and 2.2%, respectively). Among these four subgroups, the differences in EGFR, KRAS, and PTEN mutations were statistically significant.

CONCLUSIONS

The distinct features of driver gene alterations in different subgroups based on histology and smoking status were helpful in defining patients for future clinical trials that target these genes. This study also suggests that we may consider patients with infrequent alterations of driver genes as having rare or orphan diseases that should be managed with special molecularly targeted therapies.

BACKGROUND

In colorectal cancer (CRC), DNA methylation anomalies define distinct subgroups termed CpG island methylator phenotype 1 (CIMP1), CIMP2, and CIMP-negative. The role of this classification in predicting recurrence and disease-free survival (DFS) in resected stage III CRC was evaluated.

METHODS

Sporadic cancers from 161 patients were analyzed. Bisulfite pyrosequencing was used to examine the methylation of 2 global DNA methylation markers (LINE-1, Alu) and 9 loci (MINT1, MINT2, MINT31, P16, hMLH1, P14, SFRP1, SFRP2, and WNT5A). Mutations in BRAF and KRAS were assayed.

RESULTS

Gene hypermethylation clustered in discrete groups of patients, indicating the presence of CIMP. K-means clustering analysis identified 3 discrete subgroups: CIMP1 (n = 22, 13.7%), associated with proximal location and BRAF mutations; CIMP2 (n = 40, 24.8%), associated with KRAS mutations; and CIMP-negative (n = 99, 61.5%), associated with distal location. In proximal CRC, CIMP1 was correlated with a higher recurrence rate (53% for CIMP1, 18% for CIMP2, and 26% for CIMP-negative) and a worse DFS (P = .015). Also in proximal CRC, LINE-1 methylation was lower in patients whose cancer recurred compared with those whose cancer did not recur (P = .049). In multivariate analysis, CIMP1 and low LINE1 methylation were independent prognostic factors for DFS in proximal CRC (P = .008 for classification by K-means clustering analysis; P = .040 for LINE-1 methylation status).

CONCLUSIONS

DNA methylation is a useful biomarker of recurrence in resected stage III proximal but not distal CRC. However, as the number of CIMP1 cases was small in distal CRC, further study is required to validate our findings.

Anaplastic thyroid carcinoma (ATC) is a frequently lethal malignancy that is often unresponsive to available therapeutic strategies. The tumorigenesis of ATC and its relationship to the widely prevalent well-differentiated thyroid carcinomas are unclear. We have analyzed 22 cases of ATC as well as 4 established ATC cell lines using whole-exome sequencing. A total of 2674 somatic mutations (121/sample) were detected. Ontology analysis revealed that the majority of variants aggregated in the MAPK, ErbB and RAS signaling pathways. Mutations in genes related to malignancy not previously associated with thyroid tumorigenesis were observed, including mTOR, NF1, NF2, MLH1, MLH3, MSH5, MSH6, ERBB2, EIF1AX and USH2A; some of which were recurrent and were investigated in 24 additional ATC cases and 8 ATC cell lines. Somatic mutations in established thyroid cancer genes were detected in 14 of 22 (64%) tumors and included recurrent mutations in BRAF, TP53 and RAS-family genes (6 cases each), as well as PIK3CA (2 cases) and single cases of CDKN1B, CDKN2C, CTNNB1 and RET mutations. BRAF V600E and RAS mutations were mutually exclusive; all ATC cell lines exhibited a combination of mutations in either BRAF and TP53 or NRAS and TP53. A hypermutator phenotype in two cases with >8 times higher mutational burden than the remaining mean was identified; both cases harbored unique somatic mutations in MLH mismatch-repair genes. This first comprehensive exome-wide analysis of the mutational landscape of ATC identifies novel genes potentially associated with ATC tumorigenesis, some of which may be targets for future therapeutic intervention.

OBJECTIVE

To establish a series of clinically relevant orthotopic transplantation models of human pancreatic adenocarcinoma from both cell lines and primary tumors under uniform experimental conditions.

METHODS

Ten pancreatic cancer cell lines and 12 primary tumors were orthotopically transplanted in SCID mice. The cell lines and xenografts were characterized for K-ras, BRAF, p53, p16, and DPC4 aberrations employing direct sequencing, immunohistochemistry, and Western blotting.

RESULTS

All xenografts showed high intrapancreatic tumorigenicity and extensive local tumor growth, and each showed a unique behavioral and genetic profile. Tumor characteristics were retained during serial passaging. The cell line-derived xenografts represented the entire expected range of histologic differentiation. Although the overall metastatic rate was moderate to high, the metastatic pattern varied; 4 cell lines showed a high metastatic rate to the liver. The primary tumor-derived xenografts retained their similarity to the corresponding original donor tumors with regard to histologic presentation and biologic behavior. K-ras, p53, p16, and DPC4 aberrations were revealed in 80%, 70%, 50%, and 40% of cell lines and 100%, 33%, 75%, and 58% of primary tumor derived xenografts, respectively. No BRAF mutations were present. The metastatic behavior of the xenografts was significantly associated with the degree of histologic differentiation, number of genes altered, and p53 status.

CONCLUSIONS

The new models reflected the wide range of patho-biological features and genetic alterations that characterize human pancreatic cancer and may be used collectively or selectively as a markedly improved in vivo tool for preclinical and molecular studies of pancreatic cancer.

The goal of ovarian cancer screening is to detect disease when confined to the ovary (stage I) and thereby prolong survival. We believe this is an elusive goal because most ovarian cancer, at its earliest recognizable stage, is probably not confined to the ovary. We propose a new model of ovarian carcinogenesis based on clinical, pathological, and molecular genetic studies that may enable more targeted screening and therapeutic intervention to be developed. The model divides ovarian cancer into 2 groups designated type I and type II. Type I tumors are slow growing, generally confined to the ovary at diagnosis and develop from well-established precursor lesions so-called borderline tumors. Type I tumors include low-grade micropapillary serous carcinoma, mucinous, endometrioid, and clear cell carcinomas. They are genetically stable and are characterized by mutations in a number of different genes including KRAS, BRAF, PTEN, and beta-catenin. Type II tumors are rapidly growing, highly aggressive neoplasms that lack well-defined precursor lesions; most are advanced stage at, or soon after, their inception. These include high-grade serous carcinoma, malignant mixed mesodermal tumors (carcinosarcomas), and undifferentiated carcinomas. The type II tumors are characterized by mutation of TP53 and a high level of genetic instability. Screening tests that focus on stage I disease may detect low-grade type I neoplasms but miss the more aggressive type II tumors, which account for most ovarian cancers. A more rational approach to early detection of ovarian cancer should focus on low volume rather than low stage of disease.

Papillary thyroid carcinomas are the most common thyroid cancers and constitute more than 70% of thyroid malignancies. The most common etiologic factor is radiation, but genetic susceptibility and other factors also contribute to the development of papillary thyroid carcinoma. The most common variants include conventional, follicular variant and tall cell variant. However, many other uncommon variants have been described including oncocytic, columnar cell, diffuse sclerosing and solid forms. Immunohistochemical staining with TTF-1 and thyroglobulin is very useful in confirming the diagnosis of papillary thyroid carcinoma especially in metastatic sites. Markers such as HBME-1 and CITED1 can assist in separating some difficult cases of follicular variants of papillary thyroid carcinomas from follicular adenomas. Molecular studies have shown that the BRAF V600E mutation is found mainly in papillary and anaplastic thyroid carcinomas. Other molecular markers such as HMGA2 and insulin-like growth factor II mRNA binding protein 3 have been used recently as molecular tests to separate papillary thyroid carcinoma and its variants from follicular adenomas and other benign thyroid nodules.

OBJECTIVE

STAT3 is a transcription factor that is constitutively activated in some cancers. It seems to play crucial roles in cell proliferation and survival, angiogenesis, tumor-promoting inflammation, and suppression of antitumor host immune response in the tumor microenvironment. Although the STAT3 signaling pathway is a potential drug target, clinical, pathologic, molecular, or prognostic features of STAT3-activated colorectal cancer remain uncertain.

METHODS

Utilizing a database of 724 colon and rectal cancer cases, we evaluated phosphorylated STAT3 (p-STAT3) expression by immunohistochemistry. The Cox proportional hazards model was used to compute mortality HR, adjusting for clinical, pathologic, and molecular features, including microsatellite instability (MSI), the CpG island methylator phenotype (CIMP), LINE-1 methylation, 18q LOH, TP53 (p53), CTNNB1 (β-catenin), JC virus T-antigen, and KRAS, BRAF, and PIK3CA mutations.

RESULTS

Among the 724 tumors, 131 (18%) showed high-level p-STAT3 expression (p-STAT3-high), 244 (34%) showed low-level expression (p-STAT3-low), and the remaining 349 (48%) were negative for p-STAT3. p-STAT3 overexpression was associated with significantly higher colorectal cancer-specific mortality [log-rank P = 0.0020; univariate HR (p-STAT3-high vs. p-STAT3-negative): 1.85, 95% CI: 1.30-2.63, P(trend) = 0.0005; multivariate HR: 1.61, 95% CI: 1.11-2.34, P(trend) = 0.015]. p-STAT3 expression was positively associated with peritumoral lymphocytic reaction (multivariate OR: 3.23; 95% CI: 1.89-5.53, P < 0.0001). p-STAT3 expression was not associated with MSI, CIMP, or LINE-1 hypomethylation.

CONCLUSIONS

STAT3 activation in colorectal cancer is associated with adverse clinical outcome, supporting its potential roles as a prognostic biomarker and a chemoprevention and/or therapeutic target.

OBJECTIVE

In the era of DNA-guided personalized cancer treatment, it is essential to conduct predictive analysis on the tissue that matters. Here, we analyzed genetic differences between primary colorectal adenocarcinomas (CRC) and their respective hepatic metastasis.

METHODS

The primary CRC and the subsequent hepatic metastasis of 21 patients with CRC were analyzed using targeted deep-sequencing of DNA isolated from formalin-fixed, paraffin-embedded archived material.

RESULTS

We have interrogated the genetic constitution of a designed "Cancer Mini-Genome" consisting of all exons of 1,264 genes associated with pathways relevant to cancer. In total, 6,696 known and 1,305 novel variations were identified in 1,174 and 667 genes, respectively, including 817 variants that potentially altered protein function. On average, 83 (SD = 69) potentially function-impairing variations were gained in the metastasis and 70 (SD = 48) variations were lost, showing that the primary tumor and hepatic metastasis are genetically significantly different. Besides novel and known variations in genes such as KRAS, BRAF, KDR, FLT1, PTEN, and PI3KCA, aberrations in the up/downstream genes of EGFR/PI3K/VEGF-pathways and other pathways (mTOR, TGFβ, etc.) were also detected, potentially influencing therapeutic responsiveness. Chemotherapy between removal of the primary tumor and the metastasis (N = 11) did not further increase the amount of genetic variation.

CONCLUSIONS

Our study indicates that the genetic characteristics of the hepatic metastases are different from those of the primary CRC tumor. As a consequence, the choice of treatment in studies investigating targeted therapies should ideally be based on the genetic properties of the metastasis rather than on those of the primary tumor.